Oncogene (2019) 38:1136–1150 https://doi.org/10.1038/s41388-018-0501-z ARTICLE A reciprocal feedback between the PDZ binding kinase and androgen receptor drives prostate cancer 1 2 3 4 3 5,6 Anne Y. Warren ● Charlie E. Massie ● Kate Watt ● Katarina Luko ● Folake Orafidiya ● Luke A. Selth ● 7 1 8 8 1 8 Hisham Mohammed ● Brinder S. Chohan ● Suraj Menon ● Ajoeb Baridi ● Wanfeng Zhao ● Carles Escriu ● 4 8 8 8 8 8 Thanakorn Pungsrinont ● Clive D’Santos ● Xiaoping Yang ● Chris Taylor ● Arham Qureshi ● Vincent R. Zecchini ● 8 9 10,11 8 5,6 3 Greg L. Shaw ● Scott M. Dehm ● Ian G. Mills ● Jason S. Carroll ● Wayne D. Tilley ● Iain J. McEwan ● 4 8,11 8,11,12 Aria Baniahmad ● David E. Neal ● Mohammad Asim Received: 16 March 2018 / Revised: 17 July 2018 / Accepted: 16 August 2018 / Published online: 20 September 2018 © The Author(s) 2018. This article is published with open access Abstract Elucidation of mechanisms underlying the increased androgen receptor (AR) activity and subsequent development of aggressive prostate cancer (PrCa) is pivotal in developing new therapies. Using a systems biology approach, we interrogated the AR-regulated proteome and identified PDZ binding kinase (PBK) as a novel AR-regulated protein that regulates full- 1234567890();,: 1234567890();,: length AR and AR variants (ARVs) activity in PrCa. PBK overexpression in aggressive PrCa is associated with early biochemical relapse and poor clinical outcome. In addition to its carboxy terminus ligand-binding domain, PBK directly interacts with the amino terminus transactivation domain of the AR to stabilise it thereby leading to increased AR protein expression observed in PrCa. Transcriptome sequencing revealed that PBK is a mediator of global AR signalling with key roles in regulating tumour invasion and metastasis. PBK inhibition decreased growth of PrCa cell lines and clinical specimen cultured ex vivo. We uncovered a novel interplay between AR and PBK that results in increased AR and ARVs expression that executes AR-mediated growth and progression of PrCa, with implications for the development of PBK inhibitors for the treatment of aggressive PrCa. Introduction These authors contributed equally: David E. Neal, Mohammad Asim Prostate cancer (PrCa) is a major cancer responsible for Electronic supplementary material The online version of this article male fatalities worldwide [1]. The growth and survival of (https://doi.org/10.1038/s41388-018-0501-z) contains supplementary PrCa is driven by the male sex hormone androgens that act material, which is available to authorized users. * Mohammad Asim 6 Freemasons Foundation Centre for Men’s Health, School of [email protected] Medicine, Faculty of Health Sciences, University of Adelaide, [email protected] Adelaide, Australia 7 Cancer Early Detection Advanced Research Center, Knight 1 Department of Pathology, Addenbrooke’s Cambridge University Cancer Institute, OHSU, Portland, USA Hospital, Cambridge, UK 8 Cancer Research UK Cambridge Institute, University of 2 Early Detection Programme, Cancer Research UK Cambridge Cambridge, Cambridge, UK Centre, Department of Oncology, University of Cambridge, Cambridge, UK 9 Masonic Cancer Center, University of Minnesota, Minneapolis, USA 3 Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK 10 Prostate Cancer UK/Movember Centre of Excellence, CCRCB, Queens University, Belfast, UK 4 Institute of Human Genetics, Jena University Hospital, Jena, Germany 11 Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK 5 Dame Roma Mitchell Cancer Research Laboratories, School of Medicine, Faculty of Health Sciences, University of Adelaide, 12 Department of Clinical & Experimental Medicine, Faculty of Adelaide, Australia Health & Medical Sciences, University of Surrey, Guildford, UK A reciprocal feedback between the PDZ binding kinase and androgen receptor drives prostate cancer 1137 by binding to the androgen receptor (AR) [2, 3]. Conse- NTD and LBD of AR to directly regulate the stability and quently, androgen-deprivation therapy (ADT) is used as a function of both full-length AR and ARVs. Inhibition of first-line therapy for the treatment of locally advanced or PBK activity destabilises AR and decreases PrCa growth metastatic PrCa. ADT comprises; luteinizing hormone- and metastasis. These results provide novel insights into the releasing hormone (LHRH) analogues that block androgen role of androgen-induced pathways in PrCa revealing PBK production, anti-androgens such as bicalutamide and enza- as a key effector through which AR manifest its oncogenic lutamide that antagonise androgen binding to the AR, and function in PrCa. abiraterone that inhibits the androgen biosynthesis pathway [4, 5]. ADT is initially effective but resistance is common and Results this leads