Abstract
Ras-association domain family (RASSF) proteins are encoded by numerous tumor suppressor genes that frequently become silenced in human cancers. RASSF10 is downregulated by promoter hypermethylation in cancers and has been shown to inhibit cell proliferation; however, the molecular mechanism(s) remains poorly understood. Here, we demonstrate for the first time that RASSF10 inhibits Cdk1/cyclin-B kinase complex formation to maintain stable levels of cyclin-B for inducing mitotic arrest during cell cycle. Using LC-MS/MS, live cell imaging, and biochemical approaches, we identify Nucleophosmin (NPM) as a novel functional target of RASSF10 and revealed that RASSF10 expression promoted the nuclear accumulation of GADD45a and knockdown of either NPM or GADD45a, resulting in impairment of RASSF10-mediated G2/M phase arrest. Furthermore, we demonstrate that RASSF10 is a substrate for the E3 ligase ring finger protein 2 (RNF2) and show that an NPM-dependent downregulation of RNF2 expression is critical to maintain stable RASSF10 levels in cells for efficient mitotic arrest. Interestingly, the Kaplan–Meier plot analysis shows a positive correlation of RASSF10 and NPM expression with greater gastric cancer patient survival and the reverse with expression of RNF2, suggesting that they may have a role in cancer progression. Finally, our findings provide insights into the mode of action of the RASSF10/NPM/RNF2 signaling cascade on controlling cell proliferation and may represent a novel therapeutic avenue for the prevention of gastric cancer metastasis.
Highlights
RAS oncogenes are central players in many human cancers
These results suggest that K183 may be the potential ubiquitination site in RASSF10 for proteasome-mediated degradation and further indicate that NPM may regulate a novel E3 ligase responsible for RASSF10 polyubiquitination/destabilization
The present study demonstrates that RASSF10 induces mitotic arrest to control cell cycle progression by inhibiting Cdk1–cyclin-B kinase complex formation
Summary
RAS oncogenes are central players in many human cancers. Ras regulates various physiological functions through downstream molecules known as Ras effectors [1]. Our results prompted us to hypothesize that the observed mitotic arrest might be due to RASSF10-induced NPM-dependent nuclear translocation of GADD45a followed by the alteration of Cdk1–cyclin-B kinase complex formation (Fig. S3B). To further understand whether p53 play any role in RASSF10mediated nuclear translocation of GADD45a, we performed nuclear cytoplasmic fractionation followed by Western blot analysis in p53-deficient colon cancer cell line HCT116p53−/− with RASSF10 expression under NPM knockdown conditions.
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