Published OnlineFirst July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Biology of Human Tumors Clinical Cancer Research The Proteome of Prostate Cancer Bone Metastasis Reveals Heterogeneity with Prognostic Implications Diego Iglesias-Gato1,2, Elin Thysell3, Stefka Tyanova4, Sead Crnalic5, Alberto Santos6, Thiago S. Lima1,2, Tamar Geiger7,Jurgen€ Cox4, Anders Widmark8, Anders Bergh3, Matthias Mann4,6, Amilcar Flores-Morales1,2, and Pernilla Wikstrom€ 3 Abstract Purpose: Bone is the most predominant site of distant fatty acid b-oxidation; and reduced levels of proteins were metastasis in prostate cancer, and patients have limited ther- related to cell adhesion and carbohydrate metabolism. Within apeutic options at this stage. bone metastases, we identified two phenotypic subgroups: Experimental Design: We performed a system-wide BM1, expressing higher levels of AR canonical targets, and quantitative proteomic analysis of bone metastatic prostate mitochondrial and Golgi apparatus resident proteins; and tumors from 22 patients operated to relieve spinal cord BM2, with increased expression of proliferation and DNA compression. At the time of surgery, most patients had repair–related proteins. The two subgroups, validated by the relapsed after androgen-deprivation therapy, while 5 were inverse correlation between MCM3 and prostate specific anti- previously untreated. An extended cohort of prostate cancer gen immunoreactivity, were related to disease prognosis, bone metastases (n ¼ 65) was used for immunohistochem- suggesting that this molecular heterogeneity should be con- ical validation. sidered when developing personalized therapies. Results: On average, 5,067 proteins were identified Conclusions: This work is the first system-wide quantitative and quantified per tumor. Compared with primary tumors characterization of the proteome of prostate cancer bone (n ¼ 26), bone metastases were more heterogeneous and metastases and a valuable resource for understanding the showed increased levels of proteins involved in cell-cycle etiology of prostate cancer progression. Clin Cancer Res; 1–12. progression, DNA damage response, RNA processing, and Ó2018 AACR. Introduction patients with castration-resistant bone metastases (mCRPC), their prognosis remains poor with a median survival below 2 years (4). Mortality in prostate cancer is strongly associated with devel- This is in contrast to patients harboring prostate confined tumors, opment of bone metastatic disease (1, 2). Prostate cancer metas- who have a 10-year cancer-specific survival rate above 90%, even tases are generally treated with androgen ablation (3). This for patients managed by observation protocols (5, 6). Why therapy is initially effective, but most patients relapse to a cas- metastatic tumors have a more aggressive behavior than localized tration-resistant stage. Despite recent treatment developments for tumors is not fully understood. Recent genetic studies have identified only a modest increase in the number and frequency 1 of genetic aberrations in lethal, therapy-resistant metastatic cancer Department of Drug Design and Pharmacology, Faculty of Health and Medical TP53, PTEN, FOXA1 Sciences, University of Copenhagen, Copenhagen, Denmark. 2The Danish Can- as compared with localized tumors (e.g., , and PI3KCA AR fi cer Society, Copenhagen, Denmark. 3Department of Medical Biosciences, ) with the exception of androgen receptor ( ) ampli ca- Pathology, Umea University, Umea , Sweden. 4Department of Proteomics and tions, rearrangements, and mutations, seen almost exclusively in Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany. CRPC (7, 8). One possible explanation for these findings is that 5Department of Surgical and Perioperative Sciences, Orthopaedics, Umea many of the low-frequency mutations occurring in individual 6 University, Umea, Sweden. The Novo Nordisk Foundation Centre for Protein tumors may confer growth advantages through the regulation of a Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, 7 set of core signaling pathways required for metastatic growth. For Denmark. Department of Human Molecular Genetics and Biochemistry, Sackler FOXA1 MLL2 School of Medicine, Tel Aviv University, Tel Aviv, Israel. 