Here we report that “loss of 5-hmC” is a distinctive epigenetic event of neoplastic progression in melanoma that correlates with clinical relapse-free survival and melanoma staging parameters. Thus, 5-hmC holds promise as a putative molecular biomarker with predictive and prognostic value. The present study also for the first time illustrates the genome-wide 5-hmC landscape of benign nevi and melanomas and reveals the strikingly demolished 5-hmC levels and distribution along the epigenome of melanomas in comparison with benign nevi. Furthermore, loss of 5-hmC in melanoma is caused, at least in part, by the decreased expression of key enzymes, IDH2 and TET family proteins, controlling 5-hmC production. In relevant animal models, increase in 5-hmC levels via either IDH2 or TET2 overexpression is shown to suppress tumor invasion and growth and improve tumor free survival. Taken together, the present study provides multiple layers of evidence to support that genome-wide “loss of 5-hmC” is a new epigenetic hallmark of melanoma with diagnostic and prognostic advantages over global DNA hypomethylation, a recognized epigenetic mark of cancer. Of clinical and therapeutic significance, the present study also opens a new avenue for cancer prevention by targeting the cellular and biochemical pathways that can re-establish 5-hmC levels and landscape in melanoma.
The IHC staining approach to detect 5-hmC may have practical applications clinically. We and others have observed similar loss of 5-hmC in other solid tumors such as breast, ovarian, and colon carcinoma (data not shown) using the same methods (Haffner et al., 2011
; Yang et al., 2012
). The anti-5-hmC antibody-based IHC strategy could lead to the development of a new, simple, sensitive and practical adjuvant diagnostic assay. Future studies focusing on borderline lesions and with sufficient clinical outcome annotation are now indicated to evaluate the utility of “loss of 5-hmC” as a novel diagnostic and prognostic tool.
The high level of 5-hmC in differentiated and benign nevomelanocytes raises an intriguing question as to the nature of the biological role of 5-hmC in melanocyte differentiation, self-renewal and malignant transformation. Studies of embryonic stem (ES) cells indicate that 5-hmC may be involved in the regulation of cell differentiation and lineage commitment (Ito et al., 2010
; Xu et al., 2011b
). Until now, skin tissue-specific 5-hmC distribution and genome-wide mapping of 5-hmC in cancer have not been well studied. Our findings of a high level of 5-hmC in mature melanocytes and benign nevi, as well as a significantly lower level of 5-hmC associated with melanoma, provide new insights supporting a role of 5-hmC in pathways fundamental to cellular differentiation and de-differentiation. We postulate that the well-controlled dynamic level of 5-hmC during transition from ES cell to melanocyte progenitor to terminally-differentiated melanocyte is a novel epigenetic signature of melanocyte differentiation, the perturbation of which may lead to the induction of oncogenic pathways underlying melanoma progression.
There are several ways to influence 5-hmC levels in cells (). Presumably, dysfunction of TET and/or IDH enzymes, two key factors involved in 5-hmC generation, would greatly reduce 5-hmC generation. 10% of melanomas (4/39) harbor an IDH1 or IDH2 mutation Shibata et al., 2011), whereas no TET mutations have been reported in melanoma. The low penetration of IDH and TET mutations in melanoma constitutes robust evidence that other cancer pathways inactivating these 5-hmC-generating enzymes must play a major role in down-regulation of 5-hmC. Herein, we demonstrate the significant decrease in TET1, TET2, TET3 and IDH2 gene expression in melanomas compared to benign nevi, which suggests that insufficient enzymes required for the conversion of 5-mC to 5-hmC may account for one of the molecular mechanisms underlying global “loss of 5-hmC” in melanomas.
