Latest Articles Include:
- Cancer drugs: remedy required
- Nat Med 17(3):231 (2011)
Nature Medicine | Editorial Cancer drugs: remedy required Journal name:Nature MedicineVolume: 17,Page:231Year published:(2011)DOI:doi:10.1038/nm0311-231Published online07 March 2011 Cancer drugs often impair quality of life and fail to extend patient survival. Mandating increased efficacy and promoting efforts to target tumor metastasis may improve outcomes for patients with cancer. View full text Additional data - Fearful of Avastin's fate, Genentech asks for unusual hearing
- Nat Med 17(3):233 (2011)
Nature Medicine | News Fearful of Avastin's fate, Genentech asks for unusual hearing * Mark RatnerJournal name:Nature MedicineVolume: 17,Page:233Year published:(2011)DOI:doi:10.1038/nm0311-233Published online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The US Food and Drug Administration sent shockwaves through the medical community last year when it stated its plans to revoke marketing approval for the monoclonal antibody treatment Avastin (bevacizumab) in combination with the chemotherapy drug paclitaxel for first-line treatment of metastatic breast cancer. But rather than taking the blow sitting down, Genentech—which makes Avastin—is contesting the FDA's plans. The drugmaker's move, in the form of a 98-page petition submitted in January in support of its request for a public hearing, is unprecedented, and some analysts quietly worry it could jeopardize the goodwill between the company and regulators. In its arguments to the FDA, Genentech, a South San Francisco–based unit of the Swiss pharma giant Roche, is keeping the debate focused on questions of science and proof of efficacy, rather than issues of access and reimbursement, according to Cole Werble of Prevision Policy, a health care policy analysis consultancy in Washington, DC. "Genentech is using the debate with the FDA to create a public record," he says. "It allows them to make the arguments for the oncology community to see." The FDA's regulations provide for public participation at a hearing, but a Genentech spokesperson told Nature Medicine in an email that it has no plans to ask patient advocacy groups or payers to present on its behalf. The FDA gave Avastin the go-ahead for use in metastatic Her2-negative breast cancer in 2008 under its accelerated approval process based on a study showing a 5.5-month improvement in patients' median progression-free survival (J. Clin. Oncol.27, 4966–4972, 2009). Two subsequent trials examining Avastin with other types of chemotherapy showed a less striking improvement in this endpoint measure. That follow-up information led an FDA advisory committee to vote 12-1 last summer in favor of removing approval for Avastin's use in metastatic breast cancer, saying the drug's benefits did not outweigh its risks. Its approvals in other cancers are unaffected. AP Photo/Paul Sakuma Will a public hearing sway regulators to soften their stance on the cancer drug? No pharmaceutical firm has ever asked for a hearing to challenge an FDA proposal to withdraw a single indication for a drug on the basis of data gathered as part of post-marketing commitments. That said, there have only been a handful of cases where the regulator has limited marketing authorization based on post-approval data. Pfizer voluntarily withdrew the leukemia drug Mylotarg (gemtuzumab ozogamicin) last year following a high death rate in a confirmatory trial conducted as part of its post-marketing commitments after accelerated approval. And in 2005, MedImmune decided to withdraw an indication for Ethyol (amifostine), its drug to reduce the side-effects of chemotherapy and radiation, just as the FDA was about to review post-marketing study data on Ethyol's use in non-small cell lung cancer. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Mark Ratner Search for this author in: * NPG journals * PubMed * Google Scholar - Whistleblower protections for US government scientists flounder
- Nat Med 17(3):234 (2011)
Nature Medicine | News Whistleblower protections for US government scientists flounder * Megan ScudellariJournal name:Nature MedicineVolume: 17,Page:234Year published:(2011)DOI:doi:10.1038/nm0311-234aPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In December, after more than a decade of negotiations and hearings, both houses of the US Congress unanimously passed a bipartisan bill aimed at providing legal safeguards for federal employees who disclose alleged wrongdoing occurring in government. The bill, called the Whistleblower Protection Enhancement Act, seemed poised to be signed into law. But late in the afternoon on the final day of the congressional session, an anonymous Republican senator thwarted the antisecrecy reform by placing a secret 'hold' on the bill, which effectively killed the measure. Now, with a new Congress in session, the bill—which, for the first time, included specific protections for federal scientists—must be reintroduced for a new round of voting and potential roadblocks. And, until it passes, federal scientists preparing to report abuse, waste or fraud in the government may be better off holding their tongues, experts say. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Megan Scudellari Search for this author in: * NPG journals * PubMed * Google Scholar - Bill to help Canadian companies ship generics has uncertain future
- Nat Med 17(3):234 (2011)
Nature Medicine | News Bill to help Canadian companies ship generics has uncertain future * Hannah HoagJournal name:Nature MedicineVolume: 17,Page:234Year published:(2011)DOI:doi:10.1038/nm0311-234bPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Backed by nongovernmental organizations and the generics industry, the left-of-center New Democratic Party has championed a bill that set out to improve Canada's Access to Medicines Regime (CAMR), a law that enables drug manufacturers in the country to make generic medications for shipment to developing countries to treat illnesses such as tuberculosis and AIDS. The bill, C-393, was introduced to the House of Commons in 2009 and aimed to eliminate many of the CAMR procedures that its supporters consider unwieldy and extend the list of eligible drugs. But the bill has been so gutted that many global health advocates say they cannot support it in its current state, and it is floundering in Canada's parliament. Under the existing legislation, generic manufacturers that are unable to negotiate a voluntary license from the patent holders can ask the Canadian Commissioner of Patents for a compulsory license to produce an eligible product to address public health problems in another country. If the commissioner says yes, the law then authorizes a one-time license for a named product, along with the country to which it is to be shipped and order size. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Hoag Search for this author in: * NPG journals * PubMed * Google Scholar - Alzheimer's researchers call for clinical revamp
- Nat Med 17(3):235 (2011)
Nature Medicine | News Alzheimer's researchers call for clinical revamp * Melinda Wenner MoyerJournal name:Nature MedicineVolume: 17,Page:235Year published:(2011)DOI:doi:10.1038/nm0311-235aPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Current treatment options for Alzheimer's disease leave much to be desired. Existing medications, which either prevent the breakdown of neurotransmitters or modulate key receptors in the brain, can temporarily ease some of the cognitive decline associated with the disease, but they do nothing to halt or reverse its progression. And although scientists are developing new therapeutics that target the cause of the Alzheimer's more directly, even these latest experimental drugs might do little to help patients. To make headway, some Alzheimer's experts now argue that the research community must fundamentally change how it diagnoses the disease and designs clinical trials. "The way we need to get at the disease is through prevention and through presymptomatic therapy, as opposed to classic therapy," says Dale Bredesen, a neurologist at the Buck Institute for Research on Aging in Novato, California. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Melinda Wenner Moyer Search for this author in: * NPG journals * PubMed * Google Scholar - Facing budget cuts, Spain launches funding foundation
- Nat Med 17(3):235 (2011)
Nature Medicine | News Facing budget cuts, Spain launches funding foundation * Lucas LaursenJournal name:Nature MedicineVolume: 17,Page:235Year published:(2011)DOI:doi:10.1038/nm0311-235bPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. MADRID — In Spain, the government's overall spending on research is set to wither by about 8% this year, according to an analysis released last fall by the Confederation of Spanish Scientific Societies. Given the climate of budget cuts, it's perhaps no surprise that scientists there are turning to the public for funding. Historically, Spain has fallen behind other nations in Europe when it comes to private giving for research. A Eurobarometer report published last summer said that 28% of people in the country reported having donated money to fundraising campaigns for medical research, below the EU average of 39%. By comparison, 78% and 70% of individuals surveyed in the Netherlands and UK said they had given money for these campaigns. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Lucas Laursen Search for this author in: * NPG journals * PubMed * Google Scholar - Government-funded journal seen by some as waste of grant money
- Nat Med 17(3):236 (2011)
Nature Medicine | News Government-funded journal seen by some as waste of grant money * Michelle PflummJournal name:Nature MedicineVolume: 17,Page:236Year published:(2011)DOI:doi:10.1038/nm0311-236aPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. This summer, California's $3 billion stem cell agency is scheduled to launch a state-subsidized open-access research journal. With a $600,000 taxpayer-backed starting fund, the publication is intended as a forum for translational scientists and regulators geared toward moving stem cell–based therapies to the clinic. But with more than a dozen stem cell–focused journals already crowding library shelves and a limited agency budget, many critics wonder whether this is the best use of government research dollars. "They need to demonstrate a need, and I don't think they have done that," says John Simpson, stem cell project director of Consumer Watchdog, an advocacy group based in Santa Monica, California. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Michelle Pflumm Search for this author in: * NPG journals * PubMed * Google Scholar - Despite surge in orphan drug designations, approvals still lag
- Nat Med 17(3):236 (2011)
Nature Medicine | News Despite surge in orphan drug designations, approvals still lag * Monica HegerJournal name:Nature MedicineVolume: 17,Page:236Year published:(2011)DOI:doi:10.1038/nm0311-236bPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In 1983, US lawmakers passed the Orphan Drug Act to encourage pharmaceutical companies to pursue treatments for largely ignored diseases affecting small populations. And for the next 15 years or so, the number of rare diseases given orphan drug status hovered between about 40 and 80 per year. But over the last decade, that number began steadily increasing, and last year the US Food and Drug Administration (FDA) granted a record 192 designations. "There's been a substantial upsurge of interest in orphan drugs and rare disorders," says James Cloyd, director of the Center for Orphan Drug Research at the University of Minnesota–Twin Cities College of Pharmacy. According to Cloyd, reasons for this jump include a push from federal regulators and patient advocacy groups to target rare diseases, new genomic technologies that allows researchers to subtype diseases into rare niches, and the death of the blockbuster model of drug development View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Monica Heger Search for this author in: * NPG journals * PubMed * Google Scholar - Antidepressants tested for everything from hot flashes to stroke
- Nat Med 17(3):237 (2011)
Nature Medicine | News Antidepressants tested for everything from hot flashes to stroke * Michelle PflummJournal name:Nature MedicineVolume: 17,Page:237Year published:(2011)DOI:doi:10.1038/nm0311-237aPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Antidepressants known as selective serotonin reuptake inhibitors (SSRIs) have become a fixture in many medicine cabinets ever since Prozac, the first such drug to receive US approval, hit the market in 1987. According to the health analytics firm SDI, in 2009 physicians wrote an estimated 52 million SSRI prescriptions for Americans aged 45 to 65 alone. Now, as evidenced by a flurry of papers this year, SSRIs are emerging as a possible treatment for a range of ailments from mood disorders to menopause. "It's only a historical accident that these drugs were used for depression," says psychiatrist James Hudson, co-director of the Biological Psychiatry Laboratory at McLean Hospital in Belmont, Massachusetts. Although the exact mechanism for SSRIs' action against depression remains unclear, the drugs are known to block the recycling of serotonin, making this important signaling chemical more available to trigger receptors at brain cell synapses. Given that some people taking SSRIs for depression experience a loss of appetite, it's perhaps not surprising that one of the earliest alternative uses of these drugs was the treatment of bulimia. Indeed, when people with this eating disorder took Prozac (fluoxetine), they experienced a reduction in binge eating, leading to fewer bulimic episodes (Am. J. Psychiatry, 1756–1762, 1988). Gerard Karsenty of Columbia University Medical Center in New York, says that SSRIs, which keep serotinin outside of cells, might actually decrease the amount of this neurotransmitter inside certain brain cells, and this intracellular drop in serotonin might suppress appetite. In one experiment his group conducted, mice genetically engineered to produce less serotonin in neurons ate about 25% less than their control counterparts (Cell138, 976–989, 2009). A subsequent study published in January indicated that blocking serotonin signaling in certain neurons caused mice deficient in the appetite-suppressing hormone leptin to eat less (J. Exp. Med., 41–52, 2011). istockphoto Researchers repurpose antidepressants to treat other diseases. Also in January, a team of doctors led by Ellen Freeman of the University of Pennsylvania School of Medicine in Philadelphia reported that the SSRI Lexapro (escitalopram) may be an alternative to hormone replacement therapy to reduce hot flashes. In an eight-week trial involving 205 healthy, menopausal women, researchers found that 55% of women taking Lexapro experienced half as many hot flashes as before. By comparison, only 36% of those on placebo experienced the same reduction (J. Am. Med Assoc., 267–274, 2011). SSRIs, however, may contribute to bone loss, so these drugs may not be a good alternative to hormone replacement therapy for menopausal women prone to osteoporosis (Arch. Intern. Med.167, 1240–1245, 2007). View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Michelle Pflumm Search for this author in: * NPG journals * PubMed * Google Scholar - Correction: 'Genzyme, though unique, could be a bellweather for US biotech'
- Nat Med 17(3):237 (2011)
Nature Medicine | News Correction: 'Genzyme, though unique, could be a bellweather for US biotech' Journal name:Nature MedicineVolume: 17,Page:237Year published:(2011)DOI:doi:10.1038/nm0311-237bPublished online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In the February 2011 issue, the article entitled "Genzyme, though unique, could be a bellwether for US biotech" (Nat. Med.17, 145, 2011) incorrectly stated that GlaxoSmithKline had acquired AstraZeneca. Although GlaxoSmithKline has acquired medicines from AstraZeneca in the past, the two companies never merged. The error has been corrected in the HTML and PDF versions of the article. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - News in brief: Biomedical briefing
- Nat Med 17(3):238-239 (2011)
Nature Medicine | News News in brief: Biomedical briefing Journal name:Nature MedicineVolume: 17,Pages:238–239Year published:(2011)DOI:doi:10.1038/nm0311-238Published online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. POLICY Funding feud US lawmakers drew lines in the sand last month over spending for science. On 14 February, President Barack Obama released his administration's vision for next year's federal budget, which included requested increases of around $750 million for the National Institutes of Health (NIH), $900 million for the National Science Foundation (NSF) and $150 million for the US Food and Drug Administration (FDA) relative to the 2010 enacted budget. The White House also proposed to cut discretionary spending at the Centers for Disease Control and Prevention (CDC) by approximately $600 million, although the agency will see some $750 million in new money from the health care law passed last year. A bill released three days earlier from the Republican-controlled House of Representatives' Appropriations Committee proposed to trim all these agencies budgets by at least 5% relative to 2010 spending levels. NIH reshuffle The NIH is moving ahead with its plan to dissolve the National Center for Research Resources, which funds tools and training for scientists, and open a new center devoted to translational research. In mid-January, agency officials released details of the proposed reshuffle—slated for 1 October—which involves, among other measures, transferring the Clinical and Translational Science Awards program to the new National Center for Advancing Translational Sciences. Many onlookers applauded the changeover, but critics voiced concern about the haste of move. "I think the process has been rushed, and the [scientific] community is understandably concerned, frustrated and a bit angry," says Jeremy Berg, director of the National Institute of General Medical Sciences. See go.nature.com/9tMVfw for more. Hedging ties In addition to disclosing financial relationships with drug and medical device makers, study authors may soon have to divulge ties to hedge funds and other investment firms. The New England Journal of Medicine, JAMA, the Lancet and 11 other medical journals will consider the requirement at their annual meeting in June. Former NEJM editor-in-chief Jerome Kassirer, a kidney disease specialist at Tufts University School of Medicine in Boston, praised the move, but notes that disclosure "doesn't solve the problem of conflicts of interest." CDC PrEPares Two months after researchers reported that taking a daily pill can greatly reduce the risk of contracting HIV in sexually active gay and bisexual men, the US Centers for Disease Control and Prevention issued its first recommendations about how best to use so-called pre-exposure prophylaxis (PrEP). On 28 January, the agency released interim guidelines stating that only men at high risk of becoming infected with HIV—for example, those who have sex with other men and have multiple partners—should use antiretroviral drugs such as Gilead's Truvada prophylactically. But experts caution that PrEP is not a replacement for safe sex practices. "It should only be given as a part of a comprehensive HIV prevention strategy including using condoms," says Grant Colfax, director of the HIV prevention and research at the San Francisco Department of Public Health. Expedited devices For close to two decades, the US Food and Drug Administration (FDA) has had an accelerated review process for drugs in place, but no comparable expedited system for medical devices. Now, a prosthetic arm will be the first product assessed under the agency's Innovation Pathway Program, unveiled last month, which aims to review first-of-a-kind devices in under five months. "Now is the time to assure that we have a regulatory pathway to market that will help facilitate the development and assessment of the truly breakthrough technologies," says Jeffrey Shuren, director of the FDA's Center for Devices and Radiological Health. See go.nature.com/BPSxZC for more. Global Fund fraud The Global Fund to Fight AIDS, Tuberculosis and Malaria received unwanted attention in late January when news broke that its internal investigators had uncovered extensive fraud, including $3.5 million of undocumented spending in Zambia alone. The Geneva-based organization, which has distributed $13 billion in aid to 150 countries since its inception in 2002, subsequently announced that it has demanded the recovery of $34 million from countries confirmed of misappropriating funds. In response to the controversy, on 8 February the Global Fund said it would convene a panel of international experts to review its operations. The group's report is expected in May. PEOPLE Cytokine citation The 2011 Japan Prize in bioscience and medical science was awarded in January to Tadamitsu Kishimoto and Toshio Hirano from Osaka University. The two researchers, who will share the $600,000 prize, discovered that interleukin-6 (IL-6), a cytokine released by immune cells in response to injury, promotes fever and protective responses to infection. The pair also helped develop a monoclonal antibody drug, called tocilizumab, to block IL-6 activity in people with rheumatoid arthritis. CIRM hires VP Ellen Feigal After 18 months of searching for a suitable candidate, the California Institute for Regenerative Medicine (CIRM) finally hired its first vice president for research and development, a position created in 2009 to replace the job of chief scientific officer. Ellen Feigal, a former executive medical director at Amgen in Thousand Oaks, California, stepped into the role on 31 January. She is tasked with working with bench scientists, regulatory bodies and the drug industry to help translate preclinical research from the agency's science portfolio into marketable therapies. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Straight talk with...Donna Ambrosino
- Nat Med 17(3):240 (2011)
Nature Medicine | News Straight talk with...Donna Ambrosino * Elie DolginJournal name:Nature MedicineVolume: 17,Page:240Year published:(2011)DOI:doi:10.1038/nm0311-240Published online07 March 2011 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the 1890s, a diphtheria epidemic was killing thousands of children across the US, prompting many state health departments to create laboratories to start extracting antiserum from horses' blood. More than a century later, however, only one of these public labs is still in operation: the Massachusetts Biologic Laboratories, known simply as MassBiologics. Established in 1894 as the Massachusetts Public Health Laboratories, it is now the only nonprofit, licensed vaccine and biologics manufacturer and research center in the country—which gives the lab a unique position to tackle diseases that 'big pharma' isn't willing to touch. Leading the charge is Executive Director Donna Ambrosino, who took the helm in 1998 after a 20-year career at the Dana Farber Cancer Institute in Boston. On a snowy day in January, sat down to chat with Ambrosino at MassBiologics's brand new research center, opened last summer in the outskirts of Boston. View full text Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Reprogramming Rx
- Nat Med 17(3):241-243 (2011)
Nature Medicine | News Reprogramming Rx * Monya Baker1Journal name:Nature MedicineVolume: 17,Pages:241–243Year published:(2011)DOI:doi:10.1038/nm0311-241Published online07 March 2011 Patient-specific stem cells have been heralded as the next frontier in regenerative medicine. Although most therapies are probably a decade or more away, for one life-threatening skin disease the first clinical trials involving induced pluripotent stem cells may begin as early as 2014. investigates. View full text Additional data Affiliations * Monya Baker is technology editor for Nature and Nature Methods in San Francisco. Author Details * Monya Baker Search for this author in: * NPG journals * PubMed * Google Scholar - Despite steep costs, payments for new cancer drugs make economic sense
- Nat Med 17(3):244 (2011)
Nature Medicine | News Despite steep costs, payments for new cancer drugs make economic sense * Frank R Lichtenberg1Journal name:Nature MedicineVolume: 17,Page:244Year published:(2011)DOI:doi:10.1038/nm0311-244Published online07 March 2011 Cancer drugs have become more expensive over the past few years, leading many people to question whether the treatments are really worth their high costs. But despite the sticker shock, cancer medicines have provided good value for money. View full text Additional data Affiliations * Frank R. Lichtenberg is the Courtney C. Brown Professor of Business at Columbia University Graduate School of Business in New York and a research associate at the National Bureau of Economic Research in Cambridge, Massachusetts. His research has been supported by pharmaceutical and device companies as well as by government and nonprofit organizations. Author Details * Frank R Lichtenberg Search for this author in: * NPG journals * PubMed * Google Scholar - Hugging tight in Huntington's
- Nat Med 17(3):245-246 (2011)
Nature Medicine | Letter Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity * Wenjun Song1 * Jin Chen1 * Alejandra Petrilli1 * Geraldine Liot1 * Eva Klinglmayr2 * Yue Zhou1 * Patrick Poquiz3 * Jonathan Tjong3 * Mahmoud A Pouladi4 * Michael R Hayden4 * Eliezer Masliah5 * Mark Ellisman3 * Isabelle Rouiller6 * Robert Schwarzenbacher2 * Blaise Bossy1 * Guy Perkins3 * Ella Bossy-Wetzel1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:377–382Year published:(2011)DOI:doi:10.1038/nm.2313Received29 September 2010Accepted18 January 2011Published online20 February 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis1, 2. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, whi! ch, in turn, stimulates its enzymatic activity. Mutant huntingtin–mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease. View full text Figures at a glance * Figure 1: Mutant huntingtin triggers mitochondrial fragmentation, decreases in anterograde and retrograde transport and neuronal cell death, which depend on polyQ length. () Fluorescence micrographs (scale bar, 50 μm) and 6× zoom (scale bar, 8.33 μm) of boxed regions of neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito, a stain for mitochondria. () Mitochondrial fragmentation of neurons expressing HTTex1-Q17, HTTex1-Q46, or HTTex1-Q97 and DsRed2-Mito. () Cell death of neurons expressing HTTex1-Q17, -Q46 or -Q97. () Fluorescence micrographs (scale bar, 25 μm) and 3× zoom (scale bar, 8.33 μm) of boxed regions of MitoTracker Red–stained human fibroblasts from an unaffected individual (left) or an individual with adult-onset Huntington's disease (right). () Mitochondrial fragmentation in fibroblasts from an unaffected individual or an individual with Huntington's disease. () Kymographs of mitochondrial transport in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito. () Scatter plots of mitochondrial velocity in retrograde or anterograde direction as a function of distance traveled in 5 min in neu! rons (n = 10) expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito. () Anterograde and retrograde movement, motility and mean velocity of mitochondria in the same neurons analyzed in . Data are means ± s.e.m. of triplicate samples of representative experiments. Results are representative of three or more independent experiments. Statistics: one-way analysis of variance (ANOVA). * Figure 2: Mutant huntingtin increases the number of small mitochondria and cristae but decreases cristae surface area and volume in the striatum of six-month-old YAC128 mice. () Electron microscopy of control (top) and YAC128 (bottom) brain showing an elongated neuronal mitochondrion (arrowhead, top) and several short mitochondria (arrowheads, bottom), respectively. Scale bar, 500 nm for both. () Percentage of mitochondria of short length (0–1,000 nm) and long length (1,000–5,000 nm). () Electron microscopy of a control mitochondrion (arrowhead) nearly 4 μm long. Scale bar, 500 nm. () Surface-rendered volume showing the normal structure of the 84 cristae of the control mitochondrion. () Electron microscopy of a mitochondrion dividing into three parts (arrowheads). Scale bar, 500 nm. () A slice of the volume shows the separation of the three mitochondrial bodies (arrows). () Top, top view showing all 223 cristae. There are no cristae in the constriction (arrowhead) between the left and middle bodies, but a few cristae appear in the constriction between the middle and right bodies (arrow). Middle, side view of the outer membrane showing the wi! dth of the constrictions. Bottom, side view showing cristae fragmentation by how few cristae extend from top to bottom of the volume. () Number of cristae per mitochondrial cross-sectional area. (,) Cristae surface area () and volume (). Data are means ± s.e.m. Statistics: Student's t test, *P < 0.05, **P < 0.01. * Figure 3: Mutant huntingtin interacts with DRP1 in mice and humans with Huntington's disease and alters DRP1 structure and function. () Fluorescence micrographs of neurons expressing HTTex1 and DsRed2-Mito. Scale bar, 50 μm. Inset shows an 8× zoom of the boxed region. Scale bar, 6.25 μm. () Colocalization of mitochondria and huntingtin in neurons expressing HTTex1 and DsRed2-Mito. Data are means ± s.e.m. Statistics: one-way analysis of variance (ANOVA). () Fluorescence micrographs with line scan of colocalization of huntingtin, DRP1 and mitochondria in neurons expressing DsRed2-Mito, DRP1-YFP, and HTTex1. Scale bar, 10 μm. Inset shows a 5× zoom of the boxed region. Scale bar, 2 μm. () Coimmunoprecipitations (IP) of brain mitochondrial lysates from 1.5- or 2-month-old YAC18 and YAC128 mice followed with huntingtin- or Drp1-specific antibodies (Anti-HTT and Anti-DRP, respectively). The intensities of the signals are presented as arbitrary units (AU) and are normalized to input signals. () Coimmunoprecipitations of human lymphoblast lysates from unaffected individuals or individuals with Huntington's ! disease (HD) with huntingtin-specific antibodies. () Coimmunoprecipitations of human postmortem brain tissue lysates from unaffected individuals or individuals with Huntington's disease with DRP1- or huntingtin-specific antibodies. () Coimmunoprecipitations of bacterially expressed DRP1 protein and bacterially expressed huntingtinex1-Q20-GST or huntingtinex1-Q53-GST protein with DRP1-specific antibodies. () Steady-state kinetics of DRP1 GTPase activity (left), bar graph of GTPase activity at 0.05 mM GTP and apparent Michaelis-Menten constant (Km) (right) in the presence of wild-type or mutant huntingtinex1 protein and MOM liposomes. Data are means ± s.d. from three independent measurements. Statistics: Student's t test. () Negative-stain electron microscope images of baculovirus DRP1 in the absence of nucleotides (left), the presence of GTP (center) and the presence of GTP and huntingtinex1-Q53-GST protein (right). Scale bars, 10 nm. * Figure 4: Restoring mitochondrial fusion with DRP1 K38A or in combination with MFN2 RasG12V rescues neurons from neuritic trafficking defects and cell death. () Mitochondrial fragmentation of neurons expressing either HTTex1-Q17, HTTex1-Q46 or HTTex1 -Q97 alone or in combination with either DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Cell death of neurons expressing either mutant HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito alone or in combination with either DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Anterograde and retrograde movement of mitochondria in neurons expressing HTTex1-Q17HTTex1-Q46 or HTTex1-Q97 alone or in combination with DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Motility of mitochondria in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 alone or in combination with either DRP1 K38A alone or both DRP1 K38A and MFN2 RasG12V. () Mean velocity of mitochondria in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 alone or in combination with DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. Results are representative of three or more independent experiments. Statistics: Two-way AN! OVA with post hoc test. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_012062.3 * NM_005690.3 Author information * Accession codes * Author information * Supplementary information Affiliations * Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA. * Wenjun Song, * Jin Chen, * Alejandra Petrilli, * Geraldine Liot, * Yue Zhou, * Blaise Bossy & * Ella Bossy-Wetzel * Structural Biology Group, Department of Molecular Biology, University of Salzburg, Salzburg, Austria. * Eva Klinglmayr & * Robert Schwarzenbacher * National Center for Microscopy and Imaging Research, University of California–San Diego, San Diego, California, USA. * Patrick Poquiz, * Jonathan Tjong, * Mark Ellisman & * Guy Perkins * Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada. * Mahmoud A Pouladi & * Michael R Hayden * University of California–San Diego, La Jolla, California, USA. * Eliezer Masliah * Department of Anatomy and Cell Biology, McGill University, Montreal, Québec, Canada. * Isabelle Rouiller Contributions W.S. performed all imaging and participated in the mitochondrial fragmentation and cell death analyses. J.C. performed the GTPase assays, some of the immune precipitations and the electron microscopy analysis. A.P. performed some of the neuronal cell death and immune precipitations. G.L. performed the electron microscopy stereology and generated some of the preliminary data. E.K. purified, cloned and prepared the recombinant DRP1 protein. Y.Z. performed western blotting for the DRP1 knockdown. P.P. and J.T. participated in the electron microscope tomography. M.A.P. and M.R.H. provided the YAC18 and YAC128 mice and advice on huntingtin coimmunoprecipitations. E.M. provided human postmortem brain samples. R.S. led the DRP1 protein purification. M.E. and G.P. led the electron microscope tomography experiment. B.B. performed GTPase assays, prepared samples for electron microscopy and purified the huntingtin protein. I.R. performed electron microscope negative-stain experiments. ! E.B.-W. conceived the project and wrote the article. All authors participated in the data analysis and interpretation of the results. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ella Bossy-Wetzel Author Details * Wenjun Song Search for this author in: * NPG journals * PubMed * Google Scholar * Jin Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Alejandra Petrilli Search for this author in: * NPG journals * PubMed * Google Scholar * Geraldine Liot Search for this author in: * NPG journals * PubMed * Google Scholar * Eva Klinglmayr Search for this author in: * NPG journals * PubMed * Google Scholar * Yue Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Poquiz Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Tjong Search for this author in: * NPG journals * PubMed * Google Scholar * Mahmoud A Pouladi Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Hayden Search for this author in: * NPG journals * PubMed * Google Scholar * Eliezer Masliah Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Ellisman Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle Rouiller Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Schwarzenbacher Search for this author in: * NPG journals * PubMed * Google Scholar * Blaise Bossy Search for this author in: * NPG journals * PubMed * Google Scholar * Guy Perkins Search for this author in: * NPG journals * PubMed * Google Scholar * Ella Bossy-Wetzel Contact Ella Bossy-Wetzel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information Movies * Supplementary Video 1 (1M) Mitochondrial movement in a neuron expressing HTT exon1-Q17-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , top panel and shows mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 2 (967K) Mitochondrial movement in a neuron expressing HTT exon1-Q46-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , center panel and shows a clear decrease in mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 3 (623K) Mitochondrial movement in a neuron expressing HTT exon1-Q97-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , bottom panel and shows more pronounced arrest in mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 4 (8M) Electron tomography of a control mitochondrion in a medium spiny neuron. Movie showing the three-dimensional details of a mitochondrion in a medium spiny neuron reconstructed using electron tomography. These mitochondria are typically elongated along the direction of the axonal long axis. Clip 1: a rapid sequence through 190 slices (2.2 nm slice−1) of the tomographic volume that shows nearly the entire mitochondrial volume. There are 84 cristae. Clip 2: rotations and zooms of the surface-rendered volume after segmentation of the inner and outer membranes. The blue outer membrane is translucent to visualize the cristae displayed in various colors. Clip 3: rotation of the cristae after removal of the outer membrane to better distinguish the variety of shapes and sizes. * Supplementary Video 5 (10M) Electron tomography of a fissioning YAC128 mitochondrion in a medium spiny neuron. Movie showing the three-dimensional details of a mitochondrion fissioning into three parts in a medium spiny neuron reconstructed using electron tomography. Clip 1: a rapid sequence through 210 slices (2.2 nm slice−1) of the tomographic volume. There are 223 cristae, many of which are small. Clip 2: rotation showing the outer membrane and the widths of the two constriction sites. Clip 3: rotations showing the cristae in each of the three parts. Clip 4: rotations and zooms highlighting the cristae and the constriction sites. The blue outer membrane is translucent to visualize the cristae displayed in various colors. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Methods Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The other sweet face of XBP-1
- Nat Med 17(3):246-248 (2011)
