BackgroundThe 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.ResultsFrequent 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).ConclusionThese results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.

The Ras association domain family 1A (RASSF1A) tumor suppressor encodes a Sav-RASSF-Hpo domain (SARAH), which is an interaction domain characterized by hWW45 (dSAV) and MST1/2 (dHpo). In our study, the interaction between RASSF1A and RASSF1C with MST1 and MST2 was demonstrated and it was shown that this interaction depends on the SARAH domain. SARAH domain-deleted RASSF1A had a similar growth-reducing effect as full-length RASSF1A and inhibited anchorage independent growth of the lung cancer cell lines A549 significantly. In cancer cells expressing the SARAH deleted form of RASSF1A, reduced mitotic rates (P = 0.001) with abnormal metaphases (P < 0.001) were observed and a significantly increased rate of apoptosis was found (P = 0.006) compared to full-length RASSF1A. Although the association with microtubules and their stabilization was unaffected, mitotic spindle formation was altered by deletion of the SARAH domain of RASSF1A. In summary, our results suggest that the SARAH domain plays an important role in regulating the function of RASSF1A.

The glycoprotein hormone erythropoietin (EPO) is a key regulator in the production of red blood cells. EPO is produced mainly in the embryonic liver and kidney of adults. Other organs are also known to express varying amounts of EPO. In our study, we have analyzed the epigenetic regulation of EPO in human cancer cell lines by DNA methylation assays, chromatin immunoprecipitation, RT-PCR, and promoter analysis under different growth conditions. Moreover, the growth-related effects of ectopic EPO expression were analyzed in a head and neck cancer cell line. We found frequent DNA hypermethylation of the CpG island promoter and enhancer of EPO in different cancer cell lines. Aberrant methylation of EPO promoter was observed in primary lung, head and neck, breast, and liver cancers. Hypermethylation of EPO was associated with a decreased expression of EPO in cancer cells. Treatment of cancer cell lines with 5-aza-2'-deoxycytidine (Aza), an inhibitor of DNA methylation, reactivated EPO expression under hypoxia. In contrast, in the liver cancer cell line HepB3, the EPO promoter was unmethylated, and a high EPO expression was observed independently of Aza treatment. Moreover, in vitro hypermethylation of the EPO promoter and enhancer reduced expression of a reporter gene under normoxia and hypoxia. Induction of EPO under hypoxia was accompanied by increased histone H3 acetylation and reduced histone H3 lysine 9 trimethylation. In a head and neck cancer cell line, which exhibited low EPO levels, ectopic expression of EPO significantly enhanced proliferation under normoxia and hypoxia. In summary, we show that hypermethylation of regulatory sequences of EPO is frequently observed in tumors and that this aberrant methylation induces epigenetic silencing of EPO in cancer cells.

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