In support of this hypothesis, we show that increasing 5-hmC levels and partially reestablishing the 5-hmC landscape in melanoma cells by restoring expression of active TET2 enzyme but not the catalytically inactive TET2 mutant, significantly suppresses tumor growth in a murine human melanoma xenograft model. Importantly, this study excludes the possibility that the tumor-suppressive effect is due to the over-expression of TET2
itself. Rather, such an effect is due to elevated levels of 5-hmC on the genes important for key cellular processes. Moreover, we demonstrated that a forced increase in 5-hmC in an established zebrafish melanoma model via over-expression of IDH2 wt
, but not IDH2 R172K
mutant, significantly suppressed tumor growth and prolonged tumor-free survival. These data suggest that IDH2,
but not IDH1,
is specifically down-regulated in melanomas and that the wt IDH2
acts as a putative tumor suppressor in the zebrafish melanoma model although the IDH family of enzymes have been considered candidate oncogenes in various tumors (Ward et al., 2010
; Wrzeszczynski et al., 2011
). Nonetheless, while the nature of the putative tumor suppressor function of IDH2
in melanoma and other tumor types warrants future investigations, a dramatic increase in the global 5-hmC level is the most pronounced epigenetic alteration observed both in the TET2
over-expressing melanoma animal models. Thus, these data demonstrate that high levels of 5-hmC and the appropriate 5-hmC landscape in the epigenome of melanocytes and nevus cells, both potential melanoma progenitors, play a role in preserving the integrity of these indolent cells and in preventing melanoma initiation and progression. Our study supports the novel concept that an elevated level of 5-hmC can serve as a distinctive epigenetic molecular beacon for the reversal of an aggressive melanoma phenotype. It further indicates that particular TET and IDH family enzymes have putative tumor suppressor functions in melanoma progression, and spontaneously targeted down-regulation or inactivation of multiple key enzymes in 5-hmC generating pathway is one of the epigenetic mechanisms underlying melanoma development.
The present study also attempts to address the molecular mechanisms directly linking the gene specific 5-hmC loss to melanoma formation. Genome-wide mapping and comparative analyses of 5-mC and 5-hmC landscape in benign nevi, primary melanomas, MOCK, TET2- and TET2 M-over-expressing melanoma cells indicated that a program of genes involving various cancer pathways display significant reduction of 5-hmC in comparisons between benign nevi and melanoma, which can be reversed by over-expression of active TET2 but not inactive TET2 M. However, we did not find simple correlations between significant loss of 5-hmC and expression of associated genes because a reduced 5-hmC level is associated with both up- and down-regulated genes in melanoma compared with nevi. This is not surprising since we and others have shown complex roles of 5-hmC in gene transcription regulation in mouse ES cells (Ficz et al., 2011
; Xu et al., 2011b
). Thus, understanding the intricate relationship between the regulation of 5-hmC and associated gene transcription remains a challenge in 5-hmC biology (Cimmino et al., 2011
; Wu and Zhang, 2011
). Of note, we identified a subset of genes showing significant 5-hmC level decreases and simultaneous 5-mC level increases in melanomas compared to nevi. The strong association of this subset of genes with various cancer pathways suggests that gene-specific 5-hmC loss may partially contribute to the abnormal DNA methylation pattern in the epigenome of melanoma that has been linked to the progression of various cancers. However, there are a portion of genes showing significant reduction in 5-hmC levels but no obvious 5-mC level changes in melanomas compared to nevi, suggesting that other independent molecular mechanisms, such as 5-hmC-mediated regulation of cell division/replication, cell differentiation/senescence and/or genome/epigenome instability, may also be involved in linking the loss of 5-hmC to melanoma progression. Future studies aimed at identifying these potential 5-hmC-related mechanisms should enhance insights into 5-hmC function in melanoma formation and progression.
Finally, with melanoma as a paradigm of aggressive cancer, our study provides important insight for future functional studies of 5-hmC in cancer biology. Increasing 5-hmC levels via over-expressing TET2 reversed the genome-wide 5-hmC distribution from the global 5-hmC loss pattern in melanoma toward a benign nevus-like pattern. More importantly, the phenotype of melanoma was rescued by increasing 5-hmC levels via over-expressing either TET2 or IDH2 in animal models. Thus, “loss of 5-hmC” in melanoma progression is a fundamental epigenetic event that provides proof-of-principle that key factors in the 5-hmC generating pathway can be therapeutically targeted to restore 5-hmC in human melanoma, thus revealing new strategies for the design of melanoma treatment.