A245 THE ROLE OF P53 ON P2Y6R EXPRESSION IN COLORECTAL CANCER

Abstract Background The G protein-coupled P2Y6 receptor (P2Y6R) is activated by extracellular UDP. In the colonic epithelium, it participates in the maintenance of the hydric balance by regulating NaCl secretion. More recently, P2Y6R activity was reported to worsen inflammatory symptoms in a mouse model of inflammatory bowel diseases (IBD). Long-term IBD-suffering patients are at higher risk of developing colorectal cancer (CRC). In response to chronic exposure to inflammatory stressors, p53 is one of the first mutated oncogene, which leads to colitis-associated CRC. We focused on the potential role of p53 in the regulation of P2Y6R expression in CRC. Aims The hypothesis is that the presence of a mutant form of p53 will differentially regulate P2Y6R expression as compared to the wild-type protein. The general aim of our work is to characterize the molecular mechanisms associated with p53-dependent regulation of P2Y6R in CRC. More specifically, we will 1) determine and characterize the P2RY6 promoter in cancerous intestinal epithelial cells (cIEC) and 2) study the role of wild-type or mutant p53 on P2Y6R expression. Methods The P2RY6 gene encodes for 8 messenger RNA (mRNA) variants. Most of them are encoding for the P2Y6R isoform 1 which is well characterized. However, mRNA variant 9 codes for an uncharacterized form of the receptor: isoform 2. We used 5’RACE experiments to identify the different transcription start sites (TSS) corresponding to the several mRNA variants and determined the amount of each variant in cIECs. We then cloned four consensus regions (R1, R2, R3 and R4) of the P2RY6 promoter encompassing the four previously identified TSS upstream of the luciferase reporter gene in the pGL4.10 vector. The pGL4.10-P2RY6 promoter constructs were cotransfected with wild-type p53 and mutant forms and transcription activity measured by luciferase assays. Chromatin immunoprecipitation (ChIP) assays were used to confirm the luciferase results. Results We identified four TSS in the P2RY6 promoter region and showed that Caco-2 cells expressed predominantly mRNA variant 9. Using luciferase assays, we showed that wild-type p53 can activate the promoter R1 and R4 regions, whereas p53 R273H mutant (p53R273H) has no effect on the transcriptional activity of the R1 region but significantly stimulated the R4 promoter region. The ChIP experiments showed that p53R273H could occupy the R4 but not the R1 region; whereas wild-type p53 could bind both R1 and R4 regions.