In addition to genetic alterations, promoter hypomethylation has been recognized as a epigenetic mechanism that contributes to oncogene activation in cancer cells (16
). In this case, a demethylation event is supposed to occur in an inactive, methylated promoter, leading to transcriptional reactivation of an oncogene. However, experimental proof for genuine promoter hypomethylation is frequently difficult and inconclusive because the outgrowth of a subpopulation of cancer cells may confound this epigenetic observation. For example, the oncogene of interest may have never been silent in a minor population of cancer-initiating cells while the majority of other cells display promoter hypermethylation of the gene. The increased expression of this oncogene may simply result from rapid expansion of these few cells that eventually take over the whole population during tumor progression. If this scenario indeed occurs, it cannot be a bona fide
epigenetic event for oncogene activation.
In this study, we demonstrate that promoter hypermethylation can be directly associated with the activation of an oncogene. Specifically, this epigenetic event occurs in an upstream regulator, miR-129-2, that was shown to negatively regulate SOX4 oncogene in both knock-in and -out assays. miR-129-2 is embedded in a canonical CpG island on chromosome 11, which was found to be frequently hypermethylated in endometrial cancer. This epigenetic event results in miR-129-2 silencing, which in turn derepresses SOX4 expression. While we still cannot rule out hypomethylation of the SOX4 promoter CpG island as one of the causes, our present observation conclusively suggest that promoter hypermethylation of miR-129-2 is a common mechanism leading to the SOX4 over-expression in endometrial cancer.
It should be noted that a second CpG island is found 1.2-kb upstream from the first one analyzed in this study. In addition, the 5′-ends of two transcripts, BG120451
, are located in this upstream region. It is possible that these transcripts are the primary RNAs for miR-129-2
). Future mapping of putative transcription start sites located in these 5′-end areas will provide insight into the transcriptional control of this microRNA locus. Additional methylation analysis may further determine the role of this second CpG island in the silencing of miR-129-2
during endometrial tumorigenesis.
We have additionally searched the miRBase database and found that the expression of SOX4 may be regulated by at least 16 putative miRNAs, including miR-129-1 located on chromosome 7. Similar to miR-129-2, the miR129-1 precursor produces mature miR-129-5p, which negatively regulates SOX4 as described in this study. Since there is no known CpG island located near or within the miR-129-1 locus, it remains to be determined whether this miRNA is transcriptionally silenced by other epigenetic mechanisms (e.g., EZH2-mediated histone modifications, 29) in endometrial cancer.
, and miR-618
) of the other 15 miRNAs are located close to CpG islands based on our computational analysis (data not shown). Among these loci, miR-335
is the only one currently reported to be lost in primary breast tumors of recurrent patients (7
). However, it remains to be determined whether promoter hypermethylation plays a role in the silencing of this locus. Future studies will therefore be important to study the coordinate regulation of these miRNAs on SOX4
repression. It is also possible that concurrent hypermethylation of these loci contributes to a CpG island methylator phenotype (30
) and may improve the statistical power for predicting disease recurrence in our endometrial patient cohort (see ).
Extending from our present observation, epigenetically mediated silencing of other miRNAs that lead to tumor progression has recently been reported in the literature (15
). For example, ABL1
was showed to be a direct target of miR-203
). This miRNA was genetically and epigenetically down-regulated in leukemia cells expressing ABL1 or BCR-ABL1 fusion protein (32
). Restoration of miR-203 in vitro
led to reduced ABL1
expression and decreased proliferation of malignant cells (32
). Taken together, these and our studies clearly indicate that epigenetic silencing of tumor-suppressive miRNAs can be important constituents in cancer epigenomes and that the event is as significant as hypomethylation of oncogenes and hypermethylation of tumor suppressor genes.
In conclusion, our findings support a comprehensive screening of miRNA regulators at the 3′-UTR regions of all known oncogenes. High-throughout functional studies can be developed to establish the inverse relationship between these tumor-suppressive miRNA loci and their target oncogenes. This type of omics study may find that epigenetically mediated silencing of these miRNAs can be as common as genetic alterations that contribute to oncogene activation in cancer cells. As such, the combined epigenetic and miRNA-based therapies can be feasible options for future treatments in cancer patients.