NUAK2 and RCan2 participate in the p53 mutant pro-tumorigenic network

Abstract

Most inactivating mutations in TP53 gene generates neomorphic forms of p53 proteins that experimental evidence and clinical observations suggest to exert gain-of-function effects. While massive effort has been deployed in the dissection of wild type p53 transcriptional programme, p53 mutant pro-tumorigenic gene network is still largely elusive. To help dissecting the molecular basis of p53 mutant GOF, we performed an analysis of a fully annotated genomic and transcriptomic human pancreatic adenocarcinoma to select candidate players of p53 mutant network on the basis their differential expression between p53 mutant and p53 wild-type cohorts and their prognostic value. We identified NUAK2 and RCan2 whose p53 mutant GOF-dependent regulation was further validated in pancreatic cancer cellular model. Our data demonstrated that p53^R270H can physically bind RCan2 gene locus in regulatory regions corresponding to the chromatin permissive areas where known binding partners of p53 mutant, such as p63 and Srebp, bind. Overall, starting from clinically relevant data and progressing into experimental validation, our work suggests NUAK2 and RCan2 as novel candidate players of the p53 mutant pro-tumorigenic network whose prognostic and therapeutic interest might attract future studies.

Keywords: Cancer prognosis; Metastasis; Tumour progression; Tumour suppression.

Fig. 1

RCan2 and NUAK2 emerge as part of mutant p53 network in a human PDAC cohort. a Oncoprint diagram reports mutation spectrum, fraction genome, overall survival, histology and p53 mutational status in the PDAC cohort from the TCGA PanCancer Atlas. b, c Samples from p53 mutant patients display higher “aneuploidy score”, “genome altered” and “hypoxia score” if compared to the p53 wt cohort. d Kaplan–Meier plot reports overall survival of p53 mutant vs p53 wt PDAC patients from the TCGA PanCancer Atlas. e Volcano plot reports genes differentially regulated between p53 mutant versus p53 wt PDAC patients from the TCGA PanCancer Atlas. f mRNA expression level (from RNA-seq) data of RCAN2 and NUAK2 display differential expression between p53 mutant versus p53 wt PDAC patients (TCGA PanCancer Atlas). g Kaplan–Meier plot reports overall survival of patients with RCan2 and NUAK2 highly expressing PDAC (TCGA PanCancer Atlas). Source: cbioportal.org

Fig. 2

Mutant p53 regulates a RCan2 expression via GOF effects. a Oncoprint diagram reports mutational status of RCan2 and NUAK2 in full cohort of TCGA PanCancer Atlas. b–d mRNA level of RCan2, NUAK2 and p53 in KPC²⁷⁰ cells following p53 silencing. e ChIP-seq profile of histone post-translational modifications, transcriptional factors binding in the genomic region of mouse RCan2. f ChIP-qPCR for p53 binding on peak 1 and peak 2 in KPC²⁷⁰ cells

Fig. 3

RCan2 expression influences cell motility but not proliferation. a, b p53 mRNA level in KPC²⁷⁰ cells following p53 silencing. c, d In vivo live cell imaging analysis by IncuCyte platform measured cell growth (phase contrast) in c and migration (scratch closure measured as scratch area by phase contrast) in d in KPC²⁷⁰ cells following RCan2 silencing. Red boxes indicate the scratch area. Scale bars 400 microns