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In Vivo Quantitative Phosphoproteomic Profiling Identifies Novel Regulators Oncogene (2015) 34, 2764–2776 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth N Jiang1,2, K Hjorth-Jensen1, O Hekmat1, D Iglesias-Gato1, T Kruse1, C Wang2,WWei2,BKe2,BYan2, Y Niu2, JV Olsen1 and A Flores-Morales1 Prostate cancer remains a leading cause of cancer-related mortality worldwide owing to our inability to treat effectively castration- resistant tumors. To understand the signaling mechanisms sustaining castration-resistant growth, we implemented a mass spectrometry-based quantitative proteomic approach and use it to compare protein phosphorylation in orthotopic xenograft tumors grown in either intact or castrated mice. This investigation identified changes in phosphorylation of signaling proteins such as MEK, LYN, PRAS40, YAP1 and PAK2, indicating the concomitant activation of several oncogenic pathways in castration-resistant tumors, a notion that was confirmed by tumor transcriptome analysis. Further analysis demonstrated that the activation of mTORC1, PAK2 and the increased levels of YAP1 in castration-resistant tumors can be explained by the loss of androgen inhibitory actions. The analysis of clinical samples demonstrated elevated levels of PAK2 and YAP1 in castration-resistant tumors, whereas knockdown experiments in androgen-independent cells demonstrated that both YAP1 and PAK2 regulate cell colony formation and cell invasion activity. PAK2 also influenced cell proliferation and mitotic timing. Interestingly, these phenotypic changes occur in the absence of obvious alterations in the activity of AKT, MAPK or mTORC1 pathways, suggesting that PAK2 and YAP1 may represent novel targets for the treatment of castration-resistant prostate cancer. Pharmacologic inhibitors of PAK2 (PF-3758309) and YAP1 (Verteporfin) were able to inhibit the growth of androgen-independent PC3 xenografts. This work demonstrates the power of applying high-resolution mass spectrometry in the proteomic profiling of tumors grown in vivo for the identification of novel and clinically relevant regulatory proteins. Oncogene (2015) 34, 2764–2776; doi:10.1038/onc.2014.206; published online 28 July 2014 INTRODUCTION findings have been observed using the human LNCaP xenograft 11 Most patients with castration-resistant prostate cancer (CRPCa) die model, which respond better to a combined treatment of within 2 years after diagnosis.1,2 Studies of gene copy number castration and rapamycin than to the each of the treatments 12 variations, transcriptomics and most recently exon sequencing alone. In contrast to these findings, phase II clinical trials have have revealed recurrent genetic changes associated with CRPCa.3,4 shown little effects of the rapamycin analogue RAD001 as an add- Loss of phosphatase and tensin homolog (PTEN) is the most on therapy to the AR antagonist bicalutamide for the treatment of 13 common genetic alteration observed in prostate cancer irrespec- castration-resistant prostate tumors in human patients. Likewise, tive of the tumor stage, affecting approximately 45% of all tumors; mTORC1 inhibition alone is ineffective in reducing tumor load in while the amplification of the androgen receptor (AR) gene (20%) the prostate-specific PTEN knockout model,14 indicating that or point mutations within the AR (25%) are the most frequently additional pathways are critical for the growth of castration- observed genetic alteration specific to CRPCa.5–7 Also associated resistant tumors. with advanced tumors is an increased frequency of point Phosphorylation is a reversible posttranslational modification mutations within the p53 tumor suppressor gene, loss of Rb and that can inform about the activity status of kinase-driven signaling amplification of c-myc.4 How these genetic events regulate the pathways. Mass spectrometry (MS)-based proteomic profiling in pathways that promote survival and growth in CRPCa remains combination with SILAC labeling has been used to analyze growth poorly understood. factor-driven kinase-dependent signaling pathways in tissue Recent studies using genetically modified mice have demon- cultures.15,16 This approach allows for the precise and unbiased strated that prostate tumors initiated by PTEN inactivation develop quantitation of phosphorylation changes in hundreds of peptides into castration-resistant tumors that exhibit reduced expression of in a biologic sample.17 So far, this powerful methodology has been androgen-regulated genes, as a consequence of reduced levels of mainly applied to studies in vitro. Here, we take advantage of the AR expression and activity.8,9 Moreover, PTEN-negative tumors are capacity of LNCaP cells to grow both in culture and