Nature Medicine | Article Regulation of glucose homeostasis through a XBP-1–FoxO1 interaction * Yingjiang Zhou1 * Justin Lee1 * Candace M Reno2 * Cheng Sun1 * Sang Won Park1 * Jason Chung1 * Jaemin Lee1 * Simon J Fisher2 * Morris F White1 * Sudha B Biddinger1 * Umut Ozcan1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:356–365Year published:(2011)DOI:doi:10.1038/nm.2293Received15 October 2010Accepted16 December 2010Published online13 February 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To date, the only known role of the spliced form of X-box–binding protein-1 (XBP-1s) in metabolic processes has been its ability to act as a transcription factor that regulates the expression of genes that increase the endoplasmic reticulum (ER) folding capacity, thereby improving insulin sensitivity. Here we show that XBP-1s interacts with the Forkhead box O1 (FoxO1) transcription factor and directs it toward proteasome-mediated degradation. Given this new insight, we tested modest hepatic overexpression of XBP-1s in vivo in mouse models of insulin deficiency or insulin resistance and found it improved serum glucose concentrations, even without improving insulin signaling or ER folding capacity. The notion that XBP-1s can act independently of its role in the ER stress response is further supported by our finding that in the severely insulin resistant ob/ob mouse strain a DNA-binding–defective mutant of XBP-1s, which does not have the ability to increase ER folding capac! ity, is still capable of reducing serum glucose concentrations and increasing glucose tolerance. Our results thus provide the first evidence to our knowledge that XBP-1s, through its interaction with FoxO1, can bypass hepatic insulin resistance independent of its effects on ER folding capacity, suggesting a new therapeutic approach for the treatment of type 2 diabetes. View full text Figures at a glance * Figure 1: XBP-1s binds FoxO1 and promotes its degradation. () Left, XBP-1s and FoxO1 protein amounts in total cell lysates and cytoplasmic and nuclear fractions from MEFs expressing FoxO1 and increasing amounts of XBP-1s. Right, Foxo1 mRNA levels in MEFs expressing FoxO1 and increasing amounts of XBP-1s were analyzed by quantitative PCR. 18S ribosome RNA (Rn18s) was used for normalization of gene expression. () Endogenous FoxO3a and FoxO1 protein amounts in MEFs expressing increasing amounts of XBP-1s. () Nuclear FoxO1 and XBP-1s protein amounts in MEFs treated with DMSO or MG132. () Pulse-chase analysis of FoxO1 stability in MEFs overexpressing XBP-1s. () Ubiquitinylated FoxO1 amounts in MEFs expressing ubiquitin and XBP-1s after DMSO or MG132 treatment. () Immunoblotting of FoxO1 and XBP-1s in XBP-1s immunoprecipitates (IP) from MG132-treated MEFs expressing FoxO1 and XBP-1s. Total FoxO1 protein amounts were determined in whole-cell lysates (WCL). () Mammalian two-hybrid assay with pM–XBP-1s and pVP16-FoxO1 or pM-FoxO1 and pVP16! –XBP-1s. () Nuclear FoxO1 and XBP-1s protein amounts in MEFs treated with 1 μM BEZ235 for 4 h. Phosphorylated Akt Ser473 (pAkt Ser473) and total Akt amounts determined in cell lysates. () FoxO1, pAkt Ser473, total Akt and XBP-1s amounts in WT and Akt1/2 double-knockout (Akt1/2 DKO) MEFs treated with DMSO or 20 μM Akt inhibitor (Akti-VIII) for 30 min. () Phosphorylation-resistant mutant FoxO1(ADA) protein amounts in MEFs expressing FoxO1(ADA) and increasing amounts of XBP-1s. Each experiment was independently reproduced three times. Error bars are means ± s.e.m.; **P < 0.01, ***P < 0.001. * Figure 2: Medium-level expression of XBP-1s in ob/ob mice improves glucose homeostasis without altering insulin receptor signaling. Seven-week-old male ob/ob mice were injected with medium dose (4 × 107 PFU g−1) of Ad-LacZ (n = 6) or Ad-XBP-1s (n = 6) through the tail vein, except for one group of mice injected with low dose (1 × 107 PFU g−1) of Ad-XBP-1s as indicated in . () XBP-1s protein amounts in the liver of ob/ob mice injected with a low or medium dose of adenovirus. () Relative mRNA levels of XBP-1s target genes Dnajb9, Pida3 and Hspa5 in the liver of Ad-LacZ– or Ad–XBP-1s–injected mice. () Blood glucose concentrations in fed, 6-h fasted and 14-h fasted mice on the indicated days after injections. (–) Plasma insulin concentrations on day 9 (), GTT on day 5 () and ITT on day 7 () after the injections. () Insulin receptor (IR) and IRS-1 tyrosine (PY) and Akt Ser473 phosphorylations with or without insulin (Ins) stimulation in the liver on day 9 after injection. () Total and nuclear amounts of FoxO1 protein in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice. Graphs depict ! total FoxO1 / tubulin and nuclear FoxO1 / lamin A/C ratios. (,) Relative mRNA levels of Foxo1 () and Igfbp1, G6pc, Pck1 and Ppargc1a () in the liver of adenovirus-injected mice. Experiments were repeated in five independent cohorts. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 3: High-level expression of XBP-1s in the liver of ob/ob mice increases insulin sensitivity. Seven-week-old male ob/ob mice were injected with Ad-LacZ (n = 6) or Ad–XBP-1s (n = 6) (1.8 × 108 PFU g−1) through the tail vein. () XBP-1s protein amounts in the liver lysates on day 6 after injection. () Dnajb9, Pida3 and Hspa5 mRNA levels in the liver of Ad-LacZ– or Ad–XBP-1s–injected mice. (–) Fed blood glucose concentrations on day 5 (), plasma insulin concentrations on day 7 () and GTT on day 3 () after the adenovirus injections. () In vivo insulin receptor signaling in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice on day 6 after injection. Graphs depict the (phospho / total) / pIR ratios. () Relative mRNA levels of Insr in the liver of adenovirus-injected mice on day 6 after injection. () Total and nuclear FoxO1 amounts in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice. Graphs depict total FoxO1 / tubulin and nuclear FoxO1 / lamin A/C ratios. (,) Relative mRNA levels of Foxo1 () and G6pc, Pck1 and Ppargc1a () in the liver of A! d-LacZ– or Ad–XBP-1s–injected ob/ob mice. Experiments were repeated in three independent cohorts. NS, nonspecific. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 4: A DNA-binding-defective mutant XBP-1s (ΔDBD) improves glucose homeostasis in ob/ob mice. () Nuclear protein amounts of XBP-1s and ΔDBD in MEFs infected with the same dose of Ad–XBP-1s or Ad-ΔDBD. () ER stress element (ERSE)-luciferase activity and Hspa5 mRNA levels in MEFs infected with Ad–XBP-1s or Ad-ΔDBD. () Immunoblotting of FoxO1 and ΔDBD in ΔDBD immunoprecipitates from MEFs expressing FoxO1 and ΔDBD. Total FoxO1 protein amounts were determined in WCL. (,) FoxO1 protein amounts () and Foxo1 mRNA levels () in MEFs expressing FoxO1 and increasing amounts of ΔDBD. (–) Seven-week-old male ob/ob mice were injected with Ad-LacZ (n = 6), Ad–XBP-1s (n = 6) or Ad-ΔDBD (n = 6) (4 × 107 PFU g−1) through the tail vein. () Six-hour-fasted blood glucose concentrations on day 3 after the adenovirus injections. () GTT on day 5 after the adenovirus injections. () Phospho-Akt Ser473, total Akt, FoxO1, XBP-1s and ΔDBD protein amounts in the liver of Ad-LacZ–, Ad–XBP-1s– or Ad-ΔDBD–injected ob/ob mice on day 7 after the injections. Graph depicts to! tal FoxO1 / tubulin protein ratio. (,) Relative mRNA levels of Hspa5 () and Igfbp1, G6pc and Pck1 () in the liver of Ad-LacZ–, Ad–XBP-1s– or Ad-ΔDBD–injected ob/ob mice. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 5: XBP-1s can also improve glucose homeostasis in insulin independent manner. (–) Eight-week-old male streptozotocin (STZ)-treated mice were injected with Ad–XBP-1s (n = 9) or Ad-LacZ (n = 9) (1.5 × 108 PFU g−1) though the tail vein. () Plasma insulin concentrations before and after streptozotocin treatment. () Fed and 12-h–fasted blood glucose concentrations on the indicated days. (,) Total and nuclear FoxO1 protein amounts () and liver Igfbp1, G6pc and Pck1 mRNA levels () 10 d after adenovirus injections. (–) Eight-week-old male LIRKO mice were injected with Ad-LacZ (n = 8) or Ad–XBP-1s (n = 8) (4 × 107 PFU g−1) through the tail vein. () Liver insulin receptor protein amounts and GTT on day 4 after adenovirus injections. (,) FoxO1, XBP-1s, pAkt Ser473, pAkt Thr308 and total Akt amounts () and Igfbp1, G6pc and Pck1 mRNA levels () in the liver of Ad–XBP-1s– or Ad-LacZ–injected LIRKO mice 8 d after adenovirus injection. (–) Eight-week-old male IRS-1/2 double-knockout (DKO) mice were injected with Ad-LacZ (n = 8) or Ad–XBP-1s (! n = 8) (7.5 × 107 PFU g−1) through the tail vein. () Liver IRS-1 and IRS-2 protein levels in Irs1flox/flox;Irs2flox/flox (DF) and DKO mice. GTT was performed on day 4 after injection. (,) FoxO1 and XBP-1s protein amounts () and Igfbp1, G6pc and Pck1 mRNA levels () in the liver of adenovirus-injected DKO mice on day 8 after the injections. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 6: Enhanced glucose tolerance after medium-level expression of XBP-1s is FoxO1 dependent. Seven-week-old male ob/ob mice were injected with Ad-shGFP + Ad-LacZ (n = 5), Ad-shGFP + Ad–XBP-1s (n = 5), Ad-shFoxO1 + Ad-LacZ (n = 5) or Ad-shFoxO1 + Ad-LacZ (n = 5) through the tail vein. (,) Blood glucose concentrations on day 7 () and GTT on day 5 () after the adenovirus injections. () FoxO1 and XBP-1s protein amounts in the liver of adenovirus-injected ob/ob mice on day 7 after injections. (–) Relative mRNA levels of Foxo1 (), Igfbp1 and Ppargc1a () and Dnajb9 and Hspa5 () on day 7 after adenovirus injections. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Endocrinology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Yingjiang Zhou, * Justin Lee, * Cheng Sun, * Sang Won Park, * Jason Chung, * Jaemin Lee, * Morris F White, * Sudha B Biddinger & * Umut Ozcan * Division of Endocrinology, Metabolism & Lipid Research, Washington University in St. Louis, St. Louis, Missouri, USA. * Candace M Reno & * Simon J Fisher Contributions Y.Z. came up with the hypothesis, designed and performed the experiments, analyzed the data and wrote the manuscript. Justin Lee, C.M.R., C.S., S.W.P., J.C., Jaemin Lee and S.J.F. performed the experiments. M.F.W. provided liver specific IRS1/2 double-knockout mice. S.B.B. provided LIRKO mice and performed experiments. U.O. came up with the hypothesis, designed and performed the experiments, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Umut Ozcan Author Details * Yingjiang Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Justin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Candace M Reno Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Won Park Search for this author in: * NPG journals * PubMed * Google Scholar * Jason Chung Search for this author in: * NPG journals * PubMed * Google Scholar * Jaemin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Simon J Fisher Search for this author in: * NPG journals * PubMed * Google Scholar * Morris F White Search for this author in: * NPG journals * PubMed * Google Scholar * Sudha B Biddinger Search for this author in: * NPG journals * PubMed * Google Scholar * Umut Ozcan Contact Umut Ozcan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (406K) Supplementary Figures 1–7 and Supplementary Methods Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Community Corner: Opening the Pandora's box of prenatal genetic testing
- Nat Med 17(3):250-251 (2011)
Nature Medicine | Community Corner Community Corner: Opening the Pandora's box of prenatal genetic testing Journal name:Nature MedicineVolume: 17,Pages:250–251Year published:(2011)DOI:doi:10.1038/nm0311-250Published online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Trisomy 21, or Down's syndrome, is the most common autosomal aneuploidy, occurring in 1 in 600 live births in the absence of prenatal testing and selective abortion. However, current prenatal diagnosis of trisomy 21 requires invasive procedures, which are associated with a risk of miscarriage. Rossa Chiu et al.1 now report the first large-scale noninvasive prenatal assessment of trisomy 21 using multiplex sequencing to analyze fetal DNA from maternal plasma. These results raise the possibility that sequencing approaches might be integrated into current screening and diagnosis programs for Down's syndrome and perhaps even be applied to the diagnosis of other genetic diseases. We asked four experts to comment on the implications of this study for prenatal genetic screening and diagnostics. Caroline Wright The discovery that fragments of cell-free fetal DNA can be detected in maternal blood during pregnancy is now almost 15 years old2. Although noninvasive prenatal diagnosis for specific genetic traits such as fetal sex and RhD status has been integrated into specialist clinical practice, a noninvasive prenatal test for Down's syndrome could ultimately transform routine antenatal care for all pregnant women. There is little doubt that such a test would be welcomed by future parents, as it removes the risk of miscarriage associated with invasive testing and facilitates earlier decision-making. But, it also raises a complex set of social and ethical issues3. There are concerns about ensuring informed consent, and fears about widespread fetal genetic profiling leading to unchecked specification may creep into a multitude of prenatal tests. Nonetheless, the rapidly decreasing cost of next-generation sequencing technologies means that noninvasive prenatal testing for Down's syndrome may soon become a clinical reality. The recent study from Chiu et al.1 is the first to validate such a test in a sizeable cohort and forecast its potential impact within a population screening program. It is still unclear whether a noninvasive test could replace the current multistep screening protocol, be added as an intermediate step to refine the results from existing screening programs or eventually replace invasive diagnostic testing. Although large population studies are now needed to further evaluate the test proposed by Chiu et al.1, national health services should take heed: a test may become available directly to consumers before health systems are able to offer it. This raises another set of issues—well known to those familiar with the recent consumer genomics movement—about accuracy, reliability, informed consent and ! equity of access. We need to ensure that regulators, health care professionals and the public are suitably prepared. Stephen R Quake It is unfortunate that expectant parents today do not have a satisfactory prenatal genetic test. Invasive procedures such as amniocentesis and chorionic villus sampling provide fairly accurate results but have a health risk to both the mother and the baby. Noninvasive aneuploidy screening, including maternal serum biochemical markers and nuchal translucency measurements, is becoming widely used, but these are not genetic methods and they suffer from poor performance. The false positive rates are ~ 5%, and considering that the rate of genetic disease is generally below ~1%, this means that nearly all positive results reported by these approaches are incorrect. "Measurement of circulating cell-free DNA can be a powerful approach for noninvasive prenatal diagnostics." Measurement of circulating cell-free DNA can be a powerful approach for noninvasive prenatal diagnostics. My group invented the first genetic noninvasive prenatal diagnostic for aneuploidies, including Down's syndrome, which uses a molecular counting scheme and was implemented by high-throughput sequencing4. Chiu et al.1 recently published the largest-scale study of this approach yet. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Competing financial interests D.B. is chair of the clinical advisory board of Artemis Health, an early biotechnology company working in prenatal diagnosis. She has equity options in Artemis, as well. S.R.Q. is a consultant for and equity holder of Fluidigm and Artemis Health. N.J.W. holds a patent for the Integrated test. With others, he also holds a patent in connection with the use of unconjugated oestriol as a second-trimester Down's syndrome screening marker. He is a director of Logical Medical Systems, Ltd., which produces software for the interpretation of Down's syndrome screening tests. Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - A siege of hepatitis: Immune boost for viral hepatitis
- Nat Med 17(3):252-253 (2011)
Nature Medicine | Between Bedside and Bench A siege of hepatitis: Immune boost for viral hepatitis * Benoît Callendret1 * Christopher Walker1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:252–253Year published:(2011)DOI:doi:10.1038/nm0311-252Published online07 March 2011 Finding mechanisms of viral resistance and new ways to tackle chronic hepatitis will help find a cure for this disease. In 'Bench to Bedside', Christopher Walker and Benoît Callendret highlight studies showing that overcoming immune exhaustion during chronic infection by blocking several inhibitory pathways of T cells may restore an adequate immune response. In 'Bedside to Bench', Lawrence Corey, Joshua Schiffer and John Scott discuss recent advances in antiviral therapy with protease inhibitors and the findings of a mathematical model that predicts possible single and double mutations prior to antiviral therapy. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Benoît Callendret and Christopher Walker are at the Center for Vaccines and Immunity, Nationwide Children's Hospital and Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA. Competing financial interests C.W. is a consultant to Merck and Roche and is on the scientific advisory boards of Transgene and Okairos. Corresponding author Correspondence to: * Christopher Walker Author Details * Benoît Callendret Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher Walker Contact Christopher Walker Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - A siege of hepatitis: Fighting a defiant virus
- Nat Med 17(3):253-254 (2011)
Nature Medicine | Between Bedside and Bench A siege of hepatitis: Fighting a defiant virus * Joshua T Schiffer1, 2 * John Scott1 * Lawrence Corey1, 2 * Affiliations * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:253–254Year published:(2011)DOI:doi:10.1038/nm0311-253Published online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Hepatitis C is the leading cause of liver cancer in the US and Europe, where approximately 3% of the general populations are infected. At least 20% of untreated persons will progress to cirrhosis1, and 15% of individuals with cirrhosis will develop hepatocellular carcinoma within ten years. In the late 1990s, it was shown that, in contrast to HIV-1 infection, hepatitis C could be eradicated with antiviral therapy. Clinical trials with 12 months of interferon and ribavirin resulted in sustained virologic response—undetectable virus 24 weeks after last dose—in 50% of subjects2, 3, which correlated with viral cure, as well as a 60% decrease in overall mortality. But this regimen has incomplete and unpredictable clinical efficacy, frequent dose-related toxicities and treatment costs of approximately $35,000 per year—a source of frustration for patients and providers4. A consequence of interferon and ribavirin therapy for hepatitis C was the recognition that the replication dynamics of hepatitis C are rapid. Like in HIV-1 infection, in hepatitis C infection turnover of viruses is high—free viral half-life is from 11 to 19 h5—and chronic infection is maintained by rapid viral replication in the liver and other anatomic reservoirs of infection6. Hepatitis C, however, replicates via an RNA-dependent RNA polymerase in the cytosol, and, unlike HIV-1, lacks a stable reservoir within infected cells. Intracellular HCV RNA particles are fragile and short lived, and rapid replenishment is needed to sustain infection. The RNA-dependent polymerase is error prone and lacks a proofreading function, creating a diverse array of variants that allow for immune evasion. The rapid kinetics of hepatitis C infection, and the success of targeted agents for HIV-1 infection, spurred the pharmaceutical industry to develop small-molecule inhibitors of viral replication. There are numerous compounds in phase 2 or phase 3 trials, including NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase (RdRp) nucleoside analogs, RdRp nonnucleoside inhibitors and an NS5A assembly protein inhibitor. Telaprevir, an NS3/4A protease inhibitor, showed promise in two clinical trials: approximately 65% of treatment-naive, genotype 1 hepatitis C–infected patients who received only 24 weeks of pegylated interferon and ribavirin therapy with 12 weeks of telepravir treatment achieved a sustained virologic response, compared to 43% in the placebo arms7, 8. Notably, viral sequencing revealed that more than half of the therapeutic failures were associated with mutations in the protease-encoding gene that are known to confer drug resistance7, 8. These findings emphasize the formidable therapeutic hurdles that prevent elimination of highly replicating RNA viruses. Recent inroads toward understanding antiviral resistance have been made by Rong et al.9, who showed that hepatitis C resistance occurs due to preexisting mutations in circulating viruses. Using probability and dynamic mathematical models, in combination with data from four previously untreated individuals who received 14 days of telaprevir alone, the authors considered two genotypic variants that confer resistance to protease inhibitors and estimated the number of single- (8.7 × 1010), double- (4.2 × 109) and triple- (1.3 × 108) mutant viruses among the estimated 1 × 1012 circulating HCV virions in a typical chronically infected person9. Their data suggest that every possible single- or double-mutant virus was likely to have existed before therapy, and one additional mutation may have occurred after 24 h of treatment9. Viral load decreases by 99.97% within two days of therapy but often rebounds within seven to ten days during telaprevir monotherapy10. Preexisting mutant viruses avoid killing and represent 50% of circulating viruses after five to six days of antiviral initiation (Fig. 1)9. These surviving virions expand, accrue further mutations and rapidly predominate. The high error rate of hepatitis C RdRp11 in combination with the high burden of replicating virus leads to high numbers of new mutants each day. Therefore, hepatitis C continually explores a large genetic space to select for resistant variants with the highest replicative and immune evasion capabilities. This plasticity allows the virus to escape antiviral agents in a mechanism similar to that of HIV-1. Figure 1: Drug resistance dynamics during hepatitis C monotherapy. Before monotherapy with a protease inhibitor, drug-resistant mutant viruses (red) represent a minority among drug-susceptible viruses. At days 5–6 after therapy, drug-susceptible viral levels decrease substantially, whereas resistant viruses expand only slightly but become the predominant viral form. At day 10, drug-resistant clones reexpand. * Full size image (159 KB) The major implication of this study is that monotherapy results in resistance and treatment failure, underscoring a lesson learned from HIV-1, hepatitis B, influenza A and tuberculosis trials12, 13. With the availability of precisely targeted agents, interferon and ribavirin may eventually be suboptimal backbones to multidrug therapy. Yet these agents are currently necessary, given the incidence of resistance to protease inhibitors when these are used alone. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Joshua T. Schiffer, John Scott and Lawrence Corey are in the Department of Medicine, University of Washington, Seattle, Washington, USA. * Joshua T. Schiffer and Lawrence Corey are also in the Vaccine and Infectious Diseases Division, The Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. Competing financial interests J.S. has received research funding from Genentech, Tibotec and Anadys. He serves on the Speaker's Bureau for Genentech, Gilead, Merck and Vertex. He has served on advisory boards in the last year for Vertex. Corresponding authors Correspondence to: * Joshua T Schiffer or * Lawrence Corey Author Details * Joshua T Schiffer Contact Joshua T Schiffer Search for this author in: * NPG journals * PubMed * Google Scholar * John Scott Search for this author in: * NPG journals * PubMed * Google Scholar * Lawrence Corey Contact Lawrence Corey Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research Highlights
- Nat Med 17(3):255-256 (2011)
Nature Medicine | Research Highlights Research Highlights Journal name:Nature MedicineVolume: 17,Pages:255–256Year published:(2011)DOI:doi:10.1038/nm0311-255Published online07 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Neuroscience: Stroke signals The transcription factor cAMP responsive element–binding protein (CREB) mediates neuroprotection after stroke. Now, Tsutomu Sasaki et al. identify a cell signaling pathway that modulates CREB activation after ischemia (Neuron, 106–119). CREB activity can be controlled by recruitment of stimulatory cofactors such as transducer of regulated CREB activity-1 (TORC1). In cell culture experiments, the researchers showed that TORC1 translocation to the nucleus was increased after ischemia and was required for activation of CREB. TORC1 overexpression could reduce neuron death in response to ischemia. TORC1 is phosphorylated by salt-inducible kinase-2 (SIK2), which was degraded in cultured neurons after ischemia, and SIK2 phosphorylation by Ca+2/calmodulin-dependent protein kinases seemed to be responsible for this process. Increasing SIK2 expression prevented TORC1 from entering the nucleus and from activating CREB, and this enhanced cell death after ischemia. The researchers found that a SIK2 inhibitor could enhance CREB activity and prevent neuron death in response to ischemia, and SIK2-deficient mice were protected from stroke. These findings suggest that SIK2 degradation after ischemia is beneficial to neurons.—EC Stressing out Chronic stress can have various effects on people, with some becoming susceptible to psychiatric conditions, including depression. The mechanism underlying this differential vulnerability to stress was recently investigated in mice. Shusaku Uchida et al. (Neuron, 359–372) compared the levels of brain-expressed genes in a stress-vulnerable mouse strain, BALB/c, and a stress-adaptive strain, C57BL/6, in the presence and absence of chronic stress. They found that the levels of glial cell–derived neurotrophic factor (GDNF) mRNA and protein were reduced in the nucleus accumbens of BALB/c mice after stress but increased in C57BL/6 mice. The authors then characterized an epigenetic mechanism involving both histone modification and DNA methylation that led to reduced expression from the Gdnf promoter in BALB/c mice. Administration of histone deacetylase inhibitors or DNA methylase inhibitors could ameliorate the BALB/c stress-induced depressive behaviors by relieving epigenetic repression of Gdnf expression. The results of this study raise many interesting issues for further study, but one of the most pressing to address will be whether GDNF also modulates the response to stress in humans and, if so, how it is regulated. —MS Reproduction: Feeding the reproductive cycle Energy metabolism is known to affect reproductive cycles, acting as an evolutionary oversight to ensure that reproduction occurs only in favorable nutritional conditions. A recent study characterizes a mechanism through which the liver integrates metabolic responses to control ovulation (Cell Metab., 205–214). Investigating the link between estrogen and food consumption in mice, Sara Della Torre and colleagues found that caloric restriction decreased hepatic estrogen receptor-α (ER-α) activation in the liver and arrested estrous cycle progression. Interestingly, amino acid supplementation was sufficient to rescue mice from this metabolic block of the estrous cycle. The authors found that ER-α activation led to increased hepatic expression of insulin growth factor–like-1 (IGF-1) and increased the amount of circulating IGF-1. Increased IGF-1 expression was required for E2-induced proliferation of uterine lumen epithelial cells and for estrous cycle progression in vivo. The findings highlight a crucial role of hepatic ER-α as an integrator of metabolic and reproductive functions. The exact mechanisms by which IGF-1 and E2 promote progression of the estrous cycle remain to be determined, but this study might provide insights into infertility conditions, especially those linked to metabolic dysfunction. —MS Cancer: Resisting radiation Radiation can induce lethal genomic damage in tumor cells and is one of the few therapeutic options to treat gliomas. But its efficiency is limited by radioresistance—a property commonly ascribed to glioma stem cells (GSCs) that is thought to be associated with the ability to activate protective checkpoints and DNA repair mechanisms. Cheng et al. (EMBO doi:10.1038/emboj.2011.10 4 February 2010) provide functional insights into the increased radio resistance of GSCs. The authors found that the expression of L1 cell adhesion molecule (L1CAM), a surface molecule enriched in GSCs, was stimulated by radiation-like insults and was required for the recovery of normal growth in this cell population. Moreover, L1CAM downregulation impaired protective checkpoint activation and DNA repair activities in GSCs. Exposure to radiation induced L1CAM cleavage from the membrane followed by its nuclear translocation, whereby it indirectly activated the expression of NSB1, a sensor of DNA damage. GSCs with higher L1CAM expression are thus more proficient at detecting radiation damage and protecting themselves from it. Although further exploration of the involvement of this pathway in radioresistance in human tumors is warranted, L1CAM is a potential therapeutic target to improve the efficacy of radiation treatment for gliomas. —VA Infection: Evasion through evolution Viruses can evade clearance by host cells by inhibiting the host innate immune response. Upon infection, microbial components are sensed by a variety of host cell receptors, leading to activation of a multiprotein complex termed the inflammasome. Activation of the inflammasome promotes proinflammatory cytokine production and microbial clearance. Blossom Damania and her colleagues (Science, 330–334) found that Kaposi's sarcoma–associated herpesvirus (KSHV) encodes Orf63, a protein homologous to NLR family, pyrin domain–containing-1 (NLRP1), a component of the host inflammasome. Despite this homology, Orf63 lacks key domains needed for activation of the inflammasome. Instead, Orf63 binds NLRP1 and inhibits inflammasome activation, blunting the innate immune response and permitting effective viral infection and reactivation. Silencing of Orf63 in infected cells promotes inflammasome activation and inhibits viral gene expression and genome replication. Kenneth Eward / Photo Researchers, Inc. The authors propose that KSHV acquired Orf63 over the course of evolution, subverting its function to inhibit the protective host response. Further studies will tell whether Orf63 has a key role in vivo to promote KSHV persistence. —KDS View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - A close look at cancer
- Nat Med 17(3):262-265 (2011)
Nature Medicine | Introduction A close look at cancer * Alison Farrell1Journal name:Nature MedicineVolume: 17,Pages:262–265Year published:(2011)DOI:doi:10.1038/nm0311-262Published online07 March 2011 Advances in cancer research are enabling fast-paced discovery and translation of results into potential clinical tools. Here we consider some of the most influential findings of the past two years, selected by experts in the cancer field. View full text Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Alison Farrell is a senior editor of Nature Medicine. Competing financial interests The author declares no competing financial interests. Author Details * Alison Farrell Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - New technologies aim to take cancer out of circulation
- Nat Med 17(3):266 (2011)
Nature Medicine | News New technologies aim to take cancer out of circulation * Elie DolginJournal name:Nature MedicineVolume: 17,Page:266Year published:(2011)DOI:doi:10.1038/nm0311-266Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg BOSTON — A plashet of blood slowly leaks out from atop the microfluidics chip in Daniel Haber's lab here at the Massachusetts General Hospital (MGH) Cancer Center. "That's not supposed to happen," murmurs technician Jenna Lord, as she carefully pipettes the fluid into a Falcon tube. The blood, taken from a man with elevated levels of prostate-specific antigen who is suspected of having cancer, is being pushed through eight tiny channels, each coated with cancer-specific antibodies to capture any circulating tumor cells (CTCs). Four years ago, Haber and his MGH colleague Mehmet Toner first reported the effectiveness of such a microfluidics-based contraption for isolating CTCs, which are approximately one out of every billion cells in the blood (Nature450, 1235–1239, 2007). And last year, they refined their invention to create a platform that can be manufactured on a commercial scale with improved cell capture rates (Proc. Natl. Acad. Sci. USA, 18392–18397, 2010). "Now, you can draw a blood sample and say, 'aha, this is the molecular status of my tumor, and this is how it has or has not been changed by the drugs I've been given,'" Haber says. Haber and Toner's technology has garnered immense attention—and investment. In January, New Jersey–based Johnson & Johnson, the world's largest medical devices and diagnostics company, announced a five-year, $30 million deal with the MGH team to build the next-generation assay: a 'liquid biopsy' test for early cancer detection that would be as easy to use as a home pregnancy test. Clearly, the researchers aren't there yet, as the mishap with the leaky blood shows. But with dozens of competing devices under development in labs around the world, the MGH team—and its pharma backers—are facing pretty stiff competition to deliver a diagnostic cancer test. "We poked the sleeping bear," Toner says. "It has suddenly created a frenzy." Shannon Stott, MGH (left); Hsian-Rong Tseng, UCLA (right) The MGH (left) and UCLA (right) microfluidic CTC detectors. Currently, the only CTC device approved by the US Food and Drug Administration for tracking cancer progression is a technology called CellSearch. Manufactured by the Johnson & Johnson subsidiary Veridex, CellSearch, like the MGH team's device, captures CTCs on the basis of their affinity to antibodies against a protein called epithelial cell adhesion molecule (EpCAM), which is found on tumor cells but not on blood cells. Using CellSearch, researchers have shown that the number of CTCs is a good predictor of survival and disease progression in people with metastatic breast, colon and prostate cancers. But "the problem with the current platform is that the cell yields are very low, and they're not consistent," says Shivaani Kummar, a clinician at the US National Cancer Institute in Bethesda, Maryland. Moreover, "what the CellSearch system definitely doesn't do now is tell a physician what treatment to give," says the technology's inventor Leon Terstappen from the University of Twente in the Netherlands. Tech transfer Steven Soper, a bioengineer at Louisiana State University in Baton Rouge, is also working on a microfluidics device based on EpCAM binding. But unlike the MGH chip, the antibodies in Soper's device can be easily detached, allowing researchers to collect cells intact for further study. "Now, we actually have the opportunity to collect those cells and do molecular profiling," says Soper, who reported his device last month in Analytical Chemistry (doi:10.1021/ac103172y, 2011). This month, Hsian-Rong Tseng, a chemist at the University of California–Los Angeles, reported another microfluidics design that relies on EpCAM-coated nanofibers that bind like Velcro to the unusually high number of microvilli on the surface of tumor cells (Angew. Chem. doi:10.1002/ange.2010005853, 2011). "Because our surface is much stickier," says Tseng, "we can operate in a much faster blood flow through the device and also have a shorter channel length," which makes the chip smaller and cheaper to manufacture than competing designs. Meanwhile, at Stanford University in Palo Alto, California, a team led by surgeon Stefanie Jeffrey has created a technology called the MagSweeper. The device, now licensed to and further developed by sequencing giant Illumina, consists of a magnetic rod that sweeps through blood to capture CTCs. In collaboration with Microsoft, biophysicist Peter Kuhn of the Scripps Research Institute in La Jolla, California, is working on a staining and imaging approach for identifying CTCs without relying on any given specific surface marker such as EpCAM. And in December, Chwee Teck Lim from the National University of Singapore's Mechanobiology Institute reported another chip that separates CTCs on the basis of their physical qualities; they are generally larger and stiffer than blood cells (Biosens. Bioelectron., 1701–1705, 2010) "It's really exciting because the number of technologies is really exploding," says Howard Scher, an oncologist at Sloan-Kettering Memorial Cancer Center in New York. "But at the end of the day there has to be focused development on the specific decision the test is going to inform." Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - The search for child cancer drugs grows up
- Nat Med 17(3):267 (2011)
