Novel oncogenic transcriptional targets of mutant p53 in esophageal squamous cell carcinoma.

Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common "hotspot" p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, "non-hotspot" p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic-expression of certain "non-hotspot" mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription-quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild-type and several mutant p53 forms followed by RT-qPCR, chromatin affinity-purification (ChAP), and promoter-luciferase assays indicated the exclusive recruitment of p53 mutants-P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of "non-hotspot" mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor-specific p53 mutations.

Aberrant cytoplasmic localization of ARID1B activates ERK signaling and promotes oncogenesis.

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 (NLS). 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 for ARID1B, as evidenced from several cell line- and mouse xenograft-based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF (RAF1) and PPP1CA causing stimulation of RAF-ERK signaling and ß-catenin (CTNNB1) 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. Furthermore, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1 and ERK2 (also known as MAPK3 and MAPK1) and of ß-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.This article has an associated First Person interview with the first author of the paper.

Exome sequencing identifies ARID2 as a novel tumor suppressor in early-onset sporadic rectal cancer.

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.

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

Canonical Wnt/ß-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 ß-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/ß-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 ß-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/ß-catenin pathway with particular significance for CRC.

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/ß-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/ß-catenin signaling. More importantly, transcriptome profiling revealed a (non-Wnt/ß-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/ß-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 MESSAGES: Early-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.

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.

BACKGROUND: Squamous 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. METHODS: We 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. RESULTS: Our 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. CONCLUSION: Our study has therefore identified distinct features in SCCOT tumorigenesis with respect to age and tobacco exposure and revealed possible prognostic utility of p53.

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.

Distinct genetic aberrations in oesophageal adeno and squamous carcinoma.

BACKGROUND: 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. MATERIALS AND METHODS: We evaluated status of p53, EGFR, Wnt and HPV in addition to microsatellite instability and loss of heterozygosity of several chromosomal loci in the two oesophageal cancer subtypes from India. The comparative analysis was extended to two oesophageal adenosquamous mixed cancer samples. RESULTS: Our results reveal a high frequency of EGFR overexpression in ESCC as against EAC, while Wnt activation was a significantly more common event in EAC as against ESCC. Frequencies of p53 perturbations were not significantly different in the two subtypes. Interestingly, the EGFR and Wnt status in adenocarcinoma and squamous components of the two oesophageal adenosquamous cancer samples were identical to primary tumours. In addition, no common molecular aberration (including instability and loss of heterozygosity) in several microsatellites was detected in DNA isolated from the two components in both adenosquamous cancer samples. CONCLUSIONS: Our results reveal the presence of distinct aberrations in oesophageal adenocarcinoma and squamous cell carcinoma which are replicated in the respective components of adenosquamous cancers. The study therefore suggests perhaps an independent origin of the two components of oesophageal adenosquamous mixed cancer.