Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family plays a critical role in regulating tumor growth, development, and progression. CRT0066101, a PKD inhibitor, has demonstrated antitumor activity across various carcinoma types; however, its effects and underlying mechanisms in bladder cancer remain unclear. In this study, we show that CRT0066101 inhibits the proliferation and migration of four bladder cancer cell lines in vitro and suppresses tumor growth in a mouse flank xenograft model of bladder cancer.

To further investigate the role of PKD in bladder cancer, we examined the expression of the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma samples from the TCGA database. Short hairpin RNA (shRNA)-mediated knockdown of PKD2 significantly reduced bladder cancer cell growth and invasion both in vitro and in vivo, indicating that the antitumor effects of CRT0066101 in bladder cancer are mediated through PKD2 inhibition. Supporting this notion, we observed a marked reduction in phospho-PKD2 levels in bladder tumor explants treated with CRT0066101.

Cell cycle analysis by flow cytometry revealed that treatment with CRT0066101 or silencing of PKD2 induced G2/M phase arrest in bladder cancer cells. This arrest was associated with decreased levels of cyclin B1, CDK1, and phospho-CDK1 (Thr161), alongside increased levels of p27Kip1 and phospho-CDK1 (Thr14/Tyr15). Additionally, CRT0066101 downregulated the expression of Cdc25C, which activates CDK1 through dephosphorylation, while enhancing the activity of checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating and inactivating Cdc25C.

Furthermore, CRT0066101 treatment led to increased levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which contribute to the inhibition of CDK1-cyclin B1 complex activity. Collectively, these findings indicate that CRT0066101 suppresses bladder cancer growth by targeting PKD2, inducing G2/M cell cycle arrest, and thereby blocking cell cycle progression.