Nature Medicine | News The search for child cancer drugs grows up * Branwen MorganJournal name:Nature MedicineVolume: 17,Page:267Year published:(2011)DOI:doi:10.1038/nm0311-267Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg "Off the top of my head, I really can't think of a single cancer drug that was developed specifically for pediatric use," says Peter Houghton, director of the Center for Childhood Cancer at Nationwide Children's Hospital in Columbus, Ohio. "All have had their origin in adult clinical trials...wait, I lie: there is an antibody that was developed specifically for kids with neuroblastoma." Houghton is referring to ch14.18, an experimental drug in the process of seeking approval by the US Food and Drug Administration (FDA). Houghton's remarks highlight a systemic problem with how cancer drugs are developed for children. Of the approximately 120 approved cancer therapies, roughly 30 are used in children, and only half of those have pediatric information included on their labels. Moreover, new cancer therapies are usually considered for use in children only several years after the results of successful adult clinical cancer trials have been published. "This means there's a delay of two to seven years before we know if children could benefit from a new drug," Houghton explains. "That's too long." "I can't think of a single cancer drug that was developed specifically for pediatric use." As recently as last month, an international collection of scientists penned an opinion article deploring the lack of attention and coordination in child cancer research (ecancermedicalscience, 210, 2011). In their review of the scientific literature, the authors found that pediatric oncology papers had lower citation rates than those expected for the journals in which they appeared. Pediatric cancers differ substantially from those found in adults. For example, about one third of children with cancer have a form of leukemia, which is comparatively rare in adults. The most common types of solid tumors found in children are brain tumors, whereas adults tend to suffer from lung, breast and prostate cancers. And children's cancers often differ with respect to drug sensitivity and prevalence of biomarkers. Part of the problem with finding cures for child cancers has to do with identifying enough participants for clinical trials. Only 1% of people diagnosed with cancer each year are under the age of 21. To remedy the situation, in 2001 Houghton, together with Malcolm Smith, associate branch chief for pediatric oncology at the US National Cancer Institute (NCI) in Bethesda, Maryland, and Peter Adamson from the Children's Hospital of Philadelphia orchestrated a meeting to bring together twenty leading experts in the field from twelve institutions. The two-day brainstorming session produced the idea of establishing a Pediatric Preclinical Testing Program (PPTP) for new anticancer agents. Three years later, the NCI launched the PPTP, which is now partway through its second five-year funding term. The funding, currently set at $3 million per year, is split across six main testing sites: five in the US and one in Australia. The PPTP's primary goal is to help determine which of the myriad new cancer therapeutics in development should be clinically evaluated in children, given that it is not logistically possible to test them all. Paul Sondel, a pediatric oncologist at the University of Wisconsin–Madison, who has helped develop ch14.18 but is not involved with the PPTP, says the program fills an important gap. "The pharmaceutical industry is less eager to invest in the development of agents with perceived limited applicability due to the small market for rare diseases," which include many child cancers. Zeroing in on xenografts Houghton says that testing involving human tumor tissue transplanted into immune-deficient mice—known as mouse xenograftsis the PPTP effort's mainstay, because the xenografts have proved capable of accurately predicting clinical activity of anticancer drugs. "Xenograft models have been maligned over the years, but ours have several significant differences. They are created by directly grafting patient biopsies into the mice, rather than using cell culture to propagate and expand the cancer cells," he says. James Gommel Five-year old Kaylee Gommel, who suffers from neuroblastoma, received a 20-hour infusion of ch14.18 at the Cleveland Clinic in September 2010. Across the PPTP there are currently 45 xenograft mouse models of solid tumors and leukemias that represent most of the common types of childhood cancers. The leukemia-testing component of the PPTP is carried by an Australian team led by Richard Lock at the Children's Cancer Institute Australia near Sydney. Over the last six years, they have tested almost 50 investigational agents supplied by more than 30 pharmaceutical companies on their xenograft panels of eight different leukemia samples from humans. The agents tested by the PPTP include nine that are FDA-approved for use against one or more adult cancers but lack a defined role in children. Not all of these have produced good results in the xenograft tests. "For the leukemias, where the cure rate is already 80%, we aren't interested in any compound that doesn't elicit at least a 50% response rate, and more than 75% of the agents we've tested have not met that criterion," says Lock. Despite this surprising finding, Lock and the other PPTP scientists are particularly excited about a new compound manufactured by the Cambridge, Massachusetts–based Millennium Pharmaceuticals named MLN8237, which entered NCI-sponsored pediatric clinical trials in 2008, just ten months after presentation of PPTP-supported data showing its strong activity against neuroblastoma and refractory acute lymphoblastic leukemia in mice. The small-molecule drug, which blocks an enzyme called Aurora A kinase, is currently in phase 2 trials. The overall success of the PPTP will be evaluated in 2014 when the current five-year funding term ends. "In terms of cost effectiveness and life years saved, investing in therapy for pediatric cancers is very economic," Houghton says in support of the effort. "The way I look at it, if you cure an adult with a carcinoma, we extend their median lifespan by three years, whereas if we cure a six-year-old kid with leukemia, we give them another 70-plus years." Additional data Author Details * Branwen Morgan Search for this author in: * NPG journals * PubMed * Google Scholar - Companies compete over mutation-specific melanoma drugs
- Nat Med 17(3):268 (2011)
Nature Medicine | News Companies compete over mutation-specific melanoma drugs * Cassandra WillyardJournal name:Nature MedicineVolume: 17,Page:268Year published:(2011)DOI:doi:10.1038/nm0311-268aPublished online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg When a doctor spots a cancerous skin growth, simple surgery is often sufficient to rid the patient of cancer. Once melanoma has spread throughout the body, however, few treatment options exist. Neither chemotherapy nor the immune protein interleukin-2—the two therapies currently approved for treating metastatic melanoma—offer much hope. But a new generation of melanoma drugs nearing approval that target a common mutation implicated in driving tumor growth could revolutionize the way physicians treat skin cancer. These new drugs target a mutated form of the B-RAF oncoprotein, and cancer specialists say they represent a major improvement over existing therapies, which suffer from low response rates and fail to significantly extend survival. "We've never seen anything like this," says Lynn Schuchter, an oncologist at the University of Pennsylvania School of Medicine in Philadelphia who is involved in trials to test the new compounds and has been treating patients with metastatic melanoma, the deadliest form of the disease, for almost 25 years. "We really have tears of joy." Phanie / Photo Researchers, Inc. New melanoma treatments. In January, the Swiss drug maker Roche announced preliminary phase 3 trial results showing that its experimental targeted B-RAF therapy significantly extended lifespan in people with metastatic melanoma. In fact, the study was so successful, with some participants emerging nearly cancer free, that the trial was halted prematurely, and many of the 340 subjects in the study's control arm, who previously received only standard chemotherapy, are now being given the drug. Keith Flaherty, an oncologist at Massachusetts General Hospital in Boston and principle investigator on the trial, expects federal regulators to approve the small-molecule drug, developed in partnership with Berkeley, California–based Plexxikon, later this year. "It's sort of a slam dunk," he says. Full results from the trial will be presented in June at the American Society of Clinical Oncology's annual meeting in Chicago. The ubiquity of B-RAF mutations—first identified in 2002 and since found in many types of tumors, including lung and colon cancer cells, albeit at lower frequencies than in melanoma, where more than half of all cases of the disease express the aberrant protein—has made it an attractive target for many pharmaceutical companies. Thus, Roche is in the midst of a heated race to develop the first-in-class, mutation-specific therapy for melanoma. Also in January, GlaxoSmithKline (GSK), the British pharma giant, launched a phase 3 trial to test its own B-RAF inhibitor, and the Swiss drugmaker Novartis has another compound currently in phase 1 trials. Despite the buzz surrounding these drugs, they aren't without side effects. Notably, some of the study subjects developed small, cancerous skin growths that had to be surgically removed, possibly as a result of nonspecific drug targeting. But, according to Keiran Smalley, a melanoma expert at the Moffitt Cancer Center in Tampa, Florida who was not involved in any of the trials, that's a small price to pay for longer survival, as people with metastatic melanoma typically survive for less than a year. More worryingly, B-RAF inhibitors suffer from the limitation that patients nearly always develop resistance to the drugs after around six to nine months of treatment, and the tumors return or begin growing again. Researchers have just begun to sort out how this resistance occurs. Three papers published in December suggest that tumor cells can circumvent the drugs by reactivating the B-RAF pathway through other mutations or by turning on an entirely different survival pathway (Nature468, 968–972 & 973–977, 2010; Cancer Cell, 683–695, 2010). Knowing how cancer cells evade the medications may help researchers devise drug cocktails that can prevent resistance from occurring, says Roger Lo, a dermatologist at the University of California–Los Angeles who led one of the studies and served as a subinvestigator on both the GSK and the Roche trials. Researchers are now testing whether combining a B-RAF inhibitor with another experimental compound that blocks a separate enzyme in the same pathway will stave off resistance (see page 270). According to Richard Kefford, an oncologist at the University of Sydney in Australia and the primary investigator on a GSK combination trial, a therapy that targets two proteins at once should be "a much more durable inhibitor of cellular growth." Additional data Author Details * Cassandra Willyard Search for this author in: * NPG journals * PubMed * Google Scholar - Recent deal highlights hopes for cancer-killing viruses
- Nat Med 17(3):268-269 (2011)
Nature Medicine | News Recent deal highlights hopes for cancer-killing viruses * Jon EvansJournal name:Nature MedicineVolume: 17,Pages:268–269Year published:(2011)DOI:doi:10.1038/nm0311-268bPublished online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Can viruses be engineered to successfully tackle cancer? The biotechnology giant Amgen certainly hopes so. At the end of January, the California-based company announced that it was buying BioVex, a pioneer in developing so-called oncolytic viruses, for an impressive $1 billion. "Without a doubt, the Amgen deal is a validation of this field, which has often been thought of as a little bit of a backwater," says Robert Coffin, founder and chief technology officer of BioVex. Originally spun off from University College London but now headquartered in Woburn, Massachusetts, BioVex is currently conducting phase 3 trials of OncoVEX GM-CSF, a genetically modified herpes simplex virus, for treating various cancers. It hopes to submit this vaccine for US approval at the start of 2012. Other companies are advancing similar products. The Canadian company Oncolytics Biotech is conducting a phase 3 trial of Reolysin, a reovirus, for treating head and neck cancer. And Jennerex, headquartered in San Francisco, is conducting phase 2 trials of JX-594, a modified vaccinia virus, for treating liver cancer. Meanwhile, an engineered adenovirus for treating head and neck cancer, developed by Shanghai Sunway Biotech, was approved in China in 2005. Scientists have known for more than a century that viral infections occasionally lead to cancer remission. Subsequent studies revealed that not only can some viruses directly infect and kill cancer cells, but also this process releases antigens that prime the immune system to attack the tumor. Historically, scientists have had a tough time finding viruses that attack cancer cells without harming healthy tissue. But researchers can now engineer oncolytic viruses that lack certain regulatory genes—such as the E1A or E1B genes—that they use to defeat the antiviral defense mechanisms possessed by normal cells, rendering the viruses harmless in healthy tissue. These defense systems are often switched off in cancer cells, making them vulnerable to the engineered infectious agents. On top of this, genes coding for proteins that enhance the body's immune response, such as granulocyte-macrophage colony–stimulating factor, can be added to stimulate the expansion of white blood cells that enhance the immune response against tumors, as with BioVex's and Jennerex's products. So can genes coding for enzymes that turn a separately injected drug precursor into a toxic anticancer agent. "The issue of targeting oncolytic viruses selectively to cancer cells is largely solved and not as much of a barrier anymore," says Timothy Cripe, a physician at Cincinnati Children's Hospital Medical Center who develops oncolytic viruses for treating pediatric cancers. Other challenges still persist, though. The immune system, for example, can be a double-edged sword, prone to attacking the oncolytic viruses as well as the tumor. "The barriers now are getting the viruses to spread efficiently within a tumor microenvironment and to persist long enough to have their effect," says Cripe. Scientists are continuing to evaluate how viruses might join chemotherapy and radiotherapy in the arsenal of cancer treatments. "What we're going to continue to see over the next couple of years is a careful assessment of safety and some glimpses of efficacy as an add-on to existing therapeutic regimens," predicts William Phelps, program director of preclinical and translational cancer research at the American Cancer Society in Atlanta. Additional data Author Details * Jon Evans Search for this author in: * NPG journals * PubMed * Google Scholar - Treatment approaches that target tumor suppressors mutate
- Nat Med 17(3):269 (2011)
Nature Medicine | News Treatment approaches that target tumor suppressors mutate * Monica HegerJournal name:Nature MedicineVolume: 17,Page:269Year published:(2011)DOI:doi:10.1038/nm0311-269Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg For all the differences documented between cancers, they share some striking similarities. Around 80% of detected cancer-related mutations are errors in so-called 'tumor suppressor genes', and more than half of all cancers have below-normal amounts of the tumor suppressor protein p53. This makes tumor suppressors—and p53 in particular—enticing targets for drug developers. Despite failed attempts in the past, researchers have hope for new variations on this cancer treatment approach. "There has been a huge amount of interest in therapeutic targeting of p53 for over 15 years," says Alex Swarbrick at the Garvan Institute in Sydney. For example, in a small subset of cancer cases, the gene producing p53 is inhibited by the protein MDM2, murine double minute 2. A class of drugs called Nutlins, made by the Swiss pharma giant Roche, have shown promise for its ability to block MDM2 and are currently in clinical trials to treat people with tumors in their fat tissue. Meanwhile, several small molecules are being studied for their ability to restore mutant p53 proteins, but results have been mixed. So far, there are no approved anticancer agents that target p53. One reason for the absence is that cancer cells sometimes completely lack that gene, and therefore there is nothing to target. Other times, p53 is present, but mutated, and restoring the function of a protein is inherently difficult. The latest idea for targeting tumor suppressors is through microRNAs, small pieces of single-stranded, noncoding RNA that regulate an estimated 20% to 30% of all protein-coding genes, including both tumor suppressor genes and oncogenes (Cancer Res., 7027–7030, 2010). In the same way that tumor suppressor proteins are often absent or nonfunctional in cancer cells, so are certain microRNAs. A strategy known as microRNA replacement therapy aims to create the same sequence of missing RNA and restore it to the cancer cells. The approach could be more powerful than restoring the missing protein, because individual microRNAs regulate upwards of a hundred protein-coding genes at once, so "targeting microRNAs in cancer cells can have a very potent effect," says Joshua Mendell, of the Johns Hopkins University School of Medicine in Baltimore, whose group is studying these molecules in liver cancer. Pathways to success? Last year, for example, Mendell and his colleagues showed that introducing a microRNA called miR-26a could reduce the size of liver tumors in mice by about a third without any harmful effects on healthy tissue (Cell137, 1005–1017, 2009). More recently, scientists at Mirna Therapeutics, a biotech company in Austin, Texas, showed that another microRNA called miR-34a, which regulates the p53 pathway, blocks the growth of human lung tumors in mice (Cancer Res.70, 5923–5930, 2010) and inhibits prostate cancer stem cells and metastasis (Nat. Med.17, 211–215, 2011). According to Mirna's associate director of research Andreas Bader, the company intends to apply for human testing of their microRNA sometime next year. Despite these recent advances, several hurdles remain. Because miRNAs regulate so many genes, researchers worry about how they will affect healthy tissue, even though no serious adverse events have been detected in any of the mice studied so far. Jim Dowdalls / Photo Researchers, Inc. microRNAs replace mutated genes. Even more challenging is the problem of designing an effective way to deliver microRNAs to the site of the tumor without the small strands of RNA getting degraded or filtered out of the body, notes Bader. "Delivery still remains the major hurdle to bringing this to the clinic," he says. Various strategies under development include encasing microRNAs in lipid membranes that can then be coated with tumor-specific peptides to act like homing agents, as well as using nanoparticles or viral vectors as delivery mechanisms. Nevertheless, researchers are cautiously optimistic. "It would be circumspect to propose that microRNAs will be magically easier to work with than tumor suppressors," says Swarbrick, who is studying the role of microRNAs as both tumor suppressors and oncogenes. All the same, he adds, there's clear evidence that microRNAs will be important molecules to try and target in cancer. "The field is exploding." Additional data Author Details * Monica Heger Search for this author in: * NPG journals * PubMed * Google Scholar - When it takes two to tango, FDA suggests a new regulatory dance
- Nat Med 17(3):270 (2011)
Nature Medicine | News When it takes two to tango, FDA suggests a new regulatory dance * Elie DolginJournal name:Nature MedicineVolume: 17,Page:270Year published:(2011)DOI:doi:10.1038/nm0311-270Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Two drugs taken together can sometimes be greater than the sum of their parts. And for certain diseases, including cancer, drug combinations are essential to achieve any therapeutic benefit. But the existing regulatory framework for testing and approving new drugs focuses primarily on the effectiveness and safety of individual experimental compounds. This means that it's been nearly impossible to conduct early clinical trials on multiple new drugs at the same time. All that might soon change. In December, the US Food and Drug Administration (FDA) released draft guidance outlining a path toward developing combinations of unapproved drugs. "We need to enable these innovative targeted therapies in a way that isn't possible in traditional drug development," Janet Woodcock, director of the FDA's Center for Drug Evaluation and Research (CDER), told Nature Medicine. "The places we think are most likely to have interest, at least initially, are oncology and infectious diseases," says CDER deputy director for clinical science Robert Temple. That's because pathogens such as tuberculosis and HIV quickly develop drug resistance when only single-agent therapies are used. Similarly, tumors often harbor multiple gene mutations that allow cancer to grow and that need to be targeted simultaneously. Combination drug development at present usually involves phase 3 trials in which fixed doses of two drugs are tested both alone and in tandem against a placebo treatment. To get to that stage, however, experimental drugs usually need to be taken through earlier-stage trials in isolation, even if they have limited utility on their own. The new proposed guidelines would allow drug makers to forego monotherapy trials and conduct combined drug studies earlier in the development process, when there are compelling reasons and preclinical data to support such a move. Jeff Allen, executive director of Friends of Cancer Research, a Washington, DC–based think tank that cohosted a meeting on combination drug development in September 2009, says that advances in genomics and personalized medicine have precipitated the need for a new drug development model for these combinations. "It's really the direction the biology is moving," he says. "It becomes much more biologically plausible that two or more agents used in combination could have more efficacy than one agent used alone." Friends of Cancer Research Experts meet in September 2009 to discuss combination drug trials. "As our understanding of the molecular biology of cancer continues to increase, we're seeing that cancers are really being subdivided for different pathways," says Adam Clark, director of scientific and federal affairs at FasterCures, a Washington, DC–based think tank, and lead author on a study published last year outlining the need for a new regulatory approval process (Oncologist, 496–499, 2010). "Being able to develop combination therapies that can target two pathways at once or two players in the same pathway will open up more lines of effective therapies for patients based on their genotypes." Compelling rationale The concurrent testing of new drugs does present added risks to study participants, so the FDA is not taking the approach lightly. However, by asking for a nonspecific "compelling biological rationale" from companies wishing to go down the codevelopment pathway, the agency is keeping the wording intentionally vague. "We're trying definitely not to be rigid about this," says Temple. "We're talking about drugs that in the end are supposed to make a difference to a very bad disease. That tends to make one a little bit flexible." The drug industry, for the most part, is embracing the FDA's new guidance. "You'd like to push the novel combination strategy earlier in the life cycle of the drug development process," says Stuart Lutzker, vice president of BioOncology Exploratory Clinical Development at Genentech in South San Francisco and a coauthor of the Oncologist report. But even without the policy being fully formalized, last year Genentech started recruiting participants with advanced solid tumors for an early-stage trial involving two drugs, one that selectively inhibits MEK, also known as mitogen-activated protein kinase, and another that blocks the product of an oncogene called phosphoinositide 3-kinase (PI3-kinase). Trials are also planned to test the former drug in combination with a BRAF kinase inhibitor for metastatic melanoma and to try the latter drug in conjunction with an investigational targeted agent to treat breast cancer. Similarly, GlaxoSmithKline is testing its own MEK and PI3-kinase double whammy treatment as well as running trials combining the same MEK inhibitor with another drug that blocks part of the PI3-kinase pathway. "And the fact that the FDA has come out with the draft guidance document is going to encourage more companies to do the same," Lutzker says. Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Let's get physical: mechanical forces drive a new field of study
- Nat Med 17(3):271 (2011)
Nature Medicine | News Let's get physical: mechanical forces drive a new field of study * Lauren CahoonJournal name:Nature MedicineVolume: 17,Page:271Year published:(2011)DOI:doi:10.1038/nm0311-271Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg ITHACA, NEW YORK — The cells in Cynthia Reinhart-King's lab are getting a workout. Placed in a variety of man-made environments, these human cancer cells must fight their way through obstacle courses of tangled collagen fibers. Others pull their healthy companions behind them as they slog their way through a viscous terrain. The Reinhart-King lab here at Cornell University's Center on the Microenvironment and Metastasis hosts this grueling cellular boot camp to study how cells physically respond to environmental forces. In the past, science viewed cancer mostly from a genetics and chemistry view—genes turned off or on stimulating chemical signals and protein cascades—but the role of physics was largely ignored. Now, however, researchers are learning that mechanical forces hold the power to turn healthy tissue diseased or push cancerous cells to leave their home tissue and metastasize. This knowledge, researchers hope, should spur on a new generation of therapeutics to keep cancer cells glued in their tracks. "Our goal is to cure cancer. I make no bones about it," says Paolo Provenzano, a biomedical engineer at the Fred Hutchinson Cancer Research Center in Seattle who studies the proteins involved in cell adhesion. "If I didn't think this had a chance in eradicating cancer, I wouldn't be studying it." Reinhart-King and her colleagues demonstrated the importance of physical forces in regulating cancer in 2005. By manipulating tissue stiffness in vitro, the researchers showed that a more pliant environment actually reduced tumor growth (Cancer Cell8, 241–254, 2005). The paper created a buzz among bioengineers and biophysicists; for them, it was logical that physical forces could affect biological outcomes. Yet "the cancer folks pretty much ignored it," says Valerie Weaver, of the University of California–San Francisco, who led the study. Six years on, the role of mechanical forces in influencing cancer is now well accepted among cancer biologists, owing in large part to the ever-growing body of research being published in high-profile journals. Jerry Lee, deputy director of the Center for Strategic Scientific Initiatives at the US National Cancer Institute (NCI) in Bethesda, Maryland, says that this work has brought the field of cell mechanics into the mainstream. "People are finally paying attention to the academic research," he says. Michael King, Cornell University Cynthia Reinhart-King and her Cornell lab. When tissue becomes more rigid, the cells attached to the surrounding scaffolding feel this hardening via receptors in the cell membrane known as integrins, which act as a bridge between this extracellular matrix and the cells' cytoskeleton. As the tissue tugs on the integrins, the cells feel that pull and flex their cytoskeletons in response, becoming stiffer themselves. Just five years ago, researchers assumed that this mechanical messaging took place only in load-bearing 'workhorse cells', such as fibroblasts or bone cells. But thanks to recent research, scientists now know cells of every stripe respond to force. For instance, in the past year, researchers showed that both mammary cells and neural cells react to stiffness (PLoS One, e12905, 2010; Mol. Cancer, 35, 2010). Pillar of strength Researchers are now clarifying the roles of cell strength and motility in cancer. Reinhart-King's group has unpublished evidence showing that metastatic cells are far stronger than their healthy siblings. This extra strength allows these rogue cells to leave their home tissue and push their way through the fibrous extracellular matrix, even occasionally pulling normal cells behind them like reluctant accomplices. If she can find the protein responsible for this superstrength, Reinhart-King hopes to develop targeted therapies to render metastatic cells harmless. But before such therapies can be found, researchers need new laboratory tools that better mimic the microenvironment of living human tissue, argues Denis Wirtz, a biomolecular engineer at Johns Hopkins University in Baltimore. Last year, Wirtz and his colleagues showed that the structure and behavior of cells are dramatically different in two-dimensional environments (such as Petri plates) compared to three-dimensional systems such as those found in the body (Nat. Cell Biol.12, 598–604, 2010). In some cases, proteins that slowed cells down in flat settings actually sped them up once they entered the three-dimensional extracellular matrix or vasculature—a structural switch that "is shaking up the current understanding of motility," says Wirtz. To foster growth in this burgeoning field, in October 2009 the NCI launched the Physical Sciences-Oncology Centers program, a collection of a dozen scientific teams across the country collaborating on understanding the physics of cancer. Weaver, Wirtz and Reinhart-King are all participants in the project. "We want to make sure that [these labs] are not just concept shops," says Lee, who is leading the initiative. "The technology is there, the literature is there, so now the pace of new discoveries will be faster." Additional data Author Details * Lauren Cahoon Search for this author in: * NPG journals * PubMed * Google Scholar - Cancer vaccine boosted by infrastructure for HIV care in Africa
- Nat Med 17(3):272 (2011)
Nature Medicine | News Cancer vaccine boosted by infrastructure for HIV care in Africa * Esther NakkaziJournal name:Nature MedicineVolume: 17,Page:272Year published:(2011)DOI:doi:10.1038/nm0311-272aPublished online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg LWEZA, UGANDA — On 19 January, health workers at an HIV care facility run by Mildmay Uganda began vaccinating 500 HIV-positive girls between the ages of 9 and 13 against the cancer-causing human papillomavirus (HPV). The campaign makes the clinic run by Mildmay, an international not-for-profit Christian organization, the first treatment center devoted to HIV to vaccinate for HPV in Uganda. AIDS clinics are poised to have an important role in providing essential infrastructure for delivery of the vaccine, Gardasil, which is made by Merck and protects against cervical cancer. Peter Mugyenyi, a world specialist in HIV/AIDS and director at the Joint Clinical Research Centre in Kampala, Uganda says this is part of larger plan whereby governments will integrate HIV services in normal delivery of health care: "We have to be futurists." Parisa Azadi Uganda tackles cervical cancer. Cervical cancer is the leading cause of cancer deaths among women in the developing world. The International Agency for Research on Cancer estimates that more than 274,000 women die of cervical cancer each year and that 80% of these deaths occur in developing countries. In addition to the distribution of Merck's Gardasil by Mildmay, GlaxoSmithKline has committed to donating 90,000 doses of its HPV vaccine, Cervarix, to be administered in Uganda, a portion of which was delivered with help from the US-based nonprofit Path. Currently, schools and health centers are being used to administer the HPV vaccine to adolescent girls in Africa. But these facilities and the teachers that staff them are overstretched. What's more, the vaccine cannot be delivered through the national routine immunization days in most countries, as these campaigns target children younger than five years. At Mildmay, the Cleveland, Ohio–based Axios International donated 1,600 doses for the 500 HIV-positive girls. The facility already has cold-chain facilities to ensure proper preservation of the shots and trained staff members who are knowledgeable in vaccine management and can report adverse effects to the government. The facility also has a fun-hospital setting for its young HIV-positive clients: there is an area where youngsters can read books and assemble puzzles and an adolescents' club where the girls discuss various issues affecting their lives. According to Emmanuel Luyirika, a doctor and the country director of Mildmay Uganda, the clinic has taken a comprehensive approach. "We are not only looking at the vaccine; we are screening for cancer of the cervix for all women. All women found to have early signs [of cervical malignancies] are treated; those with advanced signs are referred to the Mulago hospital," she says. Additional data Author Details * Esther Nakkazi Search for this author in: * NPG journals * PubMed * Google Scholar - New guidelines could plug data gaps in India's cancer research
- Nat Med 17(3):272 (2011)
Nature Medicine | News New guidelines could plug data gaps in India's cancer research * T V PadmaJournal name:Nature MedicineVolume: 17,Page:272Year published:(2011)DOI:doi:10.1038/nm0311-272bPublished online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg NEW DELHI — In India, cancer researchers often lack comprehensive country-specific data, which makes it difficult for them to probe why some cancers predominate here and to know what prevention and treatment strategies work best. To plug this data gap, the Indian Council of Medical Research (ICMR), located in New Delhi, released its first set of recommendations on tracking and treating three kinds of cancers in late December, with instructions on 17 more cancer types in the pipeline. The ICMR, which was established in 1949 under India's health ministry to oversee biomedical research and disease control, released treatment guidelines for cancers of the stomach, cervix, and 'buccal mucosa cavity', which refers to malignancies in the area between cheeks and the tongue. The guidelines note that the new framework "may be used for more focused and planned research programs." They hope to use the feedback they receive about treatment challenges to help scientists select research topics with immediate practical applications within India, ICMR deputy director general Kishore Chaudhry told Nature Medicine. The newly issued recommendations "will give direction to local research," says Jai Prakash Agarwal, an oncologist at the Tata Memorial Centre (TMC), a cancer treatment facility in Mumbai. The guidelines, which emerged after three years of consultation with more than 130 cancer experts in India, invite feedback on cancer cases and treatment complications doctors encounter to the ICMR, which will compile the information and identify topics that need further research. Prakash and others hope that by streamlining the collection of cancer-related information from hospitals, the new rules will help shed light on differences in cancer biology and genetics between Indians and other populations. For example, breast cancer often strikes premenopausal Indian women, whereas it is more common among postmenopausal women in the West, says TMC's Vani Parmar. Parmar adds that 'triple negative' breast cancers, in which three crucial cancer cell receptors are missing, account for around a third of all cases among Indian women, compared with half that rate in Western countries. Even within India itself cancer rates vary dramatically. The ICMR guidelines state that there are few studies on factors linked to the strikingly high incidence of stomach cancer—57 per 100,000—in India's northeast, compared with the rates as low as 5 per 100,000 in other regions. Agarwal hopes that the guidelines will ultimately help researchers in India better understand the variation in chemotherapy and radiation dose response within the country's population. According to the ICMR, Indian researchers also need to strengthen the research agenda for buccal mucosa cancers, caused by extensive use of chewing tobacco in India but rare in developed countries. The agency notes that scant information exists worldwide regarding what the optimum chemotherapy and radiation dosages are for treating this particular cancer. Additional data Author Details * T V Padma Search for this author in: * NPG journals * PubMed * Google Scholar - From spinach scare to cancer care
- Nat Med 17(3):273-275 (2011)
Nature Medicine | News From spinach scare to cancer care * Elie Dolgin1Journal name:Nature MedicineVolume: 17,Pages:273–275Year published:(2011)DOI:doi:10.1038/nm0311-273Published online07 March 2011 In recent years, Salmonella has tainted foods including spinach, peanut butter and eggs, sickening thousands of people in the process. But researchers hope that these microbes will make headlines for a better reason: curing cancer. They want to harness Salmonella's special ability to thrive in oxygen-deprived conditions to target regions of solid tumors that are normally immune to conventional therapies. reports. View full text Additional data Affiliations * Elie Dolgin is a news editor with Nature Medicine in New York. Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Cancer as we know it
- Nat Med 17(3):276 (2011)
