Tumor Suppressor Ras Association Domain Family 5 (RASSF5/NORE1) Mediates Death Receptor Ligand-induced Apoptosis

Abstract

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 ...

Netrin-1 exerts oncogenic activities through enhancing Yes-associated protein stability

“…4), the regulation of the upstream signaling of MST1/2 by netrin-1 is not excluded. Moreover, RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands (31). Thus, netrin-1, as the ligand of dependence receptors, through up-regulation of YAP activity, may exert the antagonistic action against RASSF.…”

RASSF tumor suppressor gene family: Biological functions and regulation

“…However, cells from the Rassf5a À/À mice were resistant to TNFa and TRAIL-dependent cell death and reduced activation of JNK kinases in response to TNFR stimulation failed to activate MST1 in vivo [80]. In line with the role of RASSF5 as a tumor suppressor, K-Ras transfected mouse embryonic fibroblasts developed a significant amount of tumors when injected into immune compromised mice to support the role of RASSF5 in restricting excessive growth [80]. Recently it was demonstrated that RASSF5A plays a role in developing the nervous system [83].…”

“…The C57BL/6-Rassf5a À/À mice were just recently generated but do not have an overt phenotype nor evidence of tumor formation as they age [80]. However, cells from the Rassf5a À/À mice were resistant to TNFa and TRAIL-dependent cell death and reduced activation of JNK kinases in response to TNFR stimulation failed to activate MST1 in vivo [80].…”

“…RASSF5A has been shown to associate with cytoskeletal proteins through its RA domain and promote growth suppression via the ERK pathway [79]. In addition, RASSF5A can complex with MST1 kinase upon TNF-a and TNF-related apoptosis-inducing ligand (TRAIL) stimulation in order to drive apoptosis [80]. RASSF5A can also heterodimerize with RASSF1A via N-terminal interactions and RASSF5 can associate with Ras-like GTPases to promote cell death [81].…”

“…Required for apoptosis and growth inhibition [8] Inhibits MST2 activity [93] Modulates the MAP kinase signal downstream of the Ras signal [77] K-Ras [8] ST1, MST2 [177] MST1 [75] Not available 5A (Nore1A) 1q32 47.1 kDa Suppresses tumour growth via apoptosis induction or cell cycle delay [7,82] Regulates microtubule formation [182] Induces degradation of HIPK1 oncoprotein [183] Required for the TNFa mediated apoptosis and full activation of MST1 [80] Ras, Carma1 [182,184] MST1 [43,177] MST2 [177] tubulin, Aurora A [182] Mdm2 [183] Itch [185] Yes [80] resistant to TNFa-induced apoptosis, fail to activate Mst1 in vivo 5C (Nore1B, RAPL) 1q32 30.4 kDa…”