Novel oncogenic transcriptional targets of mutant p53 in Esophageal Squamous Cell Carcinoma

Abstract

Abstract 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.