Nature Medicine | Book Review Cancer as we know it * Victoria Aranda1Journal name:Nature MedicineVolume: 17,Page:276Year published:(2011)DOI:doi:10.1038/nm0311-276Published online07 March 2011 The Emperor of All Maladies: A Biography of Cancer Siddhartha Mukherjee Scribner, 2010 592 pp., hardcover, $30.00 ISBN: 1439107955 Buy this book: USUKJapan Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cancer is the second leading cause of death in many industrialized countries, and almost one in three of us will have a direct encounter with it in our lives. Although the subject of cancer fills countless pages of scientific publications, gets incessant media coverage and occupies a prevalent place in science politics, few books have approached it in a comprehensive manner accessible to most readers. The Emperor of All Maladies by Siddhartha Mukherjee is, arguably, as good as it gets. The prospect of digesting 4,000 years of medical history for a lay audience would make even the most committed cancer expert gasp. Mukherjee, a cancer clinician-scientist, sets out for this task with the right qualifications. He also proves to be an engaging storyteller. But his ability to empathize with his subjects, not only people with cancer but also doctors, scientists and advocates, is what ends up making this book an enthralling read for all audiences. Admittedly, the book can get quite complicated at times, particularly when Mukherjee delves into the complexity of cancer biology. But wading through these details is a rewarding endeavor: a clear picture emerges of where the fight against cancer currently stands, enabling readers to make an educated guess about the future. The book covers the history of cancer from antiquity to the present. Reading it is akin to riding in a time machine: prepare to spend 500 pages hopping from Persian royal chambers to scientifically marginalized labs in dark basements to dreadful hospital wards, in almost-cinematic cuts and flashbacks. This daring approach succeeds in organizing the chronologically overlapping quests for a cure and a cause as they mesh with the mainstream of cancer science, medicine and policy. The twists and turns of Mukherjee's story have a connecting thread throughout: the touching testimonies of people with cancer. Of them, Mukherjee writes recurring tales of "reckoning, fear, hope and resilience." Regardless of how much these stories resonate for each reader, they uniformly deliver the powerful message that the disease itself has not changed, and that every personal battle against it still encompasses the entire history of cancer. Mukherjee captures the reader by choosing his main characters to symbolize the many minds and bodies that have carried the burden of this scourge. We can root for all cancer patients through Carla Reed, as she pushes through her treatments under Mukherjee's care. We can almost pity the arrogance of the early radical cancer surgeons, such as William Halsted, as they try to carve away tumors that are beyond their surgical reach, disfiguring patients along the way. We fall for the deceitful simplification that propelled Mary Lasker's unfaltering activism and Sidney Farber's audacious research to find a magic bullet and blast away the core of a coreless disease. Although the book traces a comprehensive picture of cancer's history, it is anchored strongly around the iterative advances of empirical drug discovery, steered in the second half of the 20th century by the US National Cancer Institute. We follow doctors and advocates who, unfazed by the bodies piling up on the battlefield, use their partial victories to convince society into sanctioning an all-out 'war on cancer'. Mukherjee disarms this premature attack with merciless logic. His hindsight illuminates the inevitability of our ensuing failure. Rallied by technological progress and impatient lobbying, science policymakers charted the timeline and defined the terms of our victory against cancer. It may be obvious to anyone professionally involved in cancer today that this thrust was doomed by our limited understanding of the disease at the time, but it is still useful to present the reasons for our defeat to the public. Mukherjee's insights might help quiet a continuing public demand for a cure. Mukherjee juxtaposes the struggles of cancer therapy with the steady progress of the search for the biological origin of cancer. He argues that the recent wealth of scientific knowledge has generated the tools that are tilting the death toll of cancer slowly but surely: prevention, early detection and targeted therapies. The author uses poignant examples to endorse this multipronged attack on cancer, and a few lengthy digressions to illustrate the need to join our forces against it. The story wanders out of focus with the retelling of the tobacco industry's woes, and the detour into AIDS territory reads as a drawn-out narrative of the author's ambivalence toward cancer advocacy. While it has helped transform cancer drug discovery, misguided public pressure can at times derail our quest. He hints at the need to inform the public dispassionately and without bias, and we can support his attempt to do so in this book. With the merging of cancer biology and therapy, the book winds down as the author realizes that he "must confront the death of his subject." His sketch of the future is a wavering picture but is one of adamant hope. Mukherjee finds the progress of cancer science encouraging, despite the dangers of simplification to which the field has succumbed in the past. Mukherjee offers up the growing population of cancer survivors as a testimony that, although a war against cancer might not be ours to win, we can make substantial progress against the disease. With this realistic conclusion, the book outlines a truce aimed at reducing untimely cancer deaths but concedes that cancer might always remain a part of us, a harbinger of our finitude. The reality of his subjects' harrowing journeys notwithstanding, the author posits that the integration of biology and therapy provides a hopeful framework for our renewed efforts. Scientists and doctors can certainly agree that the more we know about cancer, the closer we will be to defeating it, and this book is a step toward bringing the public onboard in that direction. Additional data Affiliations * Victoria Aranda is an associate editor at Nature Medicine. Author Details * Victoria Aranda Search for this author in: * NPG journals * PubMed * Google Scholar - A passion to cure cancer
- Nat Med 17(3):277 (2011)
Nature Medicine | Book Review A passion to cure cancer * Anton Hagenbeek1Journal name:Nature MedicineVolume: 17,Page:277Year published:(2011)DOI:doi:10.1038/nm0311-277Published online07 March 2011 Henry Kaplan and the Story of Hodgkin's Disease Charlotte Jacobs Stanford General Books, 2010 456 pp., hardcover, $35.00 ISBN: 0804768668 Buy this book: USUKJapan Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Henry Seymour Kaplan (1918–1984) was a pioneer in the field of cancer research. This biography, written by Charlotte Jacobs, professor of Medicine in the Division of Oncology at Stanford University School of Medicine, describes his major achievements in the fight against Hodgkin's lymphoma over almost 65 years, but it also touches upon the man he was in relation to his family and peers. The fact that Hodgkin's lymphoma, a cancer arising in lymph nodes, seemed to be the first curable human malignancy makes this journey all the more exciting. Jacobs based the biography on hours of interviews and correspondence with people that worked closely with Kaplan within the US. In 1943, Kaplan began training in diagnostic radiology in Minneapolis, spending nights and weekends learning basic skills in cancer biology and applying these to his first studies on the induction of leukemia in mice by radiation. With his first academic appointment at Yale University in 1945, he got his own laboratory and a budget of $300 a year for his mice. At that time, it was already clear that Kaplan had a passionate desire to cure cancer. After he became a leading scientist at the US National Cancer Institute in Bethesda, Maryland, Kaplan was appointed Chairman of Radiology at Stanford University in 1948. In the years to follow, he was responsible for moving the first linear accelerator from the lab to the clinic and started to irradiate patients in 1956, with special emphasis on patients with Hodgkin's lymphoma. Kaplan realized that he needed a team of experts to cure his patients: a pathologist to come up with the diagnosis, a surgeon to perform a so-called staging laparotomy (called by some a 'living autopsy') during which the spleen and a number of lymph nodes are removed from the abdomen, a radiologist to aid in lymph nodes visualization through lymphangiography and, later on, an oncologist to introduce chemotherapy. For this he hired Saul Rosenberg in 1961. Kaplan initiated the first randomized clinical trial in Hodgkin's lymphoma in 1962. The trial compared radiation doses, resulting in a step-by-step increase in cure rate. Rosenberg, however, was opposed to aggressive treatments, so Kaplan openly criticized him. This soured the relationship between these two pioneering researchers. In 1967, a study by Vincent de Vita reported that a combination chemotherapy called MOPP could achieve a 90% complete remission rate for advanced Hodgkin's lymphoma. Because of this, Kaplan included MOPP in his next randomized trial in 1968, comparing radiation alone with radiation plus MOPP. With the combined treatment, 86% disease-free survival was achieved in patients with advanced disease. Again, Rosenberg worried about potential morbidity and late effects, but Kaplan insisted on aggressive treatment of the disease. In the meantime, Kaplan pursued his laboratory studies focusing on detecting a virus that might cause Hodgkin's lymphoma, collaborating with Robert Gallo from the National Cancer Institute on isolating the malignant cell in Hodgkin's lymphoma and on developing monoclonal antibodies ('magic bullets') against lymphoma. However, these three programs failed. One of the major disappointments in his professional career was that he failed to establish a Comprehensive Cancer Center at Stanford after the US National Cancer Act came into effect in 1971. This initiative was blocked by other department heads, who did not want Kaplan to be too much in control, revealing the tense relationships between Kaplan and his peers. He blamed Rosenberg for this failure, which made him very bitter, causing him to withdraw into his research lab. In the 1970s, the formation of secondary tumors in patients previously treated with radio- and chemotherapy became apparent. Kaplan admitted this after carefully checking Stanford patients that had finished treatment. Therefore, in the early 1980s, a new chemotherapy regimen called ABVD was introduced by Gianni Bonadonna from Milano that appeared to be much less toxic than the original MOPP regimen. Even today, ABVD is recognized worldwide as the first-choice chemotherapy regimen. During the last years of his career, Kaplan focused on reducing radiation doses and fields, to prevent late effects. Despite his achievements and recognition as one of the pioneers in developing curative treatments for Hodgkin's lymphoma, at the end he felt that he had failed to answer his major question on the cause of Hodgkin's lymphoma and to determine ways to prevent it. A considerable part of the book is devoted to Kaplan's personal life. Although he married and had children, he was never at home. His wife called his lab his mistress, and he failed to recognize his children's needs, eventually becoming estranged from them. In the Kaplan family, achievement was presumed; no one spoke of failure. Problems were not discussed, only solutions. Among his peers, Kaplan was called a saint by some and a son of a bitch by others; he always had the last word, and he could be disrespectful to colleagues. He had few real friends. During the last weeks of his life, after he was diagnosed with lung cancer, possibly due to prolonged inhalation of radioactive radon gas in the early years, he again became close to his family. This biography deals with an extraordinary man who was instrumental in developing curative treatment strategies for Hodgkin's lymphoma. Jacobs has provided a superb overview, although most of the stories are limited to Stanford and the US. This is a weak aspect of the book, which could have instead been situated in a more global environment. Nevertheless, I strongly recommend this biography to anyone interested in following the stormy journey of a true pioneer in medicine. Author information Affiliations * Anton Hagenbeek is Professor of Hematology at the University of Amsterdam, Amsterdam, The Netherlands. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Anton Hagenbeek Author Details * Anton Hagenbeek Contact Anton Hagenbeek Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Cancer's top papers
- Nat Med 17(3):278-279 (2011)
Nature Medicine | Analysis Cancer's top papers Journal name:Nature MedicineVolume: 17,Pages:278–279Year published:(2011)DOI:doi:10.1038/nm0311-278Published online07 March 2011 Which are the most relevant recent discoveries in cancer research? Which advances in cancer biology, drug discovery and clinical practice have been the most important for the field? View full text Additional data - Highly cited cancer papers, 2008–2010
- Nat Med 17(3):280-282 (2011)
Nature Medicine | Analysis Highly cited cancer papers, 2008–2010 Journal name:Nature MedicineVolume: 17,Pages:280–282Year published:(2011)DOI:doi:10.1038/nm0311-280Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The following tables show the primary research papers on cancer published between 2008 and 2010 that have had the highest number of citations in the literature. To create these tables, we queried the Scopus database (http://www.scopus.com/) to search for articles that included the term 'cancer' or related terms in the title, abstract or keywords. After sorting the results on the basis of citation number, we removed reviews and epidemiological studies. The predominance of genomic studies in these tables is remarkable. The number of citations is accurate as of 8 February 2010. The tables include papers that have been cited at least 250 (2008 table), 125 (2009 table) and 35 (2010 table) times. Table 1: Highly cited cancer research published in 2008 Full table * Tables index * Next table Table 2: Highly cited cancer research published in 2009 Full table * Previous table * Tables index * Next table Table 3: Highly cited cancer research published in 2010 Full table * Previous table * Tables index Additional data - Targeting the missing links for cancer therapy
- Nat Med 17(3):283-284 (2011)
Nature Medicine | News and Views Targeting the missing links for cancer therapy * Kornelia Polyak1 * Judy Garber1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:283–284Year published:(2011)DOI:doi:10.1038/nm0311-283Published online07 March 2011 A continuing quest in clinical oncology is to effectively eliminate tumors without major side effects. But drugs rationally tailored against specific tumors and predictive markers for patient selection are very limited, and their identification is challenging. A phase 1 study has provided proof of concept for the use of PARP inhibitors in tumors from individuals carrying BRCA mutations—a remarkable success in rational drug design and translational research. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A quote by Paracelsus, the father of toxicology, "it is the dose that determines that a thing is not a poison," highlights one crucial bottleneck of anticancer drug development: the need to identify compounds that eliminate cancer cells at concentrations harmless to the individual. Most traditional chemotherapeutic agents were identified on the basis of their ability to kill fast-growing cancer cells in in vitro models. Not surprisingly, they also eliminate rapidly dividing normal cells, such as hematopoietic and intestinal epithelial progenitors, leading to serious side effects that limit their effective dosing. But even these imperfect drugs can eradicate a subset of tumors, including testicular cancer and many childhood leukemias. Their efficacy in other tumor types, however, is fairly limited, especially in advanced disease. A major goal of cancer research in the past decades has therefore been to identify cancer-specific molecular alterations as targets for rational drug design and biomarkers to help select those people who will probably benefit from the treatment. Tumorigenesis is an evolutionary process driven by the accumulation of genetic and epigenetic alterations that create a diverse population of cancer cells, of which the most successful are continuously being selected by the tumor microenvironment1. Thus, drugs that selectively eliminate cancer cells can be developed in three general ways. First, we can identify and target mutant or abnormally expressed proteins that are only present in cancer cells. Successful agents of this type in the clinic include imatinib, targeting BCR-ABL oncoprotein in chronic myelogenous leukemia, and trastuzumab, targeting HER2 oncoprotein in a subset of breast tumors2. Recent whole-genome sequencing projects aimed to identify additional cancer-specific mutations that can be exploited therapeutically, but thus far there has been little success. Second, the microenvironment could be altered to reduce the fitness of cancer cells by limiting infiltration of leukocytes that secrete cytokines promoting ! tumor cell survival or angiogenesis with anti-inflammatory and antiangiogenic drugs. The third approach may consist of identifying and targeting genes and pathways necessary for cancer cells because of their abnormal cellular milieu caused by genetic and epigenetic abnormalities—even targeting proteins other than the mutant protein per se. The so-called synthetic lethal strategy belongs to this general approach3, where two genes show a synthetic lethal relationship if the elimination of either one is compatible with cellular survival but the inhibition of both is not. A cancer-specific mutation in one of two genes in synthetic lethality combined with the therapeutic targeting of the other one will selectively kill tumors without harming normal cells. Previous studies showed that cancer cells deficient in either BRCA1 or BRCA2 (the breast-cancer–associated proteins) are specifically sensitive to poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibition4, 5, which is a family of enzymes involved in base-excision repair, a key pathway in DNA single-strand break repair. Inhibition of PARP activity led to accumulation of nuclear RAD51 foci, indicating the presence of double-strand breaks that are normally repaired by homologous recombination6. Cells with defective homologous recombination, such as those lacking BRCA1 or BRCA2, are therefore killed by PARP inhibitors, whereas normal cells are relatively unaffected (Fig. 1). PARP inhibitors can then be used as a different therapeutic strategy for the treatment of tumors that lack BRCA function. The availability of a highly effective and well-tolerated oral PARP inhibitor, olaparib, developed by Kudos and acquired by AstraZeneca (AZD2281), allowed the rapid translation of these findings into a clinical trial7. Figure 1: Synthetic lethality in tumors from BRCA1 and BRCA2 mutation carriers treated with PARP inhibitors. DNA repair pathways are categorized into single and double-strand break–specific mechanisms, both of which are further divided into subgroups. PARP inhibitors block the repair of single-strand breaks (SSBs), which if left unrepaired are converted to double-strand breaks (DSBs) during replication. In normal cells of BRCA1 and BRCA2 mutation carriers (BRCA1/2+/−), these DSB lesions are repaired by homologous recombination because one copy of BRCA1 or BRCA2 is sufficient for repair proficiency, and the cells remain viable. However, in cells with defective homologous recombination, such as tumor cells in BRCA mutation carriers that lost the wild-type copy of BRCA by loss of heterozygosity (LOH), double-strand breaks cannot be efficiently repaired, leading to cancer cell death and elimination of the tumor. Resistance may arise because of the presence of tumor cells that retained a wild-type copy of BRCA or mutations in BRCA or other genes that restore repair proficiency or ov! ercome PARP inhibition by other mechanisms. Katie Vicari * Full size image (83 KB) One of the first studies showing the potential of synthetic lethal strategy in clinical trials in people with cancer was published in the New England Journal of Medicine by Fong et al.7. They designed a phase 1 clinical trial to test the pharmacokinetic and pharmacodynamic profiles of olaparib in a cohort of individuals enriched for BRCA1 or BRCA2 mutation carriers7 independent of their type of tumor. The authors tested six different treatment regimens using a range of drug concentrations (100–600 mg) and compared continuous daily drug administration with drug administration in two of every 3 weeks (2 weeks of daily treatment, 1 week with no treatment)7. On the basis of dose-limiting toxicity, they established 600 mg and 400 mg as the maximum administered and tolerated doses, respectively. Among the 60 patients enrolled in the study, only the 22 BRCA1 or BRCA2 mutation carriers showed an objective antitumor response, and 12 out of 19 mutation carriers who had breast, ovarian or prostate tumors, had a clinical benefit from PARP inhibitor treatment, with remarkable responses being achieved in a few cases7. The side effects, mostly nausea and vomiting, were relatively mild at doses where effective antitumor responses (decreases in tumor size) were observed, although myelosuppression was also detected. Such exciting results have led to the use of other PARP inhibitors in clinical trials alone or in combination with other chemotherapeutic drugs, such as carboplatin and cisplatin, gemcitabine, irinotecan and temazolamide, in diverse patient populations: individuals with BRCA1 and/or BRCA2 mutations, triple-negative (negative for estrogen receptor, progesterone receptor, and HER2) breast cancer, serous ovarian cancer, glioblastomas and pancreatic cancer, among others. A recent study showed impressive clinical responses in people with triple-negative breast cancer treated with a combination of the intravenous PARP inhibitor iniparib and carboplatin and gemcitabine8, 9. Unfortunately, whereas the synthetic lethality concept was relatively easy to show in BRCA1 and BRCA2 mutation carriers, finding effective and nontoxic combinations, bioavailable formulations and delivery schedules, and molecular profiles of sensitive tumors remains more empiric than would be ideal. Despite the remarkable success of PARP inhibitors in early trials, numerous questions remain unanswered. Not every tumor in BRCA mutation carriers responds to treatment, suggesting that additional factors besides the loss of these genes define cellular response. Loss of the gene encoding the phosphatase and tensin homolog (PTEN) is one of these candidates, as it seems to confer sensitivity to PARP inhibitors even in wild-type BRCA1 and BRCA2 tumor cells, at least in in vitro models10. In addition, PARP inhibitors may be used to treat non-BRCA1 or non-BRCA2 mutated tumors if similar or related defects in DNA damage repair response can be identified or if DNA damage with specific cytotoxic chemotherapeutic agents can be induced when the cell's ability to repair DNA using the PARP inhibitor is compromised. Which agents would be combined with PARP inhibitors with the best therapeutic index (that is, the most effective and least toxic) and which biomarkers would allow selection of those individuals who would benefit from this treatment remain to be determined. Most tumors eventually escape from PARP inhibition, indicating preexisting or induced resistance11. A study showed that cells from BRCA2 mutation carriers resistant to cisplatin and PARP inhibitors have an interesting mechanism of resistance that restores wild-type BRCA2 function by correcting the defect in homologous recombination, leading to loss of PARP inhibitor sensitivity12. Although several PARP inhibitors have been developed with specificities for inhibition of different PARPs and nonidentical mechanisms of action, specificity to PARP inhibition does not always correlate with the degree of clinical response, suggesting potential off-target effects. For example, in contrast to olaparib and most other PARP inhibitors, iniparib does not inhibit PARP's enzymatic activity but rather blocks the interaction of PARP with DNA13. The in vitro potency of iniparib is also much lower (micromolar range) compared to olaparib (low nanomolar range), although presumably a more active metabolite must exist, as the serum half-life of iniparib is very short (~4 min). Because this metabolite has not been conclusively identified, the in vivo targets of this drug are even less clear, although no other targets aside from PARP have been reported. There is a concern for potential long-term toxicities from exposure of healthy tissues to agents inhibiting DNA repair, such as PARP inhibitors, including the induction of secondary tumors or leukemias. These issues can be crucial, as the agents are developed to cure early-stage tumors—through adjuvant and neoadjuvant therapies—and even to reduce cancer risk in BRCA1 and BRCA2 mutation carriers, who have substantial risk of developing lethal ovarian, breast and pancreatic malignancies. In light of the large number of clinical trials ongoing or planned with various PARP inhibitors, the answers to many of these questions will begin to emerge in the near future. References * References * Author information * Merlo, L.M., Pepper, J.W., Reid, B.J. & Maley, C.C.Nat. Rev. Cancer6, 924–935 (2006). * ChemPort * ISI * PubMed * Article * Murdoch, D. & Sager, J.Curr. Opin. Oncol.20, 104–111 (2008). * ChemPort * ISI * PubMed * Article * Kaelin, W.G. Jr.Nat. Rev. Cancer5, 689–698 (2005). * ChemPort * ISI * PubMed * Article * Farmer, H.et al. Nature434, 917–921 (2005). * ChemPort * ISI * PubMed * Article * Bryant, H.E.et al. Nature434, 913–917 (2005). * ChemPort * ISI * PubMed * Article * Schultz, N., Lopez, E., Saleh-Gohari, N. & Helleday, T.Nucleic Acids Res.31, 4959–4964 (2003). * ChemPort * ISI * PubMed * Article * Fong, P.C.et al. N. Engl. J. Med.361, 123–134 (2009). * ChemPort * ISI * PubMed * Article * Carey, L.A. & Sharpless, N.E.N. Engl. J. Med.364, 277–279 (2011). * O'Shaughnessy, J.et al. N. Engl. J. Med.364, 205–214 (2011). * Dedes, K.J.et al. Sci. Transl. Med.2, 53ra75 (2010). * PubMed * Ashworth, A.Cancer Res.68, 10021–10023 (2008). * ChemPort * ISI * PubMed * Article * Edwards, S.L.et al. Nature451, 1111–1115 (2008). * ChemPort * PubMed * Article * Ferraris, D.V.J. Med. Chem.53, 4561–4584 (2010). * ChemPort * PubMed * Article Download references Author information * References * Author information Affiliations * Kornelia Polyak and Judy Garber are at the Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA and the Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA. Competing financial interests K.P. receives research support from and is a consultant to the Novartis Institute of Biomedical Research and is also a member of the Scientific Advisory Boards of Theracrine, Inc. and Metamark Genetics, Inc. J.G. is conducting clinical trials using PARP inhibitors manufactured by Abbott Laboratories and AstraZeneca Pharmaceuticals. She is a consultant to Generation Health, Inc. Corresponding author Correspondence to: * Kornelia Polyak Author Details * Kornelia Polyak Contact Kornelia Polyak Search for this author in: * NPG journals * PubMed * Google Scholar * Judy Garber Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - B cells and macrophages in cancer: yin and yang
- Nat Med 17(3):285-286 (2011)
Nature Medicine | News and Views B cells and macrophages in cancer: yin and yang * Alberto Mantovani1Journal name:Nature MedicineVolume: 17,Pages:285–286Year published:(2011)DOI:doi:10.1038/nm0311-285Published online07 March 2011 Inflammation is an important component of the tumor microenvironment; however, the mechanisms through which immune cells might promote tumorigenesis are unclear. A recent study indicates that B cells and antibodies have a key role in orchestrating macrophage-driven, tumor-promoting inflammation, suggesting that modulating the pathways involved might be of therapeutic benefit in cancers driven by chronic inflammation. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cancer-related inflammation is characterized by the recruitment of cells of the monocyte-macrophage lineage to tumor tissues1, 2, 3. Cancer-causing genetic events, such as oncogene activation and inactivation of tumor suppressor genes, elicit leukocyte recruitment and the formation of an inflammatory microenvironment. In turn, myelomonocytic cells can affect virtually all steps of carcinogenesis, including genetic instability and metastasis. In progressing tumors, tumor-associated macrophages (TAMs) generally express an M2-like phenotype3, which is characterized by low interleukin-2 (IL-2) expression, high IL-10 expression and low tumoricidal activity, and promotes tissue remodeling and angiogenesis (Fig. 1). The functional orientation of TAMs is orchestrated by tumor-derived and host-derived signals3. In most human tumors, TAM infiltration is associated with poor prognosis, as seen in Hodgkin's disease4. Figure 1: B cell–mediated orchestration of tumor-associated macrophages. Various pathways orchestrate the protumor function of M2-like myelomonocytic cells in tumors. Andreu et al.5 showed that B cells produce antibodies that can form immunocomplexes (ICs) that orient TAMs to promote cancer via Fcγ receptors on the surface of TAMs. T helper type 2 (TH2)-derived IL-4, tumor-associated fibroblasts (TAFs) and tumor cells themselves provide alternative or complementary pathways that can skew macrophages toward an M2-like protumor phenotype. Katie Vicari * Full size image (86 KB) A recent study by Andreu et al.5 using a mouse model of human papilloma virus-driven multistage squamous epithelium carcinogenesis has provided insights into the complexity of the pathways involved in skewing mononuclear phagocyte function in this type of cancer. B lymphocytes were found to orchestrate TAM functions by 'remote control' by producing antibodies that interact with and activate Fcγ receptors on both tumor-resident and recruited myeloid cells (Fig. 1). These results suggest that B cell– and Fcγ receptor–mediated pathways might be therapeutically targeted in individuals with chronic inflammatory disease who are at risk of developing cancer or even in established tumors. Andreu et al.5 showed that during carcinogenesis, with the help of CD4+ T cells, B cells produced antibodies directed against extracellular matrix components at the tumor site. Fcγ receptor–mediated recognition of immune complexes containing these antibodies, most likely in concert with myeloid differentiation factor-88–dependent signaling, led to mast cell–dependent angiogenesis and recruitment of mononuclear phagocytes. In this tumor model, TAM-mediated enhancement of carcinogenesis is complement independent, but complement components can drive myelomonocytic cell recruitment in other tumors3, 6. In the tumors studied by Andreu et al.5, mononuclear phagocyte-dependent tumor promotion is mediated by a mature, M2-like macrophage population rather than by immature cells (the myeloid-derived suppressor cells) in the myelomonocytic differentiation pathway. The immune complex-conditioned TAMs can in turn participate in yet another pathway of tumor promotion mediated by fi! broblasts7 (Fig. 1). In this pathway, via macrophage-derived IL-1, carcinoma cells direct fibroblasts to recruit more macrophages and promote angiogenesis. Functionally skewed cells of the myelomonocytic differentiation pathway are a common denominator of inflammation-promoted carcinogenesis, although the cell types and mediators involved can differ considerably. For instance, in a mouse model of breast carcinogenesis and metastasis, the culprit of M2 polarization of TAMs and tumor promotion was T helper type 2 cell–derived IL-4 (ref. 8) (Fig. 1). Similarly, the mechanisms of B cell–mediated tumor promotion need not be restricted to antibody production by these cells3, 9 (T. Schioppa and F. Balkwill, Queen Mary University of London, personal communication). B cells have also been shown to drive M2-like polarization of macrophages and to promote the growth of transplanted B16 melanomas9. In this case, the promoter of M2 macrophage polarization was a B cell subset (B-1 cells) characterized by constitutive IL-10 production. In prostate cancer, androgen ablation results in tissue damage and leukocyte recruitment, including B ce! lls. In this example, B cells produced lymphotoxin, which promoted the hormone-independent growth of prostate cancer10. Antibodies are also part of the anticancer therapeutic armamentarium, and there is strong evidence that macrophages and Fcγ receptors are key to their antitumor activity, such as for the effects of the CD20-specific monoclonal antibody rituximab. Treatment of a xenograft lymphoma model with rituximab resulted in leukocyte recruitment mediated by the chemokines CCL3 and CCL4 and tumor eradication11. However, combined treatment with a CCL3 antagonist and rituximab compromised the antitumor activity of rituximab, and an absence of CCL3 signaling led to macrophage depletion, suggesting that macrophages mediate the antitumor effects of this antibody, as also indicated by clodronate-mediated macrophage depletion11. Interestingly, rituximab elicits an M2-like phenotype in macrophages, leading to the increased phagocytosis of mouse lymphoma cells12. However, in people with lymphoma, the combination of rituximab with high-dose chemotherapy and autograft with peripheral blood progeni! tor cells was associated with an increased frequency of solid tumors in a large 20-year retrospective follow-up study13. Thus, the interplay between macrophages and B cells is complex, and, depending on the context, it can result in promotion of carcinogenesis5 or antitumor activity11. The results reported in these studies raise issues related to the diversity of the cancer-related inflammatory response and the design of therapeutic strategies to target this response. In different tissues and tumors, the pathways that orchestrate cancer-related inflammation can differ considerably, with a pivotal role of B cells and antibodies in squamous carcinogenesis5, 8. Strategies targeting B cells and IL-1 are currently in clinical use and may provide information relevant for defining the diversity of cancer-related inflammatory responses in humans, as well as innovative therapeutic strategies14. Myelomonocytic cells come in various types in different tumor contexts2, 3, but mononuclear phagocytes emerge as an essential common constituent of cancer-related inflammation. The identification of the various cellular and molecular pathways that participate in inflammation in different human cancers will be required to translate a better understanding of cancer-related inf! lammation to the bedside. References * References * Author information * Mantovani, A., Allavena, P., Sica, A. & Balkwill, F.Nature454, 436–444 (2008). * ChemPort * ISI * PubMed * Article * Pollard, J.W.Nat. Rev. Cancer4, 71–78 (2004). * ChemPort * ISI * PubMed * Article * Biswas, S.K. & Mantovani, A.Nat. Immunol.11, 889–896 (2010). * Article * Steidl, C.et al. N. Engl. J. Med.362, 875–885 (2010). * ChemPort * ISI * PubMed * Article * Andreu, P.et al. Cancer Cell17, 121–134 (2010). * ChemPort * ISI * PubMed * Article * Markiewski, M.M.et al. Nat. Immunol.9, 1225–1235 (2008). * ChemPort * PubMed * Article * Erez, N., Truitt, M., Olson, P., Arron, S.T. & Hanahan, D.Cancer Cell17, 135–147 (2010). * ChemPort * PubMed * Article * DeNardo, D.G.et al. Cancer Cell16, 91–102 (2009). * ChemPort * ISI * PubMed * Article * Wong, S.C.et al. Eur. J. Immunol.40, 2296–2307 (2010). * ChemPort * Article * Ammirante, M.et al. Nature464, 302–305 (2010). * ChemPort * PubMed * Article * Cittera, E.et al. J. Immunol.178, 6616–6623 (2007). * Leidi, M.et al. J. Immunol.182, 4415–4422 (2009). * ChemPort * PubMed * Article * Tarella, C.et al. J. Clin. Oncol. published online, doi:doi:10.1200/JCO.2010.28.9777 (28 December 2010). * Article * Dinarello, C.A.Annu. Rev. Immunol.27, 519–550 (2009). * ChemPort * ISI * PubMed * Article Download references Author information * References * Author information Affiliations * Istituto Clinico Humanitas Istituto di Ricovero e Cura a Carattere Scientifico and University of Milan, Milan, Italy. * Alberto Mantovani Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Alberto Mantovani Author Details * Alberto Mantovani Contact Alberto Mantovani Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Targeting RAF: trials and tribulations
- Nat Med 17(3):286-288 (2011)
