Serine 249 phosphorylation and Pin1-induced isomerization activate mutant p53 R249S gain-of-function and promote cross-talk with JAK/STAT1 signalling in hepatocellular carcinoma

Abstract

Evidences from basic and clinical studies clearly established that mutations of the tumour suppressor TP53 contribute to carcinogenesis. Unlike the majority of tumour suppressors, the TP53 gene is often found to undergo missense mutations, with a strong dominance in the DNA-binding domain. Mutant p53 proteins contribute to carcinogenesis by losing tumour suppressor activities, by exerting dominant negative effects over the wild-type allele and by acquiring gain-of-function properties. These oncogenic activities enforce tumour growth, EMT, metastasis and chemoresistance. It has been clearly demonstrated that mutations on p53 are not sufficient to drive tumorigenesis per se but mutant p53 needs to be activated by specific oncogenic signalling produced by a pro-tumorigenic cellular background to be active as an oncogene. Moreover, understanding what are the environmental factors that account for the selection of specific p53 mutant forms in different tumours is critical to identify novel determinants of carcinogenesis and to find new putative targets for cancer therapy. Among the frequently mutated residues in p53, Arginine 249 is replaced in about 65% of cases with a Serine (mutp53 R249S). Since Arginine 249 regulates the secondary structure of p53 loops 2 and 3, its substitution is responsible for a local distortion of the molecule, which reaches a dynamic equilibrium between a native and a native-like conformation. Although mutp53 R249S displays an overall low frequency in tumours bearing missense mutations in p53, it is detected in up to 90% of hepatocellular carcinomas (HCC) occurring in populations exposed to HBV infections and aflatoxin B1 food-contamination, which represent two major risk factors for the HCC in the east of Asia and sub-Saharan Africa. In these areas, since R249S mutation occurs early in the pathway leading to HCC, it is considered an early biomarker of hepatocarcinogenesis. Interestingly, R249S mutation is associated to a bad prognosis and to the expression of a stem cell-associated gene signature. Although in vitro studies show that mutant p53 R249S is able to promote the proliferation of HCC cell lines and that the co-expression with the HBV-oncogene HBx provides further oncogenic properties, a specific mechanism of gain of function is still missing. In this thesis, it is shown that mutation of the Arginine 249 introduces a residue phosphorylated in liver and breast cancer cells, and that the Dual-specificity Tyrosine regulated kinase 2, DYRK2, is the main kinase for Serine 249 phosphorylation. Moreover, it is provided evidence that Serine 249 phosphorylation creates a new binding site for Pin1, which is responsible for the distorted conformation of mutant p53 R249S. Cancer cells depleted of mutp53 R249S show an impairment in their proliferation, which is unleashed by Pin1 activity over mutant p53. By analyzing the transcription profile of Mahlavu cells upon knocking down of mutp53 R249S, it has been identified the JAK/STAT pathway as molecular axis which sustains the mutant p53 R249S activity. This study unveils a novel post-translational modification signalling converging in mutant p53 R249S required for the gain-of-function of the protein and suggests a possible opportunity for therapeutic intervention