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TRACING CANCER TO ITS ROOT
Imprinting May Provide Cancer Prevention Tools
At a time when sequencing of cancer cell DNA is revealing the tremendous complexity of gene mutations, some researchers are proposing an underlying theme that may precede and could unite all cancers: loss of imprinting (LOI).
Now research from several laboratories is beginning to bear out the hypothesis, indicating that imprinting status may provide a powerful new tool in cancer prevention and early detection. Several companies are rushing to create diagnostic tests for early cancer detection, even as other groups are concentrating on reversing the process to prevent cancer.
Parent-of-origin gene imprinting is part of epigenetics, the study of gene regulation though specific chemical tags called methyl groups that are added to DNA. Without changing the genetic sequence itself, these tags prevent nearby genes from being expressed. The effect is that either the maternal or paternal copy of the gene is expressed, but not both.
The loss-of-imprinting theory suggests that a disruption in the mechanism that regulates imprinting predisposes cells to cancer. Although understanding of this mechanism is far from complete, the results of its dysfunction are becoming clear: When genes lose their imprinted methyl groups, diseases, including cancer, may result.
Andrew Feinberg, M.D., the director of the center for epigenetics of common human disease at Johns Hopkins University in Baltimore, calls this precancerous state the epigenetic progenitor model of cancer. His theory, first articulated in a 2005 Nature Reviews Genetics article, ties together several observations: cancer arises from stemlike cells, the epigenetic tagging of specific genes distinguishes stem cells from other cells, and virtually all cancer cells contain a disruption in normal epigenetic patterns.
Maternal and paternal gene imprints, which are created on specific genes during the formation of sperm and eggs, lay the foundation for normal embryonic development, growth, and differentiation. Many of the proteins involved in the imprinting process are then turned off, but Feinberg suggests that some are erroneously turned back on in precancerous cells. This error leads to LOI of some genes and improper imprinting, a term called hypermethylation, of other genes, some of which are tumor suppressors.
Randy Jirtle, Ph.D., of Duke University in Durham, N.C., a pioneer in studying the role of gene imprinting in animals and humans, says the LOI does not cause cancer per se, but it may increase the susceptibility of cells to cancer because imprinted genes are almost always involved in regulating cell growth and differentiation. When this regulation is lost, cells become more resistant to death.
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"It is susceptibility we are talking about here," Jirtle said. "These are little bitty changes, but they are very important. It's like if you are off by a half a degree when you shoot to the moon, by the time you get there you are a hundred thousand miles away. When you go out in time, a lot of other things can happen to the cells, such as mutations, that would normally be taken care of [by programmed cell death] that aren't, and then you have cancer."
Epigenetic Patterns as Biomarkers
Much of the excitement about examining epigenetic patterns in cells revolves around the notion that these disrupted methylation patterns might be useful in diagnosing cancer before it becomes established.
Feinberg and his colleagues have taken a first step in this direction by showing that seemingly normal colon cells in which the growth-promoting gene IGF2 (human insulin-like growth factor II) has lost its imprint are 3.5–5 times more likely to develop into colon cancer than cells with normal gene imprinting. These cells also behave more like undifferentiated stem cells in the colon, which also suggests a precancerous state.
"To know if we can use LOI as a predictive marker we need to do the [LOI] test then see what happens to people who are positive versus negative," Feinberg said. "The way to do this is in a nested case–control study, which we are beginning." The prospective study will look at imprinting status in healthy patients and monitor them to see which patients develop cancer.
Meanwhile, biotech companies, such as Epigenomics AG of Berlin and BioCangen Inc., New York, are already developing proprietary imprinting tests that they hope to commercialize for early cancer detection. In January, BioCangen announced the completion of a small clinical study of 102 patients with solid tumors, including lung, colon, breast, and stomach cancers, and 82 healthy controls. In the unpublished study, the test correctly identified 73 samples as cancer and incorrectly identified three noncancerous samples as cancer, according to a company news release. BioCangen chief scientific officer Adam Li, M.D., Ph.D., said the company is planning a larger multicenter study at two U.S. hospitals and two hospitals in China later this year and is developing individual tests of each type of cancer.
