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 of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.
KeywordsG2/M cell cycle arrest Invasion and migration c-Jun phosphorylation Protein kinase D inhibitor RNA interference TCGA database
This research was supported by the Intramural Research Program of the U.S. National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interests.
- 6.Grossman HB, Natale RB, Tangen CM, Speights VO, Vogelzang NJ, Trump DL, deVere White RW, Sarosdy MF, Wood DP Jr, Raghavan D, Crawford ED (2003) Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 349:859–866CrossRefPubMedGoogle Scholar
- 14.Ha CH, Wang W, Jhun BS, Wong C, Hausser A, Pfizenmaier K, McKinsey TA, Olson EN, Jin ZG (2008) Protein kinase D-dependent phosphorylation and nuclear export of histone deacetylase 5 mediates vascular endothelial growth factor-induced gene expression and angiogenesis. J Biol Chem 283:14590–14599CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Harikumar KB, Kunnumakkara AB, Ochi N, Tong Z, Deorukhkar A, Sung B, Kelland L, Jamieson S, Sutherland R, Raynham T, Charles M, Bagherzadeh A, Foxton C, Boakes A, Farooq M, Maru D, Diagaradjane P, Matsuo Y, Sinnett-Smith J, Gelovani J, Krishnan S, Aggarwal BB, Rozengurt E, Ireson CR, Guha S (2010) A novel small-molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vitro and in vivo. Mol Cancer Ther 9:1136–1146CrossRefPubMedPubMedCentralGoogle Scholar
- 77.Li QQ, Hao JJ, Zhang Z, Hsu I, Liu Y, Tao Z, Lewi K, Metwalli AR, Agarwal PK (2016) Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: a proteomic approach. Int J Oncol 48:2591–2607CrossRefPubMedPubMedCentralGoogle Scholar
- 78.Li QQ, Hao JJ, Zhang Z, Krane LS, Hammerich KH, Sanford T, Trepel JB, Neckers L, Agarwal PK (2017) Proteomic analysis of proteome and histone post-translational modifications in heat shock protein 90 inhibition-mediated bladder cancer therapeutics. Sci Rep 7:201CrossRefPubMedPubMedCentralGoogle Scholar