Short hairpin RNA library-based functional screening identified ribosomal protein L31 that modulates prostate cancer cell growth via p53 pathway

Abstract

Androgen receptor is a primary transcription factor involved in the proliferation of prostate cancer cells. Thus, hormone therapy using antiandrogens, such as bicalutamide, is a first-line treatment for the disease. Although hormone therapy initially reduces the tumor burden, many patients eventually relapse, developing tumors with acquired endocrine resistance. Elucidation of the molecular mechanisms underlying endocrine resistance is therefore a fundamental issue for the understanding and development of alternative therapeutics for advanced prostate cancer. In the present study, we performed short hairpin RNA (shRNA)-mediated functional screening to identify genes involved in bicalutamide-mediated effects on LNCaP prostate cancer cells. Among such candidate genes selected by screening using volcano plot analysis, ribosomal protein L31 (RPL31) was found to be essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR) cells, based on small interfering RNA (siRNA)-mediated knockdown experiments. Of note, RPL31 mRNA is more abundantly expressed in BicR cells than in parental LNCaP cells, and clinical data from ONCOMINE and The Cancer Genome Altas showed that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues. Intriguingly, protein levels of the tumor suppressor p53 and its targets, p21 and MDM2, were increased in LNCaP and BicR cells treated with RPL31 siRNA. We observed decreased degradation of p53 protein after RPL31 knockdown. Moreover, the suppression of growth and cell cycle upon RPL31 knockdown was partially recovered with p53 siRNA treatment. These results suggest that RPL31 is involved in bicalutamide-resistant growth of prostate cancer cells. The shRNA-mediated functional screen in this study provides new insight into the molecular mechanisms and therapeutic targets of advanced prostate cancer.

Figure 1. Screening for bicalutamide response-related genes in prostate cancer cells.

(A) Schematic representation of shRNA screening. LNCaP cells were infected with a lentiviral shRNA library and further cultured with or without bicalutamide for 1 month. Individual integrated shRNA amounts were quantified by microarray. (B) Volcano plot of microarray data, as generated by clustering based on probes that were enriched or depleted (fold change <0.5; P<0.01) in bicalutamide-treated cells compared to vehicle-treated cells.

Figure 2. Effects on bicalutamide-resistant BicR cell growth by treatment with selected siRNAs that target genes determined by shRNA screening.

Growth inhibition of BicR cells by siRNA targeting RPL31 (siRPL31), HIST1H2BD (siHIST1H2BD), and ADAMTS1 (siADAMTS1) was shown. Cells were transfected with 10 nM siRNA in culture medium. Twelve hours after transfection, cells were then further cultured in medium containing 1 µM bicalutamide. WST-8 cell proliferation assays were performed at the indicated time points after transfection. The absorbance of the wells in the plates was measured using a microplate reader at 450 nm. Data are presented as mean ± s.d. (n = 3; *, P<0.05; **, P<0.01).

Figure 3. RPL31 is overexpressed in BicR cells and clinical prostate cancer tissues.

(A) Expression levels of RPL31, HIST1H2BD, and ADAMTS1 mRNA evaluated by quantitative reverse-transcription PCR analysis (qRT-PCR) with gene-specific primers. Data are normalized to GAPDH and shown as mean ± s.d. (n = 3; **, P<0.01). (B) RPL31 mRNA is abundantly expressed in clinical prostate carcinoma tissues compared with normal prostate tissues (by > 2-fold), as retrieved from datasets by Tomlins et al. in the ONCOMINE database . Normal: normal prostate tissue, PCa: prostate cancer, PIN: prostatic intraepithelial neoplasia. (C) RPL31 mRNA expression is elevated in clinical prostate cancer samples versus normal samples in a study of RNA-sequencing in The Cancer Genome Analysis , .

Figure 4. Knockdown of RPL31 inhibits cell-cycle progression.

(A) Knockdown of RPL31 in BicR cells increased the proportion of cells in G0/G1 and decreased the proportion of those in S phase. Cells were transfected with siRPL31 or siLuc in culture medium for 48 h. Cells were then washed with PBS, stained with propidium iodide, and subjected to FACS analysis. (B) The percentages of BicR cells in S, G0/G1, and G2/M phase were determined using CellQuest software and are shown as mean ± s.d. (n = 3; *, P<0.05; **, P<0.01). (c) Knockdown of RPL31 decreased the proliferation of LNCaP cells. Cells were treated the same as described in (A). (D) The percentages of LNCaP cells in S, G0/G1, and G2/M phases were determined using CellQuest software and are shown as mean ± s.d. (n = 3; **, P<0.01).

Figure 5. RPL31 regulates levels of p53 protein expression.

(A) Knockdown of RPL31 increases p53, MDM2, and p21 protein expression. LNCaP and BicR cells were transfected with siRPL31 or siLuc for 48 h. Cell extracts were subjected to SDS-PAGE and western blot analysis using the indicated antibodies. (B) RPL31 regulated the degradation of p53 protein. BicR cells were transfected with siRPL31 or siLuc for 60 h and treated with 50 µg/mL cycloheximide (CHX) for the indicated time. Cell extracts were analyzed by western blotting. (C) p53 protein levels were quantified by densitometry and normalized to the levels of the corresponding β-actin protein and shown as mean ± s.d. (n = 3; *, P<0.05).

Figure 6. p53 partially mediates the function of RPL31 in BicR cells.

(A) Knockdown effects of RPL31 and p53 on MDM2 and p21 mRNA. BicR cells were transfected with siRPL31, sip53, siRPL31 plus sip53, or siLuc. qRT-PCR for RPL31, p53, MDM2, and p21 mRNAwas performed. Experiments were performed in triplicate; mRNA expression is normalized to GAPDH and shown as mean ± s.d. (n = 3; **, P<0.01). (B) sip53 partially cancelled the repression of cell growth induced by siRPL31. BicR cells were transfected with 10 nM each siRPL31, sip53, siRPL31 plus sip53 or siLuc, and cultured with the medium containing 1 µM bicalutamide. WST-8 cell proliferation assay was performed at the indicated time points. The absorbances of the wells in the plates were measured using a microplate reader at a 450 nm. Data are presented as mean ± s.d. (n = 4; **, P<0.01).