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© Oxford University Press 2006.
NEWS |
No miR Hype: MicroRNA's Cancer Role Expands
Long considered a mere slave to DNA, carrying the genetic message from chromosomes to the protein-making machinery of the cell, RNA has come into its own.
RNA interference, discovered in 1998, is now a standard laboratory tool for knocking down gene expression. Drug therapies using small interfering RNA are now in clinical testing for treating a respiratory virus and age-related macular degeneration. And a rush of discoveries in the last 4 years have linked another class of small RNAs, known as microRNAs, to cancer. Our current knowledge of microRNAs "might be the tip of the iceberg," said Nobel Prize winner Phillip Sharp, Ph.D., of the Massachusetts Institute of Technology in Cambridge, at this year's meeting of the American Association of Cancer Research.
Research into microRNAs in cancer is exploding. MicroRNAs—miRs for short—are noncoding RNAs, about 22 nucleotides long, that bind to specific messenger RNA (mRNA) targets and either block their translation into proteins or trigger their degradation. They're well conserved through evolution, suggesting an important biological role. About 350 microRNAs have been identified in humans, with the total predicted to eventually reach 1,000 or more. Because each microRNA has dozens, perhaps hundreds, of targets, "most human genes will probably be influenced in some way by a microRNA," said Frank Slack, Ph.D., of Yale University in New Haven, Conn.
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Emerging From Obscurity
"The whole class of genes has been a surprise to many people," Slack said. "They're so small, and they were just missed for many years." Because mutations that inactivate microRNAs are rare, functional knockouts are uncommon so microRNAs went unnoticed for decades. Scientists assumed the bands at the bottom of their electrophoresis gels represented degraded RNA or other artifacts and ignored them. Lin-4, the first known microRNA (cloned by Victor Ambros, Ph.D., at Harvard in 1993), was considered a weird quirk of worm larval development. Only after Harvard's Gary Ruvkun, Ph.D., cloned the second microRNA, let-7, in 2000 did the search for more begin in earnest. In 2001 research groups in the United States and Germany reported finding dozens of new microRNAs. "That really opened the floodgates," said Slack.
The first link between microRNAs and cancer came the following year. In the early 1990s three groups had identified a region of chromosome 13 that was deleted in more than half of all cases of chronic lymphocytic leukemia (CLL). They assumed the region contained tumor suppressor genes. Ohio State University in Columbus researcher Carlo Croce, M.D., searched fruitlessly for these genes for the better part of a decade until 2002, when he finally found the genes for two microRNAs in the deleted region. Croce and postdoc George Calin, M.D., showed that both genes were absent or had reduced activity in two-thirds of CLL patients, strongly suggesting that the microRNAs—dubbed miR-15 and miR-16—were tumor suppressors. Croce's lab has since confirmed this supposition, showing that miR-15 and miR-16 induce apoptosis by targeting the key survival protein Bcl-2, which is overexpressed in CLL.
Croce's lab has linked microRNAs to solid tumors as well. "MicroRNAs are only part of the story," said Calin. "But noncoding RNAs are involved in a lot of human cancers."
In lung cancer, for example, the let-7 microRNA also acts as a tumor suppressor, with similar therapeutic implications. After helping clone the let-7 gene 5 years ago, Slack searched for let-7 targets in the roundworm, finding the worm version of human ras, a critical oncogene in lung cancer. Meanwhile, groups in Japan and at the biotech company Ambion showed that let-7 was poorly expressed in lung cancer, which suggested its tumor suppressor function. Slack confirmed this in 2004 by showing that let-7 regulates ras levels in cell culture.
New Class of Oncogenes
But microRNAs are not always tumor suppressors. They can act as cancer-causing oncogenes as well. In 2004 Masao Seto, M.D., at the Aichi Cancer Center Research Institute in Nagoya, Japan, identified a new gene on chromosome 13 that is often amplified in cancer, and he showed that it encoded a cluster of seven microRNAs. Scott Hammond, Ph.D., at the University of North Carolina in Chapel Hill had already noticed that the cluster was overexpressed in many cancer cell lines. Hammond teamed up with Greg Hannon, Ph.D., of the Cold Spring Harbor Laboratory in New York to see if the cluster, called miR-17-92, could promote cancer. It did: In a mouse model of lymphoma, expression of the microRNA cluster accelerated tumor growth. (See News, August 3, 2005, Vol. 97, No. 15, p. 1114.)
These microRNAs may act as oncogenes in other cancers besides lymphoma. At April's AACR meeting, Hannon reported similar results in a mouse model of breast cancer. "The number of animals is so far still relatively small," Hannon said, "but we are seeing acceleration of the onset of tumorigenesis."
No one yet knows how these microRNAs are promoting cancer. "It's been a tough one to figure out," Hammond said. Because each microRNA has hundreds of potential targets, demonstrating its role in biology and in cancer is enormously time-consuming. And each of the seven microRNAs in the miR17-92 cluster appears to act independently, adding to the complexity. But Hannon, Hammond, and others are making progress.
"We're trying to address whether miR17-92 is important in tumor initiation, tumor progression, tumor maintenance, or all of the above," Hannon said.
