Ras association domain family members (RASSFs) are non-enzymatic Ras effectors, controlling key signaling events. RASSF10, a recent member of this family, is downregulated by promoter hyper-methylation in a wide range of cancers, shown to inhibit cell proliferation and induce apoptosis but the underlying molecular mechanism remains elusive. The current study suggests that RASSF10 inhibits cyclin-B/Cdk1kinase formation and arrests cells at mitotic phase of the cell division cycle. To elucidate the downstream targets of RASSF10, we adopted the proteomics approach. Two-dimensional electrophoresis coupled with tandem mass spectrometry revealed Nucleophosmin (NPM), known to translocate GADD45a to the nucleus and thereby regulate G2/M phase, as a novel functional target of RASSF10. Results from our study indicated an increase in nuclear translocation of GADD45a in the presence of RASSF10 and cytoplasmic retention with NPM knockdown. Interestingly, NPM or GADD45a depletion resulted in abrogation of RASSF10 mediated mitotic arrest.
Surprisingly, NPM was also found to be critical for maintaining RASSF10 protein cellular levels at post-translational levels. Results from ubiquitination assay indicated that NPM down modulates ubiquitination status of RASSF10 protein. Screening of the expression pattern of a panel of E3 ligases using RT-qPCR suggested that NPM suppress the expression of E3 ligases RNF2 to stabilize RASSF10 protein levels in cells. Together, these data suggest that RASSF10 induces mitotic arrest via NPM and there also exists a positive feedback loop between NPM and RASSF10. Additionally, Kaplan-Meier plot analysis showed existence of a positive correlation of RASSF10 and NPM expression with better survival of gastric cancer patients and the reverse with expression of RNF2, suggesting their role in cancer progression. Finally, these findings demonstrate that a novel RASSF10/NPM/RNF2 signaling cascade regulates gastric cancer progression and may act as potential target for the development of anti-cancer therapeutics to control gastric cancer metastasis.
Publications:
1. Ch Naga Padma Lakshmi,. Ananthi S, Sebastian Raja J, Mahalingam S Molecular basis for RASSF10/NPM/RNF2 feedback cascade-mediated regulation of gastric cancer cell proliferation. Journal of Biological Chemistry, 2021 Aug; 297(2):100935. doi: 10.1016/j.jbc.2021.100935.

2. Sivagnanam Ananthi, Ch Naga Padma Lakshmi, Mahalingam S.Global quantitative proteomics reveal deregulation of Cytoskeletal and Apoptotic signaling proteins in Oral tongue squamous cell carcinoma. Scientific reports. 2018 Jan 25, 8(1), 1567. doi: 10.1038/s41598-018-19937-3.
3. Rehna Krishnan, Mariappan Murugiah, Naga Padma Lakshmi and Sundarasamy Mahalingam. Guanine Nucleotide Binding Protein like-1 (GNL1) Promotes Cancer Cell Proliferation and Survival through AKT/p21CIP1 Signaling Cascade. Molecular Biology of Cell. 2020 31(26), pp.2904–2920. doi: 10.1091/mbc.E20-04-0267.