Nature Medicine | News and Views Targeting RAF: trials and tribulations * Julian Downward1Journal name:Nature MedicineVolume: 17,Pages:286–288Year published:(2011)DOI:doi:10.1038/nm0311-286Published online07 March 2011 Although the rapid development of drug resistance is a known problem with targeted cancer therapies, recent studies have uncovered other surprises with RAF kinase inhibitors. These drugs can paradoxically activate downstream ERK signaling in some settings, with important implications for their clinical use. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg One of the most exciting developments in cancer drug discovery in recent years has been the success of Plexxikon's PLX4032—a small-molecule inhibitor of BRAF kinase—in the treatment of malignant melanoma. BRAF is activated by point mutation in roughly half of melanoma cases and drives the growth-promoting ERK pathway (Fig. 1). PLX4032 was designed to selectively target activated V600E mutant BRAF, which accounts for the majority of BRAF mutations found in melanoma. Although still in Phase 3 trials, PLX4032 looks set to join the very small group of clinically useful oncology agents targeting protein kinases, of which imatinib and trastuzumab have so far been the most impressive. Figure 1: Effects of RAF inhibitors on cells with BRAF or RAS mutations. Hatzivassiliou et al.6, Heidorn et al.7 and Poulikakos et al.8 described how RAF inhibitors can drive downstream ERK signaling in the absence of BRAF mutations, contributing to resistance in tumors treated with these drugs. On the left side of the figure, activated mutant BRAF V600E (mutation indicated by a star) acts as a homodimer to drive ERK pathway activation and melanoma growth in the absence of upstream activation by RAS proteins. BRAF inhibition by PLX4032 ablates BRAF-induced ERK activation and causes tumor regression. On the right, in cells lacking BRAF or CRAF mutation but containing a mutant form of a RAS protein, activated RAS binds and stimulates BRAF-CRAF or CRAF-CRAF dimers to switch on the ERK pathway. This promotes tumor growth in NRAS-mutant melanoma or can induce a premalignant lesion in other tissues. On treatment with PLX4032 or other RAF inhibitors, binding of the drug to one RAF subunit transactivates the other, in cooperation with RAS interaction, re! sulting in paradoxical ERK activation and tumor progression. * Full size image (64 KB) However, despite the excitement surrounding PLX4032, intense scrutiny has led to some sobering warnings about the whole basis of so-called rational drug design. The first and least unexpected of these has been the speed with which resistance develops to this inhibitor, even when its use is limited to individuals with BRAF-activating mutations. An 81% response rate with a median progression-free survival of seven months was reported in PLX4032-treated people with BRAF-mutant melanoma in a recent Phase 1 trial1, but most of these individuals do progress eventually. Many recently published studies have unearthed a handful of different molecular events explaining this, including mutation or upregulation of upstream components of the ERK pathway, such as NRAS, MAP3K8 and the PDGF and IGF-I receptor tyrosine kinases2, 3, 4. These changes side-step BRAF and induce activation of ERK and other downstream components of the pathway independently of mutant BRAF (Fig. 1). The lack of a s! ingle unifying resistance mechanism will complicate the development of treatments for PLX4032-resistant melanoma, although these studies have suggested several potentially tractable targets, including IGF-I receptor, PDGF receptor, PI 3-kinase and MEK. More troublingly, intensive research on PLX4032 and other similar RAF-inhibitory drugs has revealed the quite astonishing complexity of this pathway, which is a result of multilayered feedback and crosstalk. Does this largely unpredictable intricacy of signaling pathways mean that the emperor's new clothes of targeted therapy amount to not much more than the same old naked trial and error? In the case of the RAF pathway, the problem lies at least in part in the existence of multiple RAF isoforms—BRAF, which is activated by mutation in melanoma, and CRAF (also known as RAF1) and ARAF, which are not. These RAF isoforms are not redundant in function and, to complicate matters further, they form both homo- and heterodimers as part of their activation mechanism. One puzzling thread of evidence initially raised concerns about the linearity of the RAF pathway: a small minority of the mutations found in BRAF in melanoma inactivate its kinase activity rather than stimulate it5. Once BRAF inhibitors were developed and characterized in detail, it became clear that they can sometimes paradoxically activate downstream signaling through ERK, especially in melanoma cells with an activating mutation in the upstream component NRAS6, 7, 8. So how could the wiring of this pathway explain these counterintuitive observations? The work from three landmark papers by Hatzivassiliou et al.6, Heidorn et al.7 and Poulikakos et al.8 (all published in 2010), although not in agreement in every detail, can be distilled into a simple model, in which the effect of BRAF-selective inhibitors in melanoma depends entirely on how the ERK pathway is activated. In the ~40% of melanomas that have an activating BRAF mutation such as V600E, the pathway is driven by mutant BRAF homodimers whose activity is effectively blocked by drugs such as PLX4032, resulting in tumor cell death. However, in melanomas without BRAF-activating mutations, the ERK pathway is likely to be activated by other mechanisms that involve CRAF. This is particularly clear in the 20% of melanomas that carry an activating mutation in NRAS, a direct upstream activator of both BRAF and CRAF. In cells with mutant NRAS or its relatives KRAS or HRAS, treatment with a RAF! inhibitor promotes the formation of a BRAF-CRAF heterodimer or a CRAF-CRAF homodimer in which one drug-inactivated RAF subunit aids the activation of the other, drug-free, CRAF subunit by NRAS. PLX4032 is thus likely to exacerbate, rather than suppress, NRAS-mutant melanoma. The findings of these three studies limit the usefulness of BRAF-selective inhibitors such as PLX4032 to melanoma in which BRAF is mutationally activated. By itself, this would not be cause for undue concern, and it fits nicely within the paradigm of personalized cancer therapy. However, these observations cast a long shadow. KRAS mutations occur very frequently in many tumor types, most notably pancreas, colon, lung and ovary, and they occur early in tumorigenesis, a process that usually takes decades and most often will never progress to full malignancy. NRAS mutations are also important in several other tumors, including hematological malignancies and thyroid cancer. Could BRAF inhibitors exacerbate the progression of premalignant conditions involving RAS mutations? It is likely that everyone carries some RAS-mutant premalignant lesions, possibly at highly inaccessible internal sites, such as pancreatic intraepithelial neoplasia. The side effects seen with RAF inhibitors ! such as PLX4032 and the less-selective drug sorafenib suggest that this concern might not be unwarranted: many people treated with these drugs rapidly develop skin lesions such as benign keratoacanthomas, a tumor type characterized by HRAS mutations, and, less frequently, squamous cell carcinomas9. Although these surface lesions are easily spotted and removed, there is reason to be concerned about less visible and less easily treatable events that may be occurring in people treated with RAF inhibitors. On the positive side, the unusual wiring of the RAF pathway may underlie the impressive therapeutic window seen in BRAF-mutant melanoma with PLX4032. Although it may be possible to develop BRAF inhibitors that do not transactivate CRAF or RAF inhibitors that very potently inhibit all RAF isoforms, the likelihood is that broad specificity RAF inhibitors will inhibit RAF pathway function in normal tissues and will show much greater toxicity. Over the past decade many clinical trials have been conducted with highly specific inhibitors of MEK, the next component downstream from RAF in the ERK pathway. Although it is still too early to know how these will turn out, these drugs have yet to show signs of activity as impressive as PLX4032, and they are associated with significant toxicity. It is possible that for PLX4032, unexpected pathway complexity has worked largely in our favor to give a very effective drug. The concern for the cancer drug discovery community is that this set o! f circumstances may be relatively rare for targeted agents and requires immense knowledge to predict effectively. One final lesson that might be drawn from this unfolding story is that once again hitting mutationally activated or upregulated targets in cancer has proved effective, whereas targeting pathways at largely unmutated signaling intermediates has yet to prove its worth. This perhaps reflects the difficulty of predicting therapeutic outcome once one moves away from a directly dysregulated component, owing to the plasticity of these signaling networks. This raises the issue of how long we can afford to ignore the most frequently activated oncogene, RAS, as 'undruggable', while resources are concentrated on pursuing more accessible but less well-validated pathway intermediates. References * References * Author information * Flaherty, K.T.et al. N. Engl. J. Med.363, 809–819 (2010). * ChemPort * ISI * PubMed * Article * Johannessen, C.M.et al. Nature468, 968–972 (2010). * Article * Nazarian, R.et al. Nature468, 973–977 (2010). * Article * Villanueva, J.et al. Cancer Cell18, 683–695 (2010). * Garnett, M.J., Rana, S., Paterson, H., Barford, D. & Marais, R.Mol. Cell20, 963–969 (2005). * ChemPort * ISI * PubMed * Article * Hatzivassiliou, G.et al. Nature464, 431–435 (2010). * ChemPort * ISI * PubMed * Article * Heidorn, S.J.et al. Cell140, 209–221 (2010). * ChemPort * ISI * PubMed * Article * Poulikakos, P.I., Zhang, C., Bollag, G., Shokat, K.M. & Rosen, N.Nature464, 427–430 (2010). * ChemPort * ISI * PubMed * Article * Robert, C., Arnault, J.P. & Mateus, C.Curr. Opin. Oncol. published online, doi:10.1097/CCO.0b013e3283436e8c (29 December 2010). * Article Download references Author information * References * Author information Affiliations * Julian Downward is at the Cancer Research UK London Research Institute, London, UK. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Julian Downward Author Details * Julian Downward Contact Julian Downward Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Resistance, epigenetics and the cancer ecosystem
- Nat Med 17(3):288-289 (2011)
Nature Medicine | News and Views Resistance, epigenetics and the cancer ecosystem * Stephen B Baylin1Journal name:Nature MedicineVolume: 17,Pages:288–289Year published:(2011)DOI:doi:10.1038/nm0311-288Published online07 March 2011 Therapeutic resistance is a key roadblock to effective cancer treatment and can occur through various mechanisms. A recent study characterized a previously unknown, reversible mechanism of drug resistance mediated by an altered chromatin state, suggesting that cancer cell populations can use a dynamic strategy to ensure their survival when challenged by therapeutic intervention. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We will never control the most common cancers unless we conquer the overwhelming problem of therapeutic resistance. Resistance blunts the full curative potential of not only standard chemotherapy approaches but also emerging targeted therapies1. Such resistance can occur by multiple mechanisms. A paramount one that has generated the most interest is selection for mutations. This has been a particular problem for resistance to targeted therapies, wherein secondary mutations in initially mutated protein target genes are selected for during treatment; for example, the oncoprotein BCR-ABL2, targeted by imatinib in chronic myelogenous leukemia, and epidermal growth factor receptor (EGFR) mutations in lung cancer, targeted by drugs such as erlotinib3. Mutations that mediate antagonism to drug effects are also a prominent mechanism, such as those that occur in proteins mediating drug efflux, and, for targeted therapies, those that induce increased signaling through alternative surv! ival pathways1. Another mechanism that has been suggested by some studies but has been less focused upon is a change in drug response mediated by epigenetic rather than genetic changes4. Epigenetic states are determined by heritable patterns of changes in chromatin and gene expression without underlying alterations in DNA sequence5. As such, they can potentially be reversed, and this might invite attractive strategies to counter therapy-induced resistance. In 2010, Sharma et al.6 linked the challenge of countering drug resistance to two central themes at the core of cancer biology: epigenetics and self renewal. After exposing cancer cell lines to various anticancer drugs, the authors consistently found a subpopulation of cells in which drug resistance was transient and reversible. This resistance was mediated by global changes in the chromatin state of the cells, which required the histone demethylase jumonji AT-rich interactive domain-1A (JARID1A). The drug-resistant subpopulation could be eliminated by treatment with a histone deacetylase (HDAC) inhibitor, highlighting a potential therapeutic strategy. These findings suggest that overcoming therapeutic resistance may depend on our ever-deepening understanding that epigenetic abnormalities, alongside genetic mutations fundamentally drive tumorigenesis7. In addition, the data of Sharma et al.6 indicate that epigenetic mechanisms may regulate tumor cell subpopulations that express stem-like markers, and these cells might be those with the highest self-renewal capacities. Indeed, these 'cancer stem cells' or 'tumor-initiating cells' are thought to lie at the heart of therapeutic resistance8. Sharma et al.6 most intensely focused upon resistant cell subpopulations induced by the EGFR antagonist erlotinib in EGFR-mutant human non–small-cell lung cancer cells. They also found similar resistant subpopulations when they exposed other cultured tumor cell types to erlotinib or to standard chemotherapy agents such as carboplatin6. Several key findings suggested dependence of the drug-resistant phenotype on epigenetic rather than genetic mechanisms. No additional EGFR mutations or activation of other pathways that confer resistance to erlotinib were found in the resistant subpopulations. Moreover, the kinase activity of the mutant EGFR could still be reversed by erlotinib in the resistant cells. Intriguingly, the resistance was transient and, after a time lag, single clones of the resistant subpopulations could be reverted to sensitivity following drug withdrawal. Clones of these revertant cells could generate the cellular heterogeneity of the parent cell population, p! roducing a population in which the resistant cells were a minor subpopulation. Similarly, drug resistance could be induced in single clones of the parent cells. Using genome-wide expression analyses, the authors noted that in the resistant subpopulations, the most highly expressed genes were significantly nonrandomly localized along chromosomes6, suggesting that global chromatin changes might have a role in resistance. They focused on the JARID1A gene, which was highly expressed in the drug-resistant cells6. JARID1A catalyzes demethylation of trimethylation of lysine 4 on histone H3 (H3K4me3), a key chromatin mark for active gene expression and the maintenance of open chromatin states9. Sharma et al.6 observed a decrease in the overall levels of H3K4me3 in resistant cells, which could be reverted to a drug-sensitive phenotype by targeting JARID1A through RNAi. High levels of phosphorylated insulin growth factor-1 receptor (IGF-1R) may be involved in signaling the chromatin state mediated by JARID1A, as drugs targeting IGF-1R phosphorylation could selectively block the emergence of the resistant cell subpopulations. The findings from this paper could have profound therapeutic implications. The authors reasoned that the low levels of the active transcriptional mark H3K4me3 and the reduced acetylation of H3K14 that they observed in the resistant cells indicated a globally closed chromatin state mediated by HDACs. Indeed, HDAC inhibitors, which are being widely tested in cancer clinical trials, could alleviate the resistant phenotype. In addition, HDAC inhibitors are currently in clinical trial in combination with erlotinib to try and block the inevitable erlotinib resistance that follows dramatic initial responses in individuals harboring lung cancers with EGFR mutations3. So, where do the observations of Sharma et al.6 stand in our understanding of cancer and the means to control it? First, their study has obvious implications for future therapeutic approaches, but the clinical translation of these results must await the resolution of some puzzling aspects of the findings. Although treatment resistance was reversed by HDAC inhibitors, these drugs have dose-dependent, pleiomorphic effects. In this regard, the ultimate mechanism for HDAC inhibitor–mediated killing of the resistant cells suggested by the authors' results is DNA damage6. There is ongoing debate over whether HDAC inhibitors produce chromatin alterations to produce such damage or whether they directly cause DNA breaks10. Furthermore, as single agents, HDAC inhibitors are highly effective only in isolated cancer types, and they are not effective alone for reversing other epigenetic abnormalities, including the silencing of cancer genes associated with abnormal DNA methylation7. Mi! ght new combinatorial epigenetic therapy strategies be employed to reverse cancer therapeutic resistance? The second implication of the findings of Sharma et al.6 concerns the cancer ecosystem, with its constituent cell populations, their dynamics for renewal and the maintenance of cell phenotypes. It has been postulated that therapy-resistant, tumor-initiating cell subpopulations constitute a static population that supports the tumor8. However, another hypothesis fits better both with the data from Sharma et al.6 and for explaining the epigenetic modulation of such resistant cells (Fig. 1). In this scenario, the entire population of tumor cells is in a state of flux that can shift, especially under stress such as therapy, to allow cell survival4. This stress response could constitute the expansion of already resistant clones and/or, more intriguingly, involve an epigenetically mediated, bidirectional transition between tumor-initiating cells and their progeny. An epigenetic bidirectional shift could explain how single-cell clones give rise to therapy-resistant cells and how, af! ter a time lag and drug withdrawal, single resistant cell clones regenerate the heterogeneous parent cell population. Figure 1: Epigenetic modulation of tumor-initiating cells and therapy resistance. The chronic stress of therapy on a heterogeneous population of cancer cells is depicted. Top, therapy-resistant, tumor-initiating cells as defined by Sharma et al.6 are shown in dark blue (dark blue stars indicate increased JARID1A expression, green stars show decreased H3K4me3 and the red stars show decreased H3K14ac). More differentiated cancer cells are red and orange, and more committed cancer progenitor cells are light blue. Under chronic therapy conditions, existent clones of resistant cells can expand and/or more committed progeny (shown by the circled light blue cell) can give rise, via epigenetic changes, to a tumor-initiating, stem-like cell, which expands. Bottom, single clones of induced resistant cells (shown within the green circle) can regenerate over time after drug withdrawal into the heterogeneous, parent cancer cell populations through epigenetic reprogramming. * Full size image (123 KB) Where during tumor progression might the above epigenetic landscape emerge? Again, the response of cancer cells to stress could be a key issue. Cancer risk states such as cell renewal during chronic inflammation might challenge cells to develop resistance to death and to evolve the capacity for self-renewal to evade a toxic environment11. Epigenetic states that underlie such survival might also allow cells to circumvent oncogenic senescence bred by arising mutations that would otherwise select against tumor progression—or, alternatively, the mutations could help create or augment such an epigenetic landscape to foster cell oncogenic addiction11. These concepts would fit well with the shifting cell phenotypes observed by Sharma et al.6. In a time when we can engineer induced pluripotent stem cells from more mature cells, entailing complete reprogramming of the epigenome12, it is not farfetched to conceive that cancer cells could do the same. References * References * Author information * Hoey, T.Sci. Transl. Med.2, 28ps19 (2010). * Sawyers, C.L.Nat. Med.15, 1158–1161 (2009). * Article * Engelman, J.A. & Janne, P.A.Clin. Cancer Res.14, 2895–2899 (2008). * ISI * PubMed * Article * Cohen, A.A.et al. Science322, 1511–1516 (2008). * ChemPort * PubMed * Article * Bird, A.Genes Dev.16, 6–21 (2002). * ChemPort * ISI * PubMed * Article * Sharma, S.V.et al. Cell141, 69–80 (2010). * ChemPort * ISI * PubMed * Article * Jones, P.A. & Baylin, S.B.Cell128, 683–692 (2007). * ChemPort * ISI * PubMed * Article * Reya, T., Morrison, S.J., Clarke, M.F. & Weissman, I.L.Nature414, 105–111 (2001). * ChemPort * ISI * PubMed * Article * Klose, R.J.et al. Cell128, 889–900 (2007). * ChemPort * ISI * PubMed * Article * Gaymes, T.J.et al. Mol. Cancer Res.4, 563–573 (2006). * ISI * PubMed * Article * Johnstone, S.E. & Baylin, S.B.Nat. Rev. Genet.11, 806–812 (2010). * Article * Mikkelsen, T.S.et al. Nature454, 49–55 (2008). * ChemPort * ISI * PubMed * Article Download references Author information * References * Author information Affiliations * Stephen B. Baylin is at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, Maryland, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Stephen B Baylin Author Details * Stephen B Baylin Contact Stephen B Baylin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Hunting ALK to feed targeted cancer therapy
- Nat Med 17(3):290-291 (2011)
Nature Medicine | News and Views Hunting ALK to feed targeted cancer therapy * Anton Wellstein1 * Jeffrey A Toretsky1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:290–291Year published:(2011)DOI:doi:10.1038/nm0311-290Published online07 March 2011 Neuroblastoma is a fatal childhood cancer, but lack of knowledge about the underlying causative genes has hampered the development of effective therapies. The identification of anaplastic lymphoma kinase (ALK) mutations as drivers of neuroblastoma has indicated that targeted therapy with ALK inhibitors might be a valuable strategy in the fight against this lethal cancer. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the era of personalized medicine, understanding the molecular drivers of oncogenesis will be likely to trump morphological characteristics with regard to diagnostics, prognostics and choice of therapies. Identifying single driver mutations from billions of possibilities used to require substantial insight. However, as the cost of deep genomic sequencing goes down each month, clever tricks such as identifying family cohorts may not be as necessary. Family cohorts have driven genetic discovery for decades, including the identification of most tumor suppressor genes and many oncogenes. Neuroblastoma is a lethal cancer of early childhood that essentially comes in two forms: highly malignant and locally manageable, or, to paraphrase Audrey Evans, an early leader in the field, the 'good'ies and the 'bad'ies. The underlying genes responsible for neuroblastoma remain largely unknown, despite the discovery of a handful of genetic changes that have been implicated in neuroblastoma development. For example, Brodeur et al.1 and Look et al.2 correlated MYCN oncogene amplification with aggressive tumors that have a high propensity for metastasis and that cause mortality. Also, very small subsets of familial neuroblastoma were associated with PHOX2B (encoding paired-like homeobox-2) mutations, but these mutations accounted for only very few families (reviewed in ref. 3). To identify additional oncogenes that cause familial neuroblastoma, Mossé et al.4 returned to old-fashioned family trees. They collected the pedigrees of 20 families that showed an autosomal dominant pattern of neuroblastoma inheritance. Using a genome-wide scan for linkage at 6,000 single nucleotide polymorphisms (SNPs), these investigators identified a previously unknown series of germline mutations in the ALK gene. Some but not all of these families had genomic amplicons on chromosome 2 that also included MYCN. Among the mutations identified, most were predicted to lead to amino acid substitutions in the ALK tyrosine kinase domain (Fig. 1). Five of the eight pedigrees with germline mutation led to proteins with the same R1275Q mutation. A neuroblastoma cell line containing the R1275Q mutation showed ALK activation, which was indicated by tyrosine phosphorylation at ALK Y1604. Figure 1: The ALK receptor kinase: its domains, pathways, mutations and inhibitors. The different domains of ALK are shown within their bordering amino-acid positions. The major mutations found by Mossé et al.4, Janoueix-Lerosey et al.5, Chen et al.6 and George et al.7 in the kinase domain and their relative frequencies (somatic or germline) are shown. The percentage of tumors that show ALK amplification or contain ALK mutations is indicated. Representative small-molecule kinase inhibitors that also inhibit ALK are listed according to their current status. The ALK breakpoint that leads to fusion proteins in different cancers is indicated. SigP, signal peptide; LBD, ligand binding domain; PTN, pleiotrophin; TM, transmembrane domain; IRS-1, insulin receptor substrate 1; Grb2, growth factor receptor–bound protein-2; mTOR, mammalian target of rapamycin. * Full size image (95 KB) Three additional publications by Janoueix-Lerosey et al.5, Chen et al.6 and George et al.7 used SNP- or amplicon-based sequencing methods to identify similar mutations in ALK (Fig. 1). Each of the three studies showed the effects of a dominant oncogene with a similar pattern of results. Not all mutations resulted in constitutive kinase activity; in fact, only the minority showed clear increases in ALK autophosphorylation and downstream target activation, such as phosphorylation of AKT. The R1275Q and F1174L(V) mutations were shown to have constitutive tyrosine phosphorylation in cell lines that endogenously expressed these mutant forms of ALK, or when expressed in either 3T3 or Ba/F3 cells6, 7. ALK mutations in neuroblastoma tissues account for small survival differences in the whole population of people with neuroblastoma; this is due to their very low incidence in the population. Still, individuals who have neuroblastomas with the ALK F1174 mutation show a significantly worse outcome relative to the overall cohort, suggesting that this mutation has an important impact on malignant progression, whereas the presence of the ALK R1275Q mutation did not make a difference8. Stratification according to ALK expression showed that individuals with high ALK expression in their tumors had a significantly worse overall survival and disease-free survival than those with low tumor ALK expression. Although this type of single-gene analysis can be useful to direct treatment with a targeted therapy, genomic instability in the tumors leads to complex mixtures of clonal cells; therefore, multigene analyses can provide a more robust predictor of disease course and may ultimately pro! vide a more complex insight into pathways activated during tumorigenesis and even predict candidate drug sensitivity8. The functional role of these ALK mutations was addressed in each of the four studies4, 5, 6, 7; however, a deeper understanding will be necessary to optimize any ALK-targeted therapy. Each study used either ALK expression followed by inhibition in non-neuroblastoma cell lines, RNAi-mediated reduction of endogenous ALK expression in cell lines or small-molecule kinase inhibitors. Generally, the results from these studies supported a dominant role for mutant ALK. However, in some cases, RNAi-mediated reduction of wild-type ALK expression also reduced neuroblastoma cell growth, suggesting that the level of ALK expression might be as important as an activating mutation in driving tumorigenesis. A recent paper by Sasaki et al.9 identified ALK F1174L as a mutation that occurred naturally in an individual with inflammatory myoblastic tumor undergoing treatment with the dual MET and ALK kinase inhibitor crizotinib. This tumor contained a somatic RANBP2-ALK translocation; however, when the person experienced recurrent disease, one ALK locus contained an additional F1174L mutation. An evaluation of the inflammatory myoblastic tumor cells harboring ALK F1174L showed that they had higher levels of phosphorylated ALK along with increased downstream phosphorylation of AKT. Thus, this study supports that F1174L is an activating mutation of ALK. ALK was independently identified as a molecular target in neuroblastoma by screening >600 human cancer cell lines with pharmacological inhibitors of ALK kinase activity10. This work provided a platform from which to launch inhibitor development when a transforming EML4-ALK fusion gene was identified as a major driver in a subset of approximately 5% of non–small-cell lung cancers11. Indeed, a first clinical trial with crizotinib showed striking efficacy in individuals with non–small-cell lung cancer, providing evidence that a well-defined activated pathway may be targeted effectively12 (Fig. 1). A recent structural study of the ALK kinase domain revealed differences from the same domain in related receptor kinases in the insulin receptor family that will be helpful in designing new inhibitors as resistance to the initial ALK inhibitors emerges13. As higher ALK receptor expression in neuroblastoma coincides with poorer disease outcome8, overexpressed ALK may thus be a potentially valuable therapeutic target. Indeed, two proteins—pleiotrophin and the related midkine—have been shown to activate mammalian ALK14 through extracellular interactions with it (Fig. 1). Therefore, antibody strategies to target overexpressed ALK in neuroblastoma may be an attractive additional approach. Antibody therapy may even act synergistically with small-molecule tyrosine kinase inhibitors. It is clear that targeting ALK is now open season for the development of new therapies for neuroblastoma, as well as other cancers. The insights gained from ongoing trials are likely to directly benefit both subgroups of individuals whose tumors are driven by ALK and, in general, targeted approaches to cancer treatment. Obviously, genetic mutations can reveal new drivers and pathways activated in cancer and may present new therapeutic opportunities. However, fully validating any new target is a challenge at a new frontier of a deadly cancer. References * References * Author information * Brodeur, G.M., Seeger, R.C., Schwab, M., Varmus, H.E. & Bishop, J.M.Science224, 1121–1124 (1984). * ChemPort * ISI * PubMed * Article * Look, A.T.et al. J. Clin. Oncol.9, 581–591 (1991). * ChemPort * ISI * PubMed * Maris, J.M.N. Engl. J. Med.362, 2202–2211 (2010). * ISI * PubMed * Article * Mossé, Y.P.et al. Nature455, 930–935 (2008). * ChemPort * ISI * PubMed * Article * Janoueix-Lerosey, I.et al. Nature455, 967–970 (2008). * ChemPort * ISI * PubMed * Article * Chen, Y.et al. Nature455, 971–974 (2008). * ChemPort * ISI * PubMed * Article * George, R.E.et al. Nature455, 975–978 (2008). * ChemPort * ISI * PubMed * Article * De Brouwer, S.et al. Clin. Cancer Res.16, 4353–4362 (2010). * Sasaki, T.et al. Cancer Res.70, 10038–10043 (2010). * McDermott, U.et al. Cancer Res.68, 3389–3395 (2008). * ChemPort * PubMed * Article * Soda, M.et al. Nature448, 561–566 (2007). * ChemPort * ISI * PubMed * Article * Kwak, E.L.et al. N. Engl. J. Med.363, 1693–1703 (2010). * ChemPort * ISI * PubMed * Article * Lee, C.C.et al. Biochem. J.430, 425–437 (2010). * Stoica, G.E.et al. J. Biol. Chem.276, 16772–16779 (2001). * ChemPort * ISI * PubMed * Article Download references Author information * References * Author information Affiliations * Anton Wellstein and Jeffrey A. Toretsky are at Georgetown University, Lombardi Cancer Center, Washington, DC, USA. Competing financial interests A.W. is named as an inventor on Georgetown University patents that are related to the ALK receptor. Corresponding author Correspondence to: * Jeffrey A Toretsky Author Details * Anton Wellstein Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey A Toretsky Contact Jeffrey A Toretsky Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Metabolism unhinged: IDH mutations in cancer
- Nat Med 17(3):291-293 (2011)
Nature Medicine | News and Views Metabolism unhinged: IDH mutations in cancer * John R Prensner1 * Arul M Chinnaiyan2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:291–293Year published:(2011)DOI:doi:10.1038/nm0311-291Published online07 March 2011 Recently characterized IDH1 and IDH2 mutations in leukemia and glioblastoma have introduced a fascinating cancer-specific role for metabolic genes essential to cellular respiration. Studies also link aberrant IDH1 and IDH2 activity to an altered metabolite profile, an observation that may have broad implications for both cancer epigenetics and clinical management of disease. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the 1920s, Otto Warburg first proposed the idea that dysregulated metabolism, specifically cellular respiration, was the origin of cancer. The relatively recent discovery that the genes encoding isocitrate dehydrogenases IDH1 and IDH2—cytoplasmic and mitochondrial, respectively—are recurrently mutated in cancer represents one of the biggest success stories for cancer biology in the era of high-throughput sequencing. These metabolic enzymes, integral to cellular respiration, have subsequently unveiled a fascinating and complex biology behind the dysregulation and functional consequences of metabolites in cancers. The first reported mutation in an IDH-family gene was found in a metastatic colon cancer in 2006 and unceremoniously included in a long table of mutations observed in a set of breast and colon cancers1. Two years later, large-scale sequencing efforts in over 200 glioblastoma tumors rediscovered IDH1 and IDH2 as recurrently mutated in a minority (5%) of primary gliomas and a large majority (~60–90%) of secondary gliomas, with IDH1 Arg132 mutations and IDH2 Arg140 and Arg172 mutations accounting for >90% of aberrations2, 3. Similar studies of leukemia have found that about 12–18% of acute myeloid leukemias (AMLs) and related myeloproliferative neoplasms harbor both IDH1 and IDH2 mutations, although they tend to acquire IDH2 mutations preferentially over IDH1 (refs. 1,4). Exhaustive sequencing efforts in other human solid tumors have reported IDH1 and IDH2 mutations only rarely, suggesting that mutations of IDH1 and IDH2 are predominantly a feature of glioma and AML. Biochemical studies showed that all reported mutations of IDH1 and IDH2 negatively affect the enzymatic capacity of these proteins to bind isocitrate, their substrate, and convert it into α-ketoglutarate (α-KG), generating carbon dioxide and replenishing NADH and NADPH as side products1. As this is one of the irreversible steps in the tricarboxylic acid cycle (TCA cycle, also known as the citric acid or Krebs cycle) essential for cellular respiration, initial studies speculated that IDH1 and IDH2 mutations might represent dominant-negative loss-of-function events5. Supporting this, tumors with IDH1 or IDH2 mutations are heterozygous, with only extremely rare cases seen of a tumor harboring both an IDH1 or IDH2 mutation and loss of heterozygosity affecting the paired allele, suggesting that IDH1 and IDH2 mutants show haploinsufficiency, but homozygous inactivation is selected against1. Yet, using liquid chromatography-mass spectrometry as a discovery tool, two studies, by Dang et al.6 and Ward et al.7, identified a surprising 100-fold increase in 2-hydroxyglutarate (2-HG), another small metabolite, in glioma and leukemia patients with IDH1 or IDH2 mutations. The authors resolved the protein structures of mutant IDH1 and IDH2 proteins and showed that the mutant enzymes were not, in fact, inactive—they had instead adopted a new active site. A series of biochemical experiments confirmed that mutant IDH1 and IDH2 can metabolize α-KG—the very product of wild-type IDH1 and IDH2 activity—into 2-HG6, 7 (Fig. 1), unveiling a fascinating neomorphic enzymatic capacity for these proteins. Figure 1: Mutant IDH1 and IDH2 activity and 2-HG signaling in cancer. Mutant (mut) IDH1 (cytoplasmic) and IDH2 (mitochondrial) enzymes show a neomorphic enzymatic capacity to convert α-KG into 2-HG, a small oncometabolite. The presence of mutant IDH1 or IDH2 proteins results in increased amounts of 2-HG, which then alters a number of downstream cellular activities. 2-HG competitively inhibits α-KG binding to several histone demethylases, including KDM2a, leading to a widely aberrant histone modification profile (indicated in the illustration by several forms of H3 lysine methylation), particularly histone tail methylation. The metabolite 2-HG also inhibits the TET1 and TET2 hydroxymethylases, decreasing levels of 5-hydroxymethylcytosine. The epigenetic dysregulation caused by altered levels of 2-HG and α-KG in IDH1 and IDH2 mutant cells may contribute to aberrant regulation of gene expression in cancer. Finally, 2-HG also helps to stabilize HIF-1α, partially by decreasing levels of the HIF-1α antagonist endostatin, which results in increa! sed VEGF signaling, a driver of increased angiogenesis in human cancers. IDH1, isocitrate dehydrogenase 1; IDH2, isocitrate dehyrogenase 2; IDH3, isocitrate dehyrogenase 3; α-KG, α-ketoglutarate; 2-HG, 2-hydroxyglutarate; HIF-1α, hypoxia-inducible factor 1, alpha subunit; TET1, Tet oncogene 1; TET2, Tet oncogene family member 2; KDM2a, lysine-specific demethylase 2A; VEGF, vascular endothelial growth factor. * Full size image (111 KB) These findings suggest that the normal physiology of cellular respiration and metabolism is profoundly altered in IDH1 or IDH2 mutant cancer cells. But if IDH1 and IDH2 mutant cancers produce an overabundance of the 2-HG metabolite, these mutations are not merely loss-of-function events—instead 2-HG must have a specific role in driving a malignant phenotype in these types of tumors. But what is this overexpressed metabolite doing? A crucial clue to 2-HG function came from large-scale analyses of genome-wide DNA methylation in human gliomas showing that distinct methylation profiles associated with gene expression subtypes8. This study suggested that nearly all IDH1 and IDH2 mutations were associated with a highly specific DNA methylation profile, termed glioma CpG-island methylator phenotype (G-CIMP), which, in turn, corresponded to an oligodendrocyte-like expression subtype of glioma8. Although the mechanism underlying the association between IDH1 and IDH2 mutations and G-CIMP remained elusive, similar work in human AML samples also showed a correlation between such mutations and specific DNA methylation profiles9, supporting the findings in glioma. Moreover, in AML, IDH1 and IDH2 mutations were observed to be mutually exclusive with loss-of-function mutations in TET2—a gene encoding a dioxygenase that requires α-KG to convert 5-methylcytosine (the biochemical mark of DNA methylation) into 5-hydroxymethylcytosine, a related epigenetic modification9. These data suggested that IDH1 or IDH2 and TET2 mutations may be functionally redundant. In addition, further studies showed impaired hematopoiesis in mouse primary bone marrow cells manipulated to overexpress IDH2 mutant proteins or silence TET2, suggesting a role in leukemia9. Together, these findings in glioma and AML imply a link between cancer metabolism and epigenetic regulation in cancer. But how do IDH1 and IDH2 mutations affect the epigenetic profile of cancer? To answer this question, recent work has focused on understanding the impact of these mutations and the resulting changes in metabolite abundance on epigenetics. IDH1 and IDH2 mutations cause a decrease in α-KG production, probably owing to heterodimerization of the mutant IDH1 or IDH2 protein with the wild-type counterpart5, and an increase in 2-HG. Recent findings from Xu et al.10 suggest that, in vitro, this combination prevents α-KG from binding several histone demethylases, enzymes involved in epigenetic control. This competitive inhibition of α-KG by 2-HG causes widespread epigenetic changes, suggesting that 2-HG may function as an oncometabolite (Fig. 1). Although further investigation is still needed to understand this process, these studies provide a network of oncometabolite-driven epigenetic aberrations in IDH1 and IDH2 mutant cancers, in concordance with studies of oncometabolites promoting oncogenic functions in other cancers, such as sarcosine upregulation i! n prostate cancer11. The discovery of IDH1 and IDH2 mutations and the 2-HG metabolite also has dramatic clinical implications. People with IDH1 or IDH2 mutations have highly elevated (~100-fold) amounts of 2-HG6, 7, indicating that 2-HG may be used as a clinical biomarker. Equally important, IDH1 and IDH2 mutations stratify individuals into molecular subtypes with distinct clinical outcomes—the mutations are associated with lower-grade astrocytomas, oligodendrogliomas (grade II/III) and secondary gliomas with better overall survival, progression-free survival and chemosensitivity than glioblastomas that are wild type for both genes1, 2, 3. In AML, IDH1 and IDH2 mutations characterize a molecular subtype of disease that is more likely to be cytogenetically normal and to harbor NPM1 mutations12. In contrast to the favorable prognostic value of these mutations in glioma, in AML, IDH1 and IDH2 mutations have the opposite implication—they confer a worse prognosis12—although the reason for this notable difference between is not known. What does this all mean for cancer research and therapy? The neomorphic mutant IDH1 and IDH2 enzymes not only provide an attractive molecule for targeted therapy but also generate 2-HG, a new metabolite that may serve as a biomarker for disease stratification and prognosis. And although IDH1 and IDH2 are not the first metabolic enzymes to be implicated in neoplastic growth1, they are the first to be incorporated into the basics of cancer biology as a major genetic, biochemical and clinical alteration. As a discovery with broad implications, IDH1 and IDH2 mutations have dramatically altered our understanding of cancer biology with the introduction of a new, unconventional type of cancer gene. Metabolic enzymes, much like kinases and transcription factors, have now regained their rightful place on the growing 'oncogene hit list' revealed by high-throughput sequencing. References * References * Author information * Yen, K.E., Bittinger, M.A., Su, S.M. & Fantin, V.R.Oncogene29, 6409–6417 (2010). * Article * Parsons, D.W.et al. Science321, 1807–1812 (2008). * ChemPort * ISI * PubMed * Article * Yan, H.et al. N. Engl. J. Med.360, 765–773 (2009). * ChemPort * ISI * PubMed * Article * Mardis, E.R.et al. N. Engl. J. Med.361, 1058–1066 (2009). * ChemPort * ISI * PubMed * Article * Zhao, S.et al. Science324, 261–265 (2009). * ChemPort * ISI * PubMed * Article * Dang, L.et al. Nature462, 739–744 (2009). * ChemPort * ISI * PubMed * Article * Ward, P.S.et al. Cancer Cell17, 225–234 (2010). * ChemPort * ISI * PubMed * Article * Noushmehr, H.et al. Cancer Cell17, 510–522 (2010). * ChemPort * ISI * PubMed * Article * Figueroa, M.E.et al. Cancer Cell18, 553–567 (2010). * Xu, W.et al. Cancer Cell19, 17–30 (2011). * Sreekumar, A.et al. Nature457, 910–914 (2009). * ChemPort * ISI * PubMed * Article * Paschka, P.et al. J. Clin. Oncol.28, 3636–3643 (2010). * ChemPort * ISI * PubMed * Article Download references Author information * References * Author information Affiliations * John R. Prensner is in the Department of Pathology and the Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA * Arul M. Chinnaiyan is in the Departments of Pathology and Urology and the Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA, and the Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Arul M Chinnaiyan Author Details * John R Prensner Search for this author in: * NPG journals * PubMed * Google Scholar * Arul M Chinnaiyan Contact Arul M Chinnaiyan Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Research Highlights
