This suggests that therapeutic strategies targeting kinase cascades can overcome the compensatory signaling mechanisms that limit the effectiveness of androgen ablation. In order to identify the signaling pathways that regulate prostate cancer cell growth, we screened a panel of WZ8040 shRNAs that target the human kinome against LNCaP prostate cancer cells grown in the presence and absence of androgen. We searched for kinases that had general growth effects and kinases that compensated for androgen ablation. The screen identified multiple shRNA clones against gene targets that regulate both androgen sensitivity and cell growth. We report here the results of our screen and the detailed evaluation of a subset of kinases identified as regulators of prostate cancer cell growth. As an intermediate step to examining the activation state of the kinases, we examined kinase message levels in the Oncomine database. We found that in at least two independent studies the mRNA levels for the six kinases increased either when primary prostate cancer is compared to normal prostate or when metastatic prostate cancer is compared to primary disease or normal prostate. We validated the growth effect and knockdown of our six selected kinases using the CyQuant Assay, which measures DNA content as a surrogate for cell number, and used this technique to also extend our analysis to the castration-resistant cell line, C4-2B. The cells were transduced with lentiviral particles expressing two shRNAs specific for each kinase of interest or pLKO empty vector control in the presence or absence of androgen. As observed in Figure 2, growth was decreased in both cell lines in response to each shRNA. In general, kinase knockdown inhibited growth in the presence and absence of androgen. Furthermore, kinase knockdown affected growth equivalently in both the androgen-dependent LNCaP and castration-resistant C4-2B cell line. qPCR was used to determine kinase knockdown by shRNA in LNCaP and C4-2B cells. Hormone was added at various concentrations and RNA was isolated at 2 and 24 hours following hormone treatment. These hormone treatments were the same as those used to assess the effect of kinase knockdown on AR transcriptional activity, which is described below and presented in Table 1. Each kinase was knocked down in both cell lines with two different shRNAs and compared to the pLKO empty vector control. We did not observe an effect of hormone dose on the efficiency of kinase knockdown; thus, the data shown in Figure 3 are the qPCR values averaged across biological replicates and hormone concentrations for each shRNA or the pLKO control at 24 hours following hormone stimulation. The shRNA viruses elicit greater than 50% knockdown of the target kinase mRNA as compared to pLKO, with most knockdowns greater than 70% at both time points and in both cell lines. There was some differential knockdown of kinase mRNA by shRNA, which may account for the differential knockdown of growth.