Identification of novel oncogenic transcriptional targets of mutant p53 in Esophageal Squamous Cell Carcinoma

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. 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 harbouring 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.