Pabla N, Huang S, Mi QS, Daniel R, Dong Z. cancer remain poorly understood. To address this deficit, in this preclinical study, we used both small molecule inhibitors of CDK8/19 and genetic approaches to investigate the dependence of prostate cancer cells on CDK8/19 activity. Furthermore, we explored the biological roles of CDK8/19 in prostate cancer cells as well. RESULTS Anti-proliferative activity of CDK8/19 inhibitors in prostate cancer cells To accurately explore the function of CDK8 and CDK19, we used two structurally differentiated compounds, both of which potently inhibit NCH 51 CDK8 and CDK19, in enzyme assays (T-474; CDK8/19 IC50 = 1.6/1.9 nmol/L, T-418; CDK8/19 IC50 = 23/62 nmol/L) (Figure ?(Figure1A).1A). In a panel of 456 kinases, both compounds showed marked kinase selectivity (Figure ?(Figure1A1A and Supplementary Tables 1 and 2). Kinases inhibited by >80% in response to 300 nM T-474 were limited to CDK19 (99% inhibition), Haspin (99% inhibition), and CDK8 (90% inhibition). CDK19 was the only kinase that was inhibited by >80% in response to 300 nM T-418 (94% inhibition) (Supplementary Tables 1 and 2). In VCaP prostate cancer cells, treatment with T-474 or T-418 suppressed the phosphorylation of the known CDK8 substrate STAT1 at Ser727 both in the absence and in the presence of IFN- (Figure ?(Figure1B),1B), which stimulates CDK8-mediated STAT1 phosphorylation [23]. Furthermore, T-474 treatment reduced Wnt/-catenin-dependent NCH 51 transcriptional activity in SW480 colon cancer cells as reported previously (Supplementary Figure 1) [17]. Open in a separate window Figure 1 Anti-proliferative activity of CDK8/19 inhibitors in prostate cancer cells(A) Compound structure, potency, and kinase selectivity of T-474 or T-418. Kinase selectivity profiling was performed using 300 nmol/L T-474 or T-418. (B) VCaP cells were treated with T-474 or T-418 together with 10 ng/mL IFN- as indicated for 30 minutes. Cell lysates were analyzed by western blot. (C) mRNA expression of CDK8 or CDK19 in prostate cancer cell lines (CCLE). (D) Western blot of CDK8 or CDK19 in prostate cancer cell lines. VCaP cells were transfected with siRNA as indicated for 72 hours. Cell lysates were analyzed by western blot. The relative band intensities of CDK8 or CDK19 were quantified and are indicated as percentage (%) of control (non-treated VCaP cells). An NCH 51 arrow indicates the expected position of bands derived from CDK19. (E) LNCaP or 22Rv1 cells were treated with T-474 as indicated for 9 days (= 3, mean with = 3, mean with = 2, mean). Cell viability was measured. We then investigated the expression of CDK8 and CDK19 in several commercially available prostate cancer cell lines. In accordance with previous reports [14], CDK19 was highly expressed in some prostate cancer cells at the mRNA and protein levels (Figure 1C, 1D, and Supplementary Figure 2). We observed that CDK8 protein levels were moderately elevated in CDK19-depleted cells (Figure ?(Figure1D1D and Supplementary Figure 2). Notably, similar compensatory effects in paralogs have been reported previously [24]. CDK8/19 inhibition did not obviously impact proliferation of LNCaP, 22Rv1, PC-3, or DU 145 cells (Figure ?(Figure1E1E and ?and1F),1F), whereas we observed that treatment with T-474 or T-418 substantially inhibited the proliferation of VCaP cells (Figure ?(Figure1G).1G). Furthermore, in VCaP cells, knockdown of CDK8 or CDK19 by siRNA did not obviously impact the cell proliferation (Supplementary Figure 3A). Specifically, only one of four CDK19 siRNAs substantially suppressed cell proliferation; however, the effects appeared to be off-target considering the limited knockdown efficiency (Supplementary Figure 3A). Importantly, the simultaneous knockdown of CDK8 and CDK19 suppressed the proliferation of VCaP cells (Supplementary Figure 3B). These results suggest that inhibition of both CDK8 and CDK19 is essential for suppression of VCaP cell proliferation. Effects of CDK8/19 inhibition on cell cycle progression Given that CDK8/19 forms a subcomplex of Mediator, it was plausible that inhibition of CDK8/19 might affect the gene expression pattern. To understand the mechanism Rabbit polyclonal to SZT2 of action, we performed a microarray analysis in CDK8/19 inhibitor-sensitive VCaP cells. A comprehensive evaluation of transcriptional changes using a parametric analysis of gene set enrichment (PAGE) revealed that down-regulated genes following T-474 treatment were.