Viswakalyan Kotapalli

ARID1B, a member of the human SWI/SNF chromatin remodeling complex, exhibits tumour-suppressor activities in pancreatic cancer cell lines

Background:The human ATP-dependent SWItch/sucrose nonfermentable (SWI/SNF) complex functions as a primary chromatin remodeler during ontogeny, as well as in adult life. Several components of the complex have been suggested to function as important regulators of tumorigenesis in various cancers. In the current study, we have characterised a possible tumour suppressor role for the largest subunit of the complex, namely the AT-rich interaction domain 1B (ARID1B).Methods:We performed Azacytidine and Trichostatin A treatments, followed by bisulphite sequencing to determine the possible DNA methylation-induced transcription repression of the gene in pancreatic cancer (PaCa) cell lines. Functional characterisation of effect of ARID1B ectopic expression in MiaPaCa2 PaCa cell line, which harboured ARID1B homozygous deletion, was carried out. Finally, we evaluated ARID1B protein expression in pancreatic tumour samples using immunohistochemistry on a tissue microarray.Results:ARID1B was transcriptionally repressed due to promoter hypermethylation, and ectopic expression severely compromised the ability of MiaPaCa2 cells to form colonies in liquid culture and soft agar. In addition, ARID1B exhibited significantly reduced/loss of expression in PaCa tissue, especially in samples from advanced-stage tumours, when compared with normal pancreas.Conclusion:The results therefore suggest a possible tumour-suppressor function for ARID1B in PaCa, thus adding to the growing list of SWI/SNF components with a similar function. Given the urgent need to design efficient targeted therapies for PaCa, our study assumes significance.

Evidence for possible non-canonical pathway(s) driven early-onset colorectal cancer in India.

Two genetic instability pathways viz. chromosomal instability, driven primarily by APC mutation induced deregulated Wnt signaling, and microsatellite instability (MSI) caused by mismatch repair (MMR) inactivation, together account for >90% of late‐onset colorectal cancer (CRC). Our understanding of early‐onset sporadic CRC is however comparatively limited. In addition, most seminal studies have been performed in the western population and analyses of tumorigenesis pathway(s) causing CRC in developing nations have been rare. We performed a comparative analysis of early and late‐onset CRC from India with respect to common genetic aberrations including Wnt, KRAS, and p53 (constituting the classical CRC progression sequence) in addition to MSI. Our results revealed the absence of Wnt and MSI in a significant proportion of early‐onset as against late‐onset CRC in India. In addition, KRAS mutation frequency was significantly lower in early‐onset CRC indicating that a significant proportion of CRC in India may follow tumorigenesis pathways distinct from the classical CRC progression sequence. Our study has therefore revealed the possible existence of non‐canonical tumorigenesis pathways in early‐onset CRC in India.

TP53 Pro72 Allele Is Enriched in Oral Tongue Cancer and Frequently Mutated in Esophageal Cancer in India

Purpose The tumor suppressor p53 is known to be inactivated frequently in various cancers. In addition, germline polymorphisms in TP53 are known to affect protein function and influence risk of developing different types of cancers. In this study, we analyzed the association of TP53 Pro72Arg polymorphism with squamous cell carcinoma of oral tongue (SCCOT) and esophagus (ESCC) in India. Methods We assessed the distribution of TP53 Pro72Arg polymorphism in one hundred and fifteen and eighty two SCCOT and ESCC patients, respectively, with respect to one hundred and ten healthy controls from the same population. In addition, we analyzed association of the polymorphism with several clinico-pathological and molecular parameters. Results Pro72 allele was significantly enriched in SCCOT patients compared to the healthy control group but neither allele was enriched in ESCC. Interestingly, Pro72 allele was preferentially mutated in ESCC which was confirmed by analysis of samples heterozygous for Pro72Arg. Conclusions Our study revealed the association of Pro72 allele with SCCOT suggesting the effect of this polymorphism on SCCOT risk. Preferential mutation of Pro72 allele exclusively in ESCC indicates the need for further studies to understand the tissue specific effect of p53 polymorphism.

