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

Background: 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. 

Methods: 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. 

Results: 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. 

Conclusion: 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.

 

Comments:

The study you've described investigates the role of NUAKs (NUAK1 and NUAK2) in smooth muscle contraction and prostate function, specifically in the context of benign prostatic hyperplasia (BPH). Here's a summary of the key findings and conclusions from the study:

1. **NUAKs and Prostate Stromal Cells**: NUAK1 and NUAK2 were found to play a role in promoting the proliferation and suppressing the cell death of prostate stromal cells (WPMY-1 cells). Silencing either NUAK1 or NUAK2 resulted in a significant decrease in cell proliferation and an increase in cell death when compared to control cells transfected with scramble siRNA.

2. **Viability and Actin Organization**: Silencing of NUAK1 and NUAK2 also led to reduced cell viability and disrupted actin polymerization, indicating that these proteins are involved in maintaining the health and structural integrity of prostate stromal cells.

3. **Contractility**: The study observed that the silencing of NUAK1 and NUAK2 partially decreased smooth muscle contractility in prostate stromal cells. This suggests a potential role for NUAKs in regulating smooth muscle contraction in the prostate.

4. **NUAK Inhibitors**: The researchers also tested two presumed NUAK inhibitors, HTH01-015 and WZ4003, and found that they mimicked the effects of NUAK silencing. These inhibitors resulted in a significant increase in cell death and a decrease in proliferation, similar to the effects seen with NUAK silencing.

5. **Prostate Tissue Contraction**: In experiments with human prostate tissues, HTH01-015 was found to partly inhibit neurogenic contractions, and both HTH01-015 and WZ4003 partly inhibited U46619-induced contractions. However, contractions induced by α1-adrenergic and endothelin-1 remained unaffected or were only partially affected.

6. **Conclusion**: The study concludes that NUAK1 and NUAK2 play a role in promoting cell survival and proliferation in prostate stromal cells. These findings suggest that NUAKs may contribute to stromal hyperplasia in BPH, a condition characterized by prostate growth. The effects of NUAK silencing were mimicked by the inhibitors HTH01-015 and WZ4003, further supporting the potential therapeutic relevance of targeting NUAKs in BPH.

In summary, this research sheds light on the involvement of NUAKs in the regulation of smooth muscle contraction and cell functions in the prostate, particularly in the context of BPH. NUAKs may represent potential targets for therapeutic interventions in BPH to address issues related to prostate growth and obstruction of the urethra.