Background: There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. Results: Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. Conclusion: The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.

Background: The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis. Results: Frequent methylation of the RASSF2 and RASSF5A CpG island promoters in thyroid tumors was observed. RASSF2 was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. RASSF2 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for RASSF2 in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). RASSF2 promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated RASSF2 transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05). Conclusion: These results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.

The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth

Differentiation and malignant transformation of stem cells are regulated by epigenetic mechanisms. We analyzed promoter methylation and expression of the stem cell determining genes Brachyury, DPPA5, FGF4, FOXD3, LIN28, NESTIN and ZFP42 depending on the differentiation state in human mesenchymal stem cells (MSC), human embryonal carcinoma cells (ECC), and somatic tumor cells. Differentiation of MSC into osteoblasts and adipocytes was accompanied with a loss of expression of the Brachyury gene and down regulation of LIN28. Inactivation of Brachyury was associated with progressive methylation of its CpG island promoter. In ECC promoter methylation of stem cell markers was more frequent in the differentiated subgroup (71%) compared to undifferentiated ECC (29%) and this was associated with down regulation of Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42. DPPA5 was methylated and NESTIN was unmethylated in most tumor cells. In somatic tumor cells, methylation of stem cell markers (Brachyury, DPPA5, FGF4, FOX...

Epigenetic inactivation of tumor suppressor genes (TSG) is a fundamental event in the pathogenesis of human cancer. This silencing is accomplished by aberrant chromatin modifications including DNA hypermethylation of the gene promoter. One of the most frequently hypermethylated TSG in human cancer is the Ras Association Domain Family 1A (RASSF1A) gene. Aberrant methylation of RASSF1A has been reported in melanoma, sarcoma and carcinoma of different tissues. RASSF1A hypermethylation has been correlated with tumor progression and poor prognosis. Reactivation of epigenetically silenced TSG has been suggested as a therapy in cancer treatment. In particular, natural compounds isolated from herbal extracts have been tested for their capacity to induce RASSF1A in cancer cells, through demethylation. Here, we review the treatment of cancer cells with natural supplements (e.g., methyl donors, vitamins and polyphenols) that have been utilized to revert or prevent the epigenetic silencing of RASSF1A. Moreover, we specify pathways that were involved in RASSF1A reactivation. Several of these compounds (e.g., reseveratol and curcumin) act by inhibiting the activity or expression of DNA methyltransferases and reactive RASSF1A in cancer. Thus natural compounds could serve as important agents in tumor prevention or cancer therapy. However, the exact epigenetic reactivation mechanism is still under investigation.