Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening

Abstract

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

Figure 1. shRNA probes depleted or enriched in bicalutamide-treated LNCaP cells.

(A) Schematic of shRNA screen. Details can be found in the Materials and Methods section. (B) A heatmap was generated by clustering based on probes that were depleted or enriched (log2 bicalutamide/vehicle ≤+/−0.58 and a p-value≤0.01) in the bicalutamide (0.4 uM and 1.0 uM)-treated LNCaP cells at T = 1 and T = 2 compared to the vehicle-treated cells at the same timepoints. The shRNA target gene associated with each probe is indicated to the right of the heatmap. Target genes that appear more than one time on the heatmap indicate that more than one probe scored for that gene.

Figure 2. Silencing of a subset of genes inhibited VCaP proliferation and induced apoptosis.

Candidate target genes from LNCaP screen probes that were depleted in the presence of bicalutamide were selectively targeted using siRNAs. (A, left panel), VCaP cells were transfected with siRNAs that scored in the LNCaP drug screen and were incubated with 1 uM MDV3100 or vehicle, and the number of viable cells was measured 6 days post-treatment. (A, right panel), VCaP cells were transfected and treated as in (A, left panel) except Caspase 3/7 activity was measured after 3 days of treatment. PLK1 siRNA transfection was used as a positive control. Dashed lines indicate the level of growth inhibition or apoptosis induced by MDV3100, for comparison. NT, non-targeting siRNA. (B) Gene silencing (10–50%) was confirmed by RT-qPCR 6 days post-transfection of VCaP cells with the siRNA SMARTpools. Reactions were done in triplicate and normalized to RPL27 for each cDNA and then normalized to vehicle-treated NT. Standard error of the mean was calculated. Bic, bicalutamide.

Figure 3. Higher TTK expression is associated with biochemical recurrence.

Kaplan Meier plot of the risk of biochemical recurrence in patients (n = 131) with TTK overexpressing prostate tumors (n = 20; green line) versus those without TTK overexpressing (n = 111; blue line) tumors (p = 0.003, log-rank test).

Figure 4. Akt1/2 inhibitor cooperated with bicalutamide to induce LNCaP cell apoptosis.

(A) Number of viable LNCaP cells treated over 5 days with vehicle, bicalutamide (1 uM), Akt1/2 inhibitor (1 uM), or a combination of bicalutamide and AKT inhibitor. (B) Apoptosis was measured in LNCaP cells by immunoblotting using a PARP antibody in LNCaP cells treated as in (A). Inhibition of phosphorylated Akt (pAkt (Ser473)) and phosphorylated S6 (pS6) was also measured. Actin was used as a loading control.