Mukta Srinivasulu

PNUTS Functions as a Proto-Oncogene by Sequestering PTEN

PTEN is a well-defined tumor suppressor gene that antagonizes the PI3K/Akt pathway to regulate a multitude of cellular processes, such as survival, growth, motility, invasiveness, and angiogenesis. While the functions of PTEN have been studied extensively, the regulation of its activity during normal and disease conditions still remains incompletely understood. In this study, we identified the protein phosphatase-1 nuclear targeting subunit PNUTS (PPP1R10) as a PTEN-associated protein. PNUTS directly interacted with the lipid-binding domain (C2 domain) of PTEN and sequestered it in the nucleus. Depletion of PNUTS leads to increased apoptosis and reduced cellular proliferation in a PTEN-dependent manner. PNUTS expression was elevated in certain cancers compared with matched normal tissues. Collectively, our studies reveal PNUTS as a novel PTEN regulator and a likely oncogene.

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.

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.

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

Abstract 1216: Analysis of genetic aberrations in squamous cell carcinoma of the oral tongue.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Squamous cell carcinoma of the oral tongue (SCCOT) is a common form of head and neck squamous cell carcinoma (HNSCC). In the last few decades, a steady increase in incidence rates of SCCOT has been reported across the world including India. SCCOT is known to be an age related disease and tobacco consumption is implicated as the main etiological agent, like other HNSCC subtypes. Recent reports have indicated an increase in incidence of SCCOT in the young and in nonsmokers. Among HNSCC, SCCOT appears to be more aggressive with respect to its clinical and biological behavior. Molecular genetic studies on SCCOT are scarce and most studies have been conducted on a small cohort of patients and/or restricted to a single/few molecular marker(s). We have conducted a multipronged molecular genetic study of SCCOT and analyzed the status of known tumorigenesis pathways including TP53, epidermal growth factor receptor (EGFR), microsatellite instability (MSI), CDKN2A, FHIT and human papilloma virus (HPV) infection in surgically resected primary tumor samples and correlated with clinocopathological variables. 67% (67/100) of SCCOT samples exhibited p53 nuclear stabilization whereas a greater proportion (87/100; 87%) exhibited elevated EGFR expression, in accordance with existing literature. Interestingly, p53 nuclear stabilization was found to be more common in young (31/39; 79.5%) than in older (35/59; 59.3%) patients (p = 0.0481). As expected, patients with p53 nuclear stabilization exhibited poorer survival. Further, PCR based mutation screening of exons 5-8 of TP53 (encoding the DNA binding domain) revealed mutations in 50% of samples exhibiting nuclear stabilization. HPV infection was observed in 11/82 (13.4%) tumors while the frequencies of MSI (12/86; 14%) and loss of heterozygosity (LoH) in CDKN2A (32.6%) and FHIT (28.4%) loci were significantly higher than previous studies. In addition, LoH at FHIT locus was significantly associated with p53 nuclear stabilization (p=0.047). Analysis of genome-wide DNA copy number alterations (CNA) using array based comparative genomic hybridization revealed unique copy number alterations validated by quantitative PCR. Exome sequencing and transcriptome profiling are currently underway to understand the pathways/genes deregulated in SCCOT, especially in samples not exhibiting p53 nuclear stabilization. Citation Format: Raju Sr Adduri, Viswakalyan Kotapalli, Neha Ak Gupta, Swarnalata Gowrishankar, Mukta Srinivasulu, Subramanyeshwar Rao, Shantveer G. Uppin, Umanath Karopadi Nayak, Mohana Vamsy Chigurupati, Sujith Chayu Patnaik, NarasimhaRaju Kalidindi, Murali D. Bashyam. Analysis of genetic aberrations in squamous cell carcinoma of the oral tongue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2013-1216

Abstract 1198: Presence of non-canonical tumorigenesis pathways in early-onset sporadic rectal cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Chromosomal instability (CIN) caused by β-Catenin dependent aberrant activation of canonical Wnt signaling or Microsatellite instability (MSI) triggered by inactivation of mismatch repair (MMR) pathway, are the two major genetic instability pathways that drive classical age-related sporadic colorectal cancer (CRC). Activation of canonical Wnt signaling and MSI are primary tumor initiating events in about 80% and 15% of late-onset CRC cases, respectively. Canonical Wnt signaling is also reported as a secondary event in tumors primarily driven by MSI. These inferences are based on seminal studies that included a disproportionately greater number of colon tumors (as compared to rectal tumors). Recent genome-wide studies have suggested colon and rectal cancer to be a single entity, though several other studies appear to indicate otherwise. Exclusive studies on rectal cancer (RC), especially the early-onset sporadic subtype, have been fewer. Despite a recent trend of increased worldwide incidence, early-onset sporadic rectal cancer (EOSRC) is not well understood. We profiled canonical Wnt, KRAS and p53 (components of the classical colorectal carcinoma progression model) and MSI status in a panel of 298 colorectal cancer samples. 41% of EOSRC samples did not harbor Wnt or MSI pathways; the high proportion of a ‘double negative’ entity was neither identified in late-onset RC samples nor in colon cancer samples. KRAS mutation frequency was also significantly lower in EOSRC (24%). Since CIN is a hallmark of canonical Wnt activation driven CRC, we profiled genome-wide DNA copy number alterations (CNAs) in microsatellite stable EOSRC samples and surprisingly identified extensive chromosomal aberrations in both Wnt active and Wnt inactive subtypes suggesting the interesting possibility of presence of CIN in the absence of canonical Wnt activation. Several CNAs were detected exclusively in Wnt inactive samples (being absent in Wnt active samples) and were validated by quantitative PCR. As expected, a few CNAs, such as an amplification detected at 17q12 (ERBB2/GRB7), were present in both subtypes. Genome-wide transcript profiling performed in parallel revealed the elevated expression of genes located within the amplifications, validated by quantitative reverse transcription PCR (Q-RT-PCR). More importantly, aberrant activation of non-canonical signaling pathways was identified in a subset of the ‘double negative’ EOSRC samples, based on unsupervised (hierarchical clustering) and supervised (significance analysis of microarrays and gene set enrichment analysis) analyses of the transcriptome data. Elevation of non-canonical pathway gene transcripts was confirmed by Q-RT-PCR. Our study has therefore revealed presence of unique tumorigenesis pathways in EOSRC samples distinct from canonical pathways that drive late-onset CRC. Citation Format: Ratheesh Raman, Viswakalyan Kotapalli, Raju SR Adduri, Vasantha Kumar Bhaskara, Swarnalata Gowrishankar, Leena Bashyam, Ajay Chaudhary, Mohana Vamsy Chigurupati, Sujith Patnaik, Mukta Srinivasulu, Regulagadda Sastry, Subramanyeshwar Rao, Anjayneyulu Vasala, NarasimhaRaju Kalidindi, Jonathan Pollack, Sudha Murthy, Murali D. Bashyam. Presence of non-canonical tumorigenesis pathways in early-onset sporadic rectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1198. doi:10.1158/1538-7445.AM2013-1198

