Human RASSF6 gene is registered as one of the Ras-association domain family in the databank. RASSF6 was first referred in the paper reporting the association of SNPs in RASSF6 with susceptibility to respiratory syncytial virus bronchiolitis (Hull et al. 2004). The researchers discussed based on the structural feature and the potential function of RASSF6 in the regulation of cytoskeletal structure, gene expression, and cell-cell interactions. Thereafter, RASSF6 was identified as an interactor with a junctional protein named membrane-associated guanylate kinase with an inverted arrangement of protein-protein interaction domains (MAGI1) (Ikeda et al. 2007). RASSF6 was also identified from the EST database by tblastn using the Ras-association (RA) domain of RASSF1 (Allen et al. 2007).
Structure of RASSF6
RASSF6 and Human Cancers
It was discussed that in silico analysis predicts no CpG island in the vicinity of the first exons of RASSF6 variant 1 and variant 2 (Richter et al. 2009). However, later, CpG island methylation was detected in acute lymphocytic leukemia, chronic lymphocytic leukemia, neuroblastoma, metastatic melanoma, and gastric cardia adenocarcinoma (Hesson et al. 2009; Shinawi et al. 2012; Djos et al. 2012; Mezzanotte et al. 2014; Guo et al. 2015). RASSF6 suppression correlates with disease progression in gastric cancer, pancreatic ductal adenocarcinoma, and gastric cardia adenocarcinoma (Guo et al. 2015; Wen et al. 2011; Ye et al. 2015). Based on these findings, RASSF6 is thought to be a prognostic marker in certain cancers. Promoter hypermethylation of RASSF6 was not observed in bladder cancer and pheochromocytoma, suggesting that RASSF6 may not be important in bladder and adrenal medulla (Meng et al. 2012; Richter et al. 2015). The forkhead transcription factor FOXP1, an oncogene of B-cell lymphoma, represses RASSF6 (van Keimpema et al. 2014). Together with that the frequency of hypermethylation of RASSF6 is very high in B-cell leukemia, RASSF6 may be a main tumor suppressor in B lymphocytes (Hesson et al. 2009).
RASSF6 and Apoptosis
RASSF6 and Cell Cycle Regulation
RASSF6 overexpression causes G1/S arrest in HCT116, U2OS, and renal cancer cells (Iwasa et al. 2013; Liang et al. 2014). The G1/S arrest partially depends on p53 (Iwasa et al. 2013) (Fig. 2). RASSF6 is implicated in the UV-induced upregulation of p21 and Btg2, which are p53 target genes, in HCT116 cells (Iwasa et al. 2013). Accordingly, with RASSF6 depletion, HCT116 cells override UV-induced G1/S arrest (Iwasa et al. 2013). Importantly, when RASSF6 is suppressed, DNA repair is blocked in HCT116 cells exposed to UV or etoposide, and polyploid cells are generated (Iwasa et al. 2013).
RASSF6 and the Hippo Pathway
The tumor suppressor Hippo pathway was identified in Drosophila as the signaling that controls organ size (Pan 2010; Yu and Guan 2013; Kodaka and Hata 2015). Loss-of-mutants of Hippo, Salvador, Warts, and Mats exhibit cell overproliferation phenotype (Justice et al. 1995; Tapon et al. 2002; Harvey et al. 2003; Pantalacci et al. 2003; Udan et al. 2003; Wu et al. 2003; Lai et al. 2005). Hippo and Warts are serine/threonine protein kinases. Hippo phosphorylates and activates Warts. Salvador and Mats promote Hippo-dependent activation of Warts. Warts phosphorylates a transcription coactivator, Yorkie, and segregates it in the cytoplasm (Huang et al. 2005). As Yorkie cooperates with a transcription factor, Scaffold, in the nucleus and upregulates cell cycle-promoting and antiapoptotic genes, the deregulation of the Hippo pathway results in hyperactivation of Yorkie and leads to tumorigenesis. Drosophila RASSF (dRASSF) binds to Hippo and blocks the interaction of Hippo and Salvador, because both dRASSF and Salvador bind to the SARAH domain of Hippo (Polesello et al. 2006). Thus, at a glance, dRASSF antagonizes the Hippo pathway. RASSF6 interacts with the SARAH domain of mammalian Ste20-like kinases (MST1 and MST2), mammalian homologues of Hippo (Ikeda et al. 2009). Like dRASSF, RASSF6 inhibits MST kinases (Ikeda et al. 2009). The precise mechanism is not yet clear, but it is likely that RASSF6 blocks the oligomerization of MST kinases and inhibits the autophosphorylation, which is necessary for MST kinase activation (Rawat and Chernoff 2015). Reciprocally, MST kinases block RASSF6-induced apoptosis (Ikeda et al. 2009). When cells are exposed to stimuli, such as okadaic acid treatment, RASSF6 and MST kinases are dissociated (Ikeda et al. 2009). Consequently, the Hippo pathway is activated and RASSF6-induced apoptosis is triggered. To put this another way, RASSF6 and the Hippo pathway are resting while cells are healthy. But when cells are exposed to stress, RASSF6 and the Hippo pathway are simultaneously activated and play a tumor suppressor role in a parallel manner (Fig. 2).
RASSF6 and Other Signalings
RASSF6 suppresses serum-induced NFκ reporter activity in A549 cells (Allen et al. 2007) (Fig. 2). In renal cancer cells, RASSF6-induced increase of p21 is blocked by SP600125, an inhibitor of c-jun N-terminal kinase (JNK), suggesting that RASSF6 activates JNK (Liang et al. 2014). RASSF6 is highly expressed in differentiated 3T3-L1 cells and white adipocytes, but in 3T3-L1 cells exposed to the conditioned medium from stimulated RAW264.7 cells or TNFα, RASSF6 is suppressed (Sanada et al. 2013). The underlying mechanisms of how RASSF6 regulates NFκ and JNK and how RASSF6 expression is regulated by TNFα are currently unknown.
The frequent suppression of RASSF6 in human cancer and the correlation of low RASSF6 expression with poor clinical prognosis prove the importance of RASSF6 as a tumor suppressor. The accumulating findings suggest that RASSF6 together with the Hippo pathway regulates the checkpoint to prevent genomic instability. RASSF6 induces apoptosis and cell cycle arrest through multiple pathways. Among them, p53-dependent mechanism is best characterized, but RASSF6 also suppresses p53-negative cancer cells. This observation highlights RASSF6 as a tumor suppressor with a complementary role for p53. The presence of RA domain prompts us to speculate that the primary role of RASSF6 is to antagonize Ras and that RASSF6 is particularly important in cancers harboring Ras mutants. However, it is unreasonable to posit that RASSF6 becomes functional only after Ras is mutated. RASSF6 may play a certain role in the Ras signaling in normal cells. RASSF6 is upregulated by TNFα and suppresses NFκ. It also suggests that RASSF6 mediates a negative feedback in TNFα-NFκ pathway and that the deregulation of RASSF6 is implicated in inflammatory diseases. It is intriguing to clarify the physiological roles of RASSF6 and their pathological roles in noncancer diseases.
- Richter AM, Zimmermann T, Haag T, Walesch SK, Dammann RH. Promoter methylation status of Ras-association domain family members in pheochromocytoma. Front Endocrinol (Lausanne). 2015;6:21.Google Scholar