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Androgen-Regulation of the Protein Tyrosine Phosphatase PTPRR Activates ERK1/2 Signalling in Prostate Cancer

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Androgen-Regulation of the Protein Tyrosine Phosphatase PTPRR Activates ERK1/2 Signalling in Prostate Cancer Munkley J, Lafferty NP, Kalna G, Robson CN, Leung HY, Rajan P, Elliott DJ. Androgen-regulation of the protein tyrosine phosphatase PTPRR activates ERK1/2 signalling in prostate cancer. BMC Cancer 2015, 15, 9 Copyright: © 2015 Munkley et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. DOI link to article: http://dx.doi.org/10.1186/s12885-015-1012-8 Date deposited: 30/04/2015 This work is licensed under a Creative Commons Attribution 4.0 International License Newcastle University ePrints - eprint.ncl.ac.uk Munkley et al. BMC Cancer (2015) 15:9 DOI 10.1186/s12885-015-1012-8 RESEARCH ARTICLE Open Access Androgen-regulation of the protein tyrosine phosphatase PTPRR activates ERK1/2 signalling in prostate cancer cells Jennifer Munkley1*, Nicholas P Lafferty1, Gabriela Kalna2, Craig N Robson4, Hing Y Leung2,3, Prabhakar Rajan2,3 and David J Elliott1 Abstract Background: Androgens drive the onset and progression of prostate cancer (PCa) via androgen receptor (AR) signalling. The principal treatment for PCa is androgen deprivation therapy, although the majority of patients eventually develop a lethal castrate-resistant form of the disease, where despite low serum testosterone levels AR signalling persists. Advanced PCa often has hyper-activated RAS/ERK1/2 signalling thought to be due to loss of function of key negative regulators of the pathway, the details of which are not fully understood. Methods: We recently carried out a genome-wide study and identified a subset of 226 novel androgen-regulated genes (PLOS ONE 6:e29088, 2011). In this study we have meta-analysed this dataset with genes and pathways frequently mutated in PCa to identify androgen-responsive regulators of the RAS/ERK1/2 pathway. Results: We find the PTGER4 and TSPYL2 genes are up-regulated by androgen stimulation and the ADCY1, OPKR1, TRIB1, SPRY1 and PTPRR are down-regulated by androgens. Further characterisation of PTPRR protein in LNCaP cells revealed it is an early and direct target of the androgen receptor which negatively regulates the RAS/ERK1/2 pathway and reduces cell proliferation in response to androgens. Conclusion: Our data suggest that loss of PTPRR in clinical PCa is one factor that might contribute to activation of the RAS/ERK1/2 pathway. Keywords: PTPRR, RAS/ERK1/2, MAP Kinase, Androgens, Prostate cancer Background signatures between these different cancer types is a key Prostate cancer (PCa) is the most commonly-diagnosed goal. Androgen deprivation therapy (ADT) is the principal malignancy in men [1], and is driven by androgen hor- treatment for advanced PCa, although, over time, the mones acting via their cognate nuclear androgen receptor disease becomes castration-resistant (CRPCa) with limited (AR) transcription factor. The AR exerts its transcriptional treatment options [3]. Persistence of AR signalling and effects by binding to DNA sequences termed androgen reprogramming of the AR transcriptional landscape may response elements (AREs) within promoter regions of a underlie progression to CRPCa [4,5], and highlights the number of androgen-regulated genes, including genes importance of AR biology in advanced PCa. Hence, encoding cell cycle regulators and regulators of central increasing our understanding of the AR signalling in PCa metabolism and biosynthesis [2]. An important feature of cells should lead to more effective treatment strategies for PCa is prognostic heterogeneity: while some prostate advanced PCa. cancers can remain indolent for many years others can Recently, reciprocal cross-talk between the PI3K become much more rapidly aggressive. Distinguishing key pathway and AR signalling has been highlighted as a potential mechanism underlying CRPCa [6]. Alterations in PI3K signalling in advanced PCa are predominantly * Correspondence: [email protected] 1Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne driven by loss of the tumour suppressor gene PTEN NE1 3BZ, UK which contributes to the progression to invasive disease Full list of author information is available at the end of the article © 2015 Munkley et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Munkley et al. BMC Cancer (2015) 15:9 Page 2 of 11 [7-9]. Another common feature of advanced PCa is hyper- using NotIandXhoI for creation of the Flp-In™-293 stable activation of the RAS/ERK1/2 pathway [10-12] thought to cell line. be driven by loss of function of key negative regulators of the pathway [13]. Although RAS/ERK1/2 activation alone Cell culture cannot initiate PCa development, it