The "New Normal"
One of the biggest challenges facing researchers hoping to use LOI as a biomarker is whether epigenetic patterns will be reliable. One recent study in the November 15 issue of Clinical Cancer Research demonstrated that it is possible to map geographic zones of epigenetic changes in breast cancer radiating from the tumor into adjacent "normal" tissue. Researchers Pearlly Yan, Ph.D., and Tim Huang, Ph.D., of Ohio State University and their colleagues studied the methylation pattern of the tumor-suppressor gene RASSF1A, one of the most frequently methylated sites in human breast cancer [J Natl Cancer Inst 2001;93:691–9]. Knowing that breast cancers often recur in adjacent "normal" breast tissue, the researchers wanted to test whether premalignant epigenetic changes could be found adjacent to breast cancer tissue. They tested 144 samples, including 47 primary tumors, 69 adjacent normal tissue with increasing radius from the tumor site, and 28 healthy control samples. The study demonstrated a gradient of methylation, with the highest levels seen in tumor tissue and decreasing levels with increasing distance from the tumor site. However, the scientists point out, some patients had high levels of RASSF1A methylation even in apparently normal tissue.
"We were surprised to see this amount of DNA methylation already going on in normal tissue," Huang said. "We are seeing the pathological alteration of normal breast."
Current research in Huang's laboratory is aimed at determining what causes these epigenetic changes. Huang said he has isolated precancerous breast progenitor cells, and he has evidence in ongoing laboratory experiments that exposure to estrogen can lead to methylation of certain key genes in the cells. He said the research suggests that estrogen can drive aberrant methylation.
"We hope to identify epigenetic markers that occur really early and use them for population screening, similar to how [prostate-specific antigen] testing is used to screen for prostate cancer," Huang said.
One team appears to have taken the first step in that direction. Researchers at the University of Texas Southwestern Medical Center in Dallas have identified a panel of 45 genes that are methylated in the most common human cancers: breast, lung, prostate, and colon. They reported their findings in the Dec. 26 issue of PLoS Medicine. Many of the genes turned out to be those involved in normal cell development but had not previously been connected to cancer.
In an accompanying editorial, Joseph F. Costello, M.D., of the department of neurological surgery at the University of California, San Francisco, Comprehensive Cancer Center, and colleagues concluded that "this finding is of importance, since it highlights the possibility of identifying a common epigenetic denominator acting across tumor types, and perhaps underlying malignant transformation in general."
Adi Gazdar, M.D., chair of molecular oncology research at UT-Southwestern and one of the lead authors on the PLoS Medicine study, is collaborating on two NCI-sponsored phase II chemoprevention studies that are trying to determine whether two experimental chemopreventive agents, a green tea extract called polyphenon E and a herbal preparation called ACAPHA, can reverse methylation changes in the lungs of smokers and whether methylation patterns are associated with subsequent development of lung cancer. The methylation analysis, which Gazdar is conducting with Stephen Lam, M.D., a professor of medicine at the University of British Columbia, is one part of a larger effort by Lam to examine the effects of the experimental agents on markers of cell cycle regulation, angiogenesis, and apoptosis.
Similarly, Steven Henikoff, Ph.D., a Howard Hughes Medical Institute investigator at Fred Hutchinson Cancer Research Center in Seattle, is beginning a collaboration with his colleague Brian Reid, M.D., Ph.D., to investigate whether methylation changes in patients with Barrett esophagus are associated with development of esophageal cancer. Patients with chronic acid reflux can develop Barrett esophagus—an abnormal cellular change, called dysplasia, in the cells that line the esophagus. A small percentage of these patients go on to develop cancer, but there is currently no way to reliably predict which patients will develop cancer. Reid has developed a panel of biomarkers to help grade the disease, but the researchers hope that methylation markers will provide a risk assessment of developing cancer, which is now unknown.
In the meantime, "people have to be very careful when using normal tissue from cancer patients as their control," Huang said, because methylation changes may already be taking place in those cells. "Our whole concept has changed since publishing this paper."
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J Natl Cancer Inst 2001 93: 691-699.
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