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Colorectal cancer (CRC) appears to arise from sequential genetic lesions in tumor suppressor genes (APC, SMAD4 and TP53) and oncogenes (KRAS) leading to the classical adenoma to carcinoma progression. Biallelic APC inactivating genetic aberrations are detected in about 70% of early microadenomas implicating APC inactivation as the first genetic hit in CRC. APC is an essential protein of the Wnt ‘destruction complex’; APC inactivation is believed to cause disruption of the complex allowing stabilization and nuclear translocation of β-catenin, resulting in transcriptional activation of cancer-promoting genes. Here, we provide evidence for a surprising lack of correlation between APC mutation and β-catenin nuclear localization in early-onset sporadic rectal cancer samples. β-catenin nuclear localization and APC mutation were validated from serial FFPE sections representing same tumor regions. Several factors including status of KRAS mutation could not explain the absence of β-catenin nuclear localization in tumor samples harboring APC mutation. This lack of correlation was validated in CRC cell lines harboring various APC mutations. Our results provide evidence directly from tumor samples for possible non-canonical role(s) for mutant APC.

Early-onset sporadic rectal cancer (EOSRC) is a unique and predominant colorectal cancer (CRC) subtype in India. In order to understand the tumorigenic process in EOSRC, we performed whole-exome sequencing of 47 microsatellite stable EOSRC samples. Signature 1 was the predominant mutational signature in EOSRC, as previously shown in other CRC exome studies. More importantly, we identified TP53, KRAS, APC, PIK3R1, SMAD4 and ZNF880 as significantly mutated (q 0.1) and ARID1A and ARID2 as near-significantly mutated (restricted hypothesis testing; q 0.1) candidate drivers. Unlike the other candidates, the tumorigenic potential of ARID2, encoding a component of the SWI/SNF chromatin remodeling complex, is largely unexplored in CRC. shRNA-mediated ARID2 knockdown performed in different CRC cell lines resulted in significant alterations in transcript levels of cancer-related target genes. More importantly, ARID2 knockdown promoted several tumorigenic features including cell viability, proliferation, ability to override contact inhibition of growth, and migration besides significantly increasing tumor formation ability in nude mice. The observed gain in tumorigenic features was rescued upon ectopic expression of wild type but not mutant ARID2. Analyses of the TCGA pan-cancer dataset revealed several modes of ARID2 inactivation and of the CRC dataset revealed poorer survival in patients with ARID2 alterations. We therefore propose ARID2 as a novel tumor suppressor in CRC.

Early-onset sporadic rectal cancer (EOSRC) is a unique and predominant colorectal cancer (CRC) subtype in India. In order to understand the tumorigenic process in EOSRC, we performed whole exome sequencing of 47 microsatellite stable EOSRC samples. Signature 1 was the predominant mutational signature in EOSRC, as previously shown in other CRC exome studies. More importantly, we identifiedTP53, KRAS, APC, PIK3R1andSMAD4as significantly mutated (qARID1AandARID2as near-significantly mutated (restricted hypothesis testing; qARID2, encoding a component of the SWI/SNF chromatin remodeling complex, is largely unexplored in CRC. shRNA mediatedARID2knockdown performed in two different CRC cell lines resulted in significant alterations in transcript levels of cancer-related target genes. More importantly,ARID2knockdown promoted several tumorigenic features including cell viability, proliferation, ability to override contact inhibition of growth, and migration besides significantly increasing tumor formation ability in nude mice. The observed gain in tumorigenic features were rescued upon ectopic expression ofARID2. Analyses of the TCGA CRC dataset revealed poorer survival in patients withARID2alterations. We therefore proposeARID2as a novel tumor suppressor in CRC.

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. We employed in silico prediction, intracellular fluorescence and cellular fractionation based subcellular localization analyses to identify the ARID1B nuclear localization signal. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised in its canonical transcription activation and tumor suppressive functions, as expected. Surprisingly however, cytoplasmic localization appeared to induce a gain of oncogenic function in ARID1B as evidenced from several cell line and mouse xenograft based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAF-ERK signaling and β-catenin transcription activity. ARID1B harboring NLS mutations derived from tumor samples also exhibited aberrant cytoplasmic localization and acquired a neo-morphic oncogenic function via activation of RAF-ERK signaling. Further, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1/2 and β-catenin as well as with advanced tumor stage and lymph node positivity in human primary pancreatic tumor tissues. ARID1B therefore promotes oncogenesis through cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. Immunohistochemistry on a pancreatic cancer tissue microarray revealed significant ARID1B cytoplasmic localization that correlated with advanced tumor stage and lymph node positivity. Identification of the nuclear localization signal (NLS) using in silico prediction and subcellular localization studies facilitated evaluation of a possible cytoplasmic function for ARID1B. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised to regulate transcription activation and tumor suppression functions, as expected. Surprisingly however, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAS-RAF-ERK signaling and β-catenin transcription activity in pancreatic cancer cells. More importantly, cytoplasmic ARID1B resulted in an induction of cell growth and migration in pancreatic cancer cell lines that was dependent on ERK signaling and caused increased tumorigenesis in nude mice. NLS peptides representing mutations identified from pancreatic cancer samples exhibiting ARID1B cytoplasmic localization or curated from cancer somatic mutation database were significantly compromised to effect nuclear localization of a reporter protein. ARID1B cytoplasmic localization correlated significantly with active forms of ERK and β-catenin in primary pancreatic tumor samples. ARID1B may therefore promote oncogenesis through non-canonical cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

Though primarily a tumor suppressor, TP53 harboring specific missense mutations located in the region encoding the DNA binding domain exhibits a gain of function by transcriptional activation of oncogenes. We performed microarray-based messenger RNA profiling of squamous cell carcinoma of the oral tongue (SCCOT) and identified significant elevation of SMARCD1 in samples exhibiting p53 nuclear stabilization. Activation of SMARCD1 by mutant p53 was confirmed by evaluation of additional tongue cancer samples as well as The Cancer Genome Atlas expression datasets. SMARCD1 knockdown in HNSCC cells resulted in a significant reduction in several tumorigenic characteristics including cell viability, ability to form colonies in liquid and solid media and cell migration. We identified significantly increased SMARCD1 transcript levels in tumor versus matched normal samples in SCCOT as well as in other cancer types. Increased SMARCD1 expression predicted poor survival in HNSCC tumors harboring missense p53 mutations. Our results suggest SMARCD1 to be a novel transcriptional target of mutant p53.

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.