Overall our study presents an extensive search for the presence of and interactions between both genetic and epigenetic alterations in cancer. As it currently stands, these studies do have several limitations. First, our data do not address the biological effects of the individual mutations observed in the CAN genes. Second, the data draw on only the 13,023 subset of CCDS genes that were previously sequenced, and additional genes have now been sequenced and more mutations have been discovered [42
Despite these limitations, our study describes a valuable approach to begin to understand the biological significance of the vast amount of mutational data generated by cancer resequencing efforts. In these regards, our findings allow several important conclusions to be drawn. First, our study shows that large-scale, combined genetic and epigenetic analysis is feasible and useful for cancer gene discovery. Such combined analyses can markedly enhance links made between gene alterations and key clinical parameters for cancer. It is becoming increasingly clear that examination and interpretation of mutations to identify cancer genes on a genome-wide scale can be significantly complicated by passenger mutations [43
]. Furthermore, as we mentioned above in the Results, it is very unlikely that a given gene that in breast and/or colon cancer has evidence for mutations, promoter hypermethylation, reduced expression, and is localized to chromosome regions harboring frequent deletions in tumors is not important for tumor development. Consistent with this hypothesis, by beginning with a large pool of genes harboring mostly low incidence heterozygous missense mutations and then characterizing the methylation and expression status of these genes, our approach allowed us to identify genes that possess potentially prognostic value.
Our results also confirm that our microarray strategy is an effective approach to identify genes that are silenced by hypermethylation in colon and breast cancer. Other methods have been developed to identify hypermethylated genes in cancer, including restriction landmark genomic scanning, promoter CpG island microarrays, and methylation-specific digital karyotyping [28
]. However, the sensitivity of these techniques is restricted by the locations of methylation-sensitive restriction sites in the genome.
Several of the common target genes have been noted to undergo methylation-associated silencing in cancers by other investigators. Lund et al. noted that oncogenic RAS
can lead to the hypermethylation of the MMP2
has been found to be hypermethylated in colon carcinoma [47
has been found to be hypermethylated in lung cancer in a survey of methylated genes described by Shames et al. [48
]. The presence of methylation of the common target genes in other tumor types suggests that these genes may be targets of inactivation in a broader range of cancers, a hypothesis that warrants future investigation. In particular, it would be of value to directly compare our results with those derived by other strategies for analyzing the hypermethylome from the same as well as from other types of malignancies [48
]. Together with these studies, our data strongly suggest that a compendium of epigenetic changes underlie the progression of human cancers.
Second, our results suggest that tumors may be less biologically heterogeneous with respect to denoting key tumor suppressor pathway disruptions when consideration is given to both genetic and epigenetic changes. To our knowledge, this study represents the most comprehensive analysis of genes targeted by both mutation and hypermethylation. Prior to the present study, only a small number of genes had been found to be frequently affected by both mutations and promoter hypermethylation. Most of these genes were the initial classic tumor suppressor genes where the epigenetic event was first defined as meaningfully functional. These genes are closely linked to cancer initiation and include those for which germ-line mutations occur, such as VHL
, and STK11
in familial forms of renal, breast, and colon cancer, respectively [10
]. These tumor suppressors are frequently hypermethylated in sporadic forms of the corresponding tumor types [54
]. Furthermore, methylation-associated silencing can act as a “second genetic hit” in these genes in tumors from individuals harboring germline mutations, resulting in functional LOH [57
]. Our current findings now indicate that, particularly for tumor suppressor genes with a low incidence of mutations, it may be the rule rather than the exception that epigenetic inactivation is a more frequent event than genetic disruption. Tumor suppressors that are important for tumorigenesis may, then, often be targeted by multiple methods of functional inactivation.
A third important conclusion is that there may be more similarity among individual breast and colon tumors than is apparent from analysis of the mutational spectrum only, and, therefore, any comparison of biological changes between tumors may need to account for epigenetic effects in addition to genetic ones. Clearly, the same tumor suppressor genes in different cancers may undergo different modes of inactivation. This scenario is analogous to the situation that is observed for oncogenes such as MYC
. In hematopoietic malignancies, aberrant activation of MYC
results frequently from translocations whereas the gene is more often subject to amplifications and mutations in solid tumors [58
]. The processes underlying these differences are fundamentally important for understanding cancer and are worthy of future study.
Finally, it is important to reiterate that our findings have allowed us to begin querying the clinical significance of genes targeted by mutation and hypermethylation. By correlating our data to expression changes in cancer microarray databases and relating these to important clinical parameters, we have identified genes that may track with disease prognosis. Indeed, previously, the discovery of hypermethylated genes such as MGMT
have proven very useful for predicting clinical prognosis and response to therapy in diseases such as malignant glioma [60
], gastric cancer [61
], and lung cancer [62
]. A recent study showed that a polycomb repression signature in metastatic prostate cancer predicts for cancer outcome [64
]. Our study suggests that matching large-scale mutational and epigenetic analysis will be useful for advancing our knowledge of the biology of human cancers. These results may be useful for the development of new, more effective biomarkers and therapeutics.