Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma

Abstract

One of the most frequently mutated genes reported in human cancers is TP53. Esophageal squamous cell carcinoma (ESCC) has an extremely high frequency of p53 mutations of 60-90% in ESCC patients. Evidence of gain-of-function (GOF) p53 mutations that exert wild-type (WT) function-independent oncogenic effects are emerging. We aim to determine the functional role of several candidate GOF p53 mutations in ESCC and explore their clinical utility for prognostication and as potential treatment targets. In our recent study of Hong Kong ESCC specimens, we detected TP53 alterations in over 90% of ESCC tumors, ~36% of which are candidate GOF mutations verified in other cancers. By analyzing published ESCC sequencing data, we showed that patients from the high-risk ESCC region show frequent candidate GOF p53 mutants. We applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to functionally knockout (fKO) mutant p53 protein expression in various ESCC cell lines with different endogenous p53 mutants and subject them to in vivo and in vitro functional assays. Interestingly, only KYSE150 cells with an endogenous p53R248Q mutation show reduced tumor growth in nude mice after p53-fKO, suggesting that there is a specific mutant p53 that confers oncogenic features in ESCC. Consistently, only KYSE150 p53-fKO cells show reduced colony formation in three-dimension in vitro culture. Tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) is significantly reduced in KYSE150 p53-fKO cells, suggesting that regulation of the STAT3 pathway contributed to the oncogenic feature of the GOF p53 mutant. p53R248Q exhibits concentrated subcellular localization to the membrane fraction (including ER, mitochondria, etc.) and shows increased resistance to HSP90/HDAC inhibition-induced protein degradation, as compared to other p53 mutants. Together, these results suggest a GOF role of certain mutant p53 proteins with specific characteristics. Targeting such mutants and/or related oncogenic pathways may be of therapeutic benefit. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for General Research Fund support (17109515 to M.L.L).