- Nat Med 17(3):294-295 (2011)
Nature Medicine | Research Highlights Research Highlights * Victoria Aranda * Alison Farrell * Carolina Pola * Meera SwamiJournal name:Nature MedicineVolume: 17,Pages:294–295Year published:(2011)DOI:doi:10.1038/nm0311-294Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg More common than thought? The cancer stem cell hypothesis postulates that only a fraction of cells in a tumor have tumorigenic potential, suggesting that therapies that target these cells will effectively eliminate the tumor. Elsa Quintana and her colleagues set out to address how common cancer stem cells are in human melanoma (Nature456, 593–598, 2008). Classically, immunodeficient mouse hosts had been used to quantify the proportion of cancer-initiating cells from human tumor samples by limiting dilution assays. Under these conditions, 0.0001–0.1% of cells have tumorigenic potential. But when Quintana et al. coinjected Matrigel and human melanoma cells into a more severely immunocompromised model, the proportion of cells able to form tumors increased to ~25%. Moreover, when the authors tested primary and metastatic melanoma samples from 12 affected individuals, they found substantial variability in the prevalence and phenotype of the cancer stem cells. These findings rekindled an ongoing debate about how to define cancer stem cells. The study also uncovered the importance of studying the regulation of tumor initiation properties by suggesting they might vary depending on the assay and according to cancer type. This information will be crucial to developing therapeutic strategies that appropriately target the cells responsible for tumor initiation. —MS Convergent cancer path After previous reports of whole genome–sequencing efforts for breast, colon and brain cancers, this group comprehensively characterized the genetic alterations in pancreatic tumors (Science321, 1801–1806, 2008). Their integrative approach parsed coding region sequence, copy number alterations and expression profiling data from 24 advanced pancreatic tumors. After compiling a catalog of genomic alterations and expression differences between normal and tumor tissues, Jones et al. used computational modeling to pinpoint previously unrecognized genetic alterations driving pancreatic cancer. The study demonstrated that individual genetic modifications can be grouped into 12 major signaling pathways that contribute to tumorigenesis. The concept of redundant pathway activation derived from this study has since been expanded beyond pancreatic cancer. The convergence of independent genetic changes into discrete oncogenic pathways provides a rational framework to delimit the ever-growing complexity of cancer with manageable parameters. These findings could potentially be translated to personalized medicine in the areas of drug development, management of drug resistance and new biomarkers. —VA Stemming EMT Two previously unrelated properties of malignant tumor cells—mesenchymal features and stemness—were shown to be linked in this study by Mani et al. (Cell133, 704–715, 2008). Mesenchymal features are associated with cell migration and invasion, whereas stemness may confer the self-renewal ability required to initiate tumors at primary and distant sites. By comparing mammary gland cells that had undergone epithelial-mesenchymal transition (EMT) to stem cells from normal tissues, the authors uncovered their overlapping features: they both expressed stem cell markers and displayed mesenchymal properties. This implies not only that stem cells are more mesenchymal than their differentiated counterparts but also that EMT could revert differentiated cells into a progenitor-like state. Furthermore, stem-like mesenchymal cells were found in human samples from normal and neoplastic breast tissue. Mani et al. then showed that inducing EMT in transformed mammary epithelial cells increased tumorigenicity and promoted self-renewal and tumor-initiation properties. The correlation between mesenchymal and stem cell properties provided insights into the dynamics of stemness in cancer and how cancer stem cells contribute to different aspects of malignancy. It also highlighted the plasticity of the tumor-initiating phenotype. This landmark report has prompted research to assess the clinical relevance of EMT in cancer stem cells, the mechanistic underpinnings of this process and its therapeutic implications. —VA Tales from the crypt Finding what cell acquires the first genetic hit to initiate cancer is still a challenge. Driver mutations in colorectal cancer occur in genes such as adenomatous polyposis coli (APC) that constitutively activate the Wnt pathway, but the tumor-initiating cell where this occurs had been elusive. Using lineage-tracing studies, Nick Barker et al. pinpointed colonic stem cells in the intestinal crypt as intestinal cancer cells of origin (Nature457, 608–611, 2009). Deletion of Apc in mice in a subset of long-lived stem cells at the bottom of the intestinal crypt that express Lgr5 (leucine-rich repeat–containing G protein–coupled receptor 5) increased cellular β-catenin levels, a surrogate for Wnt activation, and led to the development of adenomas. These transformed stem cells remained at the bottom of the crypt, fueling the growing adenoma, and composed 6.5% of the tumor population. The tumors did not form, however, when Apc was deleted in Lgr5-negative cells that transit the crypt to differentiate. Although progenitor or stem cells have been suggested as cells of origin in many tumor types, this may not be always the case. These findings, however, shed light on the role of stem cells in tumor initiation, suggesting that a stem cell hierarchy can be maintained while the tumor progresses. Identifying these cell populations may open new avenues for early diagnosis and cancer therapies to target cells of origin. —CP Glioblastoma genetics Glioblastoma multiforme is a devastating tumor with extremely poor prognosis. Whether these tumors can be classified into subsets with distinct prognoses or responses to therapies is unclear, but genomic and transcriptomic analyses might help address this issue by providing new insight into brain cancer biology, as illustrated by two studies. Parsons et al. sequenced more than 20,000 genes in 22 human samples from primary tumors and from glioblastoma multiforme cells passaged as xenografts in mice to identify somatic mutations and candidate drivers of glioblastoma tumorigenesis (Science321, 1807–1812, 2008). They also analyzed copy number alterations and the transcriptional profiles of the tumors. IDH1, which converts isocitrate to a-ketoglutarate, was one of the most frequently mutated candidate drivers. IDH1 mutations occurred more often in tumors from younger individuals and were associated with better prognosis. In a second study, the Cancer Genome Atlas Research Network analyzed the nucleotide sequence and the DNA methylation, transcription and copy number alterations in 91 and 206 tumors, respectively (Nature455, 1061–1068, 2008). The study reported a high frequency of glioblastomas with mutated NF1, as well as with mutations in the gene encoding the regulatory subunit of PI3 kinase, PI3KR1. Also of note, the identification of chemotherapy-associated mutations in DNA repair genes in conjunction with methylation of the gene encoding MGMT, a dealkylating enzyme, suggested a path by which treatment with DNA alkylating agents may lead to resistance. Combined, these two studies provide a wealth of data for researchers to mine that has the potential to lead to new classifications of glioblastomas and new therapeutic avenues for one of the most treatment-resistant human tumors. —AF Charting the cancer landscape An important issue in cancer is determining which genetic alterations are required for cancer initiation, maintenance and progression and which are passengers. A comprehensive genomic analysis of human breast and colorectal cancers by Laura D. Wood et al. (Science318, 1108–1113, 2007) provided a new and provocative perspective on the landscape of cancer mutations. The authors catalogued 11 breast and 11 colorectal tumors, encompassing over 20,000 transcripts from ~18,000 genes. They found that, on average, a tumor contains 80 mutations, but the mutations varied according to cancer type. Using computational modeling to estimate the rate of passenger mutations, the authors found that fewer than 15 of the mutated genes in each tumor were likely to drive the neoplastic process. The landscape of cancer mutations could be viewed as a small number of 'mountains'—genes mutated in a large proportion of tumors—and many 'hills', genes that are only mutated in a small proportion of tumors. Despite the wide range of genes and mutations they detected, the authors homed in on a limited set of signaling pathways that influence cancer development. This work showed that although the general genomic landscapes of breast and colorectal cancers share similarities, there is considerable genetic heterogeneity in cancer. The comprehensive catalog of mutations also provided new candidate oncogenes and tumor suppressor genes, but the major challenge is now to identify exactly how they contribute to tumor growth and progression. Functional genomic information could then be applied to guide cancer therapeutics. —MS Mutant needles in a haystack Alfred Pasieka / Photo Researchers, Inc. Next-generation DNA sequencing techniques can uncover the mutations necessary for a cell to become malignant and induce cancer—the 'holy grail' for cancer genomics. But finding the driver mutations relevant to tumor pathogenesis in a specific tumor type requires functional studies and numerous human samples to confirm the mutations' recurrence. Elaine Mardis et al. sequenced the tumor genome of an individual with acute myeloid leukemia (AML) with minimal maturation but no chromosomal alterations and compared it with the normal skin genome from the same individual to identify somatic mutations (N. Engl. J. Med.361, 1058–1066, 2009). They found about 750 new AML-specific mutations, some of which appeared in almost every tumor cell in the sample, including 12 in coding sequences and 52 in regulatory regions. Four of these mutations, which affected the known cancer genes NPM1 and NRAS, the glioblastoma-related gene IDH and a region in chromosome 10, were deleterious and present in other AML tumor samples, suggesting that they are recurring and are probably driver mutations. This study unmasked frequent mutations and AML-relevant genes that may be used to develop better diagnostic tools and personalized therapies to tackle this cancer. Further investigation, however, will be required to crack the complex code behind the cancer genome. —CP Modulating macrophage-assisted metastasis David M. Phillips/Photo Researchers, Inc. Tumor-associated macrophages (TAMs) can promote tumor growth and metastasis in mouse models of breast cancer, but whether T and B cells are similarly protumorigenic in these models was unclear. Investigating this issue, David DeNardo and his colleagues reported that CD4+ TH2 cells can promote metastasis by modulating the differentiation of TAMs (Cancer Cell16, 91–102, 2009). Using a transgenic mouse model that develops aggressive mammary adenocarcinoma, DeNardo and colleagues showed that mice lacking CD4+ T cells—but not B cells or CD8+ T cells—had fewer lung metastatic foci. TAMs in wild-type mice could promote greater invasive behavior of mammary epithelial cells and produced more epidermal growth factor (EGF) than their counterparts from CD4+ T cell–deficient mice. The researchers found that interleukin-4 produced by CD4+ TH2 cells triggered the differentiation of high EGF–secreting and metastasis-promoting TAMs. In the absence of interleukin-4 signaling, or of CD4+ T cells, expression of EGF by TAMs was reduced, coincident with decreased tumor metastasis. Directly blocking EGF receptor signaling in tumor-bearing mice reduced the number of circulating tumor cells. These findings add to our understanding of how myeloid cells can switch from a tumor-suppressive phenotype to an alternatively activated phenotype that enhances tumor cell invasion and dissemination to distant sites. If these results are recapitulated in humans, inhibiting TH2 cytokines may help reduce tumor cell metastasis. —AF Written by Victoria Aranda, Alison Farrell, Carolina Pola and Meera Swami Additional data Author Details * Victoria Aranda Search for this author in: * NPG journals * PubMed * Google Scholar * Alison Farrell Search for this author in: * NPG journals * PubMed * Google Scholar * Carolina Pola Search for this author in: * NPG journals * PubMed * Google Scholar * Meera Swami Search for this author in: * NPG journals * PubMed * Google Scholar - Timeline: a decade of advances in immunotherapy
- Nat Med 17(3):296 (2011)
Nature Medicine | Timeline Timeline: a decade of advances in immunotherapy * Drew Pardoll1Journal name:Nature MedicineVolume: 17,Page:296Year published:(2011)DOI:doi:10.1038/nm0311-296Published online07 March 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Figure 1 * Full size image (319 KB) References * References * Author information * Nishimura, H., Nose, M., Hiai, H., Minato, N. & Honjo, T.Immunity11, 141–151 (1999). * ChemPort * ISI * PubMed * Article * Nishimura, H.et al. Science291, 319–322 (2001). * ChemPort * ISI * PubMed * Article * Dong, H., Zhu, G., Tamada, K. & Chen, L.Nat. Med.5, 1365–1369 (1999). * ChemPort * ISI * PubMed * Article * Latchman, Y.et al. Nat. Immunol.2, 261–268 (2001). * ChemPort * ISI * PubMed * Article * Tseng, S.Y.et al. J. Exp. Med.193, 836–846 (2001). * Dong, H.et al. Nat. Med.8, 793–800 (2002). * ChemPort * ISI * PubMed * Article * Iwai, Y.et al. Proc. Natl. Acad. Sci. USA99, 12293–12297 (2002). * ChemPort * PubMed * Article * Thompson, R.H.et al. Proc. Natl. Acad. Sci. USA101, 17174–17179 (2004). * ChemPort * PubMed * Article * Hodi, F.S.et al. Proc. Natl. Acad. Sci. USA100, 4712–4717 (2003). * ChemPort * PubMed * Article * Phan, G.Q.et al. Proc. Natl. Acad. Sci. USA100, 8372–8377 (2003). * ChemPort * PubMed * Article * Attia, P.et al. J. Clin. Oncol.23, 6043–6053 (2005). * ChemPort * PubMed * Article * Beck, K.E.et al. J. Clin. Oncol.24, 2283–2289 (2006). * ChemPort * PubMed * Article * Small, E.J.et al. Clin. Cancer Res.13, 1810–1815 (2007). * ChemPort * PubMed * Article * Yang, J.C.et al. J. Immunother.30, 825–830 (2007). * ChemPort * PubMed * Article * Kenter, G.G.et al. N. Engl. J. Med.361, 1838–1847 (2009). * ChemPort * ISI * PubMed * Article * Kantoff, P.W.et al.; IMPACT Study Investigators. N. Engl. J. Med.363, 411–422 (2010). * ChemPort * ISI * PubMed * Article * Slamon, D.J.et al. N. Engl. J. Med.344, 783–792 (2001). * ChemPort * ISI * PubMed * Article * Cunningham, D.et al. N. Engl. J. Med.351, 337–345 (2004). * ChemPort * ISI * PubMed * Article * Hurwitz, H.I.et al. J. Clin. Oncol.23, 3502–3508 (2005). * ChemPort * ISI * PubMed * Article * Witzig, T.E.et al. J. Clin. Oncol.20, 2453–2463 (2002). * ChemPort * ISI * PubMed * Article * Cartron, G.et al. Blood99, 754–758 (2002). * ChemPort * ISI * PubMed * Article * Zhang, W.et al. J. Clin. Oncol.25, 3712–3718 (2007). * ChemPort * ISI * PubMed * Article * Hodi, F.S.et al. N. Engl. J. Med.363, 711–723 (2010). * ChemPort * ISI * PubMed * Article * Yee, C.et al. Proc. Natl. Acad. Sci. USA99, 16168–16173 (2002). * ChemPort * PubMed * Article * Dudley, M.E.et al. Science298, 850–854 (2002). * ChemPort * ISI * PubMed * Article * Morgan, R.A.et al. Science314, 126–129 (2006). * ChemPort * ISI * PubMed * Article * Till, B.G.et al. Blood112, 2261–2271 (2008). * ChemPort * ISI * PubMed * Article * Kochenderfer, J.N.et al. Blood116, 4099–4102 (2010). * ISI * PubMed * Article * Brahmer, J.R.et al. J. Clin. Oncol.28, 3167–3175 (2010). * ChemPort * ISI * PubMed * Article * Krop, I.E.et al. J. Clin. Oncol.28, 2698–2704 (2010). * ISI * PubMed * Article Download references Author information * References * Author information Affiliations * Drew Pardoll is at the Sidney Kimmel Cancer Center, Johns Hopkins University, Baltimore, Maryland, USA. Corresponding author Correspondence to: * Drew Pardoll Author Details * Drew Pardoll Contact Drew Pardoll Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Cancer genomics: from discovery science to personalized medicine
- Nat Med 17(3):297-303 (2011)
Nature Medicine | Perspective Cancer genomics: from discovery science to personalized medicine * Lynda Chin1, 2, 3 * Jannik N Andersen1 * P Andrew Futreal4 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:297–303Year published:(2011)DOI:doi:10.1038/nm.2323Published online07 March 2011 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Recent advances in genome technologies and the ensuing outpouring of genomic information related to cancer have accelerated the convergence of discovery science and clinical medicine. Successful examples of translating cancer genomics into therapeutics and diagnostics reinforce its potential to make possible personalized cancer medicine. However, the bottlenecks along the path of converting a genome discovery into a tangible clinical endpoint are numerous and formidable. In this Perspective, we emphasize the importance of establishing the biological relevance of a cancer genomic discovery in realizing its clinical potential and discuss some of the major obstacles to moving from the bench to the bedside. View full text Figures at a glance * Figure 1: Cancer genetics is accelerating the time from 'driver mutation discovery' to 'clinical proof-of-concept' and the approval of new drugs. The historical timelines for developing targeted therapies discussed in the text are highlighted as examples. Gleevec received FDA approval long after the discovery of the Philadelphia chromosome mutation and hyperactive BCR-ABL protein in chronic myelogenous leukemia (CML). By contrast, the more recent discovery of chromosomal rearrangements (translocations) of ALK in NSCLC has rapidly translated into registration trials for Crizotinib, a 'cMET-turned-ALK' inhibitor, based on tantalizing response rates in ALK-fusion-positive tumors (Phase I and II trial results)63. Likewise, the development paradigm for selective BRAF inhibitors, as exemplified by PLX4032, underlines the much faster pace of translation (8 years, compared with Gleevec or Herceptin) once the driver status (in this case BRAF mutations) had been established for an indication (malignant melanoma). Such accelerated development times are enabled by the broader body of knowledge of cancer biology and mechanisms of ! actions that have been generated in the cancer field (Fig. 2). The FDA approval of Herceptin and the accompanying diagnostic test for HER2 expression (HercepTest) proved the value of biomarker-driven trials that are informed by mechanistic insights gained from cancer genetics. In a similar vein, it is the functional understanding of DNA-repair mechanisms, and the role of BRCA1 and BRCA2 mutations in sensitizing tumors to PARP inhibition, that inform current registration trials of PARP inhibitors in BRCA-associated cancer types and patients that carry the BRCA mutation. * Figure 2: From cancer genomics to personalized medicine. Some major logistical, regulatory and scientific hurdles, including sample acquisition and patient consent (to data generation and computational analyses) and functional and mechanistic studies (to clinical and commercial development). Venn diagram in the middle emphasizes key scientific challenges and their interdependencies. * Figure 3: Trade-off among commonly used experimental systems for functional validation. Model or assay selection is often a compromise between simplicity with high throughput (for example, cell line models) and complexity with high content information (for example, animal models). In general, genetic and pharmacological studies are fast (days–weeks) when using in vitro models compared with in vivo studies, where the relevant genetically engineered mouse model (GEMM) can take years to develop and optimize and human primary tumor tissues can require years to collect. In vivo studies tend to generate richer biological information content (for example, level of immune infiltrate or angiogenesis in addition to evidence of apoptosis in cancer cells) but in vitro high-throughput assays offer statistical power derived from assaying hundreds of cell lines. Therefore, complementary and reinforcing results from multiple systems can provide the strongest biological evidence. Author information * Abstract * Author information Affiliations * Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Lynda Chin & * Jannik N Andersen * Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA. * Lynda Chin * The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. * Lynda Chin * Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK. * P Andrew Futreal Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lynda Chin Author Details * Lynda Chin Contact Lynda Chin Search for this author in: * NPG journals * PubMed * Google Scholar * Jannik N Andersen Search for this author in: * NPG journals * PubMed * Google Scholar * P Andrew Futreal Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Taming the dragon: genomic biomarkers to individualize the treatment of cancer
- Nat Med 17(3):304-312 (2011)
Nature Medicine | Perspective Taming the dragon: genomic biomarkers to individualize the treatment of cancer * Ian J Majewski1 * René Bernards1 * Affiliations * Corresponding authorJournal name:Nature MedicinePages:304–312Year published:(2011)DOI:doi:10.1038/nm.2311Published online07 March 2011 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The gradual shift from cytotoxic drugs to highly selective, targeted therapeutic agents for cancer requires a parallel effort to characterize cancers at the molecular level to guide the choice of therapy for the individual patient. Here we review the genomic technologies that can be used to develop these drug response indicators, or biomarkers. We also discuss hurdles in their development and the implementation of biomarkers in clinical practice. View full text Figures at a glance * Figure 1: Types of biomarker. Prognostic tests help to identify individuals who are at high risk of recurrence of their cancer and should receive further (adjuvant) therapy. Predictive biomarkers help to identify those drugs to which patients are most responsive (or unresponsive). Pharmacodynamic biomarkers can help to identify which drug dose to use for an individual. * Figure 2: Development of a gene expression biomarker. () Unbiased discovery of a gene expression profile starts with the large-scale analysis of gene expression on a series of tumor samples of known clinical outcome. () Using bioinformatics, the set of genes is identified that correlates best with the relevant clinical parameter. (,) In the next step, this 'gene signature' is validated on a large cohort of additional clinical samples of known outcome (), and the clinical performance is evaluated in comparison with the generally accepted clinical parameters (). () Regulatory approval is still underdeveloped but might involve clearance by CMS under the CLIA guidelines and the College of American Pathologists (CAP) in the US. In Europe, both an ISO17025 accreditation of the laboratory and a CE-marking (indicating that it has met EU consumer safety, health or environmental requirements) of the diagnostic equipment are required. () Only after this process is completed should these tests be used to stratify patients by molecular sign! atures. * Figure 3: Pathway-targeted cancer therapies. () Routine sequencing of cancer genomes will identify many new genes that are involved in cancer. Detailed mechanistic studies will be required to determine how these genes contribute to tumorigenesis and how they influence therapeutic response. () The PI3K pathway is frequently mutated in cancer. Pathway components that are activated by mutations or gene amplification are marked in green, whereas those that are inactivated are marked in red86. Targeted therapeutic agents are available for multiple pathway components (boxed). It will be important to survey the genetic alterations present in the entire pathway so that an optimal therapeutic agent can be selected. For example, activating mutations in PIK3CA, or loss of the tumor suppressor PTEN, impairs response to Herceptin; in this case, a small-molecule inhibitor of AKT (protein kinase B) or mammalian target of rapamycin (mTOR) may be more effective. * Figure 4: Biomarker-driven selection of targeted therapies. A hypothetical situation is presented in which cancer therapy is adapted to counter the emergence of drug resistance. HER2-positive breast cancers are routinely treated with Herceptin, which results in inhibition of the PI3K pathway. Pathway biomarkers, such as phosphorylation sites (for example, phospho-RPS6K), could be used to monitor therapeutic response. Resistant cells emerge as a result of underlying heterogeneity in the tumor (for example, PIK3CA mutation) or because of de novo mutations (for example, AKT1 mutation). Once a resistant population is identified, a therapeutic agent should be selected that acts downstream of the oncogenic mutation. Patient relapse could be detected by monitoring genomic rearrangements specific to the tumor cells. Author information * Abstract * Author information Affiliations * Division of Molecular Carcinogenesis, Centre for Biomedical Genetics and Cancer Genomics Centre, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * Ian J Majewski & * René Bernards Competing financial interests René Bernards is founder and chief scientific officer of Agendia, a molecular diagnostics company. Corresponding author Correspondence to: * René Bernards Author Details * Ian J Majewski Search for this author in: * NPG journals * PubMed * Google Scholar * René Bernards Contact René Bernards Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The cancer stem cell: premises, promises and challenges
- Nat Med 17(3):313-319 (2011)
Nature Medicine | Review The cancer stem cell: premises, promises and challenges * Hans Clevers1Journal name:Nature MedicinePages:313–319Year published:(2011)DOI:doi:10.1038/nm.2304Published online07 March 2011 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Over the last decade, the notion that tumors are maintained by their own stem cells, the so-called cancer stem cells, has created great excitement in the research community. This review attempts to summarize the underlying concepts of this notion, to distinguish hard facts from beliefs and to define the future challenges of the field. View full text Figures at a glance * Figure 1: A timeline of the important discoveries in the fields of clonal evolution and cancer stem cells. Clonal evolution is shown on the left, and CSCs are shown on the right. * Figure 2: A theoretical synthesis of the clonal evolution and CSC concepts. Top to bottom: clonal evolution drives tumor progression. (1) The first oncogenic mutation (lightning arrow) occurs in a stem cell (or, alternatively, in a progenitor or even a differentiated cell) of a healthy epithelium, resulting in the growth of a genetically homogeneous benign lesion. (2) The second hit targets one of the cells in the benign lesion, which leads to the growth of a more malignant and invasive clone within the primary tumor. (3) A third hit in a cell within the malignant subclone causes further transformation, visualized as entry into a blood vessel for distant metastasis. Genetically independent subclones can coexist within the tumor. (4) A final mutational hit leads to tumor being entirely taken over by cells that behave as cancer stem cells. Shown, left to right: at each stage of this clonal evolution process, tumors and subclones within tumors contain some cells that behave as CSCs. The final hit (4) causes all cells to behave as CSCs, rendering the CS! C concept meaningless at this stage. Author information * Abstract * Author information Affiliations * Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, The Netherlands. * Hans Clevers Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Hans Clevers Author Details * Hans Clevers Contact Hans Clevers Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression
- Nat Med 17(3):320-329 (2011)
Nature Medicine | Review Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression * Mina J Bissell1 * William C Hines1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:320–329Year published:(2011)DOI:doi:10.1038/nm.2328Published online07 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Tumors are like new organs and are made of multiple cell types and components. The tumor competes with the normal microenvironment to overcome antitumorigenic pressures. Before that battle is won, the tumor may exist within the organ unnoticed by the host, referred to as 'occult cancer'. We review how normal tissue homeostasis and architecture inhibit progression of cancer and how changes in the microenvironment can shift the balance of these signals to the procancerous state. We also include a discussion of how this information is being tailored for clinical use. View full text Author information * Abstract * Author information * Supplementary information Affiliations * Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Mina J Bissell & * William C Hines Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mina J Bissell Author Details * Mina J Bissell Contact Mina J Bissell Search for this author in: * NPG journals * PubMed * Google Scholar * William C Hines Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (193k) Supplementary Table 1 Additional data - Cancer epigenetics reaches mainstream oncology
- Nat Med 17(3):330-339 (2011)
Nature Medicine | Review Cancer epigenetics reaches mainstream oncology * Manuel Rodríguez-Paredes1 * Manel Esteller1, 2, 3 * Affiliations * Corresponding authorJournal name:Nature MedicinePages:330–339Year published:(2011)DOI:doi:10.1038/nm.2305Published online07 March 2011 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Epigenetics is one of the most promising and expanding fields in the current biomedical research landscape. Since the inception of epigenetics in the 1940s, the discoveries regarding its implications in normal and disease biology have not stopped, compiling a vast amount of knowledge in the past decade. The field has moved from just one recognized marker, DNA methylation, to a variety of others, including a wide spectrum of histone modifications. From the methodological standpoint, the successful initial single gene candidate approaches have been complemented by the current comprehensive epigenomic approaches that allow the interrogation of genomes to search for translational applications in an unbiased manner. Most important, the discovery of mutations in the epigenetic machinery and the approval of the first epigenetic drugs for the treatment of subtypes of leukemias and lymphomas has been an eye-opener for many biomedical scientists and clinicians. Herein, we will summari! ze the progress in the field of cancer epigenetics research that has reached mainstream oncology in the development of new biomarkers of the disease and new pharmacological strategies. View full text Figures at a glance * Figure 1: DNA methylation patterns in normal and cancer cells. DNA methylation takes place along the whole genome, and its disruption is a typical hallmark of cancer. () In normal cells (top), CpG islands and CpG island shores usually remain unmethylated, allowing gene transcription. Additionally, DNA methylation within the gene bodies avoids spurious transcription initiations. In cancer cells (bottom), by contrast, although both CpG islands and CpG island shores may be strongly methylated, gene bodies lack this modification. As a result, transcription of many genes gets blocked, and aberrant transcription may occur from incorrect transcription start sites (TSSs). () In normal cells (top), methylation of repetitive sequences prevents genomic instability and, again, spurious transcription initiations. Moreover, transposable elements cannot be activated in a methylated environment. In cancer cells (bottom), global hypomethylation triggers genomic instability and aberrant transcription initiations. Concomitant activation of transposons may! lead to gene disruption. * Figure 2: Histone modification patterns in normal and cancer cells. Mainly along their protruding N-terminal tails, but also within their C-terminal regions, histones can undergo diverse post-translational modifications. In the right combination and translated by the appropriate effectors, these modifications contribute to establishing the global and local condensed or decondensed chromatin states that eventually determine gene expression. This figure depicts the main modifications of the four core histones in normal cells (type and position in the amino acid sequence). Furthermore, and because disruption of their normal patterns is related to cancer, histone modifications typically associated with the disease have also been highlighted. Ac, acetylation; Me, methylation; P, phosphorylation; Ub, ubiquitination. * Figure 3: Selection of epigenetic genes disrupted in human tumors. Mutation, deletion and/or altered expression of genes encoding components of the various epigenetic machineries are typically observed in human tumors. The figure shows a selection of genes encoding enzymes that add, remove and recognize histone modifications, as well as members of the DNA methylation machinery, whose deregulation is connected to cancer. CRCs, chromatin remodeling complexes; Ac, acetylation; Me, methylation. * Figure 4: Epigenetic biomarkers in oncology. From all types of samples obtained from individuals with cancer, single and global epigenetic screenings have been developed to identify new molecular markers to manage the disease. To predict malignancy in prostate tumorigenesis and response to temozolomide in gliomas, the study of hypermethylation events in GSTP1 and MGMT, respectively, is reaching the clinical stage. * Figure 5: Epigenetic drugs for cancer therapy. Numerous compounds have been reported to be effective against cancer cells by inhibiting components of the epigenetic machineries. This figure shows the most important epigenetic drugs classified depending on their particular epigenetic targets. Author information * Abstract * Author information Affiliations * Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, L'Hospitalet, Barcelona, Spain. * Manuel Rodríguez-Paredes & * Manel Esteller * Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain. * Manel Esteller * Institucio Catalana de Recerca i Estudis Avançats, Barcelona, Spain. * Manel Esteller Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Manel Esteller Author Details * Manuel Rodríguez-Paredes Search for this author in: * NPG journals * PubMed * Google Scholar * Manel Esteller Contact Manel Esteller Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections
- Nat Med 17(3):341-346 (2011)
