Investigatng the Role of Calcium Signalling Genes in the Progression of Neuroendocrine Prostate Cancer

Prostate cancer (PCa) can manifest as a multi-stage, incurable, progressive malignancy called NEPC (Neuroendocrine PCa) that may not respond to established therapeutic interventions. This project aims to understand the role of cell-signalling genes in the progression of NEPC. Using the OMIM database, we identified 3708 cell-signalling genes. Of these genes, 200 were significantly upregulated in a clinical dataset comprising gene expression data from 15 NEPC and 34 prostate adenocarcinoma patient samples. A pathway analysis on differentially expressed genes indicated calcium ion transport was the most highly upregulated signalling modality in NEPC versus prostate adenocarcinoma. Calcium imaging experiments reported a significant alteration in intracellular calcium signalling in response to ATP-stimulation in prostate adenocarcinoma versus NEPC cell lines. Expression of the 12 upregulated calcium ion transport genes was analysed using RT-qPCR and western blotting in a panel of human and murine PCa cell lines. Our data showed an upregulation of ANO6, a calcium-activated chloride channel, in human and murine NEPC cell lines, aligning with our bioinformatics result. ANO6 was also overexpressed in hypoxia-induced transdifferentiated LNCaP cells. Although it has not been previously studied in NEPC, ANO6 drives proliferation and migration in other cancers. We used benzbromarone, a pharmacological inhibitor of ANO6, to study the functional role of ANO6 in NEPC cells. High concentrations inhibited NEPC cell proliferation, while clinically relevant doses significantly reduced cell migration in a wound healing assay. Ano6 knockdown using siRNAs confirmed that ANO6 mainly affects NEPC cell migration and not proliferation. Single-cell RNA sequencing in patient samples revealed higher ANO6 expression in the endothelial cells of NEPC patients than those with adenocarcinoma, suggesting a role related to the tumour microenvironment and angiogenesis for ANO6. Therefore, further exploration of the ANO6 gene offers new opportunities for targeting NEPC therapeutically