When good genes go bad and cancer arises, mutation normally gets the blame. But, newer evidence is challenging that one-dimensional view. Epigenetic modifications, such as DNA methylation, can also shut down tumor suppressor genes by causing changes in the chromosomal structure surrounding the gene as noted in this issue's Hot Papers.1,2
John Minna, director of the Hamon Center for Therapeutic Oncology Research at the University of Texas Southwestern Medical Center, Dallas, demonstrated that the gene RASSF1A is lost to methylation, not mutation, in many tumor samples.1 The first clues to this change, which had no apparent relationship to methylation, came from a chromosomal region, known as 3p21.3, that is frequently lost in a variety of tumors, including lung, breast, kidney, head, and neck. The 3p21.3 deletion "was probably the earliest [change] we could detect in preneoplastic tissues, in hyperplasia and dysplasia," says Minna.
Manel Esteller and his group from the Spanish National Cancer Center took a more global view, tracking methylation of 12 tumor-associated genes in a large sample of tumors, and demonstrating that tumors could be classified according to methylation patterns.2 The finding could be the basis of a powerful diagnostic screen, says Esteller.
Both Hot Papers "really made it hard to ignore that [methylation] is affecting important genes and the cancer phenotype," says James G. Herman, an associate professor of oncology, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, and a coauthor with Esteller. Often an inheritable chemical modification, DNA methylation is the addition of a hydrophobic methyl group to the cytosine in a CG sequence. In vertebrates, methylation generally silences genes.
Minna was not the only one to make the methylation connection. A year prior, Gerd Pfeifer and colleagues from City of Hope National Medical Center, Duarte, Calif., demonstrated a cancer-causing event at 3p21.3. Their expertise in methylation analysis allowed them to link the lack of RASSF1A expression to methylation of its promoter region.3 Recognizing the location's significance, Pfeifer analyzed lung tumors and normal tissue and found expression frequently lacking in cancer cells.
PASSING THE TEST Minna's team identified 25 genes in the region and systematically analyzed them for mutations in about 70 tumor samples. Only a few turned up as mutants, including RASSF1, but the gene often had loss of expression when mutations weren't present. So, Minna's group looked for DNA-promoter methylation or other changes that could explain the functional loss. They also reintroduced normal copies of the genes into tumors to see if any acted as tumor suppressors once restored to the cell; five of the genes passed the test.4 "It looks like many of them are inactivated not by mutation but by promoter methylation," says Minna.
RASSF1 has two predominant transcript products derived from alternate splicing and promoter usage, RASSF1A and RASSF1C, but only the former is a tumor suppressor. Minna also found that the RASSF1A-specific promoter was hypermethylated in lung and breast cancers but not in normal tissues. Of the tumors sampled, the gene's messenger RNA was absent in 100% of non-small-cell lung carcinomas, 100% of small-cell lung carcinomas, and 60% of breast cancer lines.
Minna's team has gone on to characterize RASSF1 expression in more than 1,000 tumor samples. "I would say that after p53, it's the most frequently inactivated tumor suppressor gene," says Minna. Some already have begun to link RASSF1A status with prognosis. "Several studies have now shown that the presence of RASSF1A methylation confers worse prognosis on non-small-cell lung cancer patients," he says.5
UNIQUE PROFILES RASSF1A is by no means the only methylated gene linked to cancer. Other reports have connected bladder cancer to methylated genes, such as CDH1, CDKN2A, and DAPK1, isolated from urine.6 Also, methylation-specific PCR can detect breast cancer cells in ductal lavage fluid.7
To get a handle on the emerging picture, Esteller and colleagues approached the problem from a different angle. They selected 12 genes from major pathways in the cell, including cell cycle, DNA repair, cell adherence, and most of the known apoptosis pathways. Each gene has C-G rich areas, which are methyltransferase targets. These CpG islands are associated with genetic diseases that cause predisposition to cancer.
They next screened 600 tumor samples, drawn from more than 15 tumor types, to identify genes that were hypermethylated. It turned out that every major tumor type had at least one of the genes hypermethylated, but each had a unique methylation profile, making it possible to distinguish different tumor types.