as xenografts resistant to androgen deprivation but exhibit enhanced sensitivity to perform a quantitative proteomic profiling of LNCaP tumors to treatments with PI3 kinase and mTORC1 inhibitors, thereby grown orthotopically in mice. To gain a systemic understanding demonstrating a dependency for the mTORC1 pathway for growth of the mechanisms that contribute to the growth of CRPCa, a in conditions of low androgen circulating levels.8–10 Similar comparative analysis was performed of tumors grown in intact or 1The Novo Nordisk Foundation Center for Protein Research, Department of Health Science, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark and 2Tianjin Institute of Urology, Tianjin Medical University, Tianjin, China. Correspondence: Professor Y Niu, Tianjin Institute of Urology, 23 Pingjiang Road, Hexi District, 300211 Tianjin, China or Professor A Flores-Morales, The Novo Nordisk Foundation Center for Protein Research, Department of Health Science, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, Building 6.1, Copenhagen 2200, Denmark. E-mail: [email protected] or [email protected] Received 4 December 2013; revised 21 April 2014; accepted 28 May 2014; published online 28 July 2014 Castration-resistant prostate cancer phosphoproteomics N Jiang et al 2765 castrated mice. SILAC-based quantitation identified phosphoryla- antibodies (Figure 2a). We also analyzed the status of the key tion sites that changed significantly depending on the hormonal signaling proteins within the mTORC1 and MAPK pathways and status of the host, thereby allowing for the identification of novel found that both mTORC1 and its downstream target p70S6 kinase pathways sustaining CRPCa growth. exhibited enhanced phosphorylation in CR-LNCAP, indicative of increased activity.24 The phosphorylation levels of the upstream fi RESULTS negative regulator AMPK were not signi cantly changed, whereas AKT phosphorylation changes exhibited a more complex pattern; Phosphoproteomic analysis of castration-resistant LNCAP tumors the phosphorylation of AKT on Thr308 increased, whereas AKT- LNCaP xenografts mimic many of the features of human prostate Ser473 phosphorylation was reduced in CR-LNCaP tumors in tumors, which make them a valuable model to study the comparison with androgen-sensitive xenografts (Figure 2). These 11,18 mechanism of progression to CRPCa. To obtain an overview results suggest that the two distinct pathways that contribute to of these mechanisms LNCaP xenografts were grown orthotopically maximal activation of AKT through increased PDK1 (AKT Thr308) in intact and castrated mice. Analysis of tumor growth showed and mTORC2 (AKT Ser473) activity thus appear to be oppositely that castration-resistant LNCaP (CR-LNCaP) tumors proliferate at a regulated in the transition from the androgen-sensitive to the higher rate than androgen-sensitive tumors (hormone-sensitive castration-resistant state.28 The activities of additional positive LNCaP (HS-LNCaP)) at the time of the harvest, 60 days after regulators of mTORC1, the extracellular-signal-regulated kinase fi surgical orthotopic implantation (Figures 1a and b). These ndings (ERK) kinases 1/2, were also increased in CR-LNCaP tumors, mimic the situation with human CRPCa, which proliferate at a suggesting that ERKs may contribute to growth in these fi 19,20 higher rate than castration-naive, prostate-con ned tumors. conditions possibly influencing mTORC1 activation.29 Finally, In addition, CR-LNCAP tumors showed an increased number of we also confirmed the upregulation of phospho-PAK2 levels Ki67-positive cells and diminished levels of active caspase-3, (Figure 2). Immunohistochemical analysis showed that both PAK2 indicating that an enhanced proliferative rate and reduced and YAP1 exhibit significant increased expression in CR-LNCaP apoptosis contribute to the increased growth rate of tumors xenograft models, with both proteins showing a predominantly grown in castrated mice (Figures 1c and d). cytosolic expression and YAP1 occasionally staining the nucleus Next, we performed a comprehensive quantitative phospho- proteomic profiling using SILAC-labeled LNCaP-FGC cells as (Supplementary Figure S1). internal, spiked-in controls in all samples analyzed.21 To account for biologic variation, four individual tumors were analyzed for Transcript profiling reveals pathways deregulated in CR-LNCaP each experimental group. In parallel, we also performed a tumors genome-wide transcriptomic analysis. A summary of the metho- If the pathways identified as regulated in castration-resistant dology is presented in Figure 1e. cancer by phosphoproteomic analysis are functionally relevant, we A total of 2782 phosphorylated peptides from
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