Nature Medicine | Article Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections * Yang Zhang1, 3 * Xiang Li1, 3 * Alexander Carpinteiro1 * Jeremy A Goettel2 * Matthias Soddemann1 * Erich Gulbins1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:341–346Year published:(2011)DOI:doi:10.1038/nm.2296Received02 February 2010Accepted22 December 2010Published online06 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffol! d that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections. View full text Figures at a glance * Figure 1: Ksr1 deficiency results in hypersusceptibility to pulmonary P. aeruginosa infection. (–) Displayed are the mortality rates after infection of wild-type and Ksr1-deficient mice with 5 × 108 CFU of P. aeruginosa strain ATCC 27853 (), the number of the bacteria in the lungs (), the release of TNF-α in the lungs (), the neutrophil counts in the bronchoalveolar lavage fluid () and H&E staining of lungs before and 20 h after pulmonary P. aeruginosa infection (). () CFU counts in the spleens of wild-type and Ksr1-deficient mice 20 h after pulmonary infection. Scale bar, 80 μm. Data in are presented as Kaplan-Meyer curves from three independent experiments with three mice per group (*P < 0.05; log-rank test). Data in – are means ± s.d. from six independent experiments or are representative of six independent experiments (*P < 0.05; **P < 0.005; ***P < 0.001). * Figure 2: Decreased bacterial-killing capability in Ksr1-deficient alveolar macrophages. () Left, the percentage of intracellular P. aeruginosa in wild-type and Ksr1-deficient alveolar macrophages immediately (set at 100%) and 60 min after termination of a 30-min infection with P. aeruginosa. Right, the number of P. aeruginosa that survived for 90 min in the presence of wild-type or Ksr1-deficient polymorphonuclear leukocytes (PMNs) over that in samples with P. aeruginosa only. () Quantitative analysis (left) of fluorescence microscopy studies (right) showing that phagocytosis of P. aeruginosa, FITC-zymosan or latex beads by macrophages is independent of Ksr1 expression. Scale bar, 2.5 μm. () Shown is the influence of Erk1/2 inhibitor U0126 on survival of intracellular P. aeruginosa in wild-type macrophages, given as percentage of bacteria 60 min after a 30-min infection compared to the number immediately after termination of the 30-min infection. Phosphorylation and expression of Erk1/2 after infection is shown in the immunoblot insets. () Confocal microscopy ! to determine nuclear localization of the p65 subunit of nuclear factor-κB. () TNF-α release measured after short-term infection with P. aeruginosa in wild-type and Ksr1-deficient macrophages. Scale bar, 5 μm. Images in and are representative of three separate experiments. Data are presented as means ± s.d. of four independent experiments. Asterisks indicate significant differences from results obtained from wild-type cells at the respective time points (***P < 0.001). * Figure 3: Reduction of P. aeruginosa–induced NO production and peroxynitrite formation in Ksr1-deficient alveolar macrophages. (,) NO production () and peroxynitrite formation and tyrosine nitrosylation () in wild-type and Ksr1-deficient alveolar macrophages upon infection with P. aeruginosa. NO production is given in arbitrary units (AU) of the fluorescence change. Tyrosine nitrosylation was determined by immunoblotting. () The percentage of intracellular P. aeruginosa in untreated and treated alveolar macrophages immediately (set at 100%) and 60 min after termination of the 30-min infection with P. aeruginosa in the presence of the iNOS inhibitors L-NAME or aminoguanidine (AG). (,) NO release () and bacterial killing () upon P. aeruginosa infection after knockdown of Ksr1 in J774 macrophages via RNA interference. (,) NO production () and bacterial killing of P. aeruginosa () by PMNs either lacking Ksr1 or treated with aminoguanidine. Data are presented as means ± s.d. or as representative blots from at least four independent experiments. Asterisks indicate significant differences from results obt! ained from wild-type cells or control cells at the respective time points (*P < 0.05; ***P < 0.001). Blots in are representative of four independent experiments. * Figure 4: Ksr1 anchors and enhances the activity of iNOS. () Expression of iNOS in wild-type and Ksr1-deficient alveolar macrophages before and after P. aeruginosa infections, as determined by immunoblotting. (,) Coimmunoprecipitation (IP) () and confocal microscopy () experiments indicate that Ksr1 in macrophages interacts with iNOS 2 h after infection with P. aeruginosa. Immunoprecipitates were analyzed by immunoblotting (IB) with the corresponding antibodies to Ksr1 (left) or iNOS (right). Aliquots of the immunoprecipitates were blotted with the same antibody used for immunoprecipitation as a control for loading of similar amounts of protein. Arrows in indicate the sites of colocalization of Ksr1 and iNOS in the confocal microcopy studies. Scale bar, 2.5 μm. () The activity of iNOS as measured in Ksr1 and iNOS immunoprecipitates and upon incubation of Ksr1 immunoprecipitates from wild-type cells (IP-Ksr1) with iNOS immunoprecipitates (IP-iNOS) from Ksr1-deficient macrophages infected with P. aeruginosa. Data are representative ! of four independent experiments or are presented as means ± s.d. from four independent experiments (**P < 0.005). * Figure 5: Ksr1 assembles Hsp90 and iNOS to activate iNOS. () Left, immobilized, recombinant Flag-tagged Ksr1 or kinase-inactive Ksr1 interact with soluble iNOS, as revealed by immunoblotting with antibodies to iNOS. Recombinant iNOS alone was loaded as control. Right, immobilized, recombinant iNOS binds Flag-tagged Ksr1 or kinase-inactive Ksr1 expressed in bacterial lysates. The blots were developed with Ksr1-specific antibodies. The bottom blots show the loading controls. () Measurements of the activity of recombinant iNOS showing a stimulation by Hsp90 alone and a maximal augmentation by recombinant Ksr1 or kinase-inactive Ksr1. () Ksr1 (left) or Hsp90 (right) immunoprecipitates from infected or noninfected macrophages immunoblotted with Hsp90-specific or Ksr1-specific antibodies, respectively. The bottom blots are loading controls. () NO release determined in geldanamycin-treated, L-NAME–treated or untreated control J774 macrophages by measuring DAFFM-NO fluorescence. The blots and images in and are representative of four inde! pendent experiments. Data in and are means ± s.d. from three independent experiments (*P < 0.05; ***P < 0.001). * Figure 6: NO is crucial for the elimination of pulmonary P. aeruginosa. (,) Bacterial killing of P. aeruginosa by alveolar macrophages () and PMNs () treated with iNOS inhibitor aminoguanidine or the NO-donor DETA-NONOate. (,) Survival of Ksr1-deficient () or wild-type () mice treated with DETA-NONOate, aminoguanidine or saline after pulmonary P. aeruginosa infection. () Ksr1-deficient or wild-type mice were bone marrow–transplanted as indicated, and survival after P. aeruginosa infection was determined. Data in and are presented as means ± s.d. from six independent experiments (**P < 0.005; ***P < 0.001), and data in – are presented as Kaplan-Meyer curves (*P < 0.05; log-rank test). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Yang Zhang & * Xiang Li Affiliations * Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany. * Yang Zhang, * Xiang Li, * Alexander Carpinteiro, * Matthias Soddemann & * Erich Gulbins * Department of Cell and Developmental Biology, Vanderbilt University School of Medicine and Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee, USA. * Jeremy A Goettel Contributions Y.Z. performed the internalization assays, confocal and fluorescence microscopy, immunoprecipitation studies, flow cytometry analysis and protein overexpression and iNOS activity assays. X.L. conducted the mouse infection experiments, determined cytokine abundance in the lung and performed the bacterial-killing assay and immunoblot analysis. A.C. performed bone marrow transplantation and determined the neutrophil count. M.S. characterized the genotypes of Ksr1-deficient mice. J.A.G. cloned recombinant Ksr proteins. Y.Z., X.L. and E.G. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Erich Gulbins Author Details * Yang Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Xiang Li Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Carpinteiro Search for this author in: * NPG journals * PubMed * Google Scholar * Jeremy A Goettel Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Soddemann Search for this author in: * NPG journals * PubMed * Google Scholar * Erich Gulbins Contact Erich Gulbins Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (475K) Supplementary Notes 1–3, Supplementary Figures 1–6 and Supplementary Methods Additional data - UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53
- Nat Med 17(3):347-355 (2011)
Nature Medicine | Article UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53 * Hong Wu1 * Scott L Pomeroy2 * Manuel Ferreira3 * Natalia Teider2 * Juliana Mariani2 * Keiichi I Nakayama4 * Shigetsugu Hatakeyama5 * Victor A Tron6 * Linda F Saltibus7 * Leo Spyracopoulos7 * Roger P Leng1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:347–355Year published:(2011)DOI:doi:10.1038/nm.2283Received08 April 2009Accepted30 November 2010Published online13 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undesc! ribed molecular mechanisms of inactivation of p53 in brain tumors. View full text Figures at a glance * Figure 1: UBE4B interacts with Hdm2 and p53. () Western blot with UBE4B-specific or Hdm2-specific (2A10) antibodies after coimmunoprecipitation of Hdm2 from BJT whole cell lysates using UBE4B-specific (UFD2/E4) or β-gal–specific antibodies. () Western blot with UBE4B-specific and Hdm2-specific (2A10) antibodies after coimmunoprecipitation of UBE4B from BJT whole cell lysates using Hdm2-specific (2A10) or β-gal–specific antibodies. () Western blot of immunoprecipitates from BJT lysates with p53-specific (DO-1) and UBE4B-specific antibodies. BJT cells were treated with 10 μM of the proteasome inhibitor AdaAhx3L3VS (Ada) for 6 h or mock treated. Total lysates were immunoprecipitated with UBE4B-specific or β-gal–specific antibodies. () Western blot of BJT lysates with UBE4B-specific and p53-specific (DO-1) antibodies after BJT cells were treated with Ada for 6 h or mock treated. Total lysates were immunoprecipitated with p53-specific (DO-1) or β-gal–specific antibodies. () The in vitro interaction of Ube4b, Md! m2 and p53 was evaluated with GST pull-down assays and western blotting using an antibody against histidine (His-Ube4b). () BJT cell lysates were subjected to size-exclusion chromatography. Fractions were analyzed by western blotting for the presence of p53, Hdm2, UBE4B and PRP19 (control) with p53-specific (DO-1), Hdm2-specifc (2A10), UBE4B-specific (UFD2/E4) and PRP19-specific antibodies. The elution position of the molecular size markers is shown. In panels – are the eluted proteins probed by antibodies as shown. IB, immunoblot; IP, immunoprecipitation; WB, western blot. * Figure 2: Negative regulation of p53 by UBE4B. () Flag-Ube4b or Flag-Ube4bΔU or Myc-Mdm2 or Myc-Mdm2ΔRING was cotransfected with p53 into H1299 cells and analyzed by western blotting with p53-specific (Pab421), Flag-specific (M5) and Myc-specific antibodies. () Plasmids expressing human Flag-UBE4B, Myc-Hdm2, HA-Hdm4 and p53 were used in studies similar to those shown in . The p53 immunoblot was probed with a p53-specific antibody (DO-1). () H1299 cells were transfected with plasmids expressing HA-tagged ubiquitin (Ub), p53, Flag-Ube4b, Flag-Ube4bΔU, Myc-Mdm2 and Myc-Mdm2ΔRING. Equal amounts of extract were immunoprecipitated with a p53-specific antibody (Pab421) and analyzed by western blotting with HA-specific, p53-specific (Pab421), Flag-specific (M5) and Myc-specific antibodies. () H1299 cells were transfected with p53 expression plasmid alone or in combination with Flag-Ube4b, Myc-Mdm2, Flag-Ube4a, Flag-Cyc4 or Flag-PRP19 expression plasmids. The transfected cells were analyzed by western blotting with p53-specif! ic (Pab421), Flag-specific (M2) and Myc-specific antibodies. () H1299 cells were transfected with a p53 expression plasmid alone or with a Flag-UBE4B or a Flag-UBE4BΔU expression plasmid in the presence or absence of MG132. The transfected cells were analyzed by western blotting with p53-specific (DO-1) or Flag-specific (M5) antibodies. () Extracts from cells treated with MG132 were immunoprecipitated with a p53-specific antibody (DO-1) and analyzed by western blotting with p53-specific (Ab-7), UBE4B-specific and Hdm2-specific (2A10) antibodies. Asterisks (*) indicate the migration positions of p53-ubiquitin (Ub-p53) conjugates. An antibody to β-actin (actin) was used as a loading control in panels –. IgG (H), IgG heavy chain. * Figure 3: The interdependence of UBE4B and Hdm2 in promoting the degradation of p53. () H283 cells were transfected with increasing amount of the UBE4B expression plasmid. Western blot of whole-cell lysates with UBE4B-specific, p53-specific (DO-1) and Hdm2-specific (2A10) antibodies. (,) Neuro2A () or BJT () cells were transfected with the siRNA constructs shown. The amounts of endogenous Ube4b (or UBE4B), p53, p21 and Mdm2 (or Hdm2) proteins were determined by western blotting with UBE4B-specific, p53-specific (Pab421 for , DO-1 for ), p21-specific and Mdm2-specific (MD-129) or Hdm2-specific (2A10) antibodies. () Trp53−/−, Mdm2−/− (double knockout) MEFs were transfected with a p53 expression construct alone or in combination with a Myc-Mdm2 or Flag-Ube4b expression construct. The transfected cells were analyzed by western blot with p53-specific (Pab421), Flag-specific (for Ube4b) and Myc-specific (for Mdm2) antibodies. () Additionally, the extracts from the transfected cells were immunoprecipitated with a p53-specific antibody (Pab421) and analyzed ! by western blotting with HA-specific or p53-specific (Pab421) antibodies. MG132, a proteasome inhibitor, was added for 6 h before harvest as shown in the right panel. () Neuro2A cells were transfected with the Ube4b-siRNA2 construct or a control siRNA construct. Thirty hours later, the cells were further transfected with the Mdm2 expression plasmid and analyzed by western blot with Ube4b-specific (UFD2/E4), p53-specific (Pab421) and Mdm2-specific (MD-219) antibodies. () Cell extracts were prepared from Ube4b−/− and Ube4b+/+ MEFs. Western blot analysis of endogenous Ube4b, p53 and Mdm2 proteins with Ube4b-specific (UFD2/E4), p53-specific (CM5) and Mdm2-specific (MD-219) antibodies. () Ube4b−/− (Ube4b KO) MEFs were transfected with the indicated plasmids and analyzed by western blot with Flag-specific (for Ube4b and Ube4bΔU), Mdm2-specific (MD-219) and p53-specific (CM5) antibodies. () Ube4b−/− MEFs were transfected with control-siRNA or Mdm2-siRNA and analyzed b! y western blot with Mdm2-specific (MD-129), p53-specific (CM5)! and Ube4b-specific antibodies. () Ube4b−/− MEFs or wild-type MEFs were cotransfected with plasmids expressing p53, or in combination with Myc-Mdm2, or Flag-Ube4b or Flag-Ube4bΔU as well as HA-Ub. Whole-cell lysates were immunoprecipitated with a p53-specific antibody (Pab421) and analyzed by western blot with HA-specific, p53-specific (Pab421), Flag-specific and Myc-specific antibodies. Ube4b KO, Ube4b−/− MEFs; Ube4b WT, Ube4b+/+ MEFs. An antibody to β-actin (actin) was used as a loading control in all panels except and . * Figure 4: UBE4B mediates p53 polyubiquitination in vivo and in vitro. () H283 cells were transfected with control-siRNA or UBE4B-siRNA2. Forty hours later, cells were further transfected with the plasmid expressing HA-Ub. Lysates were immunoprecipitated with a p53-specific antibody (DO-1) and analyzed by immunoblotting with HA-specific, p53-specific (DO-1) and UBE4B-specific antibodies. () Similar to except that after the second transfection of the HA-Ub expression plasmid, cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. () Stable depletion of UBE4B by siRNA in H283 cells. Similar to except that the cell extracts were from the stably expressing UBE4B-siRNA2 or control-siRNA clones in H283 cells. () His-Ube4b was evaluated for E3 activity in the presence of recombinant E1, E2 (UbcH5b) and ubiquitin. Following the ubiquitination reaction, the samples were analyzed by western blotting with ubiquitin-specific and p53-specific (Pab421) antibodies. Direct western blots for Ube4b, Mdm2, UBE1 and UbcH5b ar! e shown in the lower panel. () Western blot analysis of a coupled in vitro ubiquitination-immunoprecipitation. After the in vitro ubiquitination, the samples were immunoprecipitated with a p53-specific antibody (Pab421) and analyzed by western blotting with ubiquitin-specific and p53-specific antibodies. Direct western blots for Ube4b, Mdm2, UBE1 and UbcH5b are shown in the bottom blots. () Western blot of the in vitro ubiquitination reaction probed with a p53-specific (Pab421) antibody. Direct western blots for Ube4b, Mdm2, UBE1 and UbcH5b are shown in the lower panel. () Western blot similar to except that the copurified Mdm2-p53 complexes were incubated with the purified Ube4bΔU, or the purified Ube4b was incubated with the purified Mdm2ΔRING. () Western blot of the in vitro ubiquitination reaction with a p53-specific antibody (Pab421). Direct western blots for Ube4b and Ube4bΔU (Ube4b polyclonal antibodies21), Mdm2 and Mdm2ΔRING (SMP14), UBE1 and UbcH5b are shown in! the lower panel. () Purified HA-p53 was ubiquitinated by Flag! -Hdm2 (ref. 31), then immunoprecipitated with HA antibody-conjugated beads to remove Hdm2 and nontagged ubiquitin. The immobilized p53 was then mixed in a second ubiquitination reaction, along with purified Flag-UBE4B and Flag-Ub. Western blot showing HA-p53-conjugates with a Flag-specific antibody (M2). In addition, we added Flag-Hdm2ΔRING as a negative control and Flag-CBP as a positive control. () U2OS cells were transfected plasmids expressing Flag-UBE4B or Flag-UBE4B 1001-1173. Cell lysates were immunoprecipitated with a p53-specific antibody (FL-393) and analyzed by western blotting with Hdm2-specific (2A10) and p53-specific (DO-1) antibodies. Direct western blot of the samples used is shown in the lower panel. Asterisks (*) indicate the migration positions of p53-ubiquitin conjugates. An antibody to β-actin (actin) was used as a loading control. * Figure 5: UBE4B inhibits p53-dependent transactivation and apoptosis. () Saos-2 cells were cotransfected with a p21-luciferase (Luc) reporter plasmid and a p53 expression construct in combination with Hdm2, UBE4B or UBE4BΔU expression constructs or an empty vector (pcDNA3.1). Transcriptional activity of p53 is shown; error bars indicate the s.e.m. (n = 3). Western blot of p53, Hdm2 and UBE4B with p53-specific (DO-1), Myc-specific for Hdm2 and Flag-specific for UBE4B antibodies. () H1299 cells were cotransfected with a CD20 expression construct and with pcDNA3-p53 (3 μg) and pcDNA3-UBE4B (15 μg), pcDNA3-Hdm2 (15 μg) or pcDNA3-UBE4BΔU (15 μg). The inhibitory effect of UBE4B on p53-dependent apoptosis was determined by annexin V staining of CD20-positive cells and flow cytometry. Error bars indicate the s.e.m. (n = 3). Western blot of p53, UBE4B, Hdm2 and UBE4BΔU with p53-specific (DO-1), UBE4B-specific, Myc-specific for Hdm2 and Flag-specific for UBE4BΔU antibodies. () H1299 cells were transfected with the UBE4B-siRNA or control-siRNA fo! r 30 h. The cells were then transfected with the p53 expression construct alone or in combination with the Hdm2 and CD20 expression plasmids. The number of surviving CD20-positive cells was measured by flow cytometry 24 h after transfection. Error bars indicate the s.e.m. (n = 3). Western blot of UBE4B, p53 and Hdm2 with UBE4B-specific, p53-specific (DO-1) and Myc-specific antibodies. () Depletion of UBE4B by siRNA in BJT and BJT/DD cells, analyzed by western blotting with UBE4B-specific, p53-specific (DO-1) and p21-specific antibodies. (,). BJT () or BJT/DD () cells were treated with the LacZ-siRNA or UBE4B-siRNA2, and cell-cycle profile was determined by propidium iodide staining and flow cytometry. G1/s ratio is the ratio of subpopulations and G1-S phase fractions; it indicates the degree of G1 arrest. An antibody to β-actin (actin) was used as a loading control in panels –. Results represent the average of triplicate experiments. * Figure 6: UBE4B promotes tumorigenesis in a p53-dependent manner and is overexpressed in brain tumors. () 1 × 107 NIH3T3 cells expressing Flag-Ube4b or Flag-Ube4bΔU or an empty vector were subcutaneously injected into SCID mice. A portion of the transfected NIH3T3 cells was lysed and analyzed by western blotting as indicated. The tumor volume (mm3) was estimated from caliper measurements. The difference of the population means of the Ube4b-injected group is significantly different compared to the empty vector– and Ube4bΔU-injected groups (P < 0.01, one-way ANOVA). Error bars show s.d. ± the mean volume of ten xenografts. () 1 × 107 HCT116 TP53−/− cells expressing the control siRNA or UBE4B-siRNA2 were subcutaneously injected into SCID mice as indicated (left). A portion of the transfected HCT116 TP53−/− cells was lysed and analyzed by western blotting as indicated. Similarly, HCT116-WT cells were used as the tumor cells (right). Tumor size volumes from four different injected groups were analyzed by two-way ANOVA (P < 0.01). Error bars show the s.d. from the me! an volume of ten xenografts. (–) Six tumor samples from each group mice were dissected 35 d () or 30 d (,) after injection and analyzed by western blotting with the indicated antibodies. () Representative western blot analysis of mouse medulloblastoma tissues derived from Ptch+/− mice. Proteins were extracted from mouse cerebellum (CRB) and cortex (CTX) and then analyzed by western blotting with indicated antibodies. N denotes normal mouse tissue, and T denotes tumor tissue. The inverse correlation between the amounts of p53 protein that of Ube4b protein was tested with a Pearson correlation test. y = −0.14881x + 4525.41; Pearson correlation: −0.96716; significance (two-tailed test): 4.89362 × 10−6. () Whole-cell extracts of medulloblastoma cell lines were analyzed by western blotting with the indicated antibodies. The inverse correlation between the amounts of p53 protein and the amounts of UBE4B protein was tested with a Pearson correlation test. y = −0.31433! x + 8103.88862; Pearson correlation: −0.98241; significance ! (two-tailed test): 1.3423 × 10−5. () Similar to and except that proteins were prepared from human medulloblastoma tissues. NS is the prefix for the USA national tissue bank number. The inverse correlation between p53 and UBE4B protein levels was tested with a Pearson correlation test. y = −0.17304x + 4440.7514; Pearson correlation: −0.95033; significance (two-tailed test): 8.56558 × 10−5. () Similar to except that proteins were prepared from human ependymoma. The inverse correlation between amount of p53 and UBE4B protein was tested with a Pearson correlation test. y = −0.17442x + 4192.60377; Pearson correlation: −0.93287; significance (two-tailed test): 2.41633 × 10−4. () Similar to and except that proteins were prepared from pediatric astrocytoma. The inverse correlation between p53 protein level and UBE4B protein level was tested with a Pearson correlation test. y = −0.75587x + 29275.47726; Pearson correlation: −0.98423; significance (two-tailed test! ): 1.59542 × 10−6. An antibody to β-actin (actin) was used as a loading control in panels –. Author information * Abstract * Author information * Supplementary information Affiliations * Heritage Medical Research Center, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada. * Hong Wu & * Roger P Leng * Department of Neurology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Scott L Pomeroy, * Natalia Teider & * Juliana Mariani * Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Manuel Ferreira * Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan. * Keiichi I Nakayama * Department of Molecular Biochemistry, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan. * Shigetsugu Hatakeyama * Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada. * Victor A Tron * Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. * Linda F Saltibus & * Leo Spyracopoulos Contributions H.W. and R.P.L. contributed to study design, performed most of the experiments, analyzed and interpreted the data and wrote the manuscript. S.L.P. provided logistical support and all tumor samples and interpreted and discussed the data. M.F. provided technical support and experimental assistance. N.T. conducted the western blotting for the pediatric astrocytoma tissues, isolated genomic DNAs from various tumor samples, carried out mutation detection for p53 in various tumor tissues and medulloblastoma cell lines and did long-term colony assays. J.M. collected tissue samples from various types of human brain tumors, cared for Ptch+/− mice, conducted all the mouse genotyping and isolated the cerebellum and cortex from the Ptch+/− mice. K.I.N. and S.H. provided the study material and technical support. V.A.T. provided technical support. L.F.S. conducted the FPLC protein purification experiments. L.S. provided technical support for the gel filtration. R.P.L. supervised and d! irected the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Roger P Leng Author Details * Hong Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Scott L Pomeroy Search for this author in: * NPG journals * PubMed * Google Scholar * Manuel Ferreira Search for this author in: * NPG journals * PubMed * Google Scholar * Natalia Teider Search for this author in: * NPG journals * PubMed * Google Scholar * Juliana Mariani Search for this author in: * NPG journals * PubMed * Google Scholar * Keiichi I Nakayama Search for this author in: * NPG journals * PubMed * Google Scholar * Shigetsugu Hatakeyama Search for this author in: * NPG journals * PubMed * Google Scholar * Victor A Tron Search for this author in: * NPG journals * PubMed * Google Scholar * Linda F Saltibus Search for this author in: * NPG journals * PubMed * Google Scholar * Leo Spyracopoulos Search for this author in: * NPG journals * PubMed * Google Scholar * Roger P Leng Contact Roger P Leng Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods Additional data - Regulation of glucose homeostasis through a XBP-1–FoxO1 interaction
- Nat Med 17(3):356-365 (2011)
Nature Medicine | Article Regulation of glucose homeostasis through a XBP-1–FoxO1 interaction * Yingjiang Zhou1 * Justin Lee1 * Candace M Reno2 * Cheng Sun1 * Sang Won Park1 * Jason Chung1 * Jaemin Lee1 * Simon J Fisher2 * Morris F White1 * Sudha B Biddinger1 * Umut Ozcan1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:356–365Year published:(2011)DOI:doi:10.1038/nm.2293Received15 October 2010Accepted16 December 2010Published online13 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To date, the only known role of the spliced form of X-box–binding protein-1 (XBP-1s) in metabolic processes has been its ability to act as a transcription factor that regulates the expression of genes that increase the endoplasmic reticulum (ER) folding capacity, thereby improving insulin sensitivity. Here we show that XBP-1s interacts with the Forkhead box O1 (FoxO1) transcription factor and directs it toward proteasome-mediated degradation. Given this new insight, we tested modest hepatic overexpression of XBP-1s in vivo in mouse models of insulin deficiency or insulin resistance and found it improved serum glucose concentrations, even without improving insulin signaling or ER folding capacity. The notion that XBP-1s can act independently of its role in the ER stress response is further supported by our finding that in the severely insulin resistant ob/ob mouse strain a DNA-binding–defective mutant of XBP-1s, which does not have the ability to increase ER folding capac! ity, is still capable of reducing serum glucose concentrations and increasing glucose tolerance. Our results thus provide the first evidence to our knowledge that XBP-1s, through its interaction with FoxO1, can bypass hepatic insulin resistance independent of its effects on ER folding capacity, suggesting a new therapeutic approach for the treatment of type 2 diabetes. View full text Figures at a glance * Figure 1: XBP-1s binds FoxO1 and promotes its degradation. () Left, XBP-1s and FoxO1 protein amounts in total cell lysates and cytoplasmic and nuclear fractions from MEFs expressing FoxO1 and increasing amounts of XBP-1s. Right, Foxo1 mRNA levels in MEFs expressing FoxO1 and increasing amounts of XBP-1s were analyzed by quantitative PCR. 18S ribosome RNA (Rn18s) was used for normalization of gene expression. () Endogenous FoxO3a and FoxO1 protein amounts in MEFs expressing increasing amounts of XBP-1s. () Nuclear FoxO1 and XBP-1s protein amounts in MEFs treated with DMSO or MG132. () Pulse-chase analysis of FoxO1 stability in MEFs overexpressing XBP-1s. () Ubiquitinylated FoxO1 amounts in MEFs expressing ubiquitin and XBP-1s after DMSO or MG132 treatment. () Immunoblotting of FoxO1 and XBP-1s in XBP-1s immunoprecipitates (IP) from MG132-treated MEFs expressing FoxO1 and XBP-1s. Total FoxO1 protein amounts were determined in whole-cell lysates (WCL). () Mammalian two-hybrid assay with pM–XBP-1s and pVP16-FoxO1 or pM-FoxO1 and pVP16! –XBP-1s. () Nuclear FoxO1 and XBP-1s protein amounts in MEFs treated with 1 μM BEZ235 for 4 h. Phosphorylated Akt Ser473 (pAkt Ser473) and total Akt amounts determined in cell lysates. () FoxO1, pAkt Ser473, total Akt and XBP-1s amounts in WT and Akt1/2 double-knockout (Akt1/2 DKO) MEFs treated with DMSO or 20 μM Akt inhibitor (Akti-VIII) for 30 min. () Phosphorylation-resistant mutant FoxO1(ADA) protein amounts in MEFs expressing FoxO1(ADA) and increasing amounts of XBP-1s. Each experiment was independently reproduced three times. Error bars are means ± s.e.m.; **P < 0.01, ***P < 0.001. * Figure 2: Medium-level expression of XBP-1s in ob/ob mice improves glucose homeostasis without altering insulin receptor signaling. Seven-week-old male ob/ob mice were injected with medium dose (4 × 107 PFU g−1) of Ad-LacZ (n = 6) or Ad-XBP-1s (n = 6) through the tail vein, except for one group of mice injected with low dose (1 × 107 PFU g−1) of Ad-XBP-1s as indicated in . () XBP-1s protein amounts in the liver of ob/ob mice injected with a low or medium dose of adenovirus. () Relative mRNA levels of XBP-1s target genes Dnajb9, Pida3 and Hspa5 in the liver of Ad-LacZ– or Ad–XBP-1s–injected mice. () Blood glucose concentrations in fed, 6-h fasted and 14-h fasted mice on the indicated days after injections. (–) Plasma insulin concentrations on day 9 (), GTT on day 5 () and ITT on day 7 () after the injections. () Insulin receptor (IR) and IRS-1 tyrosine (PY) and Akt Ser473 phosphorylations with or without insulin (Ins) stimulation in the liver on day 9 after injection. () Total and nuclear amounts of FoxO1 protein in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice. Graphs depict ! total FoxO1 / tubulin and nuclear FoxO1 / lamin A/C ratios. (,) Relative mRNA levels of Foxo1 () and Igfbp1, G6pc, Pck1 and Ppargc1a () in the liver of adenovirus-injected mice. Experiments were repeated in five independent cohorts. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 3: High-level expression of XBP-1s in the liver of ob/ob mice increases insulin sensitivity. Seven-week-old male ob/ob mice were injected with Ad-LacZ (n = 6) or Ad–XBP-1s (n = 6) (1.8 × 108 PFU g−1) through the tail vein. () XBP-1s protein amounts in the liver lysates on day 6 after injection. () Dnajb9, Pida3 and Hspa5 mRNA levels in the liver of Ad-LacZ– or Ad–XBP-1s–injected mice. (–) Fed blood glucose concentrations on day 5 (), plasma insulin concentrations on day 7 () and GTT on day 3 () after the adenovirus injections. () In vivo insulin receptor signaling in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice on day 6 after injection. Graphs depict the (phospho / total) / pIR ratios. () Relative mRNA levels of Insr in the liver of adenovirus-injected mice on day 6 after injection. () Total and nuclear FoxO1 amounts in the liver of Ad-LacZ– or Ad–XBP-1s–injected ob/ob mice. Graphs depict total FoxO1 / tubulin and nuclear FoxO1 / lamin A/C ratios. (,) Relative mRNA levels of Foxo1 () and G6pc, Pck1 and Ppargc1a () in the liver of A! d-LacZ– or Ad–XBP-1s–injected ob/ob mice. Experiments were repeated in three independent cohorts. NS, nonspecific. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 4: A DNA-binding-defective mutant XBP-1s (ΔDBD) improves glucose homeostasis in ob/ob mice. () Nuclear protein amounts of XBP-1s and ΔDBD in MEFs infected with the same dose of Ad–XBP-1s or Ad-ΔDBD. () ER stress element (ERSE)-luciferase activity and Hspa5 mRNA levels in MEFs infected with Ad–XBP-1s or Ad-ΔDBD. () Immunoblotting of FoxO1 and ΔDBD in ΔDBD immunoprecipitates from MEFs expressing FoxO1 and ΔDBD. Total FoxO1 protein amounts were determined in WCL. (,) FoxO1 protein amounts () and Foxo1 mRNA levels () in MEFs expressing FoxO1 and increasing amounts of ΔDBD. (–) Seven-week-old male ob/ob mice were injected with Ad-LacZ (n = 6), Ad–XBP-1s (n = 6) or Ad-ΔDBD (n = 6) (4 × 107 PFU g−1) through the tail vein. () Six-hour-fasted blood glucose concentrations on day 3 after the adenovirus injections. () GTT on day 5 after the adenovirus injections. () Phospho-Akt Ser473, total Akt, FoxO1, XBP-1s and ΔDBD protein amounts in the liver of Ad-LacZ–, Ad–XBP-1s– or Ad-ΔDBD–injected ob/ob mice on day 7 after the injections. Graph depicts to! tal FoxO1 / tubulin protein ratio. (,) Relative mRNA levels of Hspa5 () and Igfbp1, G6pc and Pck1 () in the liver of Ad-LacZ–, Ad–XBP-1s– or Ad-ΔDBD–injected ob/ob mice. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 5: XBP-1s can also improve glucose homeostasis in insulin independent manner. (–) Eight-week-old male streptozotocin (STZ)-treated mice were injected with Ad–XBP-1s (n = 9) or Ad-LacZ (n = 9) (1.5 × 108 PFU g−1) though the tail vein. () Plasma insulin concentrations before and after streptozotocin treatment. () Fed and 12-h–fasted blood glucose concentrations on the indicated days. (,) Total and nuclear FoxO1 protein amounts () and liver Igfbp1, G6pc and Pck1 mRNA levels () 10 d after adenovirus injections. (–) Eight-week-old male LIRKO mice were injected with Ad-LacZ (n = 8) or Ad–XBP-1s (n = 8) (4 × 107 PFU g−1) through the tail vein. () Liver insulin receptor protein amounts and GTT on day 4 after adenovirus injections. (,) FoxO1, XBP-1s, pAkt Ser473, pAkt Thr308 and total Akt amounts () and Igfbp1, G6pc and Pck1 mRNA levels () in the liver of Ad–XBP-1s– or Ad-LacZ–injected LIRKO mice 8 d after adenovirus injection. (–) Eight-week-old male IRS-1/2 double-knockout (DKO) mice were injected with Ad-LacZ (n = 8) or Ad–XBP-1s (! n = 8) (7.5 × 107 PFU g−1) through the tail vein. () Liver IRS-1 and IRS-2 protein levels in Irs1flox/flox;Irs2flox/flox (DF) and DKO mice. GTT was performed on day 4 after injection. (,) FoxO1 and XBP-1s protein amounts () and Igfbp1, G6pc and Pck1 mRNA levels () in the liver of adenovirus-injected DKO mice on day 8 after the injections. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 6: Enhanced glucose tolerance after medium-level expression of XBP-1s is FoxO1 dependent. Seven-week-old male ob/ob mice were injected with Ad-shGFP + Ad-LacZ (n = 5), Ad-shGFP + Ad–XBP-1s (n = 5), Ad-shFoxO1 + Ad-LacZ (n = 5) or Ad-shFoxO1 + Ad-LacZ (n = 5) through the tail vein. (,) Blood glucose concentrations on day 7 () and GTT on day 5 () after the adenovirus injections. () FoxO1 and XBP-1s protein amounts in the liver of adenovirus-injected ob/ob mice on day 7 after injections. (–) Relative mRNA levels of Foxo1 (), Igfbp1 and Ppargc1a () and Dnajb9 and Hspa5 () on day 7 after adenovirus injections. Error bars are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Endocrinology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Yingjiang Zhou, * Justin Lee, * Cheng Sun, * Sang Won Park, * Jason Chung, * Jaemin Lee, * Morris F White, * Sudha B Biddinger & * Umut Ozcan * Division of Endocrinology, Metabolism & Lipid Research, Washington University in St. Louis, St. Louis, Missouri, USA. * Candace M Reno & * Simon J Fisher Contributions Y.Z. came up with the hypothesis, designed and performed the experiments, analyzed the data and wrote the manuscript. Justin Lee, C.M.R., C.S., S.W.P., J.C., Jaemin Lee and S.J.F. performed the experiments. M.F.W. provided liver specific IRS1/2 double-knockout mice. S.B.B. provided LIRKO mice and performed experiments. U.O. came up with the hypothesis, designed and performed the experiments, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Umut Ozcan Author Details * Yingjiang Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Justin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Candace M Reno Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Won Park Search for this author in: * NPG journals * PubMed * Google Scholar * Jason Chung Search for this author in: * NPG journals * PubMed * Google Scholar * Jaemin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Simon J Fisher Search for this author in: * NPG journals * PubMed * Google Scholar * Morris F White Search for this author in: * NPG journals * PubMed * Google Scholar * Sudha B Biddinger Search for this author in: * NPG journals * PubMed * Google Scholar * Umut Ozcan Contact Umut Ozcan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (406K) Supplementary Figures 1–7 and Supplementary Methods Additional data - Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial
- Nat Med 17(3):366-371 (2011)
