Cancer Biology: A New Cancer Player Takes the Stage
11/4/2005
Science
By Jennifer Couzin
MicroRNAs are being implicated in various human cancers, and scientists are
trying to sort out just how culpable they are.
For Frank Slack, the story began when his worms exploded through their
vulvas.
It was 1997, and the developmental biologist, now at Yale, had been tinkering
with microRNAs (miRNAs), tiny RNA molecules that regulate gene expression.
Slack is a worm man, and in his wriggly subjects he had deleted the gene for
just one of the 120 known worm miRNAs.
The developing animals' stem cells failed to morph into specialized cells as
they normally do and instead kept dividing. "The worms looked extended,
weirdly floppy; they kind of looked uncoordinated," he says. The vulvas
didn't develop properly and ruptured. A worm skeleton is under hydrostatic
pressure, and with the rupture, "the animals burst through," an experience
that killed roughly half of them.
When Slack probed the underlying genetics, he uncovered something tantalizing
that linked these unfortunate animals to human biology. Deletion of this miRNA,
called let-7, prompted overexpression of a gene, Ras, that's strongly
associated with many cancers. In other words, when let-7 is expressed normally,
it seemed, it blunts Ras. Since Slack's find, the let-7-Ras story has unfolded
rapidly, one of a growing bundle of strands tying miRNAs to cancer.
More than a dozen papers have shown that miRNAs are expressed differently in
cancerous tissue. Braided together, the latest miRNA discoveries suggest
potentially vast roles for the tiny molecules in malignancy; they have also
sparked spirited debate over whether miRNAs are driving cancer or are simply a
marker of it. Either way, the nascent field could eventually assist doctors in
diagnosis, prognosis, and possibly treatment. Last week, a paper in the New
England Journal of Medicine (NEJM) reported that 13 miRNAs form a signature
associated with prognosis and disease progression in patients with chronic
lymphocytic leukemia (CLL), a cancer of blood.
Sorry fate. A worm without a microRNA bursts through its vulva; replacing the
microRNA keeps the worm intact.
"There is a whole other world out there, which I don't think we know
anything about," says Phillip Sharp of the Massachusetts Institute of
Technology (MIT) in Cambridge,
who has studied small RNA molecules for years and is examining their influence
on tumors.
Cancer Connections
With rare exceptions, it's far from clear which genes the miRNAs are
targeting, how many miRNAs are involved in cancer--and how they're
involved--and what governs miRNA behavior. Uncertainties aside, however, Sharp
and others are not surprised that miRNAs are being implicated. Many of the
dozen or so animal miRNAs of known function play a big role in early
development. In fruit flies, some miRNAs govern apoptosis, or cell death; in
worms, as Slack witnessed to dramatic effect, they control cell
differentiation. Both processes, like many others in development, are critical
components of tumor formation and spread. "There were these clues,"
says Joshua Mendell, a geneticist and molecular biologist at Johns Hopkins
University in Baltimore, Maryland,
who set up his own lab last year and began exploring the miRNA-cancer
connection.
Mendell chose to focus on a proto-oncogene called c-Myc; proto-oncogenes (Ras
is another) are often highly expressed in cancerous tissue and implicated in
initiating malignancy. "Even though Myc has been studied for several
decades, [it's] still not fully understood how it causes tumors," says
Mendell. Examining a human cell line in which c-Myc expression could be
manipulated, Mendell and his colleagues found that when expressed, c-Myc
activates a cluster of six miRNAs. More important, another gene that's both a
target of c-Myc and drives cell division damps down its expression when two
miRNAs in Mendell's cluster are active. That suggested that this miRNA pair
could control the balance of cell death and proliferation driven by c-Myc.
While Mendell and his team were sifting through their cell samples, a cell
biologist at the University of North Carolina, Chapel Hill,
was studying how miRNAs might drive lymphoma. Unaware of Mendell's findings,
Scott Hammond hit on seven relevant miRNAs in human cancer cells; the cluster
was nearly identical to Mendell's list. "We both kind of came to the same
group of miRNAs," says Hammond.
But Hammond
recognized a problem. Cancerous cells contain abundant abnormalities, many a
result of cancer rather than a cause. Hammond
didn't know into which category his miRNAs, which were strikingly overabundant
in cancer tissue compared with normal tissue, fell.
Teaming up with Greg Hannon at Cold Spring Harbor Laboratory in New York, the pair and
colleagues forced overexpression of six of the miRNAs together in 14 mice
predisposed to a form of lymphoma. Cancer accelerated dramatically. After 100
days, all the treated mice had cancer, compared with about a quarter of
controls. The work is "precedent setting," says Sharp, one of the
first times miRNAs have been shown to spark cancer. If other miRNAs are found
to target either proto-oncogenes, which can trigger cancer, or tumor
suppressors, which squelch it, that would further incriminate them.
Hammond and Hannon's work appeared in Nature this past June, along with the
studies from Mendell's lab and from Todd Golub of Harvard
Medical School
and the Dana-Farber Cancer Institute in Boston
and his colleagues. Golub's research used expression of miRNAs to classify
different types of tumors.
