Abstract 2500: Identification and characterization of a bipartite nuclear localization signal reveals non-canonical 'oncogenic' role for cytoplasm-restricted ARID1B

The AT-rich interaction domain-containing protein 1B (ARID1B/ BAF250b) is a component of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex that functions as the primary chromatin remodeler during ontogeny and adult life. ARID1B is classified as a tumor suppressor in melanoma as well as in cancers of the uterus, ovary, lung, breast, colon and pancreas. Though human ARID1B is a nuclear protein, the molecular basis of its nuclear import is not understood. Using protein truncation analysis, in silico prediction and sequence comparison within forty three metazoan species, we identified a putative bipartite Nuclear localization signal (NLS) sequence, further confirmed by immunofluorescence analyses and cell fractionation followed by immunoblotting. An immunoprecipitation-mass spectrometry based screen for identifying ARID1B interacting proteins revealed members of the importin family (IPO5, KPNA2 and KPNB1). In addition, ARID1B nuclear import was inhibited by Importazole (general importin β inhibitor). Transcription activation function of the cytoplasm-restricted ARID1B-NLS mutant, as measured by quantifying CDKN1A and CDKN1B transcript and protein levels as well as promoter activity following ectopic expression in cancer cell lines, was significantly compromised when compared to wild type ARID1B. The NLS-mutant was also restricted in its ability to induce senescence. Surprisingly however, cytoplasmic localization of ARID1B appeared to result in an ‘oncogenic9 gain of function as it boosted cell proliferation, promoted ERK signaling and activated β-catenin/TCF-LEF function. A detailed analysis of the oncogenic role of cytoplasm-restricted ARID1B is ongoing. A careful scrutiny of various cancer specific somatic mutation databases revealed several mutations located within the ARID1B NLS sequence. In addition, ARID1B immunohistochemistry on breast cancer tissue microarrays (TMAs) revealed significant cytoplasmic localization. These observations suggest possible clinical relevance of cytoplasm-restricted ARID1B. Functional validation of NLS specific mutations and TMA analysis of other cancers is currently underway. Non-canonical oncogenic gain of function due to aberrant cytoplasmic localization has been shown previously for classical tumor suppressors such as pRB, p27, p21 and p53. Our results have revealed a novel ‘oncogenic9 facet of ARID1B resulting from its aberrant cytoplasmic localization. Citation Format: Srinivas Animireddy, Padmavathi Kavadipula, Viswakalyan Kotapalli, Swarnalata Gowrishankar, Satish Rao, Murali D. Bashyam. Identification and characterization of a bipartite nuclear localization signal reveals non-canonical 9oncogenic9 role for cytoplasm-restricted ARID1B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2500.