Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-␣ and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-␣-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-␣-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-␣-and TNF-related apoptosis-inducing ligandmediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-␣-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.A small family of genes termed RASSF (Ras association domain family) has been recently described, the members of which are characterized by the presence of a Ras association (RA) 4 domain and a novel motif named the SARAH (Salvador, Rassf, Hippo) domain at the C terminus (reviewed in Refs. 1-3). Among the members of the RASSF family, RASSF1 and RASSF5 (also known as NORE1, for novel Ras effector 1) share the closest homology, displaying 49% identity (66% similarity) at the protein level, and are frequently inactivated by CpG hypermethylation in human cancer cell lines and primary tumors (4 -6). RASSF1 has been studied extensively (reviewed in Ref. 7) and shown to play important roles in mitosis (8), microtubule and genomic stability (9 -11), apoptosis (12, 13), and cell cycle (14), which are consistent with its function as a tumor suppressor. However, not much is known about the physiological roles of RASSF5.RASSF5 encodes multiple isoforms because of dual promoter usage and alternative splicing (15). The longest isoform, designated RASSF5A (NORE1A), is transcribed from the 5Ј-most promoter region containing CpG island and encodes a 418-amino acid protein containing the cysteine-rich diacylglycerol/ phorbol ester-binding domain (also called protein kinase C conserved region 1, C1), the RA domain, and the C-terminal SARAH domain. Through alternative splicing, another isoform RASSF5B (NORE1A␤), lacking the SARAH domain is produced. The shortest isoform, RASSF5C (NORE1B, also called RAPL), lacking the N-terminal C1 domain, is produced from a downstream CpG-containing ...

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.