Session: PD02: Benign Prostatic Hyperplasia: Basic Research & Pathophysiology
PD02-01: NUAK1 and -2 promote contraction, proliferation and suppression of cell death in human prostate stromal cells: evidence from isoform-specific silencing
Introduction: In benign prostatic hyperplasia (BPH), contraction and growth in the prostate contribute to urethral obstruction and voiding symptoms. NUAK1 and -2 are serine/threonine kinases and promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. Here, we examined effects of NUAK silencing on contraction and growth-related functions of human prostate stromal cells (WPMY-1). Methods: Effects of NUAK1 and -2 silencing on matrix plug contraction, proliferation (EdU, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin) were examined in cultured WPMY-1 cells. Effects of NUAK inhibitors were examined in matrix contraction and in colony formation assays. Results: Silencing of NUAK1 and -2 by transfection of WPMY-1 cells with isoform-specific siRNAs was confirmed by RT-PCR. Effects of silencing were most pronounced on proliferation and cell death. Proliferation rates were decreased by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls, 48 h after transfection), paralleled by decreases in Ki-67 mRNA by 75% and 77%. Numbers of dead cells amounted to 2.8 fold of scramble-transfected controls after silencing of NUAK1, and 4.9 fold of scramble controls after NUAK2 silencing, while no effect on apoptosis was observed. Viability was reduced by 31% and 49%, respectively, 24 h after transfection with NUAK1 and NUAK2 siRNA, compared to scramble controls. Polymerized actin was decreased by 66% and 70% by silencing of NUAK1 and -2. Time-dependent contractions in matrix contraction assays were reduced up to 45% by silencing of NUAK1, and up to 58% by silencing of NUAK2. The presumed NUAK inhibitors HTH01-015 and WZ4003 (both 10 µM) reduced contractions of WPMY-1 cells by up to 54% and 57%, and reduced the number of colonies in colony formation assays up to 78% and 64%, respectively. Conclusions: NUAK1 and -2 suppress cell death, and promote proliferation and contraction in prostate stromal cells. A role of NUAKs for BPH-relevant prostate functions driving voiding symptoms appears possible. NUAK inhibitors mimic effects of silencing on contraction, and inhibit the growth of stromal cells. SOURCE OF Funding: Deutsche Forschungsgemeinschaft (DFG), grant HE 5825/9-1; China Scholarship Council (CSC), grant 201908440478. Conflicts of Interest: Nothing to disclose.