P53 nuclear stabilization is associated with FHIT loss and younger age of onset in squamous cell carcinoma of oral tongue

BackgroundSquamous cell carcinoma of tongue (SCCT) is expected to harbor unique clinico-pathological and molecular genetic features since a significant proportion of patients are young and exhibit no association with tobacco or alcohol.MethodsWe determined P53, epidermal growth factor receptor, microsatellite instability, human papilloma virus infection and loss of heterozygosity status at several tumor suppressor loci in one hundred and twenty one oral SCCT (SSCOT) samples and analyzed their association with clinico-pathological features and patient survival.ResultsOur results revealed a significantly higher incidence of p53 nuclear stabilization in early (as against late) onset SCCOT. FHIT loss was significantly associated with p53 nuclear stabilization and the association was stronger in patients with no history of tobacco use. Samples harboring mutation in p53 DNA binding domain or exhibiting p53 nuclear stabilization, were significantly associated with poor survival.ConclusionOur study has therefore identified distinct features in SCCOT tumorigenesis with respect to age and tobacco exposure and revealed possible prognostic utility of p53.

Distinct genetic aberrations in oesophageal adeno and squamous carcinoma

The two main oesophageal cancer subtypes namely adenocarcinoma and squamous cell carcinoma exhibit interesting clinical, pathological and geographical variations with the former being more common in the West and the latter in Asia.

Ca2+/nuclear factor of activated T cells signaling is enriched in early-onset rectal tumors devoid of canonical Wnt activation

Our previous extensive analysis revealed a significant proportion of early-onset colorectal tumors from India to be localized to the rectum in younger individuals and devoid of deregulated Wnt/β-catenin signaling. In the current study, we performed a comprehensive genome-wide analysis of clinically well-annotated microsatellite stable early-onset sporadic rectal cancer (EOSRC) samples. Results revealed extensive DNA copy number alterations in rectal tumors in the absence of deregulated Wnt/β-catenin signaling. More importantly, transcriptome profiling revealed a (non-Wnt/β-catenin, non-MSI) genetic signature that could efficiently and specifically identify Wnt− rectal cancer. The genetic signature included a significant representation of genes belonging to Ca2+/NFAT signaling pathways that were validated in additional samples. The validated NFAT target genes exhibited significantly higher expression levels than canonical Wnt/β-catenin targets in Wnt− samples, an observation confirmed in other CRC expression data sets as well. We confirmed the validated genes to be transcriptionally regulated by NFATc1 by (a) evaluating their respective transcript levels and (b) performing promoter-luciferase and chromatin immunoprecipitation assays following ectopic expression as well as knockdown of NFATc1 in CRC cells. NFATc1 and its targets RUNX2 and GSN could drive increased migration in CRC cells. Finally, the validated genes were associated with poor survival in the cancer genome atlas CRC expression data set. This study is the first comprehensive molecular characterization of EOSRC that appears to be driven by noncanonical tumorigenesis pathways.Key messagesEarly-onset sporadic rectal cancer exhibits DNA gain and loss without Wnt activation.Ca2+/NFAT signaling appears to be activated in the absence of Wnt activation.An eight-gene genetic signature distinguishes Wnt+ and Wnt− rectal tumors.NFAT and its target genes regulate tumorigenic properties in CRC cells.

Evidence for presence of mismatch repair gene expression positive Lynch syndrome cases in India

Lynch syndrome (LS), the most common form of familial CRC predisposition that causes tumor onset at a young age, is characterized by the presence of microsatellite instability (MSI) in tumors due to germline inactivation of mismatch repair (MMR) system. Two MMR genes namely MLH1 and MSH2 account for majority of LS cases while MSH6 and PMS2 may account for a minor proportion. In order to identify MMR genes causing LS in India, we analyzed MSI and determined expression status of the four MMR genes in forty eight suspected LS patient colorectal tumor samples. Though a majority exhibited MSI, only 58% exhibited loss of MMR expression, a significantly low proportion compared to reports from other populations. PCR‐DNA sequencing and MLPA‐based mutation and exonic deletion/duplication screening respectively, revealed genetic lesions in samples with and without MMR gene expression. Interestingly, tumor samples with and without MMR expression exhibited significant differences with respect to histological (mucin content) and molecular (instability exhibited by mononucleotide microsatellites) features. The study has revealed for the first time a significant proportion of LS tumors not exhibiting loss of MMR expression.

