Defining the role of P2X4 receptor in prostate cancer

Prostate cancer (PCa) is the most frequently diagnosed cancer in men, causing considerable morbidity and mortality. The P2X4 receptor (P2X4R) is a receptor for ATP that is highly expressed in many cancer types and is positively associated with tumourigenesis. However, the role of P2X4R in PCa progression is not well understood. I hypothesized that P2X4R plays a critical role of pro-tumourigenesis in PCa. Firstly in the project, the effect of pharmacologically inhibiting P2X4R, with the selective P2XR4 antagonists 5-BDBD and PSB-12062, on PCa (PC3 and C4-2B4 cells) migration, invasion, and apoptosis were examined in insert assay and Caspase 3/7 assays in vitro. The results demonstrated that inhibiting P2X4R impaired the growth and mobility of PCa cells but not apoptosis. In BALB/c immunocompromised nude mice inoculated with human PC3 cells subcutaneously (106 cells/injection), 5-BDBD showed anti-tumourigenic effects. These studies suggest that P2X4R enhances PCa tumour formation and is a clinically targetable candidate for which inhibitors are already available and can potentially suppress disease progression. Secondly, to further understand the role of P2X4R in PCa biology, particularly in PCa bone metastasis, gene P2RX4 was knocked out in human PC3 cells using the CRISPR/Cas9 system (Santa Cruz:sc-401779). Successful P2X4R knock-out (KO) clones were confirmed and verified using RT-PCR, Sanger sequencing, and functional calcium assay (Fluo-4 Direct™). Cell proliferation, viability, migration, invasion and apoptosis were examined using CyQUANT®, AlamarBlue, Cell MeterTM Caspase 3/7 assays, scratch assay and insert transwell respectively. To test metastatic bone potential in vivo, PC3 wildtype (WT) and KO cells (105 cells/injection) were intracardiacally injected into 6-week-old male BALB/c immunocompromised mice (10 mice/group). Mice were euthanized 25 days post-inoculation and bone micro-architectural changes in tibias were analysed using micro-CT ex vivo, with tumour burden quantified in the metaphysis region of tibias using H&E stained sections and osteoMeasure. Results showed that depleting P2X4R significantly reduced cell proliferation, invasion and increased apoptosis compared to WT controls in vitro. Micro-CT analysis suggested KO cells caused less bone destruction than WT cells, including higher trabecular bone volume fraction, trabecular thickness, trabecular number, and cortical bone volume. There was almost no presence of tumours in KO cell injected mouse tibias, while over 50% of bone marrow areas were occupied by WT PCa cells. The body weight of mice injected with KO cells was significantly heavier than mice injected with WT cells at the endpoint. These studies demonstrated that targeting P2X4R suppresses the process of tumour bone metastasis and indicates a potential pharmacological target. Thirdly, RNA-seq was performed to understand further the mechanism for P2X4R deficiency correlated reduction in PCa tumourigenicity. Total RNA was isolated from PC3 WT and KO cells by RNA Miniprep System, then the RNA samples were sent out to Source BioScience (Nottingham, UK) for RNA sequencing. Bioinformatics tools, including Galaxy European, David, and Gorilla performed bioinformatics analysis of the RNA-seq. The results demonstrated that P2X4R deficiency in PCa cells caused dramatically up-regulate the anti-tumour genes, and it also significantly increased cell adhesion function and characteristic of the epithelia gene, which may delay the process of PCa. Regulation of the WNT and PEG2 signals might also reduce cell proliferation, enhance apoptosis, and affect angiogenesis. These findings provide important insights into the underlying biology of P2X4R and PCa progression. These results highlight the P2X4R as a potential therapeutic target for the treatment of aggressive PCa, especially for PCa bone metastasis.