Affiliations of authors: The Johns Hopkins Comprehensive Cancer Center and The Johns Hopkins Medical Institutions, Baltimore, MD.
Correspondence to: Stephen B. Baylin, M.D., The Johns Hopkins Comprehensive Cancer Center and The Johns Hopkins Medical Institutions, Cancer Research Bldg., 1650 E. Orleans St., Rm. 540, Baltimore, MD 21231 (e-mail: sbaylin{at}jhmi.edu).
We recently commented on the burgeoning evidence that epigenetically mediated gene silencing can selectively produce loss of key gene functions in cancer (1). In this issue of the Journal, Burbee et al. (2) report that yet another gene, RASSF1A, is silenced in association with promoter hypermethylation in small-cell lung cancer (SCLC), in non-small-cell lung cancer (NSCLC), and in breast cancers. Dammann et al. (3) recently made a similar finding in lung tumors. Burbee et al. (2) demonstrate that the frequency of RASSF1A gene silencing virtually parallels that of the incidence of the loss of heterozygosity (LOH) for chromosome 3p21, the region that harbors the gene. Both groups (2,3) find that RASSF1A promoter hypermethylation is an extremely frequent occurrence in SCLC, a tumor in which chromosome 3p LOH is almost always present and may be a seminal event for this disease (2,4). In both studies (2,3), reinsertion of the RASSF1A gene into cultured lung cancer cells reverts various parameters of tumorigenicity, further suggesting it as the target, or one of the targets, of chromosome 3p21 LOH for loss of tumor suppressor gene function.
Although the findings of the Burbee et al. (2) and Dammann et al. (3) are certainly of great interest, they also raise a critical question concerning epigenetically mediated gene silencing in cancer that heavily impacts future research in this arena. As more and more groups search for important cancer genes by including screens for promoter hypermethylation, one must question whether this change alone can ever identify a true tumor suppressor gene? In recent reviews (1,5), we have stressed the fundamental differences between epigenetic changes and loss of function mutations that make the burden of proof for a tumor suppressor function much harder for the former than for the latter. Promoter hypermethylation may be part of a chromatin-altering process in individual tumor types that affects multiple genes simultaneously, some of which may be, and others not, vital for tumor formation and/or progression (5,6). To date, the strongest evidence that epigenetic changes can mediate loss of tumor suppressor gene function relate to bona fide tumor suppressor genes where mutations also occur either in the germline and/or in the somatic forms of cancer (5). However, the RASSF1A gene does not yet have such proven mutations. Although the data for the reversion of tumorigenicity with RASSF1A gene reinsertion are strong in both studies (2,3), there are caveats to such experimental findings constituting absolute proof of tumor suppressor function in a native tumor setting. Furthermore, questions arise even regarding the relationship between RASSF1A promoter hypermethylation and chromosome 3p21 LOH. Certainly, this promoter hypermethylation is the first consistent mark for a loss of gene function in the long and arduous search for tumor suppressor genes in the chromosome 3p21 region. However, with chromosome 3p21 LOH in NSCLC and breast cancers, there is an equal frequency for the remaining RASSF1A allele to be unmethylated or hypermethylated (2). This finding can be interpreted in several ways: 1) RASSF1A is not the suppressor gene in the region, 2) mechanisms other than promoter hypermethylation affect the function of RASSF1A in some tumors, 3) the pathway in which RASSF1A is operative is blunted by another gene lesion, or 4) RASSF1A is only one of several tumor suppressor genes in the chromosome 3p21 region.
Thus, each gene identified as a candidate tumor suppressor gene by virtue of promoter hypermethylation alone must be investigated intensively for true tumorigenic function. How can this best be accomplished? Initially, one must consider gene function and determine if a hypermethylated gene is reactivated by demethylation. Such validation would define molecular event(s) that can be monitored to explain a tumor suppressor role. Such experiments have been done for MLH1, a gene that has both mutations and promoter hypermethylation in cancer (7). Although few studies have been done with candidate suppressor genes identified only with promoter epigenetic changes, one example is death-associated protein kinase in leukemia cells, where demethylation and reactivation of gene expression restores an apoptotic response to interferon (8). Certainly, RASSF1A encodes a protein with several interesting domains, including one that potentially interacts with the RAS family of proteins, a domain that is potentially phosphorylated via the ataxia telangiectasia mutated kinase pathway, and a DAG-binding domain that potentially links it to tumor-promoting activities (2,3). In a yeast two-hybrid system, RASSF1A can bind to a DNA repair protein (3). Which of these potential functions inhibit tumorigenesis remains to be worked out.
It is our bias that one of the key steps in documenting the true tumor suppressor status for genes that only have loss of function associated with promoter hypermethylation in cancer must include gene knockout strategies in mice. Such investigations can evaluate tumor susceptibility associated with the deletion either of only the gene in question or of paired deletions to test for complementation with classic tumor suppressor genes. Although such studies are not foolproof, they often reveal tumor suppressor effects of truly important cancer genes. We predict that these types of studies will reveal important cancer genes that have only epigenetically mediated loss of function. This conjecture is fueled by our own current studies of a gene in an extremely high-frequency LOH area in chromosome 17p13.3, which has similar methylation abnormalities to those for RASS1FA. HIC-1 is a gene that encodes for a zinc-finger transcription factor. The HIC-1 promoter is hypermethylated in multiple tumor types, but the gene contains no known mutations (9). However, HIC-1 is emerging in ongoing studies of our recently created hic-1 knockout mouse model (10) as a potent tumor suppressor gene (Chen W, Baylin SB: unpublished data). RASS1FA and other genes will certainly be interesting to study in a similar manner.
If searching for epigenetically silenced genes in cancer is a fertile path to the identification of key genes for tumorigenesis, what are the best search strategies? As discussed above, finding promoter-hypermethylation patterns in tumor suppressor genes known to be mutated in some cancers constitutes a proven approach for defining important loss of gene function situations. Searching for promoter hypermethylation in candidate genes lacking mutations, but residing in frequent LOH regions, as was used to identify RASSF1A, is also a promising approach. In addition, more random genomic searches for hypermethylated loci are appearing in the literature with increasing frequency. We identified HIC-1 by using such an approach for the LOH regions of chromosome 17p13.3 (9). Techniques such as restriction landmark genomic scanning survey the whole genome by tracking altered DNA fragments that result from tumor-associated methylation abnormalities occurring at rare base cutting, methylation-sensitive restriction sites (11). Although this approach is excellent for identifying large numbers of methylation differences between normal and tumor DNA, many of the sites identified may not be true CpG islands or are islands that are not located directly in gene-promoter regions (and thus altering the methylation status would not influence gene expression) (12). Methylated CpG amplification-restriction digest analysis (6) is a related approach that maps differences in the methylation of CpGs within two closely spaced restriction sites that are highly likely to reside in CpG-rich regions. This strategy is excellent for identifying hypermethylated CpG islands, but walking from these to nearby genes can be laborious (13). Microarray approaches could also be used to map hypermethylated promoter regions in tumor cells by examining those genes re-expressed after inhibiting methylation.
Regardless of the approach used, the choice must be tailored to fit the question being asked. Furthermore, we stress that the information generated with respect to sequences, genes, and chromosomal regions should be incorporated rapidly into genomic databases. In this way, very valuable DNA markers will be compiled to mark the sites of potential loss of key gene functions in cancers. In addition, sites of altered DNA methylation constitute some of the most promising molecular markers for use in early cancer diagnosis, predicting cancer risk status, and monitoring prognosis (14). The above databases could markedly enrich the possibilities attendant to these approaches.
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