NEWS

Silence of the Genes: Cancer Epigenetics Arrives

Ken Garber

A surging interest in epigenetics—changes in gene expression that occur without changes in DNA sequence—stems from a series of stunning discoveries over the past decade that have elevated the field from backwater status to the forefront of cancer research.

The packed talks at the recent American Association for Cancer Research annual meeting proved that epigenetics has arrived. "It clearly looks like this field has gained acceptance," remarked Rudolph Jaenisch, M.D., of MIT’s Whitehead Institute, surveying hundreds of people jammed into his lecture hall. "About 5, 6 years ago at a similar symposium, I think there were about double as many listeners as speakers."

Epigenetics is now firmly linked to cancer progression. Just in the last 6 months, the two main branches of epigenetic research, DNA methylation and chromatin remodeling, have merged amid a flurry of unifying discoveries.

"There’s nothing as exciting in science as when two fields that were separate come together," said Peter Jones, Ph.D., D.Sc., director of the University of Southern California Norris Comprehensive Cancer Center, Los Angeles.



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Dr. Peter Jones

 
Making the Cancer Connection

The epigenetics idea originated in studies by maize geneticist Barbara McClintock, Ph.D., at Cold Spring Harbor Laboratory in the 1950s. McClintock demonstrated a heritable yet reversible modification of gene expression across generations. The mechanism remained unknown, but in the mid-1970s, Art Riggs, Ph.D., and Robin Holliday, Ph.D., separately proposed that methylation—the addition of a methyl group to the cytosine base in DNA—might act as a universal "off" switch for gene expression during development.

This idea, which is still controversial, led circuitously to a cancer connection. In 1980 Jones, at USC, accidentally changed cultured fibroblasts into muscle cells using a methylation-inhibiting drug, which suggested that methylation did in fact regulate gene expression. Jones stumbled upon the cancer connection when he observed methylation of normal mouse genes immortalized in culture. "Since this process of immortalization has a role in cancer development," he recalled, "we realized that [methylation] might have great importance in the cancer phenotype."

Meanwhile, Steve Baylin, M.D., at the Johns Hopkins School of Medicine, Baltimore, was aggressively pursuing the cancer connection. Baylin thought abnormal methylation might be silencing tumor suppressor genes, and he and others eventually showed this for the Rb gene, p16, the estrogen receptor gene, the Von Hippel-Lindau gene, and others. Hypermethylation of so-called CpG islands in the promoter region of these genes causes silencing.

"We still don’t know how many genes are methylated in cancer," said Jean-Pierre Issa, M.D., of the University of Texas M. D. Anderson Cancer Center, Houston. "The estimates are hundreds." There is now persuasive evidence that methylation, by silencing tumor suppressor and mismatch-repair genes, has a major causal role in early cancer development.

But mainstream acceptance hasn’t come easy. "It was an uphill battle," said Jones. "It’s been really, really tough to convince people, particularly those that come from a strong genetics background, that [methylation] really has a role in cancer, it’s not just something which is a consequence of cancer."

But how methylation silences genes remained a mystery. Only rarely does the methyl group seem to directly interfere with gene transcription. Somehow methylation was blocking transcription factors from reaching DNA. Clues began to emerge in the early 1990s that would, by linking methylation with chromatin changes, eventually explain what was going on.

Chromatin Takes Center Stage

Loops of chromatin fiber, coiled like a very long Slinky, make up the chromosome. In the late 19th century abundant proteins in the nucleus, called histones, were identified as a major component of chromatin. For a long time, histones were suspected of being the genetic material itself, then, with the discovery of DNA, they faded from interest as merely "passive structural platforms upon which the DNA seemed to wrap," recalled biochemist David Allis, Ph.D., of the University of Virginia, Charlottesville.

There things stood until 1974, when Roger Kornberg, Ph.D., working at the Medical Research Council in Cambridge, England, discovered the nucleosome, the basic building block of chromatin. Consisting of four pairs of histones, the nucleosome is a protein "bead" wrapped by two turns of DNA. The DNA, like the string of a necklace, connects the nucleosome beads together in fibers that are coiled into chromosomes. It soon became clear that nucleosome packaging kept genes from being expressed. But how?

The right answer had already been guessed in 1964, by Vincent Allfrey, Ph.D., of Rockefeller University, although more than 30 years would pass before proof emerged. Allfrey discovered histone acetylation—the addition of acetyl groups—and suspected that it caused chromatin to open to allow DNA to be transcribed. Genetic work in yeast in the late 1980s and early 1990s gave support to this idea.

Then, in 1996, Allis and Harvard University’s Stuart Schreiber, Ph.D., a month apart, cloned the first HAT and HDAC (histone acetyltransferase and histone deacetylase), enzymes that acetylate and deacetylate histones, respectively. Stunningly, these enzymes were already well known to be gene activators and repressors. "That just changed everything," recalled Allis. "That turned the coin." Suddenly it was clear even to the skeptics that histones were more than passive DNA platforms and were actively involved in gene activation.

Meanwhile, links between histones and DNA methylation had begun to emerge. Adrian Bird, Ph.D., of the University of Edinburgh, Scotland, and Alan Wolffe, Ph.D., of the National Institutes of Health, in the early 1990s showed that methylated DNA recruits methyl binding proteins, which in turn attract a chromatin-remodeling complex, along with proteins that modify histones by deacetylating them, closing down DNA to transcription. So methylation clearly used histones to silence genes.

Something was missing from the picture, though, because histone deacetylase-inhibiting drugs did not restore gene expression in tumor cells (although adding methylation inhibitors did the trick). Something more complicated was going on. In 2000 Allis and postdoc Brian Strahl, Ph.D., proposed the "histone code," the idea that modification of the protruding histone "tails" by acetylation, methylation, phosphorylation, and other signaling generates a code that is read by other proteins, which then tell genes whether to express or shut down. While the details of the code remain to be worked out, the implications of this new gene regulatory system are enormous. "This is probably a major, major mechanism for everything DNA does," Allis said.

The epigenetics puzzle is still being put together. Last November, Eric Selker, Ph.D., of the University of Oregon, Eugene, working with filamentous fungi, showed that DNA methylation depends on histone methylation. Although gene silencing via histone methylation has yet to be verified in humans, the discovery cemented the bond between the DNA methylation and chromatin remodeling fields, and may finally lead to an understanding of what causes DNA methylation—the black box of epigenetics.

But the general role played by methylation through gene silencing is now clear. "It’s not a switch, but a lock," said Jones. "And what happens is, this locking mechanism becomes misappropriated in cancer."

The good news is that tumor suppressor genes silenced by methylation and histone deacetylation can, in theory, be reactivated, unlike genes harboring disabling genetic mutations. "Driving all this excitement about epigenetics is the idea that a gene that is silenced is an intact gene," said Issa. "So, at least in principle, anything that reverses silencing has the potential to change the gene expression pattern of a cancer cell and bring it back closer to what normal cells look like. And that will then presumably cause the cells to go into apoptosis or senescence."

This cancer-killing potential has not been lost on the pharmaceutical and biotechnology industries. They are now busy developing epigenetics-based drugs which they hope will provide the next wave of cancer therapies.

In the June 19 Journal: The race for an epigenetic cancer therapy.


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