Besides miR17-92, three other microRNAs are confirmed oncogenes. There will probably be more, since many microRNAs are overexpressed in various human cancers. "It's ... hard to imagine that at least some of these don't have a functional role in cancer," Hammond said.
In 2004, Croce's group reported that more than half of known microRNA genes were located in cancer-associated genomic regions or in fragile sites—areas of chromosomes prone to breakage, amplification, and fusion with other chromosomes.
"Their paper was kind of mind-blowing," said Slack. "That really suggests [that] those microRNAs are all playing a role in cancer."
Arrested Development
What exactly are these tiny RNAs doing in cancer? There are two schools of thought: Either they are activating or inhibiting specific cancer gene targets, with a direct impact on tumor growth, or they're mopping up many genes overexpressed in cancer to reduce the stress on genetically unstable cancer cells. "I suspect we'll find microRNAs that function at each end of the spectrum, and everywhere in between," said Hannon at the AACR meeting.
One theory of microRNAs and cancer focuses on the important role of microRNAs in development. For example, lin-4 and let-7, the first identified microRNAs, regulate the timing of development in roundworms. Since cancer cells have many characteristics of undifferentiated cells, it's possible that microRNA expression in cancer causes cells to recreate the development process but without moving past the undifferentiated, proliferating stage.
MicroRNAs "weren't put on the earth, or designed, to function in cancer," Slack observed. "They were designed to function during development to maybe shut off cell division or shut off the cell cycle so cells could differentiate."
In cancer, this developmental process may somehow start and then stop, due to mutations or misregulation of key microRNAs. The result: uncontrolled cell proliferation.
The recent microRNA discoveries have obvious clinical implications. CLL, a disease of white blood cells that won't die, is the most common leukemia. It is poorly understood at the molecular level. MicroRNAs provide a new window on the disease and could prove useful for prognosis and treatment. Last year Croce's group reported in the New England Journal of Medicine that a 13-microRNA signature could distinguish aggressive from slow-growing CLL. In theory, delivering miR-15 and miR-16 to tumors would trigger apoptosis.
| MicroRNAs currently implicated in cancer
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The same theory could prove true for let-7 in lung cancer. "Our hope is that we can use let-7 as a potential diagnostic tool to diagnose lung cancers early in patients," Slack said. "And, secondly, potentially use let-7 as a way to knock out activated ras in those lung cancers."
Detection and Treatment
Although no microRNA diagnostics or therapies yet exist, companies are working on them. In March, Ambion spun off a new molecular diagnostics company, Asuragen, based in Austin, Texas. Ambion spent 4 years developing techniques for manipulating and expressing microRNAs and analyzing their function, techniques that Asuragen is now using for potential diagnostic tests in cancer and other diseases.
"There are some clear opportunities to apply microRNAs to detect cancer in individual patients," said David Brown, Ph.D., Asuragen's director of discovery. Prognostic tests could also be in the works. "MicroRNA expression could tell you a lot about [patients], whether they're going to respond to therapy."
Brown pointed out that microRNAs are much more stable than mRNAs, which makes detecting them relatively simple. And microRNAs, unlike mRNAs, can be easily recovered from the formalin-fixed tumor samples typically stored in U.S. hospitals. Although there are far fewer individual microRNAs than mRNAs, "their importance in biology is probably as great," Brown said. "They're the functional counterpart of transcription factors ... and in fact they might be more important." Asuragen is currently evaluating different diagnostic test approaches using microRNAs.
MicroRNA therapy is probably farther off, but the early signs are hopeful. A group led by Rockefeller University researcher Markus Stoffel, M.D., reported last December in Nature that chemically modified antisense oligonucleotides—short strings of DNA bases complementary in sequence to their targets—injected into mice potently silenced a target microRNA in the liver. The Stoffel group dubbed these oligonucleotides "antagomirs." Hammond thinks antagomirs should be more effective against cancer-causing microRNAs than classic antisense therapy has been against protein-coding mRNAs, because antagomirs compete with microRNA targets for binding. That's an easier task than interfering with the protein translation machinery, which is the classic antisense mechanism. And antagomirs should benefit from the antisense field's long struggle to overcome problems of delivery, stability, and cellular uptake.
"You should be able to accelerate the development of these [microRNA] inhibitors just by ... borrowing the techniques that antisense has already developed," Hammond said.
But the microRNA field is still too new to expect microRNA cancer therapies anytime soon. "The roles of these [microRNA] molecules are still far from clear," said Nagesh Mahanthappa, Ph.D., director of business development and strategy for Alnylam Pharmaceuticals, a Cambridge, Mass., biotech company. "Until we have greater clarity on that I don't think you'll see antagomirs as the focus of today's therapeutic efforts per se."
Alnylam specializes in RNA therapy. The company has an small interfering RNA treatment in early clinical trials, and it made the antagomirs used in the Stoffel experiments. Mahanthappa says he views the potential of microRNA cancer therapies with "measured optimism." Their real therapeutic potential, he noted, will depend on future revelations about how microRNAs function in the biology of cancer and other diseases.
"The antagomir technology is definitely going to be part of the long-term therapeutic vision of [Alnylam]," he said. "And undoubtedly there are going to be even more discoveries in the coming months and years relating to the roles of small RNAs."
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