Short Hairpin RNA Library-Based Functional Screening Identified Ribosomal Protein L31 That Modulates Prostate Cancer Cell Growth via p53 Pathway

<div><p>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, <i>RPL31</i> 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 <i>RPL31</i> siRNA. We observed decreased degradation of p53 protein after <i>RPL31</i> knockdown. Moreover, the suppression of growth and cell cycle upon <i>RPL31</i> knockdown was partially recovered with <i>p53</i> 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.</p></div

<i>RPL31</i> is overexpressed in BicR cells and clinical prostate cancer tissues.

<p>(A) Expression levels of <i>RPL31</i>, <i>HIST1H2BD</i>, and <i>ADAMTS1</i> mRNA evaluated by quantitative reverse-transcription PCR analysis (qRT-PCR) with gene-specific primers. Data are normalized to <i>GAPDH</i> and shown as mean ± s.d. (n = 3; **, <i>P</i><0.01). (B) <i>RPL31</i> mRNA is abundantly expressed in clinical prostate carcinoma tissues compared with normal prostate tissues (by > 2-fold), as retrieved from datasets by Tomlins <i>et al.</i> in the ONCOMINE database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Rhodes1" target="_blank">[30]</a>. Normal: normal prostate tissue, PCa: prostate cancer, PIN: prostatic intraepithelial neoplasia. (C) <i>RPL31</i> mRNA expression is elevated in clinical prostate cancer samples <i>versus</i> normal samples in a study of RNA-sequencing in The Cancer Genome Analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Chin1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108743#pone.0108743-Chin2" target="_blank">[32]</a>.</p

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

<p>Growth inhibition of BicR cells by siRNA targeting <i>RPL31</i> (siRPL31), <i>HIST1H2BD</i> (siHIST1H2BD), and <i>ADAMTS1</i> (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; *, <i>P</i><0.05; **, <i>P</i><0.01).</p

p53 partially mediates the function of RPL31 in BicR cells.

<p>(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; **, <i>P</i><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; **, <i>P</i><0.01).</p

Knockdown of <i>RPL31</i> inhibits cell-cycle progression.

<p>(A) Knockdown of <i>RPL31</i> 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; *, <i>P</i><0.05; **, <i>P</i><0.01). (c) Knockdown of <i>RPL31</i> 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; **, <i>P</i><0.01).</p

RPL31 regulates levels of p53 protein expression.

<p>(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; *, <i>P</i><0.05).</p

The list of genes targeted by shRNAs exhibiting bicalutamide-mediated downregulation in lentiviral library-transduced LNCaP cells and the knockdown efficiency of each siRNA chosen for validation.

a)<p>Silencer select pre-designed siRNAs targeting the candidate genes were obtained from Ambion.</p>b)<p>Knockdown efficiency of siRNA was determined by qRT-PCR and normalized to that of siControl.</p><p>The list of genes targeted by shRNAs exhibiting bicalutamide-mediated downregulation in lentiviral library-transduced LNCaP cells and the knockdown efficiency of each siRNA chosen for validation.</p

Bleeding diathesis in <i>Ggcx^{Δliver/Δliver}</i> mice.

<p>A, Decreased activity of coagulation factor in <i>Ggcx^{Δliver/Δliver}</i> mice. Activities of factors II and IX were significantly decreased in <i>Ggcx^{Δliver/Δliver}</i> mice compared with wild type (<i>Ggcx^+/+</i>) mice. Bars represent the mean value ± SEM (n = 8). Differences between the mean values were analyzed using the unpaired Student's <i>t</i>-test. ***<i>P</i><0.001. B, Prolonged bleeding time in <i>Ggcx^{Δliver/Δliver}</i> mice. Tail bleeding time was measured by a filter paper method. <i>Ggcx^{Δliver/Δliver}</i> mice continued bleeding for more than 30 min. Gray triangle: 0 min; Black triangle: 10 min. A representative figure is shown. C. Platelet counts of <i>Ggcx^{Δliver/Δliver}</i> mice. Hematological examination of male <i>Ggcx^{Δliver/Δliver}</i> mice (n = 6), female <i>Ggcx^{Δliver/Δliver}</i> mice (n = 12), male wild type mice (n = 10), and female wild type mice (n = 10) was performed. The platelet count was not significantly different between wild type mice and <i>Ggcx^{Δliver/Δliver}</i> mice. Bars represent the mean value ± SEM. NS: not significant.</p

Shorter life span of <i>Ggcx^{Δliver/Δliver}</i> mice.

<p>Cumulative life spans of male <i>Ggcx^{Δliver/Δliver}</i> mice (n = 10), female <i>Ggcx^{Δliver/Δliver}</i> mice (n = 11), male heterozygous littermates (<i>Ggcx^+/Δliver</i> mice) (n = 12), and female heterozygous littermates (<i>Ggcx^+/Δliver</i> mice) (n = 12) were calculated by the Kaplan-Meier method and compared using the log-rank test. P-values were adjusted by the Bonferroni method. Life span of male <i>Ggcx^{Δliver/Δliver}</i> mice was significantly shorter (<i>P</i><0.001) compared with that of heterozygous mice. The life spans of female <i>Ggcx^{Δliver/Δliver}</i> mice were significantly longer than those of male <i>Ggcx^{Δliver/Δliver}</i> mice (<i>P</i> = 0.044).</p

Liver-specific ablation of <i>Ggcx</i> gene.

<p>A, The albumin promoter is active only in hepatocytes. Alb-Cre or wild type (WT) mice were mated with ROSA26-LacZ mice. Livers were obtained postnatally from mice and stained with X-gal. β-galactosidase activity was detected in the hepatocytes of the mouse born from mating of Alb-Cre and ROSA26-LacZ mice (right panel). Liver of the mouse born from wild type was shown as a negative control (left panel). Representative figures are shown for each group. B, Genotyping using genomic tail DNA. Expected bands from Cre recombinase, <i>loxP</i> sequence (floxed allele), and wild type allele are shown. In mouse #2, #4 and #5, both alleles were replaced with <i>loxP</i> containing recombinant alleles, with at least one copy of Cre recombinase. A representative result is shown. C, Liver-specific ablation of <i>Ggcx</i> gene was confirmed with PCR analysis. DNA samples derived from liver, spleen, kidney and heart of 6-month week old <i>Ggcx^{Δliver/Δliver}</i> mice (4 male mice and 4 female mice) and control <i>Ggcx^+/+</i> mice (4 male mice and 4 female mice) were used as templates. D, Activity of Ggcx in the liver-specific Ggcx-deficient mice. Microsome was prepared from the livers of 6-week old <i>Ggcx^{Δliver/Δliver}</i> mice and control <i>Ggcx^+/+</i> mice of both sexes. The activity of Ggcx was measured by ¹⁴CO₂ incorporated into exogenous substrate in the presence of reduced vitamin K (222 µM). Bars represent the mean value ± SEM (n = 4). Differences between the mean values were analyzed using the unpaired Student's <i>t</i>-test. **<i>P</i><0.01.</p