P2X4 purinergic receptors offer a therapeutic target for aggressive prostate cancer.

Prostate cancer (PCa) remains a leading cause of cancer-related deaths in American men and treatment options for metastatic PCa are limited. There is a critical need to identify new mechanisms that contribute to PCa progression, that distinguish benign from lethal disease, and that have potential for therapeutic targeting. P2X4 belongs to the P2 purinergic receptor family that is commonly upregulated in cancer and is associated with poorer outcomes. We observed P2X4 protein expression primarily in epithelial cells of the prostate, a subset of CD66+ neutrophils, and most CD68+ macrophages. Our analysis of tissue microarrays representing 491 PCa cases demonstrated significantly elevated P2X4 expression in cancer- compared with benign-tissue spots, in prostatic intraepithelial neoplasia, and in PCa with ERG positivity or with PTEN loss. High-level P2X4 expression in benign tissues was likewise associated with the development of metastasis after radical prostatectomy. Treatment with the P2X4-specific agonist cytidine 5'-triphosphate (CTP) increased Transwell migration and invasion of PC3, DU145, and CWR22Rv1 PCa cells. The P2X4 antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) resulted in a dose-dependent decrease in viability of PC3, DU145, LNCaP, CWR22Rv1, TRAMP-C2, Myc-CaP, BMPC1, and BMPC2 cells and decreased DU145 cell migration and invasion. Knockdown of P2X4 attenuated growth, migration, and invasion of PCa cells. Finally, knockdown of P2X4 in Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in FVB/NJ mice. Collectively, these data strongly support a role for the P2X4 purinergic receptor in PCa aggressiveness and identify P2X4 as a candidate for therapeutic targeting. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Modeling Human Prostate Cancer Metastasis in Mice via Resection of Subcutaneous Allografts.

The 5-year survival rate for patients diagnosed with distant metastatic prostate cancer in the United States is 30.6%. Therefore, there is a great need to develop in vivo model systems to study prostate cancer metastasis and to test potential therapeutics. Most murine prostate cancer metastatic models involve intracardiac or intraosseous implantation of cancer cells, which bypass the early stages of tumor cell migration and invasion. Herein we provide a detailed protocol for a novel method of resecting subcutaneous prostate cancer allografts in immunocompetent mice to produce spontaneous metastases and describe a pilot study using this method of tumor resection. Intact male FVB/NCrl mice (n = 9) were inoculated subcutaneously with Myc-CaP cells. Tumors were surgically resected, and mice were monitored for tumor recurrence. Animals were euthanized or died, and a full set of tissues was collected for histopathologic examination. Tumors took an average of 44 days (range 23-61) to reach 1.7 cm in any direction. All tumors were resectable, and resection of the tumors increased the study length by 70 days (range 30-121). One mouse was euthanized early of an unrelated cause, and of eight remaining mice, four developed tumor recurrence at the site of resection. One mouse developed bone metastases, one mouse developed metastases to the abdominal cavity, and two mice showed signs of local invasion. This study demonstrates that resection of subcutaneous Myc-CaP cell allografts in mice results in local tumor recurrence and the development of distant metastases, providing a new model system to study prostate cancer metastasis in vivo.