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Global Characterization of Signalling Networks Associated with Tamoxifen Resistance in Breast Cancer Brigid C

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Global Characterization of Signalling Networks Associated with Tamoxifen Resistance in Breast Cancer Brigid C Global characterization of signalling networks associated with tamoxifen resistance in breast cancer Brigid C. Browne1, Falko Hochgrafe€ 2, Jianmin Wu1,3, Ewan K. A. Millar1,4,5,6, Jane Barraclough1, Andrew Stone1, Rachael A. McCloy1, Christine S. Lee1, Caroline Roberts1, Naveid A. Ali1, Alice Boulghourjian1, Fabian Schmich7,8, Rune Linding9, Lynn Farrow10, Julia M. W. Gee10, Robert I. Nicholson10, Sandra A. O’Toole1,3,11,12, Robert L. Sutherland1,3,†, Elizabeth A. Musgrove1,3, Alison J. Butt1,3,*,‡ and Roger J. Daly1,3,13,‡ 1 Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia 2 Competence Center – Functional Genomics, Junior Research Group Pathoproteomics, University of Greifswald, Germany 3 St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Darlinghurst, New South Wales, Australia 4 School of Medical Sciences, Faculty of Medicine, University of New South Wales, Darlinghurst, New South Wales, Australia 5 Department of Anatomical Pathology, South Eastern Area Laboratory Service, St George Hospital, Kogarah, New South Wales, Australia 6 School of Medicine and Health Sciences, University of Western Sydney, Campbelltown, New South Wales, Australia 7 Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland 8 Swiss Institute of Bioinformatics, Basel, Switzerland 9 Cellular Signal Integration Group, Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark 10 Cardiff School of Pharmacy & Pharmaceutical Sciences, Cardiff University, UK 11 Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia 12 Sydney Medical School, University of Sydney, Westmead,New South Wales, Australia 13 Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne,Victoria, Australia Keywords Acquired resistance to the anti-estrogen tamoxifen remains a significant endocrine resistance; MARCKS; MCF7; challenge in breast cancer management. In this study, we used an integrative phosphoproteomics; Yes kinase approach to characterize global protein expression and tyrosine phos- phorylation events in tamoxifen-resistant MCF7 breast cancer cells (TamR) Correspondence compared with parental controls. Quantitative mass spectrometry and compu- R. J. Daly, Department of Biochemistry and tational approaches were combined to identify perturbed signalling networks, Molecular Biology, School of Biomedical and candidate regulatory proteins were functionally interrogated by siRNA- Sciences, Level 1, Building 77, Monash mediated knockdown. Network analysis revealed that cellular metabolism was University, Melbourne, Victoria 3800, perturbed in TamR cells, together with pathways enriched for proteins associ- Australia ated with growth factor, cell–cell and cell matrix-initiated signalling. Consistent Fax: +61 3 990 29500 with known roles for Ras/MAPK and PI3-kinase signalling in tamoxifen resis- Tel: +61 3 990 29301 tance, tyrosine-phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in E-mail: [email protected] TamR cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) *Present address were increased two- and eightfold in TamR cells respectively, and these pro- Research Investment, National Breast teins were selected for further analysis. Knockdown of either protein in TamR Cancer Foundation, 50 Pitt Street, Sydney, cells had no effect on anti-estrogen sensitivity, but significantly decreased cell New South Wales, Australia motility. MARCKS expression was significantly higher in breast cancer cell lines than normal mammary epithelial cells and in ER-negative versus ER-posi- †Deceased tive breast cancer cell lines. In primary breast cancers, cytoplasmic MARCKS ‡These authors contributed equally to this staining was significantly higher in basal-like and HER2 cancers than in lumi- work nal cancers, and was independently predictive of poor survival in multivariate analyses of the whole cohort (P < 0.0001) and in ER-positive patients (Received 18 April 2013, revised 27 June (P = 0.0005). These findings provide network-level insights into the molecular 2013, accepted 17 July 2013) alterations associated with the tamoxifen-resistant phenotype, and identify doi:10.1111/febs.12441 MARCKS as a potential biomarker of therapeutic responsiveness that may assist in stratification of patients for optimal therapy. Abbreviations EGFR, epidermal growth factor receptor; ER, estrogen receptor; MARCKS, myristoylated alanine-rich C-kinase substrate; OHT, 4-hydroxytamoxifen; pY, phosphotyrosine; SFK, Src family kinase; SILAC, stable isotopic