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The Proteome of Prostate Cancer Bone Metastasis Reveals Heterogeneity With Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 The proteome of prostate cancer bone metastasis reveals heterogeneity with 2 prognostic implications 3 4 * 5 Diego Iglesias-Gatoa,b , Elin Thysellc, Stefka Tyanovad, Sead Crnalice, Alberto Santosf, 6 Thiago S. Limaa,b, Tamar Geigerg, Jürgen Coxd, Anders Widmarkh, Anders Berghc, 7 Matthias Mannd,f, Amilcar Flores-Moralesa, † , Pernilla Wikströmc, † 8 Affiliations: 9 aDepartment of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 10 University of Copenhagen, Copenhagen, Denmark 11 bThe Danish Cancer Society. Copenhagen. Denmark. 12 cDepartment of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden 13 dDepartment of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 14 Martinsried, Germany 15 eDepartment of Surgical and Perioperative Sciences, Orthopaedics, Umeå University, 16 Umeå, Sweden. 17 18 fThe Novo Nordisk Foundation Centre for Protein Research, Faculty of Health Sciences, 19 University of Copenhagen, Copenhagen, Denmark. 20 gDepartment of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, 21 Tel Aviv University, Tel Aviv, Israel. 22 hDepartment of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden 23 †Shared senior authorship 1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. * 24 Correspondence: Diego Iglesias-Gato ([email protected]); telephone: 25 +4535330640, fax: +4535325001. 26 Keywords: Bone metastasis; quantitative proteomics; castration resistant prostate cancer; 27 MCM3; PSA; prognosis metastatic prostate cancer. 28 29 Running title: The proteome of prostate cancer bone metastasis 30 31 2 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 32 Translational relevance 33 We performed a global quantification of the proteins expressed in prostate cancer bone 34 metastatic tumors and found wide variation in their proliferative and metabolic features. 35 This variability is related to disease prognosis and should be accounted for when 36 stratifying patients into different treatments. 37 38 Abstract 39 Background: Bone is the most predominant site of distant metastasis in prostate cancer 40 (PCa) and patients have limited therapeutic options at this stage. 41 Experimental Design: We performed a system-wide quantitative proteomic analysis of 42 bone metastatic prostate tumors from 22 patients operated to relief spinal cord 43 compression. At the time of surgery, most patients had relapsed after androgen- 44 deprivation therapy, while 5 were previously untreated. An extended cohort of prostate 45 cancer bone metastases (n=65) was used for immunohistochemical validation. 46 Results: 5067 proteins on average were identified and quantified per tumor. Compared to 47 primary tumors (n=26), bone metastases were more heterogeneous than and showed 48 increased levels of proteins involved in cell cycle progression, DNA damage response, 49 RNA processing, and fatty acid -oxidation; and reduced levels of proteins related to cell 50 adhesion and carbohydrate metabolism. Within bone metastases, we identified two 51 phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and 52 mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression 53 of proliferation and DNA repair related proteins. The two subgroups, validated by the 54 inverse correlation between MCM3 and PSA immunoreactivity, were related to disease 55 prognosis, suggesting that this molecular heterogeneity should be considered when 3 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 56 developing personalized therapies. 57 Conclusions: This work is the first system-wide quantitative characterization of the 58 proteome of PCa bone metastases and a valuable resource for understanding the etiology 59 of PCa progression. 60 4 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 61 Introduction 62 63 Mortality in prostate cancer (PCa) is strongly associated with development of bone 64 metastatic disease (1,2). PCa metastases are generally treated with androgen ablation(3). 65 This therapy is initially effective, but most patients relapse to a castration resistant stage. 66 Despite recent treatment developments for patients with castration resistant bone 67 metastases (mCRPC), their prognosis remains poor with a median survival below 2 years 68 (4). This is in contrast to patients harboring prostate confined tumors, who have a 10-year 69 cancer specific survival rate above 90%, even for patients managed by observation 70 protocols (5,6). Why metastatic tumors have a more aggressive behavior than localized 71 tumors is not fully understood. Recent genetic studies have identified only a modest 72 increase in the number and frequency of genetic aberrations in lethal, therapy-resistant 73 metastatic cancer as compared to localized tumors (e.g. TP53, PTEN, FOXA1, PI3KCA) 74 with the exception of androgen receptor (AR) amplifications, rearrangements, and 75 mutations, seen almost exclusively in CRPC (7,8). One possible explanation for these 76 findings is that many of the low frequency mutations occurring in individual tumors may 77 confer growth advantages through the regulation of a set of core signaling pathways 78 required for metastatic growth. For example, mutations in genes such as FOXA1 or MLL2, 79 with apparently unrelated functions may influence tumor growth through a common 80 signaling mechanism: the regulation of AR activity (9). Many of the alterations in protein 81 signaling associated to metastatic progression have remained undiscovered, as only few 82 studies have actually examined prostate bone metastases in depth at the proteome level 83 (10). 84 5 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 85 Transcriptomics or genetic analysis provides a partial view of the signaling pathways 86 driving tumor growth because of their limited capacity to predict concentration changes at 87 the protein level or inform about status of protein driven mechanisms (11,12). Quantitative 88 proteomic analysis of tumor samples may have a large potential to unveil novel signaling 89 mechanisms of clinical relevance. We have recently analyzed the proteome of primary 90 prostate tumors and demonstrated the upregulation of various proteins involved in 91 metabolic processes in malignant compared to non-malignant tissue (13). Remarkably, we 92 did not observe a general increase of proteins involved in cell cycle progression or DNA 93 synthesis, perhaps reflecting the low proliferative capacity of most localized prostate 94 tumors. 95 96 The objective of this first-in-class study was to characterize PCa bone metastases in depth 97 at the protein level and, by integrating these results with the previously described 98 proteomic profiles of localized prostate tumors (13), to describe the proteome evolution 99 during PCa progression. Furthermore, we aimed to identify putative targets for novel 100 therapies of metastatic CRPC and also to explore whether subgroups of PC bone 101 metastasis exist that could potentially guide therapy decisions. We used system-wide 102 quantitative mass spectrometry-based proteomic analysis to describe the proteome of PCa 103 bone metastases and we found a high degree of heterogeneity among the analyzed 104 samples with at least two phenotypically distinct subgroups of bone metastases. We 105 propose that these subgroups should be accounted for when designing novel therapeutic 106 alternatives. 107 108 Materials and Methods 6 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 24, 2018; DOI: 10.1158/1078-0432.CCR-18-1229 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 109 110 Cohort description 111 112 Bone metastasis samples were obtained from men with PCa, who underwent surgery for 113 metastatic spinal cord compression at Umeå University Hospital between 2003 and 2014. 114 Metastasis biopsies were fixed in 4% buffered formalin and decalcified in 20% formic acid 115 for 1-3 days, according
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