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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 ‘oncogenic' 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 ‘oncogenic' 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 'oncogenic' 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.

The p53 oncoprotein is a tumor suppressor that is stabilized upon various forms of cellular stresses to induce transcription of genes regulating cell cycle arrest or apoptosis. TP53 is the most frequently mutated gene in human cancers and majority of mutations are located in the region encoding the DNA binding domain compromising thereby its transcription activation ability and resulting in loss of function. Recent studies however suggest mutant p53 proteins to exhibit a gain of function property. Specific p53 missense mutations can result in an altered transcription program causing positive regulation of cell proliferation, metastasis and chemoresistance. In our previous studies, we performed comprehensive characterization of squamous cell carcinoma of the oral tongue (SCCOT) with respect to p53, EGFR, Wnt, MSI, LoH of several tumor suppressor loci and HPV status. Mutant p53 was a significant predictor of overall survival and the TP53 codon 72 Proline allele was significantly associated with SCCOT. In order to dissect the role of mutant p53 in tongue cancer, we performed genome wide DNA and RNA profiling of 26 and 40 SCCOT samples, respectively. Both mutant and wild type tumor samples appeared to exhibit comparable levels of DNA copy number alterations. Transcriptome data analyses using a combination of single sample gene set enrichment analysis and comparative marker selection revealed gene sets that could significantly distinguish p53 mutant and wild type tumor samples. Significance analysis of microarrays performed on all genes constituting the differentially enriched gene sets surprisingly identified only two genes to be upregulated in p53 mutant samples at a false discovery rate significantly lower than 10% namely TP53 itself and SMARCD1; the latter a member of the SWI/SNF chromatin remodelling complex. Elevated levels of TP53 transcript in tumors harbouring mutant p53 significantly correlated with levels of ZMAT3, itself induced by p53 and known to stabilize the TP53 transcript. In addition, the analysis revealed several known (ATF3 and others) and novel (GCHFR and others) targets of wild type p53. Differential expression of all targets was validated in additional tongue cancer samples. Ectopic expression of certain (but not all) p53 mutant proteins in p53 null cells induced SMARCD1 (but not canonical wild type p53 targets) while expression of wild type p53 induced GCHFR, ATF3, CDKN1A, etc. (but not SMARCD1). In contrast, p53 stabilization in cells harboring wild type p53 caused elevation of GCHFR, ATF3, CDKN1A, etc., but not of SMARCD1. Validation of novel targets using promoter-luciferase constructs, chromatin immunoprecipitation PCR and a tongue cancer tissue microarray is underway. This is perhaps the first evidence from Head and Neck tumor samples for a gain of function activity of mutant p53. Thus wild type and mutant p53 may support distinct transcription programs in tongue cancer. Citation Format: Raju SR Adduri, Padmavathi Kavadipula, Viswakalyan Kotapalli, Leena Bashyam, Anupama Shirke, Arun kumar Paripati, Swarnalata Gowrishankar, Mukta Srinivasulu, Mohammed Mujtaba Ali, Subramanyeshwar Rao, Snehalatha Dhagam, Mohana Vamsy Chigurupati, Shantveer G. Uppin, Vijaya Tourani, Murali D. Bashyam. Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3692.