labelling using amino acids in culture; TamR, tamoxifen-resistant MCF7 cell line. FEBS Journal 280 (2013) 5237–5257 ª 2013 FEBS 5237 Phosphoproteomic analysis of tamoxifen resistance B. C. Browne et al. Introduction The anti-estrogen tamoxifen has been the most widely When combined with appropriate enrichment tech- used endocrine treatment for breast cancer for more niques, mass spectrometry may also be used to charac- than 30 years, and has significantly improved survival terize changes in particular sub-proteomes, such as the for patients with estrogen receptor (ER)-positive dis- fraction of cellular proteins that are tyrosine-phosphor- ease [1]. However, approximately one-third of patients ylated [29], providing global insights into perturbations treated with tamoxifen for 5 years develop recurrent in particular signalling and regulatory processes. In this disease within 15 years [2]. Responses to second-line study, we have used an integrated approach that com- therapeutic strategies, including sequential delivery of bines quantitative proteomics and phosphoproteomics alternative endocrine therapies, therapies targeted to with bioinformatics, functional interrogation and anal- growth factor receptors, and, in some instances, che- ysis of breast cancer cohorts in order to characterize motherapy, are often short-lived, and disease progres- changes in cellular signalling networks associated with sion is inevitable [3–6]. Further deciphering the acquisition of tamoxifen resistance. Our work high- molecular basis of tamoxifen resistance is therefore crit- lights key regulatory pathways associated with this phe- ical in order to identify robust biomarkers of response, notype, as well as the complexity of the associated as well as targets for potential therapeutic intervention. network perturbations, and identifies myristoylated To this end, early candidate-based studies of tamoxi- alanine-rich C-kinase substrate (MARCKS) as a candi- fen resistance revealed roles for receptor tyrosine kin- date prognostic marker in ER-positive breast cancer. ases such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 Results (HER2/ERBB2) and insulin-like growth factor 1 receptor [7–9], as well as numerous intracellular signal- Workflow for SILAC and MS identification of ling mediators [10–14]. Genome-wide gene expression peptides analyses performed on both experimental models of endocrine resistance and ER-positive patient samples The tamoxifen-resistant (TamR) cell line was devel- have also been used to identify potential mechanisms oped by long-term culture of MCF7 cells in the pres- of resistance and generate gene signatures that predict ence of 4-hydroxytamoxifen (OHT) [9]. Figure 1 tamoxifen response [15–25]. We have previously describes the workflow for SILAC (stable isotopic defined functionally distinct gene signatures represent- labelling using amino acids in culture) and MS analy- ing cell proliferation, apoptosis and cell growth, each ses of MCF7 and TamR cells. Briefly, equal propor- of which was predictive of response in a cohort of tions of TamR and MCF7 cells labelled with heavy tamoxifen-treated patients [19]. Gene signatures, such and light isotopes, respectively, were mixed, and iso- as those included in the commercially available Onco- lated proteins were separated by SDS/PAGE. This was typeDx (Genomic Health, Redwood City, CA, USA) followed by in-gel digestion with trypsin, extraction of [20] and Prosigna (Nanostring Technologies, Seattle, peptides and MS analysis (Fig. 1A). In parallel, equal WA, USA) [25] gene assays predict residual risk of dis- proportions of differentially SILAC-labelled MCF7 tant relapse in specific subsets of ER-positive patients, and TamR cells were mixed and protein lysates were and thus guide treatment choices. However, it is well digested overnight. Phosphotyrosine (pY) peptides understood that cellular mRNA levels do not always were eluted after sequential immunoprecipitation with reflect the level of the encoded protein nor the vast pY100 and pY20 antibodies, and MS analyses were array of post-translational modifications that individ- performed (Fig. 1B). Each assay was performed in ual proteins undergo during regulation of cellular duplicate, with reversed labelling of each cell line. In processes. total, 2059 proteins were identified by total proteomics MS-based proteomic technology is being increasingly analysis, and 116 pY sites were identified by phospho- used to study and compare the proteomes of in vitro proteomic analysis (Tables S1B and S2B). and in vivo models of cancer as well as patient tumours, and has augmented knowledge obtained from Protein expression changes associated with gene expression
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