can serve as a Cell culture and androgen treatment of cells was as de- potentiating second hit to loss of PTEN to accelerate PCa scribed previously [14,15]. All cells were grown at 37°C progression [13]. in 5% CO2. LNCaP cells (CRL-1740, ATCC) were main- Because of its established importance in clinical prostate tained in RPMI-1640 with L-Glutamine (PAA Laborator- cancer, the identification of new mechanisms through ies, R15-802) supplemented with 10% Fetal Bovine which the RAS/RAF/MAPK/ERK pathway is regulated is Serum (FBS) (PAA Laboratories, A15-101). For andro- of great interest. We recently carried out genome-wide gen treatment of LNCaP cells, medium was supple- exon-specific profiling of PCa cells to identify novel mented with 10% dextran charcoal stripped FBS (PAA androgen-regulated transcriptional events [14]. As well Laboratories, A15-119) to produce a steroid-deplete me- as identifying a number of alternative mRNA isoforms dium. Following culture for 72 hours, 10 nM synthetic [15], we also identified a subset of 226 novel androgen- androgen analogue methyltrienolone (R1881) (Perkin– regulated genes [14]. In the light of evidence implica- Elmer, NLP005005MG) was added (Androgen +) or ting cross-talk with AR, we searched this dataset for absent (Steroid deplete) for the times indicated. Where novel androgen-regulated genes associated with RAS/ indicated, LNCaP cells were pre-treated for 1 hour with ERK1/2 signalling. vehicle (dimethylsulfoxide; DMSO) (Sigma, C1988) or 1 μg/ml cycloheximide (Sigma, D2438) prior to addition Methods of 10 nM R1881 for 24 hours as previously described IPA Pathway analysis [16]. Similarly, LNCaP cells were pre-treated with with μ Gene lists from Rajan et al. [14] were uploaded to the 10 M bicalutamide (Casodex, AstraZeneca) or ethanol web-based Ingenuity Pathway Analysis (IPA; Ingenuity (vehicle) for 2 hours prior to addition of 10 nM R1881 Systems) software programme, and the “Core Analysis” for 24 hours. function was used to study direct and indirect regulatory PC-3 (CRL-1435, ATCC), PC-3 M [17], CWR22Rv1 relationships between genes and their known biological (CRL-2505, ATCC), DU145 (HTB-81, ATCC), and functions. BPH-1 cells [18] were maintained in RPMI-1640 with L-Glutamine supplemented with 10% FBS. LNCaP-AI and LNCaP-cdxR were derived from LNCaP parental Antibodies cells and maintained as previously described [19,20]. The following antibodies were used in the study: anti- Stable LNCaP cell lines were generated by transfecting PTPRR rabbit polyclonal (17937 Proteintech), anti- cells using Lipofectamine 2000 (11668-027, Invitrogen), phospho-p44/42 MAPK mouse monoclonal (Erk1/2 followed by selection with 300 μg/ml Geneticin (Invitrogen, Thr202/Tyr204) (Cell signalling 9106), anti-ERK2 mouse 10131019) (reduced to 150 μg/ml following the death of monoclonal (1647 Santa Cruz), anti-actin rabbit polyclonal untransfected cells) for at least four weeks. Flp-In™-293 (A2668, Sigma), anti-AR mouse antibody (BD Bioscience, cells (R750-07, Invitrogen) were maintained in DMEM 554226), anti-FLAG mouse monoclonal (F3165, Sigma), GlutaMax (Invitrogen, 10566-040), supplemented with anti-PTEN rabbit polyclonal (Cell Signalling 138G6), anti- 10% FBS (PAA Laboratories, A15-101) and stable cell lines α-Tubulin mouse monoclonal (Sigma T5168), normal generated using the Flp-In T-Rex Core Kit (K6500-01, rabbit IgG (711-035-152 Jackson labs) and normal mouse Invitrogen) according to the manufacturer’s instructions. IgG (715-036-150 Jackson labs). The specificity of the Protein expression was induced using 1 μg/ml tetracycline PTPRR antibody was confirmed by blocking with the (T7660, Sigma) for 72 hours. immunising peptide (ag12145 Proteintech) (Additional file 1: Figure S2). RT-qPCR Cells were harvested and total RNA extracted using TRI- esiRNA zol (Invitrogen, 15596-026), according to manufacturer’s esiRNAs PTPRR and AR were obtained from Sigma- instructions. RNA was treated with DNase 1 (Ambion) Aldrich (EHU078991 and EHU025951). and cDNA was generated by reverse transcription of 1 μg of total RNA using the Superscript VILO cDNA DNA constructs synthesis kit (Invitrogen, 11754-050). Quantitative PCR PTPRR cloned into pCDNA3.1 was a kind gift from (qPCR) was performed in triplicate on cDNA using SYBR® Mirco Menigatti, University of Zurich. The PTPRR open Green PCR Master Mix (Invitrogen, 4309155) using reading frame was subsequently cloned into pCDNA5 Applied Biosystems 7900HT. Samples were normalised Munkley et al. BMC Cancer (2015) 15:9 Page 3 of 11 using the average of three reference genes: GAPDH, Figure S1). To test whether the PTPRR gene might be β –tubulin and actin. All primer sequences are listed in under direct control of androgens through AR regulation, Additional file 2: Table S2.
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