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.

PurposeThe 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.MethodsWe 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.ResultsPro72 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.ConclusionsOur 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.

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. We had previously identified SMARCD1 as an oncogenic transcriptional target of rare non-hotspot p53 mutants detected from squamous cell carcinoma of the oral tongue (SCCOT). We now report the characterization of non-hotspot p53 mutations from ESCC. In-vitro tumorigenic assays performed following ectopic-expression of 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 due to their previously reported pro-oncogenic roles. Reverse transcription quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumor samples 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 and Promoter-luciferase assays indicated the exclusive recruitment of p53 mutants - P190T and P278L, to the target genes leading to 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 effect of the genes was confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of rare non-hotspot mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor specific p53 mutations.

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 tumour samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumours harbouring 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 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 effect of the genes was 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.

Background: Mutations in the TP53 gene are observed at a high frequency in Esophageal Squamous Cell Carcinoma (ESCC) with a majority of alterations being of the missense type affecting residues located in the region encoding the DNA binding domain. These mutations are associated with poor patient survival. However, characterization of the transcriptional networks regulated by p53 mutant proteins is limited to only a few frequent ‘hotspot' mutants, while comparatively rarer mutants have not been characterized. Objectives: Identification and characterization of differentially expressed genes associated with p53 mutation status in ESCC tumor samples. Elucidation of the mechanism of transcriptional regulation of mutant p53 target genes. Methods: A defined set of ESCC tumor samples with known p53 mutation status was selected for this study. The ability of each 'rare' p53 mutation to modulate tumor-related phenotypes was evaluated by performing phenotypic assays such as proliferation, colony formation and migration, in head and neck cancer cell-lines. Gene expression microarray analysis was performed on 36 ESCC tumors to determine putative target genes of 'rare' p53 mutant forms. Following dimension reduction, differentially expressed (putative mutant p53 target) genes were identified using Significance Analysis of Microarrays (SAM) and validated using reverse transcription quantitative PCR (RT-qPCR). Transcription induction of the putative target genes by ectopically expressed wild-type and mutant p53, in p53-null H1299 cell-line, were also evaluated by RT-qPCR. Chromatin immuno-precipitation (ChIP) assay was used to determine localization of mutant p53 proteins to chromosomal loci harboring the putative target genes. Similarly, ability to activate target promoters by mutant p53 was validated through promoter-luciferase assay. Results: Genome-wide analysis revealed 27 differentially expressed genes, of which 9 exhibited up-regulated transcript levels in p53 mutant tumors. Three putative mutant p53 target genes - C1QBP, ARF6 and TRIM23 - were selected for further analysis due to previous reports of their association with cancer. TP53 transcript levels positively correlated with transcript levels of the identified potential mutant targets. The ectopically-expressed p53 mutants - P190T and P278L, induced the increased expression of the target genes, were localized to and activated the promoters of the target genes. Further characterization of the target genes is currently underway. Conclusions: Three novel potential oncogenic targets of mutant p53 proteins were identified. The study further strengthened the heterogeneity in the functioning of different p53 mutants. Citation Format: Sara A. George, Viswakalyan Kotapalli, Raju S. Adduri, Raju Kumar, Sanjana Sarkar, Ramaswamy Pandilla, Murali D. Bashyam. Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2494.