to the development of aggressive incurable dis- ease known as castration-resistant prostate cancer (CRPC) Identification of PBK as a novel AR target in PrCa [6]. CRPC is often driven by the reactivation of AR sig- nalling through multiple molecular mechanisms, which Mass spectrometry identified the AR-regulated proteome of include amplification and gain-of-function mutations of the the CRPC cells following AR knockdown using a pre- AR gene, gain-of-function mutations in the androgen sig- validated set of four different siRNAs as used earlier [14]or nalling pathway [7], and the emergence of constitutively AR inhibition with the AR antagonist, bicalutamide. Sup- active AR splice forms known as AR variants (ARVs; e.g., porting the robustness of the data, a significant correlation ARV7). ARVs are devoid of the ligand-binding domain was found between proteins regulated by AR knockdown (LBD) and thus such variants cannot be targeted by ADT and bicalutamide treatment (Pearson r2 = 0.51). The protein [8, 9]. Notably, the AR amino terminus domain (NTD) is levels of known androgen-upregulated targets were down- the prime driver of PrCa growth, thus making it a potential regulated by both AR knockdown and bicalutamide treat- drug target [10, 11]. ment, whereas androgen-repressed proteins were In addition to direct changes to the AR, another upregulated by both AR treatments (n = 514) (Fig. 1a and mechanism underlying CRPC growth is increased activity Table S1). and/or expression of AR co-activators [12–14]. Yet another A core set of 77 AR-regulated proteins common between hallmark of AR signalling in PrCa is the integrated feed- AR knockdown and bicalutamide treatment was identified forward and feedback circuits that facilitate pro-growth that showed consistent changes in the same direction, signalling as exemplified by the reciprocal positive feed- including known AR-regulated targets such as FKBP5 and back between AR and choline kinase alpha signalling [14, FASN (Figure S1A and Table S2). This analysis also pro- 15] and reciprocal negative feedback between AR and poly vided evidence that the vesicle transport protein NIPS- ADP-Ribose polymerase-1 signalling [16]. Androgens can NAP3A is an AR-repressed target. Among the factors in the also negatively regulate AR mRNA expression by recruit- proteome data that were commonly downregulated by both ment of AR to a repressive element in intron 2 of the AR AR knockdown and bicalutamide treatment was PBK (Fig. gene [17, 18]. Despite decreasing AR mRNA levels, 1a and Figure S1A). Supporting these findings, PBK tran- androgens promote AR stabilisation leading to overall script and protein levels decreased following AR knock- increase in AR protein expression. This example highlights down (Fig. 2b, Figures S1B and S5A) and AR-binding sites the critical need to analyse the androgen-regulated proteome were identified at the enhancer upstream of the PBK gene in order to characterise androgen-regulated changes at (Figure S1C). PBK expression in situ was also androgen- protein level in PrCa. regulated in a series [19] of human prostate tissue com- In order to understand how AR is stabilised and pro- prising benign prostate tissues and PrCa samples from motes PrCa growth, it is imperative to identify clinically untreated men (n = 20) or those treated with androgen relevant AR targets, both up- and downstream of AR sig- inhibitor degarelix (a LHRH agonist that blocks testicular nalling. Here, we generated androgen-regulated proteome androgen synthesis) for 7 days (n = 27) (Fig. 1c). Nuclear data sets to comprehensively identify effectors of androgen PBK staining was significantly higher in PrCa tissues signalling and identified PDZ binding kinase (PBK) as an compared with benign control tissues from untreated men AR-regulated factor in vitro and in vivo in PrCa that inte- ((p < 0.0001) (Fig. 1c, d). Degarelix treatment decreased the grates AR signalling with PrCa growth. In men with PrCa, nuclear levels of PBK in the treated tumours compared with PBK is androgen-regulated and localised in the tumour cell untreated patients (p < 0.01), with 45% of degarelix-treated nucleus. PBK overexpression is associated with poor dis- cases having no detectable expression of PBK in compar- ease outcome and clinical progression. We further uncover ison to 15% of untreated cases. Overall, cytosolic PBK PBK interactome and identify a crucial reciprocal feedback levels were lower compared to nuclear levels in all tissues between PBK and AR, whereby PBK interacts with both the tested, whereas there was no significant difference in 1138 A. Y. Warren et al. A B Protein profiles (log2 ratio) AR siNT si 1.0 Cor = 0.51 1.0 0.32 AR 0.5 β-Actin 0.0 1.0 0.31 Bicalutamide PBK -0.5 Ponceau -1.5-1.0 -0.5