XPNPEP3 is a novel transcriptional target of canonical Wnt/β-catenin signaling

Canonical Wnt/β‐catenin signaling plays important roles in embryonic development and adult tissue regeneration while aberrant Wnt activation is the major driver of sporadic colorectal cancer (CRC). Thus, it is important to characterize the complete β‐catenin target transcriptome. We previously performed microarray‐based mRNA profiling of rectal cancer samples stratified for Wnt status. In addition to AXIN2 and EPHB2, XPNPEP3 transcripts were significantly elevated in tumors exhibiting activated Wnt/β‐catenin signaling, validated by Q‐PCR. Three different cell lines supported elevated XPNPEP3 transcript levels upon activation of Wnt signaling, confirmed using promoter‐luciferase assays. Ectopic expression of XPNPEP3 promoted tumorigenic properties in CRC cells. Immunohistochemistry on a CRC tissue microarray revealed significant correlation between β‐catenin nuclear localization and XPNPEP3 levels. More importantly, XPNPEP3 expression was upregulated compared to normal samples in published expression data sets from several cancers including CRC. Finally, XPNPEP3 expression correlated with poor survival in many cancers. Our results therefore suggest XPNPEP3 to be a transcriptional target of Wnt/β‐catenin pathway with particular significance for CRC.

Abstract 3692: Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program

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.

Abstract 1883: Clinical and molecular genetic analysis of squamous cell carcinoma of the oral tongue

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Squamous cell carcinoma of tongue (SCCT) is a common form of head and neck squamous cell carcinoma (HNSCC) in developing countries, mainly in India. In the last few decades, a steady increase in incidence rate of SCCT has been reported across the world. More importantly, though SCCT is considered to be a tobacco-related late-onset cancer, recent reports indicate an increase in incidence of SCCT in the young and in non-smokers. We analyzed the status of known tumorigenic pathways/genes including TP53, epidermal growth factor receptor (EGFR), microsatellite instability (MSI), CDKN2A, FHIT and human papilloma virus (HPV) infection in 120 surgically resected primary oral SCCT (SCCOT) samples and correlated with clinico-pathological variables and disease specific survival. 78 of 121 (65%) samples exhibited p53 nuclear stabilization confirming earlier reports. Interestingly, p53 nuclear stabilization was more common in young (36/46; 78%) than in older (44/75; 56%) patients (p = 0.0184). Further, PCR based mutation screening of exons 5-8 (encoding the DNA binding domain of p53) revealed mutations in ten of nineteen samples (52.6%) that exhibited p53 nuclear stabilization and in three of fifteen tumors (20%) that did not. We identified a novel 33bp deletion, c.616-648del33, located in exon 5 in a p53 positive tumor from a chronic tobacco chewer. Case control analysis revealed that Proline at TP53 codon 72 increased the risk of SCCOT. Majority of samples (97/121; 80%) exhibited significant EGFR expression though HPV infection was rare (14/106; 13%). MSI was observed in 14/106 (13%) samples, a frequency higher than reported for other populations. Loss of Heterozygosity (LOH) was more frequently observed in CDKN2A (28%) and FHIT (26%). In addition, LOH at FHIT locus was significantly associated with p53 nuclear stabilization (p = 0.0508), especially in non-smokers. A significant difference in survival rate between p53 positive and negative group (p = 0.0056) (Hazard ratio 2.5595) was observed. Though associated with p53 stabilization, FHIT loss did not exhibit significant effect on patient survival. Interestingly, patients exhibiting p53 nuclear stabilization as well as FHIT loss exhibited worse survival. We performed genome-wide DNA copy number and transcript profiling in several SCCOT samples. Interestingly, there was no significant difference in extent and profile of chromosomal instability in p53 positive and negative tumors. Amplifications were detected at chromosomal regions 3q26.1 (PIK3CA), 5p, 8q22 (MYC, RUNX1T1), 11q13 (CCND1) and 20q13 (HNFα). Genome-wide transcript profiling identified novel pathways that appear to drive tumorigenesis in tumors not exhibiting p53 inactivation. Our comprehensive analysis has therefore revealed important insights into the molecular basis for SCCOT and identified prognostic indicators in patients not associated with tobacco use. Citation Format: Raju S. Adduri, Viswakalyan Kotapalli, Rajender K K, Swarnalata Gowrishankar, Saumyadipta Pyne, Mukta Srinivasulu, Subramanyeshwar Rao, Shantveer G. Uppin, Mohammed Mujtaba Ali, Umanath K. Nayak, Snehalatha Dhagam, Mohana Vamsy Chigurupati, Murali D. Bashyam. Clinical and molecular genetic analysis of squamous cell carcinoma of the oral tongue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1883. doi:10.1158/1538-7445.AM2014-1883