8Department of Radi- example, mutations in genes such as or , with fl ation Sciences, Oncology, Umea University, Umea, Sweden. apparently unrelated functions, may in uence tumor growth through a common signaling mechanism: the regulation of AR Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). activity (9). Many of the alterations in protein signaling associated with metastatic progression have remained undiscovered, as only A. Flores-Morales and P. Wikstrom€ share senior authorship of this article. few studies have actually examined prostate bone metastases in Corresponding Author: Diego Iglesias-Gato, University of Copenhagen, Uni- depth at the proteome level (10). versitetsparken 2, Copenhagen 2100, Denmark. Phone: 4535330640; Fax: Transcriptomics or genetic analysis provides a partial view 4535325001; E-mail: [email protected] of the signaling pathways driving tumor growth because of their doi: 10.1158/1078-0432.CCR-18-1229 limited capacity to predict concentration changes at the protein Ó2018 American Association for Cancer Research. level or inform about status of protein-driven mechanisms www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Iglesias-Gato et al. 24, 2007, Dnr 2013-372-32M) and conducted following the Translational Relevance principles of the Declaration of Helsinki. All participants gave We performed a global quantification of the proteins written or verbal consent. Due to the acute situation, when expressed in prostate cancer bone metastatic tumors and bone metastasis surgery is performed in order to relieve spine found wide variation in their proliferative and metabolic symptoms and paresis, logistics do not always allow written features. This variability is related to disease prognosis and consent and the local ethic review board therefore specifically should be accounted for when stratifying patients into differ- approved verbal consent as well. Verbal consent is documented ent treatments. by the physician in the patient journal. Proteomic analysis The effects of formic acid-based decalcification in the proteomic (11, 12). Quantitative proteomic analysis of tumor samples may analysis were evaluated in a separate set of samples. No obvious have a large potential to unveil novel signaling mechanisms of quantitative or qualitative protein alterations were observed clinical relevance. We have recently analyzed the proteome of (Supplementary Fig. S1A). Protein extracts were obtained from primary prostate tumors and demonstrated the upregulation of approximately 20 microtome sections of 10-mm thick 25- to 50- various proteins involved in metabolic processes in malignant mm2 tumor-enriched areas. Mass spectrometry–based proteomic compared with nonmalignant tissue (13). Remarkably, we did analysis of the bone metastasis samples was performed as previ- not observe a general increase of proteins involved in cell-cycle ously described using SuperSILAC spike-in standards for accurate progression or DNA synthesis, perhaps reflecting the low prolif- quantification (ref. 13; and extended Materials and Methods). The erative capacity of most localized prostate tumors. proteomic pipeline is summarized in Supplementary Fig. S1B. The objective of this first-in-class study was to characterize Raw data files were uploaded to ProteomeXchange. Accession prostate cancer bone metastases in depth at the protein level and, references for published primary tumors and nonmalignant adja- by integrating these results with the previously described prote- cent tissue are PXD004159, PXD004132, PXD003636, PXD003615, omic profiles of localized prostate tumors (13), to describe the PXD003515, PXD003452, and PXD003430 (13). Accession refer- proteome evolution during prostate cancer progression. Further- ence for metastatic samples is PXD009868. more, we aimed to identify putative targets for novel therapies of metastatic CRPC and also to explore whether subgroups of Bioinformatic tools and statistics prostate cancer bone metastasis exist that could potentially guide Unsupervised hierarchical clustering of metastasis samples was therapy decisions. We used system-wide quantitative mass spec- performed based on Pearson correlation coefficients as distance – trometry based proteomic analysis to describe the proteome of metrics (Fig. 1E). Gene ontology (GO) classification and enrich- prostate cancer bone metastases, and we found a high degree of ment analysis was performed using DAVID (14), and results were heterogeneity among the analyzed samples with at least two graphically represented with Cytoscape (ref. 15; Figs. 2 and 3). phenotypically distinct subgroups of bone metastases. We pro- Metastatic subgroups were defined by the Pearson correla- pose that these subgroups should be accounted for when design- tion coefficients of the SILAC ratios of each protein across the ing novel therapeutic alternatives. cohort with the average SILAC ratio of the minichromosome maintenance complex components (MCM2-7) of each indi- Materials and Methods vidual tumor (Fig. 3A). Only proteins showing 2 of