Abstract 51: The codon 72 polymorphism in p53 is a gain-of-function alteration leading to activation of map kinase signaling and epithelial-to-mesenchymal transition in colon cancer

Abstract

Abstract Background: The codon 72 polymorphism in p53 has been implicated in several human malignancies and cancer health disparities. In this study, we determine the functional role of this frequently occurring polymorphism using an in-vitro cell based system in colon cancer. Experimental Design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72wt), R72wt with mutation at codon 273 cysteine (R72273Cys), p53 mutation at codon 72 (P72wt) and P72wt with mutation at codon 273 (P72273Cys)] were constructed. We selected a colon cancer cell line Caco2, which does not express p53. After transfection, the expression of p53 phenotypes was confirmed by western blot analysis. Clones resistant to geneticin were assessed for cell survival; colonies were stained with 0.5% crystal violet and counted. Western blot or immunostaining analyses for proteins of MAP kinase signaling, Akt and/or epithelial mesenchymal transition mechanisms were performed to assess the gain-of-function (GOF) of P72 phenotype of p53. Results: Our study demonstrated that there was a significant increased cell survival in cells that express P72wt phenotype compared to cells that express R72wt phenotype. This cell survival was also increased in cells that express R72273Cys mutant phenotype. The P72wt phenotype induces morphological changes including cell polarization and extension of invadopodia associated with tumor progression. Western blot analyses revealed that P72wt and mutant phenotypes effectively induced the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. Up-regulation of phosphorylated SEK1/MKK4, or phosphorylated MEK1/2, upstream kinases of p38 MAPK, p44/42 MAPK respectively has been found to be associated with P72wt or mutant phenotypes. This activation was accompanied by up-regulation of phosphorylated-MAPKAPK-2 and phosphorylated-Hsp27, downstream targets of p38 MAPK. Furthermore, suppression of Akt activation was significantly higher in cells that express R72wt phenotype compared to cells that express P72wt or mutant phenotypes. This suppression was accompanied by down-regulation of phosphorylated glycogen synthase kinase-3β, a downstream target of p-Akt. The expression of P72wt and mutant phenotypes displayed decreased expression of E cadherin (loss of epithelial nature) and/or an increased expression of vimentin, fibrinectin (gain of mesenchymal nature). Conclusions: Collectively, these findings show that the P72 polymorphism of p53 in colon cancer is a GOF alteration leading to activation of tumor promoting phenotype. The possibility of P72 blockade may be considered in patients with colon cancer that are unresponsive to conventional treatments. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat Katkoori, Lashmishankar Chaturvedi, Marc D. Basson, Upender Manne, Harvey L. Bumpers. The codon 72 polymorphism in p53 is a gain-of-function alteration leading to activation of map kinase signaling and epithelial-to-mesenchymal transition in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 51. doi:10.1158/1538-7445.AM2015-51