Esteller thinks that the paper garnered a high number of citations because it offers a "panoramic view of what's going on in the CpG islands of important genes involved in cancer. If you're interested in a particular tumor, you can look here and see that these genes are methylated," he says.
Courtesy of Manel Esteller
Joe Costello, assistant professor of neurological surgery at the University of California, San Francisco, examined brain tumors for mutation and methylation at tumor suppressor genes, and found that the two mechanisms often weren't interrelated at all.8 These mechanisms sometimes affected the same gene; that is, one copy could be deleted and the other could be methylated. But, what happened more often was that the methylation was affecting a different set of genes. Costello says that these Hot Papers and a host of others suggest that the classic genetic picture may be just part of the story. "Possibly there is a whole other set of genes that are primarily affected by methylation, and only through these integrated approaches can we get closer to knowing the total number and identity of cancer genes."
It's impossible to say whether mutation or methylation is the lynchpin, explains Costello. "Methylation is definitely much more prevalent in some cancers, but that's part of the problem; sorting out what's important is difficult."
Regardless, Pfeifer says he thinks that methylation will turn out to be a significant player. "[It] is now being recognized as probably more important, or at least more common, than mutation of genes in cancer. The methylation phenomenon is being studied by a lot of people, whereas five or 10 years ago nobody even thought about it."
Esteller says that he envisions methylation as a powerful diagnostic tool. It can be gauged using PCR techniques, raising the possibility that even the small number of escaped cells in a blood sample might be enough to catch a tumor early in its development. Such a test might even reveal the tumor type. Yet, it's still unknown how much of tumor development is attributable to methylation and other epigenetic (nonmutational) changes.
Drugs such as the methylation inhibitor Azacitidine (being submitted by Pharmion of Boulder, Colo., to treat preleukemia conditions known as myelodysplastic syndromes) appear to target hypermethylated cells over normal cells, though the reason is not clear. The evolving science and emerging clinical success have researchers "very excited about the possibility of [demethylation and other nonmutational changes] as an approach to cancer therapy," says Peter Jones, director, University of Southern California/Norris Comprehensive Cancer Center in Los Angeles.
Yet, as researchers work to develop drugs that target methylated genes, two recent studies sound a word of caution.9,10 Apparently, aggressive T-cell lymphomas develop in 80% of mice engineered to produce low levels of methyltransferase. The studies confirm that methylation plays a key role in cancer, but they also show that the knife cuts both ways, as methylation is also important in maintaining the health of a cell.
Jim Kling (firstname.lastname@example.org) is a freelance writer in Washington, DC.
1. D.G. Burbee et al., "Epigenetic inactivation of RASSF1A in lung and breast cancer and malignant phenotype suppression," J Natl Cancer Inst, 93:691-9, May 2, 2001. (Cited in 105 papers)
2. M. Esteller et al., "A gene hypermethylation profile of human cancer," Cancer Res, 61:3225-9, April 15, 2001. (Cited in 164 papers)
3. R. Dammann et al., "Epigenetic inactivation of an RAS association domain family protein from the lung tumour suppressor locus 3p21.3," Nat Gen, 25:315-9, 2000.
4. Y. Tomizawa et al., "Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B," Proc Nat Acad Sci, 98:13954-9, 2001.
5. Y. Tomizawa et al., "Clinicopathological significance of epigenetic inactivation of RASSF1A at 3p21.3 in stage I lung adenocarcinoma." Clin Cancer Res, 8:2362-8, 2002.
6. M.W. Chan et al., "Hypermethylation of multiple genes in tumor tissues and voided urine in urinary bladder cancer patients," Clin Cancer Res, 8:464-70, 2002.
7. C. Evron et al., "Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR," Lancet, 357:1335-6, 2001.
8. G. Zardo et al., "Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors," Nat Genet, 32:453-8, 2002.
9. A. Eden et al., "Chromosomal instability and tumors promoted by DNA hypomethylation," Science, 300:455, April 18, 2003.
10. F. Gaudet et al., "Induction of tumors in mice by genomic hypomethylation," Science, 300:489-92, April 18, 2003.