P2X4 purinergic receptors offer a therapeutic target for aggressive prostate cancer

Abstract

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.

Keywords: ERG; Myc-CaP cells; P2 purinergic receptors; P2X4; PTEN loss; advanced prostate cancer; extracellular ATP; macrophages; neutrophils; prostatic intraepithelial neoplasia.

Figure 1.

P2X4 purinergic receptor protein expression in prostate tissues. P2X4 purinergic receptor protein is expressed (A) modestly in the epithelium of prostate glands and more intensely on immune cells of organ donor prostates (C) modestly in benign glands and more intensely in prostatic intraepithelial neoplasia (PIN) and cancer in prostatectomy samples and (D) on immune cells (arrow indicates P2X4 positive immune cell). (B) H&E staining confirmed tissue types. (E) Dual staining showed that tryptase positive mast cells (red) do not express P2X4 protein (brown), but some CD66ce positive neutrophils (brown) and most CD68 positive macrophages (brown) express P2X4 receptors (red) (arrow indicates double-stained immune cell). Images captured with 40x objective magnification unless otherwise stated.

Figure 2.

Quantitative image analysis of P2X4 purinergic receptor expression. There was elevated P2X4 receptor protein expression in (A) cancer compared to benign tissues (n = 456; p < 0.0001) (B) from both high grade and low grade cases, and (C) in cancer TMA spots from White men compared to those from Black men. (D) There was elevated P2RX4 mRNA expression in prostatic intraepithelial neoplasia (PIN) and cancer while there is a wide range of expression across metastatic sites. (E) There is a positive correlation between P2RX4 mRNA expression and CD68 mRNA expression. (F) There was elevated P2X4 protein expression in tumors with Erg expression and in tumors with PTEN loss. (A–C, E,F) Each dot represents the mean expression per patient (3 or 4 TMA spots per patient). (D) Each dot represents the expression per TMA core. *p <0.05; *** p <0.001; **** p < 0.0001

Figure 3.

P2X4 purinergic receptor expression in metastatic prostate cancer tissues. (A, B) P2X4 protein expression was detected in cancer and immune cells and in various metastatic sites. (C) P2RX4 mRNA expression (brown, arrows) detected on CD68⁺ (red) macrophages in metastatic tissues. (D) There is a positive correlation between CD68 and P2RX4 mRNA expression in metastatic tissues. *p <0.05; *** p <0.001, **** p < 0.0001

Figure 4.

P2X4 purinergic receptor function affects PCa cell motility. (A) Nucleotide treatment and (B) P2X4 knock down in DU145 cells decreased E-cadherin protein expression and increased vimentin protein expression as assessed by western blotting. (C) CTP treatment increased cell migration and invasion while 5-BDBD and P2X4 knock down decreased cell migration and invasion in Transwell assays. *p <0.05 (P = parental, KD = knockdown)

Figure 5.

P2X4 purinergic receptor function affects PCa cell viability. (A) P2X4 antagonist 5-BDBD resulted in a dose-dependent decrease in PCa cell viability, *p<0.0001 for each cell line compared to no treatment. Each point represents 12 wells of the respective cell line. Each cell line was assessed in different experiments. (B) P2X4 knockdown in LNCaP cells resulted attenuated cell growth over time. Each point represents 24 wells. * p<0.05 for KD-6 and KD-8 compared to LNCaP; #p<0.05 for KD-6 only compared to LNCaP. (C) Treatment with 5-BDBD resulted in a dose-dependent induction of cleaved caspase-3 in PCa cells.

Figure 6

P2X4 purinergic receptor protein expression in mouse prostate tissues. (A) Robust P2rx4 mRNA expression is detected in mouse prostate tumors. (B) CRISPR knockdown of P2X4 receptor expression in Myc-CaP cells (n=10) resulted in attenuated allograft growth in FVB/NJ mice compared to control Myc-CaP cells (n=10). Gray lines represent tumor volume for individual mice and solid lines represent average tumor volume for the group, **** p < 0.0001. (C) Human MYC ISH and P2X4 and AR IHC analysis confirmed reduced P2X4 expression in Myc-CaP allograft tissue from mice. (D) P2X4 positive regions lacking AR and human MYC stain positive for CD11b and CD3 in Myc-CaP allograft tissue from mice.