No abstract

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

The Ras association domain family (RASSF) comprises a group of tumor suppressors that are frequently epigenetically inactivated in various tumor entities and linked to apoptosis, cell cycle control and microtubule stability. In this work, we concentrated on the newly identified putative tumor suppressor RASSF10. Methylation analysis reveals RASSF10 promoter hypermethylation in lung cancer, head and neck (HN) cancer, sarcoma and pancreatic cancer. An increase in RASSF10 methylation from normal tissues, primary tumors to cancer cell lines was observed. Methylation was reversed by 5-aza-2'-deoxycytidine treatment leading to reexpression of RASSF10. We further show that overexpression of RASSF10 suppresses colony formation in cancer cell lines. In addition, RASSF10 is upregulated by cell–cell contact and regulated on promoter level as well as endogenously by forskolin, protein kinase A (PKA) and activator Protein 1 (AP-1), linking RASSF10 to the cAMP signaling pathway. Knockdown of the AP-1 member JunD interfered with contact inhibition induced RASSF10 expression. In summary, we found RASSF10 to be epigenetically inactivated by hypermethylation of its CpG island promoter in lung, HN, sarcoma and pancreatic cancer. Furthermore, our novel findings suggest that tumor suppressor RASSF10 is upregulated by PKA and JunD signaling upon contact inhibition and that RASSF10 suppresses growth of cancer cells.

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.