Nature Medicine | Article Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial * Morgane Rolland1, 7 * Sodsai Tovanabutra2, 7 * Allan C deCamp3, 7 * Nicole Frahm3, 7 * Peter B Gilbert3 * Eric Sanders-Buell2 * Laura Heath1 * Craig A Magaret3 * Meera Bose2 * Andrea Bradfield2 * Annemarie O'Sullivan2 * Jacqueline Crossler2 * Teresa Jones2 * Marty Nau2 * Kim Wong1 * Hong Zhao1 * Dana N Raugi1 * Stephanie Sorensen1 * Julia N Stoddard1 * Brandon S Maust1 * Wenjie Deng1 * John Hural3 * Sheri Dubey4 * Nelson L Michael2 * John Shiver4 * Lawrence Corey3 * Fusheng Li3 * Steve G Self3 * Jerome Kim2 * Susan Buchbinder5 * Danilo R Casimiro4 * Michael N Robertson4 * Ann Duerr3 * M Juliana McElrath3 * Francine E McCutchan2, 6 * James I Mullins1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:366–371Year published:(2011)DOI:doi:10.1038/nm.2316Received28 October 2010Accepted31 January 2011Published online27 February 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans. View full text Figures at a glance * Figure 1: Maximum-likelihood phylogenetic tree of gag sequences. The tree comprises nucleotide sequences from each individual along with the MRKAd5, HXB2 and CON_B04 sequences and is rooted with sequences from the only subject not infected with a subtype B virus (CRF02-AG). Sequences from placebo recipients are in blue, whereas sequences from vaccine recipients are in red. Sequences from individuals with two or more founder variants are highlighted in yellow. The sequences from related viruses found in two individuals are labeled with the two subjects' identification numbers. The scale bar at the bottom refers to the degree of the sequence mismatch. * Figure 2: Epitope-specific protein distances between epitopes from founder sequences and the MRKAd5 HIV-1 vaccine sequence. (–) Comparison of epitopic distances across treatment assignment based on NetMHC predictions using Gag, Pol and Nef epitopes combined (), or separately (), respectively. Mean distance values are indicated. * Figure 3: Protein distances between epitopes from founder sequences and HXB2 or HIV-1 CON_B04. (–) Epitopic distances based on NetMHC predictions are compared across treatment groups, separately for epitopes derived from Gag, Pol and Nef (vaccine insert) (,) and for epitopes derived from the six other HIV-1 proteins (,). and are compared to HXB2; and are compared to CON_B04. Mean distance values are indicated. * Figure 4: Amino acid signature sites. Ten amino acid signature sites distinguishing vaccine and placebo recipients are represented with bar graphs. Each bar represents the amino acid found in one individual: the upper, black bars represent the individuals with an amino acid matching the MRKAd5 HIV-1 insert at that position and the lower, red bars correspond to individuals with a mismatched amino acid. * Figure 5: Summary of sequence analyses and IFN-γ ELISPOT data. (–) Data from Gag (,), Nef (,) and Pol (,). For each protein, the top graph (,,) represents the location of predicted epitopes based on NetMHC in vaccine and placebo groups (light and medium gray columns, respectively), amino acid signature sites (dashed lines) and signature k-mers (horizontal bars). The bottom graph (,,) corresponds to IFN-γ ELISPOT responses detected in 21 vaccine recipients before infection. Fifteen–amino-acid peptides overlapping by 11 amino acids were used to assess responses. The region covered by each responsive peptide is shown as the box width, whereas the number of subjects reacting with that peptide is given by the box height. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * JF320002 * JF320643 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Morgane Rolland, * Sodsai Tovanabutra, * Allan C deCamp & * Nicole Frahm Affiliations * Department of Microbiology, University of Washington, Seattle, Washington, USA. * Morgane Rolland, * Laura Heath, * Kim Wong, * Hong Zhao, * Dana N Raugi, * Stephanie Sorensen, * Julia N Stoddard, * Brandon S Maust, * Wenjie Deng & * James I Mullins * US Military HIV Research Program, Rockville, Maryland, USA. * Sodsai Tovanabutra, * Eric Sanders-Buell, * Meera Bose, * Andrea Bradfield, * Annemarie O'Sullivan, * Jacqueline Crossler, * Teresa Jones, * Marty Nau, * Nelson L Michael, * Jerome Kim & * Francine E McCutchan * Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. * Allan C deCamp, * Nicole Frahm, * Peter B Gilbert, * Craig A Magaret, * John Hural, * Lawrence Corey, * Fusheng Li, * Steve G Self, * Ann Duerr & * M Juliana McElrath * Merck Research Laboratories, West Point, Pennsylvania, USA. * Sheri Dubey, * John Shiver, * Danilo R Casimiro & * Michael N Robertson * San Francisco Department of Health, San Francisco, California, USA. * Susan Buchbinder * Present address: Bill and Melinda Gates Foundation, Seattle, Washington, USA. * Francine E McCutchan Contributions M.R., A.C.D., P.B.G., and J.I.M. designed the sequence analysis. M.R., A.C.D., P.B.G., C.A.M., L.H., B.S.M., W.D., F.L. and J.I.M. conducted the analyses. M.R., A.C.D., N.F., P.B.G. and J.I.M. analyzed the data. M.R., A.C.D., N.F., P.B.G. and J.I.M. wrote the manuscript. J.I.M., F.E.M., S.T., E.S.-B. and N.F. designed lab experiments. M.B., A.B., A.O., J.C., T.J., M.N., K.W., H.Z., D.N.R., S.S., J.N.S. and N.F. performed lab experiments. J.H., L.C., S.B., D.R.C., M.N.R., A.D., M.J.M., S.G.S., S.D., N.L.M., J.K. and J.S. conducted the STEP trial, provided material and oversaw laboratories. Competing financial interests M.N.R. and D.R.C. are paid employees of Merck, own Merck stock and have Merck stock options. Corresponding author Correspondence to: * James I Mullins Author Details * Morgane Rolland Search for this author in: * NPG journals * PubMed * Google Scholar * Sodsai Tovanabutra Search for this author in: * NPG journals * PubMed * Google Scholar * Allan C deCamp Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Frahm Search for this author in: * NPG journals * PubMed * Google Scholar * Peter B Gilbert Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Sanders-Buell Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Heath Search for this author in: * NPG journals * PubMed * Google Scholar * Craig A Magaret Search for this author in: * NPG journals * PubMed * Google Scholar * Meera Bose Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Bradfield Search for this author in: * NPG journals * PubMed * Google Scholar * Annemarie O'Sullivan Search for this author in: * NPG journals * PubMed * Google Scholar * Jacqueline Crossler Search for this author in: * NPG journals * PubMed * Google Scholar * Teresa Jones Search for this author in: * NPG journals * PubMed * Google Scholar * Marty Nau Search for this author in: * NPG journals * PubMed * Google Scholar * Kim Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Hong Zhao Search for this author in: * NPG journals * PubMed * Google Scholar * Dana N Raugi Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie Sorensen Search for this author in: * NPG journals * PubMed * Google Scholar * Julia N Stoddard Search for this author in: * NPG journals * PubMed * Google Scholar * Brandon S Maust Search for this author in: * NPG journals * PubMed * Google Scholar * Wenjie Deng Search for this author in: * NPG journals * PubMed * Google Scholar * John Hural Search for this author in: * NPG journals * PubMed * Google Scholar * Sheri Dubey Search for this author in: * NPG journals * PubMed * Google Scholar * Nelson L Michael Search for this author in: * NPG journals * PubMed * Google Scholar * John Shiver Search for this author in: * NPG journals * PubMed * Google Scholar * Lawrence Corey Search for this author in: * NPG journals * PubMed * Google Scholar * Fusheng Li Search for this author in: * NPG journals * PubMed * Google Scholar * Steve G Self Search for this author in: * NPG journals * PubMed * Google Scholar * Jerome Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Buchbinder Search for this author in: * NPG journals * PubMed * Google Scholar * Danilo R Casimiro Search for this author in: * NPG journals * PubMed * Google Scholar * Michael N Robertson Search for this author in: * NPG journals * PubMed * Google Scholar * Ann Duerr Search for this author in: * NPG journals * PubMed * Google Scholar * M Juliana McElrath Search for this author in: * NPG journals * PubMed * Google Scholar * Francine E McCutchan Search for this author in: * NPG journals * PubMed * Google Scholar * James I Mullins Contact James I Mullins Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Methods, Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–5 Additional data - Dominant TNF-α+Mycobacterium tuberculosis–specific CD4+ T cell responses discriminate between latent infection and active disease
- Nat Med 17(3):372-376 (2011)
Nature Medicine | Letter Dominant TNF-α+Mycobacterium tuberculosis–specific CD4+ T cell responses discriminate between latent infection and active disease * Alexandre Harari1, 2 * Virginie Rozot1 * Felicitas Bellutti Enders1 * Matthieu Perreau1 * Jesica Mazza Stalder3 * Laurent P Nicod3 * Matthias Cavassini4 * Thierry Calandra4 * Catherine Lazor Blanchet5 * Katia Jaton6 * Mohamed Faouzi7 * Cheryl L Day8 * Willem A Hanekom8 * Pierre-Alexandre Bart1 * Giuseppe Pantaleo1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:372–376Year published:(2011)DOI:doi:10.1038/nm.2299Received09 November 2010Accepted05 January 2011Published online20 February 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-αMtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry–based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion ! of single-positive TNF-αMtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease. View full text Figures at a glance * Figure 1: Quantitative and qualitative analysis of Mtb-specific T cell responses in the test cohort. () IFN-γ ELISPOT responses after stimulation with ESAT-6 or CFP-10 peptide pools in a cohort of 283 participants with latent Mtb infection (n = 272) or active tuberculosis disease (n = 11, Supplementary Fig. 1). Shown are the numbers of spot-forming units (SFU) per 106 mononuclear cells. Statistical significance (P values) of the results was calculated by unpaired two-tailed Student's t test using GraphPad Prism 5. Bonferroni's correction for multiples analyses was applied. () Qualitative analysis of Mtb-specific CD4+ T cell responses by polychromatic flow cytometry. Shown are representative flow cytometry analyses of the functional profile of Mtb-specific CD4+ T cell responses in participants with either latent Mtb infection (Subject L5, left) or active tuberculosis disease (Subject A2, right). Profiles are gated on live CD3+CD4+ T cells, and the various combinations of IFN-γ, IL-2 and TNF-α are shown following stimulation with ESAT-6 and CFP-10 peptide pools or PPD. NS,! not significant; Neg, negative control (unstimulated). () Simultaneous analysis of the functional profile of Mtb-specific CD4+ T cells on the basis of IFN-γ, IL-2 or TNF-α production. ESAT-6–, CFP-10– and PPD-specific CD4+ T cell responses are shown (as indicated by the six colored boxes to the right of the panel) from 48 participants with latent Mtb infection and eight participants with active tuberculosis (TB) disease. Representative examples from subject L5 and subject A2 are also identified. All the possible combinations of the various functions are shown on the x axis, whereas the percentages of the distinct cytokine-producing cell subsets within Mtb-specific CD4+ T cells are shown on the y axis. The pie charts summarize the data, and each slice corresponds to the proportion of Mtb-specific CD4+ T cells positive for a certain combination of functions. Colors in the pie charts are indicated by the seven colored boxes at the bottom of the panel. () Distribution of! CFP-10– and/or ESAT-6–specific CD4+ T cell responses amon! g subjects with latent Mtb infection or active tuberculosis disease. * Figure 2: Analysis of Mtb-specific T cell responses in the validation cohort after unblinding of the clinical status. () IFN-γ ELISPOT responses after stimulation with ESAT-6 or CFP-10 peptide pools. Shown are the numbers of SFU per 106 mononuclear cells. Statistical significance (P values) of the results was calculated by unpaired two-tailed Student's t test in GraphPad Prism 5. Bonferroni's correction for multiples analyses was applied. () Analysis of Mtb-specific IFN-γ ELISPOT T cell responses in subjects enrolled in Switzerland (CH) and SA. () Distribution of CFP-10– and/or ESAT-6–specific CD4+ T cell responses among subjects from the validation cohort with positive and concordant Mtb-specific CD4+ T cell responses (Supplementary Fig. 5). * Figure 3: Percentages of CFP-10– or ESAT-6–specific single-positive TNF-α–producing CD4+ T cells of the 94 subjects from the validation cohort with concordant responses against CFP-10 and ESAT-6. Dashed line represents the cutoff of 37.4% of single-positive TNF-α. () Subjects with active disease or latent infection are identified with blue and red dots, respectively. () Subjects from South Africa (SA) or Switzerland (CH) are identified with orange and green dots, respectively. * Figure 4: Longitudinal analysis of the functional profile of Mtb-specific CD4+ T cells from five subjects analyzed during untreated active tuberculosis disease and then after tuberculosis treatment. Shown is the full functional profile on the basis of IFN-γ, IL-2 and TNF-α production of a total of 7 Mtb-specific CD4+ T cell responses. All the possible combinations of the different functions are shown on the x axis, whereas the percentages of the distinct cytokine-producing cell subsets within Mtb-specific CD4+ T cells are shown on the y axis. The pie charts summarize the data, and each slice corresponds to the proportion of Mtb-specific CD4+ T cells positive for a certain combination of functions. Author information * Author information * Supplementary information Affiliations * Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Alexandre Harari, * Virginie Rozot, * Felicitas Bellutti Enders, * Matthieu Perreau, * Pierre-Alexandre Bart & * Giuseppe Pantaleo * Swiss Vaccine Research Institute, Lausanne, Switzerland. * Alexandre Harari & * Giuseppe Pantaleo * Division of Pneumology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Jesica Mazza Stalder & * Laurent P Nicod * Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Matthias Cavassini & * Thierry Calandra * Division of Occupational Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Catherine Lazor Blanchet * Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Katia Jaton * Center of Clinical Epidemiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland. * Mohamed Faouzi * South African Tuberculosis Vaccine Initiative, University of Cape Town, Cape Town, South Africa. * Cheryl L Day & * Willem A Hanekom Contributions A.H. designed the study, performed the analyses and wrote the manuscript; V.R., F.B.E. and M.P. generated data and performed analyses; J.M.S., L.P.N., M.C., T.C., C.L.B., C.L.D. and W.A.H. recruited study participants; K.J. performed analyses; M.F. performed the statistical analyses; P.-A.B. contributed to the design of the study, performed analyses and wrote the manuscript; G.P. designed the study, supervised the analyses and wrote the paper. All authors have read and approved the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Giuseppe Pantaleo Author Details * Alexandre Harari Search for this author in: * NPG journals * PubMed * Google Scholar * Virginie Rozot Search for this author in: * NPG journals * PubMed * Google Scholar * Felicitas Bellutti Enders Search for this author in: * NPG journals * PubMed * Google Scholar * Matthieu Perreau Search for this author in: * NPG journals * PubMed * Google Scholar * Jesica Mazza Stalder Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent P Nicod Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Cavassini Search for this author in: * NPG journals * PubMed * Google Scholar * Thierry Calandra Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Lazor Blanchet Search for this author in: * NPG journals * PubMed * Google Scholar * Katia Jaton Search for this author in: * NPG journals * PubMed * Google Scholar * Mohamed Faouzi Search for this author in: * NPG journals * PubMed * Google Scholar * Cheryl L Day Search for this author in: * NPG journals * PubMed * Google Scholar * Willem A Hanekom Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre-Alexandre Bart Search for this author in: * NPG journals * PubMed * Google Scholar * Giuseppe Pantaleo Contact Giuseppe Pantaleo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (401K) Supplementary Tables 1 and 2 and Supplementary Figures 1–7 Additional data - Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity
- Nat Med 17(3):377-382 (2011)
Nature Medicine | Letter Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity * Wenjun Song1 * Jin Chen1 * Alejandra Petrilli1 * Geraldine Liot1 * Eva Klinglmayr2 * Yue Zhou1 * Patrick Poquiz3 * Jonathan Tjong3 * Mahmoud A Pouladi4 * Michael R Hayden4 * Eliezer Masliah5 * Mark Ellisman3 * Isabelle Rouiller6 * Robert Schwarzenbacher2 * Blaise Bossy1 * Guy Perkins3 * Ella Bossy-Wetzel1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:377–382Year published:(2011)DOI:doi:10.1038/nm.2313Received29 September 2010Accepted18 January 2011Published online20 February 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis1, 2. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, whi! ch, in turn, stimulates its enzymatic activity. Mutant huntingtin–mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease. View full text Figures at a glance * Figure 1: Mutant huntingtin triggers mitochondrial fragmentation, decreases in anterograde and retrograde transport and neuronal cell death, which depend on polyQ length. () Fluorescence micrographs (scale bar, 50 μm) and 6× zoom (scale bar, 8.33 μm) of boxed regions of neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito, a stain for mitochondria. () Mitochondrial fragmentation of neurons expressing HTTex1-Q17, HTTex1-Q46, or HTTex1-Q97 and DsRed2-Mito. () Cell death of neurons expressing HTTex1-Q17, -Q46 or -Q97. () Fluorescence micrographs (scale bar, 25 μm) and 3× zoom (scale bar, 8.33 μm) of boxed regions of MitoTracker Red–stained human fibroblasts from an unaffected individual (left) or an individual with adult-onset Huntington's disease (right). () Mitochondrial fragmentation in fibroblasts from an unaffected individual or an individual with Huntington's disease. () Kymographs of mitochondrial transport in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito. () Scatter plots of mitochondrial velocity in retrograde or anterograde direction as a function of distance traveled in 5 min in neu! rons (n = 10) expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito. () Anterograde and retrograde movement, motility and mean velocity of mitochondria in the same neurons analyzed in . Data are means ± s.e.m. of triplicate samples of representative experiments. Results are representative of three or more independent experiments. Statistics: one-way analysis of variance (ANOVA). * Figure 2: Mutant huntingtin increases the number of small mitochondria and cristae but decreases cristae surface area and volume in the striatum of six-month-old YAC128 mice. () Electron microscopy of control (top) and YAC128 (bottom) brain showing an elongated neuronal mitochondrion (arrowhead, top) and several short mitochondria (arrowheads, bottom), respectively. Scale bar, 500 nm for both. () Percentage of mitochondria of short length (0–1,000 nm) and long length (1,000–5,000 nm). () Electron microscopy of a control mitochondrion (arrowhead) nearly 4 μm long. Scale bar, 500 nm. () Surface-rendered volume showing the normal structure of the 84 cristae of the control mitochondrion. () Electron microscopy of a mitochondrion dividing into three parts (arrowheads). Scale bar, 500 nm. () A slice of the volume shows the separation of the three mitochondrial bodies (arrows). () Top, top view showing all 223 cristae. There are no cristae in the constriction (arrowhead) between the left and middle bodies, but a few cristae appear in the constriction between the middle and right bodies (arrow). Middle, side view of the outer membrane showing the wi! dth of the constrictions. Bottom, side view showing cristae fragmentation by how few cristae extend from top to bottom of the volume. () Number of cristae per mitochondrial cross-sectional area. (,) Cristae surface area () and volume (). Data are means ± s.e.m. Statistics: Student's t test, *P < 0.05, **P < 0.01. * Figure 3: Mutant huntingtin interacts with DRP1 in mice and humans with Huntington's disease and alters DRP1 structure and function. () Fluorescence micrographs of neurons expressing HTTex1 and DsRed2-Mito. Scale bar, 50 μm. Inset shows an 8× zoom of the boxed region. Scale bar, 6.25 μm. () Colocalization of mitochondria and huntingtin in neurons expressing HTTex1 and DsRed2-Mito. Data are means ± s.e.m. Statistics: one-way analysis of variance (ANOVA). () Fluorescence micrographs with line scan of colocalization of huntingtin, DRP1 and mitochondria in neurons expressing DsRed2-Mito, DRP1-YFP, and HTTex1. Scale bar, 10 μm. Inset shows a 5× zoom of the boxed region. Scale bar, 2 μm. () Coimmunoprecipitations (IP) of brain mitochondrial lysates from 1.5- or 2-month-old YAC18 and YAC128 mice followed with huntingtin- or Drp1-specific antibodies (Anti-HTT and Anti-DRP, respectively). The intensities of the signals are presented as arbitrary units (AU) and are normalized to input signals. () Coimmunoprecipitations of human lymphoblast lysates from unaffected individuals or individuals with Huntington's ! disease (HD) with huntingtin-specific antibodies. () Coimmunoprecipitations of human postmortem brain tissue lysates from unaffected individuals or individuals with Huntington's disease with DRP1- or huntingtin-specific antibodies. () Coimmunoprecipitations of bacterially expressed DRP1 protein and bacterially expressed huntingtinex1-Q20-GST or huntingtinex1-Q53-GST protein with DRP1-specific antibodies. () Steady-state kinetics of DRP1 GTPase activity (left), bar graph of GTPase activity at 0.05 mM GTP and apparent Michaelis-Menten constant (Km) (right) in the presence of wild-type or mutant huntingtinex1 protein and MOM liposomes. Data are means ± s.d. from three independent measurements. Statistics: Student's t test. () Negative-stain electron microscope images of baculovirus DRP1 in the absence of nucleotides (left), the presence of GTP (center) and the presence of GTP and huntingtinex1-Q53-GST protein (right). Scale bars, 10 nm. * Figure 4: Restoring mitochondrial fusion with DRP1 K38A or in combination with MFN2 RasG12V rescues neurons from neuritic trafficking defects and cell death. () Mitochondrial fragmentation of neurons expressing either HTTex1-Q17, HTTex1-Q46 or HTTex1 -Q97 alone or in combination with either DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Cell death of neurons expressing either mutant HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 and DsRed2-Mito alone or in combination with either DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Anterograde and retrograde movement of mitochondria in neurons expressing HTTex1-Q17HTTex1-Q46 or HTTex1-Q97 alone or in combination with DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. () Motility of mitochondria in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 alone or in combination with either DRP1 K38A alone or both DRP1 K38A and MFN2 RasG12V. () Mean velocity of mitochondria in neurons expressing HTTex1-Q17, HTTex1-Q46 or HTTex1-Q97 alone or in combination with DRP1 K38A alone or DRP1 K38A and MFN2 RasG12V. Results are representative of three or more independent experiments. Statistics: Two-way AN! OVA with post hoc test. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_012062.3 * NM_005690.3 Author information * Accession codes * Author information * Supplementary information Affiliations * Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA. * Wenjun Song, * Jin Chen, * Alejandra Petrilli, * Geraldine Liot, * Yue Zhou, * Blaise Bossy & * Ella Bossy-Wetzel * Structural Biology Group, Department of Molecular Biology, University of Salzburg, Salzburg, Austria. * Eva Klinglmayr & * Robert Schwarzenbacher * National Center for Microscopy and Imaging Research, University of California–San Diego, San Diego, California, USA. * Patrick Poquiz, * Jonathan Tjong, * Mark Ellisman & * Guy Perkins * Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada. * Mahmoud A Pouladi & * Michael R Hayden * University of California–San Diego, La Jolla, California, USA. * Eliezer Masliah * Department of Anatomy and Cell Biology, McGill University, Montreal, Québec, Canada. * Isabelle Rouiller Contributions W.S. performed all imaging and participated in the mitochondrial fragmentation and cell death analyses. J.C. performed the GTPase assays, some of the immune precipitations and the electron microscopy analysis. A.P. performed some of the neuronal cell death and immune precipitations. G.L. performed the electron microscopy stereology and generated some of the preliminary data. E.K. purified, cloned and prepared the recombinant DRP1 protein. Y.Z. performed western blotting for the DRP1 knockdown. P.P. and J.T. participated in the electron microscope tomography. M.A.P. and M.R.H. provided the YAC18 and YAC128 mice and advice on huntingtin coimmunoprecipitations. E.M. provided human postmortem brain samples. R.S. led the DRP1 protein purification. M.E. and G.P. led the electron microscope tomography experiment. B.B. performed GTPase assays, prepared samples for electron microscopy and purified the huntingtin protein. I.R. performed electron microscope negative-stain experiments. ! E.B.-W. conceived the project and wrote the article. All authors participated in the data analysis and interpretation of the results. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ella Bossy-Wetzel Author Details * Wenjun Song Search for this author in: * NPG journals * PubMed * Google Scholar * Jin Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Alejandra Petrilli Search for this author in: * NPG journals * PubMed * Google Scholar * Geraldine Liot Search for this author in: * NPG journals * PubMed * Google Scholar * Eva Klinglmayr Search for this author in: * NPG journals * PubMed * Google Scholar * Yue Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Poquiz Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Tjong Search for this author in: * NPG journals * PubMed * Google Scholar * Mahmoud A Pouladi Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Hayden Search for this author in: * NPG journals * PubMed * Google Scholar * Eliezer Masliah Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Ellisman Search for this author in: * NPG journals * PubMed * Google Scholar * Isabelle Rouiller Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Schwarzenbacher Search for this author in: * NPG journals * PubMed * Google Scholar * Blaise Bossy Search for this author in: * NPG journals * PubMed * Google Scholar * Guy Perkins Search for this author in: * NPG journals * PubMed * Google Scholar * Ella Bossy-Wetzel Contact Ella Bossy-Wetzel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information Movies * Supplementary Video 1 (1M) Mitochondrial movement in a neuron expressing HTT exon1-Q17-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , top panel and shows mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 2 (967K) Mitochondrial movement in a neuron expressing HTT exon1-Q46-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , center panel and shows a clear decrease in mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 3 (623K) Mitochondrial movement in a neuron expressing HTT exon1-Q97-GFP and DsRed2-Mito. Movie corresponds to the kymograph in , bottom panel and shows more pronounced arrest in mitochondrial transport. The movie lasts 5 min and is played back accelerated (original: 5 s frame−1, playback: 1/6 s frame−1). * Supplementary Video 4 (8M) Electron tomography of a control mitochondrion in a medium spiny neuron. Movie showing the three-dimensional details of a mitochondrion in a medium spiny neuron reconstructed using electron tomography. These mitochondria are typically elongated along the direction of the axonal long axis. Clip 1: a rapid sequence through 190 slices (2.2 nm slice−1) of the tomographic volume that shows nearly the entire mitochondrial volume. There are 84 cristae. Clip 2: rotations and zooms of the surface-rendered volume after segmentation of the inner and outer membranes. The blue outer membrane is translucent to visualize the cristae displayed in various colors. Clip 3: rotation of the cristae after removal of the outer membrane to better distinguish the variety of shapes and sizes. * Supplementary Video 5 (10M) Electron tomography of a fissioning YAC128 mitochondrion in a medium spiny neuron. Movie showing the three-dimensional details of a mitochondrion fissioning into three parts in a medium spiny neuron reconstructed using electron tomography. Clip 1: a rapid sequence through 210 slices (2.2 nm slice−1) of the tomographic volume. There are 223 cristae, many of which are small. Clip 2: rotation showing the outer membrane and the widths of the two constriction sites. Clip 3: rotations showing the cristae in each of the three parts. Clip 4: rotations and zooms highlighting the cristae and the constriction sites. The blue outer membrane is translucent to visualize the cristae displayed in various colors. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Methods Additional data - Assessment of atherosclerotic plaque burden with an elastin-specific magnetic resonance contrast agent
- Nat Med 17(3):383-388 (2011)
Nature Medicine | Technical Report Assessment of atherosclerotic plaque burden with an elastin-specific magnetic resonance contrast agent * Marcus R Makowski1, 2, 3 * Andrea J Wiethoff1, 4 * Ulrike Blume1 * Friederike Cuello2, 5 * Alice Warley6 * Christian H P Jansen1 * Eike Nagel1, 2, 7, 8 * Reza Razavi1, 2, 7, 8 * David C Onthank9 * Richard R Cesati9 * Michael S Marber2, 5, 8 * Tobias Schaeffter1, 2, 7, 8 * Alberto Smith5, 8, 10 * Simon P Robinson9 * René M Botnar1, 2, 7, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:383–388Year published:(2011)DOI:doi:10.1038/nm.2310Received19 January 2010Accepted25 October 2010Published online20 February 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Atherosclerosis and its consequences remain the main cause of mortality in industrialized and developing nations. Plaque burden and progression have been shown to be independent predictors for future cardiac events by intravascular ultrasound. Routine prospective imaging is hampered by the invasive nature of intravascular ultrasound. A noninvasive technique would therefore be more suitable for screening of atherosclerosis in large populations. Here we introduce an elastin-specific magnetic resonance contrast agent (ESMA) for noninvasive quantification of plaque burden in a mouse model of atherosclerosis. The strong signal provided by ESMA allows for imaging with high spatial resolution, resulting in accurate assessment of plaque burden. Additionally, plaque characterization by quantifying intraplaque elastin content using signal intensity measurements is possible. Changes in elastin content and the high abundance of elastin during plaque development, in combination with the ! imaging properties of ESMA, provide potential for noninvasive assessment of plaque burden by molecular magnetic resonance imaging (MRI). View full text Figures at a glance * Figure 1: Chemical structure of ESMA and binding characteristics determined by high-resolution DE-MRI. () Structure and molecular mass of ESMA. () Cross-sectional views of the brachiocephalic artery of a male Apoe−/− mouse 12 weeks after beginning an HFD. The time-of-flight (TOF) angiogram of the aortic arch and brachiocephalic artery was used to align the scans perpendicular to the course of the vessel. Fusion of TOF and high-resolution DE-MRI provides spatial registration of contrast uptake and luminal anatomy. Scale bars, 250 μm. * Figure 2: In vivo MRI signal measurements and ex vivo quantification of contrast agent. () Comparison of average atherosclerotic plaque CNR from precontrast, post–Gd-DTPA and post–158Gd-ESMA DE-MRI scans for control, HFD and pravastatin groups. () Scatter plot shows the correlation between plaque CNR and results from ICP-MS of collected brachiocephalic arteries (n = 15). (,) Corresponding average R1 values () and scatter plot () show correlation between plaque R1 values and results from ICP-MS of collected brachiocephalic arteries. Values are expressed as means ± s.d. * Figure 3: In vivo assessment of plaque burden by morphometric measurements. () Cross-sectional views of brachiocephalic arteries by MRI of control and Apoe−/− mice 4, 8 and 12 weeks after the onset of HFD (n = 8 per group). High-resolution delayed-enhancement images overlaid on TOF images with corresponding sections from histology (H&E and EvG stain). () Comparison of average PAMV, calculated from morphometric measurement on high-resolution DE images after the injection of ESMA (n = 8 per group). (,) Scatter plots showing significant (P < 0.05) correlation between morphometric PAMV measurements () and lumen cross-sectional area (CSA) measurements () on high-resolution DE-MRI images and on corresponding EvG-stained histological sections (n = 15). Scale bars: white, 250 μm; black, 100 μm. Values are expressed as means ± s.d. * Figure 4: In vivo assessment of plaque elastin composition using signal intensity measurements. () Immunoblots (IB) of brachiocephalic arteries of control C57BL/6J and 4-week, 8-week and 12-week HFD Apoe−/− mice with a (tropo-)elastin-specific antibody (n = 5 per group). All immunoblot measurements were performed with 10 μg plaque protein (equal protein loading was assured by the GAPDH plaque protein content). (,) Scatter plots showing the correlation between CNR and elastin protein content (per 10 μg plaque protein) from immunoblot analysis () (n = 10) and percentage positive EvG stain per vessel area from histological analysis () (n = 15). (,) Response to therapy was investigated by immunoblot analysis () and T1 mapping (). Values are expressed as means ± s.d. * Figure 5: In and ex vivo characterization of binding of ESMA to elastin. () In vivo comparison between average CNR derived from high-resolution DE–MRI scans before contrast and after the injection of 0.2 mmol kg−1158Gd-ESMA on day 1. On day 2, preinjection of a tenfold higher dose (2 mmol kg−1) of nonparamagnetic La-ESMA resulted in a marked decrease of CNR after 158Gd-ESMA (0.2 mmol kg−1) injection compared to 158Gd-ESMA injection (0.2 mmol kg−1, day 1) alone (n = 3 per group). Scale bars, 250 μm. () Increasing concentrations of nonradioactive ESMA were incubated with 158Gd-ESMA and plaque-laden aorta (rabbit) in the ex vivo tissue-binding assay, decreasing binding of 158Gd-ESMA in a concentration-dependent manner. () Typical gadolinium spectra measured in areas colocalizing with elastic fibers. No gadolinium spectra could be acquired in regions not associated with elastic fibers. The specific distribution of gadolinium was mapped in the vessel wall sample (bottom right). Colocalization of targeted gadolinium with elastic fibers could! be found (n = 3). () Inhibition of 153Gd-ESMA binding to elastin by nonradioactive ESMA. Increasing concentrations of ESMA competed the binding of 153Gd-ESMA with a half-maximal inhibitory concentration (IC50) of 0.33 mM. Values are expressed as means ± s.d. Author information * Abstract * Author information * Supplementary information Affiliations * King's College London, Division of Imaging Sciences and Biomedical Engineering, London, UK. * Marcus R Makowski, * Andrea J Wiethoff, * Ulrike Blume, * Christian H P Jansen, * Eike Nagel, * Reza Razavi, * Tobias Schaeffter & * René M Botnar * British Heart Foundation (BHF) Centre of Excellence, King's College London, London, UK. * Marcus R Makowski, * Friederike Cuello, * Eike Nagel, * Reza Razavi, * Michael S Marber, * Tobias Schaeffter & * René M Botnar * Department of Radiology, Charite, Berlin, Germany. * Marcus R Makowski * Philips Healthcare, Guildford, UK. * Andrea J Wiethoff * Cardiovascular Division, King's College London, London, UK. * Friederike Cuello, * Michael S Marber & * Alberto Smith * Centre for Ultrastructural Imaging, King's College London, London, UK. * Alice Warley * Wellcome Trust and Engineering and Physical Sciences Research Council Medical Engineering Center, King's College London, London, UK. * Eike Nagel, * Reza Razavi, * Tobias Schaeffter & * René M Botnar * National Institute of Health Research Biomedical Research Centre, King's College London, London, UK. * Eike Nagel, * Reza Razavi, * Michael S Marber, * Tobias Schaeffter, * Alberto Smith & * René M Botnar * Lantheus Medical Imaging, North Billerica, Massachusetts, USA. * David C Onthank, * Richard R Cesati & * Simon P Robinson * Academic Surgery, King's College London, London, UK. * Alberto Smith Contributions M.R.M. and R.M.B. are responsible for the overall study design and implemented and optimized the magnetic resonance imaging protocols. D.C.O., R.R.C. and S.P.R. designed and manufactured the contrast agent. U.B. and T.S. developed and implemented the T1 mapping sequence and analysis tools. M.R.M., R.M.B., A.J.W., A.S. and F.C. designed, conducted and analyzed the in vitro and in vivo experiments. A.W. performed the electron microscopy experiments. M.R.M., R.M.B., A.J.W., F.C., M.S.M., E.N., T.S., A.S., R.R. and C.H.P.J. contributed to the writing of the manuscript. All authors discussed and refined the manuscript. Competing financial interests The magnetic resonance imaging scanner is partly supported by Philips Healthcare. A.J.W.is an employee of Philips Healthcare. D.C.O., R.R.C. and S.P.R. are employees of Lantheus Medical Imaging. The study was funded by the British Heart Foundation (PG/09/061), and the contrast agent was provided by Lantheus Medical Imaging. Corresponding author Correspondence to: * Marcus R Makowski Author Details * Marcus R Makowski Contact Marcus R Makowski Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea J Wiethoff Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrike Blume Search for this author in: * NPG journals * PubMed * Google Scholar * Friederike Cuello Search for this author in: * NPG journals * PubMed * Google Scholar * Alice Warley Search for this author in: * NPG journals * PubMed * Google Scholar * Christian H P Jansen Search for this author in: * NPG journals * PubMed * Google Scholar * Eike Nagel Search for this author in: * NPG journals * PubMed * Google Scholar * Reza Razavi Search for this author in: * NPG journals * PubMed * Google Scholar * David C Onthank Search for this author in: * NPG journals * PubMed * Google Scholar * Richard R Cesati Search for this author in: * NPG journals * PubMed * Google Scholar * Michael S Marber Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Schaeffter Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Smith Search for this author in: * NPG journals * PubMed * Google Scholar * Simon P Robinson Search for this author in: * NPG journals * PubMed * Google Scholar * René M Botnar Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (266K) 3D reconstruction (volume rendering) of elastin signal in the brachiocephalic artery of an Apoe−/− mouse. PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–3 and Supplementary Methods Additional data
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