Inhibition of growth and contraction in human prostate stromal cells by silencing of NUAK1 and -2, and by the presumed NUAK inhibitors HTH01-015 and WZ4003

NUAKs 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. In benign prostatic hyperplasia (BPH), contraction and growth in the prostate drive urethral obstruction and voiding symptoms. However, a role of NUAKs in smooth muscle contraction or prostate functions are unknown. Here, we examined effects of NUAK silencing and the presumed NUAK inhibitors, HTH01-015 and WZ4003 on contraction and growth-related functions in prostate stromal cells (WPMY-1) and in human prostate tissues.

NUAK1 and -2 suppress cell death and promote proliferation in prostate stromal cells. A role in stromal hyperplasia appears possible in BPH. Effects of NUAK silencing are mimicked by HTH01-015 and WZ4003.

Effects of NUAK1 and -2 silencing, HTH01-015 and WZ4003 on matrix plug contraction, proliferation (EdU assay, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin staining) were examined in cultured WPMY-1 cells. Effects of HTH01-015 and WZ4003 on smooth muscle contraction were assessed in organ bath experirments with human prostate tissues.

Effects of silencing were most pronounced on proliferation and cell death, resulting in decreases of proliferation rate by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls), decreases in Ki-67 by 75% and 77%, while numbers of dead cells after silencing of NUAK1 and NUAK2 amounted to 2.8 and 4.9 fold of scramble-transfected controls. Silencing of each isoform was paralleled by reduced viability, breakdown in actin polymerization, and partial decreases in contractility (maximally 45% by NUAK1 silencing, 58% by NUAK2 silencing). Effects of silencing were mimicked by HTH01-015 and WZ4003, with numbers of dead cells amounting up to 16.1 fold or 7.8 fold with HTH01-015 or WZ4003, compared to solvent-treated controls. Using concentrations of 500 nM, neurogenic contractions of prostate tissues were inhibited partly by HTH01-015 and U46619-induced contractions were inhibited partly by HTH01-015 and WZ4003, while α1-adrenergic and endothelin-1-induced contractions remained unaffected. Using 10 μM, inhibition of endothelin-1-induced contractions by both inhibitors and inhibition of α1-adrenergic contractions by HTH01-015 added to effects seen by 500 nM.