But the Hammond-Hannon work remains the exception; nearly all the research
implicating miRNAs in cancer does so indirectly. One of the only other studies
showing potential causality comes from Carlo Croce of Ohio State University in Columbus, the first cancer
geneticist to publish on miRNAs. In September, Croce reported that in patients
with CLL, the loss of two miRNAs boosts expression of a gene promoting cell
survival. The gene is believed to help drive the leukemia in its earliest
stages. Without the miRNAs that mediate it, leukemia can set in.
Elusive Quarry
In retrospect, says Harvard RNA expert Gary Ruvkun, given the broad roles
being assigned to miRNAs in cancer, it's amazing that cancer geneticists so
thoroughly missed miRNAs. "I just find it hard to believe that the cancer
people were that lame," says Ruvkun, who is just now starting to back a
miRNA-cancer connection.
"We share a collective guilt as a community," agrees Ren Bernards, a
cancer geneticist at the Netherlands Cancer Institute in Amsterdam who is not studying miRNAs. With a
laugh, he recalls his graduate school days, when he tossed "anything
small, degraded, uninteresting" in the trash. At the time, miRNAs fell
squarely in that category. Furthermore, miRNAs are generated by genes that
don't produce proteins--long derided as "junk" DNA.
Indeed, Croce, now a consummate miRNA fan, admits being dragged into the field
unwittingly. Ten years ago, he grew convinced that a CLL tumor-suppressor gene
was nestled in a certain stretch of DNA--but he couldn't spot it. Baffled and
stubbornly determined, Croce turned to colleagues in the CLL field, who handed
over additional leukemia samples to scour. Only when Croce stopped looking for
a coding gene 3 years ago did he settle on the two miRNA genes he's been
studying ever since.
With the outlines of a miRNA-cancer connection taking shape, researchers are
now beginning to tackle some of the toughest questions. Perhaps the most vexing
involves finding miRNA targets. Like other types of small RNA molecules, miRNAs
influence genes with a similar sequence--but the match need not be exact,
making the targets maddeningly hard to pin down.
Rainbow dysfunction. Profiles of 218 tumor samples from various cancers show
miRNA expression as colored "hills."
No experiment "can hand you a target on a silver plate," says
Nikolaus Rajewsky, a biologist and mathematician at New York University.
These days, says Rajewsky, the best target-finding melds two tactics. The more
traditional compares putative miRNA targets in mammals with known targets for
the same miRNA in other species. The other calls for over- or underexpressing a
miRNA, then running microarray studies to spot affected genes. But "the
computational approaches are still evolving; the experimental approaches are
labor-intensive," says Victor Ambros, a geneticist at Dartmouth Medical
School in Hanover, New Hampshire.
"What we're not sure about is how many targets we're missing."
Several labs are conducting massive miRNA knockout studies to delineate the
targets and functions of individual miRNAs. At the University
of California, San Francisco, RNA biologist Michael McManus
is leading a six-person mouse miRNA consortium; it plans to delete each of the
350 known miRNAs in mice, one at a time.
Under the influence. Chronic lymphocytic leukemia cells (above) appear to be
driven by miRNAs.
But in cancer especially, biologists warn, painting a comprehensive miRNA
picture will likely be exceedingly complex. When miRNAs "get overexpressed
or underexpressed or deleted, lots of things can happen," says Tyler Jacks,
director of MIT's Center for Cancer Research. "And trying to figure out
exactly which of those things is contributing to tumorigenesis or prognosis or
what have you" calls for "a lot of detective work."
Nor is it clear what prompts miRNAs to misbehave in the first place. "We'd
really like to know," says Slack, who theorizes that mutations in miRNAs
could be at fault, as could defects in transcription factors, proteins that
control gene expression. Croce has found two leukemia patients born with the
miRNA mutations implicated in CLL.
Looking Ahead
Given all the unknowns, miRNAs are a long way from the clinic. But some drug
companies are dabbling in them nonetheless. Jan Weiler, a chemist at Novartis
in Basel, Switzerland, has been studying the
role of miRNAs in disease for 2 years. (In addition to cancer, the molecules
are tentatively linked to neurological disorders and diabetes.) "It's a
lot of speculation, a lot of hope," says Weiler, who envisions perhaps
delivering miRNAs to patients lacking them. "If we don't look at it now,
we're probably too late," he says, while acknowledging the risk that
"maybe … in 3 years' time, the whole thing is dropped."
If therapeutics remain distant, diagnostics are closer to reality. Croce
co-authored last week's NEJM paper that reported on a 13-miRNA signature in
CLL. His group also found that among 94 CLL patients, many of those lacking
Croce's original two miRNAs have a milder form of CLL, whereas most with the
two functioning miRNAs suffer a more aggressive form. "It looks like CLL
is not one disease but two," he says, and the distinction could be useful
in diagnosing and treating the leukemia.
Other cancers, too, are being eyed as harboring miRNA culprits. One of the very
first miRNAs tied to cancer--let-7 with its exploding worms--was last year
found to be lacking in lung cancer tissue taken from patients in Japan. Those
with the lowest levels fared the worst--suggesting once again that flawed miRNA
expression bodes poorly for one's health.
Copyright © 2005 by the American
Association for the Advancement of Science
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