The HDL receptor SR-BI is associated with human prostate cancer progression and plays a possible role in establishing androgen independence

Schörghofer et al. Reproductive Biology and Endocrinology (2015) 13:88 DOI 10.1186/s12958-015-0087-z RESEARCH Open Access The HDL receptor SR-BI is associated with human prostate cancer progression and plays a possible role in establishing androgen independence David Schörghofer1†, Katharina Kinslechner1†, Andrea Preitschopf1, Birgit Schütz1, Clemens Röhrl2, Markus Hengstschläger1, Herbert Stangl2 and Mario Mikula1* Abstract Background: Human prostate cancer represents one of the most frequently diagnosed cancers in men worldwide. Currently, diagnostic methods are insufficient to identify patients at risk for aggressive prostate cancer, which is essential for early treatment. Recent data indicate that elevated cholesterol levels in the plasma are a prerequisite for the progression of prostate cancer. Here, we analyzed clinical prostate cancer samples for the expression of receptors involved in cellular cholesterol uptake. Methods: We screened mRNA microarray files of prostate cancer samples for alterations in the expression levels of cholesterol transporters. Furthermore, we performed immunohistochemistry analysis on human primary prostate cancer tissue sections derived from patients to investigate the correlation of SR-BI with clinicopathological parameters and the mTOR target pS6. Results: In contrast to LDLR, we identified SR-BI mRNA and protein expression to be induced in high Gleason grade primary prostate cancers. Histologic analysis of prostate biopsies revealed that 53.6 % of all cancer samples and none of the non-cancer samples showed high SR-BI staining intensity. The disease-free survival time was reduced (P = 0.02) in patients expressing high intra-tumor levels of SR-BI. SR-BI mRNA correlated with HSD17B1 and HSD3B1 and SR-BI protein staining showed correlation with active ribosomal protein S6 (RS = 0.828, P < 0.00001). Conclusions: We identified SR-BI to indicate human prostate cancer formation, suggesting that increased levels of SR-BI may be involved in the generation of a castration-resistant phenotype. Keywords: SCARB1, LDLR, Cholesterol, mTOR, Androgen synthesis Background Prostate cancer is one of the most common solid organ tumors in males. It is a slow growing type of tumor, but can potentially give rise to aggressive and metastasizing forms of cancer [1]. The risk for prostate cancer increases with consumption of a high fat, high cholesterol diet or the presence of hypercholesterolemia [2–4]. Very recently, it was shown that the accumulation of esterified cholesterol underlies the aggressiveness of human prostate cancer [5]. Cellular cholesterol is either synthesized by the * Correspondence: † Equal contributors 1 Institute of Medical Genetics, Medical University of Vienna, Währinger Strasse 10, 1090 Vienna, Austria Full list of author information is available at the end of the article cells themselves, or exogenous cholesterol is taken up and utilized by the cancer cells. Cholesterol uptake is mainly mediated by the high density lipoprotein receptor SR-BI and the low density lipoprotein receptor LDLR [6–9]. In normal tissue, SR-BI is expressed in the liver and in steroidogenic tissues, where cholesterol uptake is necessary for steroid hormone synthesis [10–13]. Notably, patients suffering from mutations in cla-1, the human homolog to SR-BI, display impaired steroid hormone synthesis [14]. There is evidence that SR-BI plays a role in prostate cancer development, specific antigen secretion and the viability of prostate cancer cells because it was shown that SR-BI-specific knockdown in LNCaP and C4-2 prostate carcinoma cells reduced PSA secretion and the viability of © 2015 Schörghofer et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Schörghofer et al. Reproductive Biology and Endocrinology (2015) 13:88 Page 2 of 10 prostate cancer cell lines [15]. Therefore, this study aimed to evaluate the expression of receptors involved in cellular cholesterol uptake in clinical prostate cancer samples. LDLR (Santa Cruz Biotechnology, Santa Cruz, CA) and pS6 (Cell Signaling Technology, Beverly, MA), diluted 1:200. For negative control staining, sections were incubated with matched isotope control antibodies instead of primary antibodies. Next, slides were washed and the corresponding secondary, biotinylated antibodies (Vector Laboratories) were added for 45 min at room temperature. After a washing step, sections were incubated for 30 min with Streptavidin-HRP (Leica, Wetzlar, Germany). For detection, tissue sections were incubated with AEC+ High Sensitivity Substrate Chromogen (Dako). Counterstaining with hematoxylin solution was performed according to Mayer (Carl Roth, Karlsruhe, Germany); tissue sections were mounted with Aquatex® (Merck Millipore, Billerica, MA). Material and methods Bioinformatic analysis For Gleason score analysis, the GSE2109 and GSE3933 datasets from the International Genomics Consortium Expression Project for Oncology were used [16]. The sample sizes were as follows: GSE2109, n = 56 (Gleason score ≤ 6 n = 20, Gleason score ≥ 7 n = 36), GSE3933, n = 58 for SR-BI (Gleason score ≤ 6 n = 24, Gleason score ≥ 7 n = 34) and n = 60 for LDLR (Gleason score ≤ 6 n = 24, Gleason score ≥ 7 n = 36). For metastasis analysis, the GSE35988, GSE3933 and GSE6919 datasets were used [16–19]. The sample sizes were as follows: GSE35988, n = 94 (primary site n = 59, metastasis n = 35), GSE3933, n = 68 for SR-BI (primary site n = 59, metastasis n = 9) and n = 68 for LDLR (primary site n = 61, metastasis n = 7) and GSE6919, n = 88 (primary site n = 64, metastasis n = 24). For Kaplan-Meier analysis, the GSE40272 dataset was used (sample size: n = 85) [20]. Evaluation of immunohistochemical staining Evaluation of tissue sections was performed by two independent researchers who were blinded to the patients’ details. Immunostaining of the anti-SR-BI antibody was scored on at least duplicate tissues using the following arbitrary scale: no staining (0), low staining (1), medium staining (2) and high staining (3). Patient cohort and pathology With institutional review board approval from the Medical University of Vienna (EK Nr: 1734/2014), tissue microarrays were obtained from US Biomax (Rockville, MD). All samples were formalin-fixed less than 10 min after surgery, paraffin embedded and assembled as cores with a diameter of 1.5 mm. Tissue sections were quality controlled and contained normal prostate tissue and pros (...truncated)