Abstract 2151: Molecular screening of CRC from India suggests the existence of non-canonical pathway(s) of tumorigenesis

A major risk factor for Colorectal Cancer (CRC) is advanced age, with a majority of cases reported in the West being above 60 years of age. However, a significantly high proportion of CRC patients from India belong to a younger age group (less than 50 years) and a majority of them have no family history of cancer. A deregulated Wnt signaling pathway is the hallmark of CRC in the West and has been indicted in 70-80% of older patients. Another significant genetic aberration is Microsatellite Instability (MSI), which may contribute to 15-20% of the cases. In addition, the simultaneous occurrence of Wnt activation and p53 inactivation has been substantiated in a significantly high proportion (about 70%) of CRC cases. Sporadic CRC occurring in the young however has not been subjected to detailed analysis so far. In order to identify important deregulated pathways that drive tumor progression in young patients we have initiated a multipronged comparative study on CRC occurring in the young and older patients from India. Our results have revealed several unique features among Indian CRC patients. Firstly, Wnt activation and p53 inactivation appeared to co-exist in a significantly less proportion of samples (34%), indicating existence of alternate pathway(s) of tumor initiation. Secondly, a significantly low proportion (35%) of young patients appeared to harbor a deregulated Wnt signaling pathway, when compared to older patients (64%). Thirdly, we did not find a significant difference in other variables between the two patient age groups including gender, grade, MSI status, etc. Using array-based Comparative Genomic Hybridization (aCGH) and genome-wide transcript profiling, we identified several copy number alterations (CNA) that may harbor genes important for tumor progression in young patients. A novel amplification located at 17q11.2-21.1 was identified which included genes involved in the MAP kinase pathway. The present study is expected to yield invaluable insights into the molecular basis for sporadic CRC in the young. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2151.

Abstract A7: Identification of novel genetic alterations in GI tumors using whole-genome profiling

Abstracts: Frontiers in Cancer Prevention Research 2008 A7 In our previous studies, we identified novel pancreatic cancer genes using a combination of array-based comparative genomic hybridization (aCGH) and gene expression microarrays (Neoplasia, 2007 and Kwei and PLOS Genet, 2008). In the current study, we have used a multipronged approach to address two important GI cancers prevalent in India. Colorectal cancer (CRC) is a major health problem worldwide and is usually an age-related disease. A small proportion of patients present at an early age due to familial cancer syndromes. Surprisingly, there appears to be a high proportion of patients in India who present at an early age without a family background and succumb to aggressive metastatic tumors. Using immunohistochemistry, mutation screening and microsatellite instability analysis of more than 150 samples, we show that Wnt signaling, a hallmark of CRC in the West, is not a major feature in a majority of young patients in India unlike older patients. There does not appear to be a significant difference between the two categories of CRC patients in other parameters including gender, lifestyle, diet, tumor location, frequency of microsatellite instability, and Loss of Heterozygosity at the BRCAI locus. In addition, we have identified several novel mutations in the APC gene in Wnt positive tumors. Determination of genome-wide copy number alterations and transcript profiles is expected to delineate the genetic aberrations that may lead to CRC in the young in India. Squamous cell carcinoma of the esophagus (ESCC) is common in India and Asia but rare in the West, as against esophagus adenocarcinoma which is common in the West but not in India. ESCC is a poorly studied highly aggressive cancer with survival rates below 15% in most populations. We have carried out molecular analysis of about 75 ESCC samples and 26 adenocarcinoma samples using archived as well as fresh resected tumors. Results indicate that unlike the ESCC samples, a significant proportion of adenocarcinoma samples exhibited an activated Wnt signaling pathway. Inactivation of p53 however appeared to be a common event in both Esophagus cancer subtypes. Analysis of copy number alterations using array-based CGH has revealed a recurrent amplification at 10q21 in ESCC samples. The amplification harbored genes that exhibited comparably altered transcript levels, as determined by array-based transcript profiling. We are also carrying out molecular analyses of mixed adeno-squamous tumors of the esophagus; results are expected to reveal whether the squamous and adeno components have an independent origin or vice versa. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A7.