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

Yojiro Maruyama,Koji Okamoto,Toshiyuki Okumura,Kuniko Horie-Inoue,Satoru Takeda,Satoshi Inoue,Kazuhiro Ikeda,Wataru Sato,Toshiaki Miyazaki,Chih-Pin Chuu

doi:10.1371/journal.pone.0108743

Yojiro Maruyama, Koji Okamoto + Show 8 more

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https://doi.org/10.1371/journal.pone.0108743

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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.

Highlights

Prostate cancer is the fourth most common cause of cancerrelated deaths, and the incidence of prostate cancer in Japan is increasing, with .11,000 deaths per year from the disease
Validation of candidate genes To evaluate the effects of the individual genes selected by short hairpin RNA (shRNA) screening on prostate cancer cell biology, the knockdown efficacy of available small interfering RNA (siRNA) targeted to 19 of the 25 candidate genes was evaluated by quantitative reverse transcriptionPCR
The results indicated that silencing of ribosomal protein L31 (RPL31), HIST1H2BD, and ADAMTS1significantly repressed cell proliferation in bicalutamideresistant LNCaP (BicR) cells by .50% compared to control siRNA

Summary

Introduction

Prostate cancer is the fourth most common cause of cancerrelated deaths, and the incidence of prostate cancer in Japan is increasing, with .11,000 deaths per year from the disease. While most early-stage, localized disease can be successfully treated by radiation therapy and/or surgery, as many as 50% of patients treated for localized disease will have local recurrence or distant metastases [1,2]. The current first-line treatments for recurrent or metastatic prostate cancer are hormone therapies, including those that target androgen receptor (AR) signaling such as bicalutamide, and drugs such as gonadotropin-releasing hormone agonists that prevent androgen production in the testicles and adrenal glands. Hormone therapies initially reduce the tumor burden, many patients become resistant to these therapies and develop a terminal form of the disease, termed castration-resistant prostate cancer (CRPC) [3]. Recent studies have revealed that CRPC is commonly associated with increased AR signaling due to AR amplification, AR mutation, transcription cofactor activation, ligand-independent phosphorylation of AR, and other processes [4,5,6,7]

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