MASARYK UNIVERSITY FACULTY OF SCIENCE Molecular interactions in low-grade breast cancer metastasis Ph.D. Dissertation JOSEF MARYÁŠ Supervisor: Doc. Mgr. Pavel Bouchal, Ph.D. DEPARTMENT OF BIOCHEMISTRY Brno 2020 Bibliografický záznam Autor: Mgr. Josef Maryáš Přírodovědecká fakulta, Masarykova univerzita Ústav Biochemie Název práce: Molekulární interakce v metastazování low-grade nádorů prsu Studijní program: Biochemie Specializace: Biochemie Vedoucí práce: Doc. Mgr. Pavel Bouchal, Ph.D. Akademický rok: 2019/2020 Počet stran: 108+109 Klíčová slova: PDLIM2, TRAF3IP2, RNF25, epiteliálně-mezenchymální tranzice, metastazování, karcinom prsu Bibliographic Entry Author: Mgr. Josef Maryáš Faculty of Science, Masaryk University Department of Biochemistry Title of Thesis: Molecular interactions in low-grade breast cancer metastasis Degree programme: Biochemistry Specialization: Biochemistry Supervisor: Doc. Mgr. Pavel Bouchal, Ph.D. Academic Year: 2019/2020 Number of Pages: 108+109 Keywords: PDLIM2, TRAF3IP2, RNF25, epithelial-mesenchymal transition, metastasis, breast carcinoma Abstrakt Nádory prsu (BrCa) představují heterogenní onemocnění, které je klasifikováno na základě histologických, klinických a/nebo imunohistochemických (IHC) markerů, které rovněž slouží jako prognostické a prediktivní faktory. Hlavní příčinou úmrtí pacientů s BrCa jsou však metastázy, které vznikají v procesu metastatické kaskády, multifaktoriálního a vícestupňového procesu zahrnujícího tzv. epiteliálně-mesenchymální tranzici (EMT). Je zřejmé, že metastatická kaskáda včetně EMT zahrnuje velké množství proteinů, na které lze cílit a modulovat tím proces vzniku metastáz. Disertační práce sumarizuje poznatky o proteinech PDZ and LIM domain protein 2 (PDLIM2), ring finger protein 25 (RNF25) a TRAF3 interacting protein 2 (TRAF3IP2), které byly identifikovány jako proteiny související s metastazováním do lymfatických uzlin u luminálních-A nádorů prsu pomocí kombinace proteomických a transkriptomických přístupů. Za účelem pochopení molekulární podstaty tohoto vztahu jsme se nejprve zaměřili na protein-proteinový interaktom a srovnali konvenční pull-down assay se surface plasmon resonance (SPR) čipy v kombinaci se “sequential window acquisition of all theoretical fragment ion spectra mass spectrometry” (SWATH-MS) pro identifikaci interakčních partnerů proteinu PDLIM2. Naše výsledky ukazují, že afinitní matrice a mechanismus promytí a eluce jsou kritickými kroky assaye, a ukazují SPR v kombinaci se SWATH-MS jako relevantní nástroj pro detekci protein-protein interakčních partnerů. Zadruhé, funkční potvrdily vztah proteinu PDLIM2 k EMT, hypoxii, proliferaci a adhezi nádorové buněčné linie MCF7, modelu odvozeném od luminálních-A nádorů prsu. Paralelní experimenty na imortalizovaných buňkách odvozených od normálního prsního epitelu MCF10A však odhalily jeho spíše tumor supresivní funkci, což vede k hypotéze o duální a na biologickém kontextu závislé roli PDLIM2 v procesu vzniku a vývoje nádorů prsu. Zatřetí, širokorozsahová celoproteomová analýza spojená s analýzou protein-proteinové interakční sítě, “gene set enrichment analysis” (GSEA) a analýzou migrace a invaze buněčné linie MCF7 prokázala souvislost zvýšených hladin proteinů PDLIM2, RNF25 a TRAF3IP2 s cytoskeletárními změnami, zvýšenou proliferací, migrací, invazí a s nádorovou progresí. Práce jako celek přispívá k pochopení molekulární sítě zapojené do metastazování luminálních-A nádorů prsu a jejího vlivu na fenotyp nádorových buněk. Abstract Breast cancer (BrCa) is a heterogeneous disease currently classified based on histological, clinical and/or immunohistochemical (IHC) markers that also serve as prognostic and predictive factors. However, the most common cause of death of BrCa patients are metastases, which are formed during metastatic cascade, the multistep and multifactorial process involving epithelial-to-mesenchymal transition (EMT). It is evident that metastatic cascade and EMT itself involve a large number of potentially targetable proteins that might modulate the process of metastasis formation. The dissertation thesis focuses on PDZ and LIM domain protein 2 (PDLIM2), ring finger protein 25 (RNF25) and TRAF3 interacting protein 2 (TRAF3IP2) which have been previously identified as proteins related to lymph node metastasis in luminal-A breast tumors using a combination of proteomic and transcriptomic approaches. To understand the molecular basis of this phenomenon, we first focused on protein-protein interactome and compared conventional pull-down assays on streptavidin agarose beads with surface plasmon resonance chips (SPR) in combination with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to identify PDLIM2 interactors. Our results show that solid support and mechanics of wash and elution are the critical aspects of the assay, and highlight SPR with SWATH-MS as a relevant tool for the detection of PDLIM2 protein-protein interactions. Second, a series of hypothesis-driven functional experiments confirmed association of PDLIM2 with EMT, hypoxia, proliferation and adhesion of MCF7 breast cancer cell line, a model of luminal-A breast cancer. However, the parallel experiments in immortalized normal epithelial breast cells MCF10A revealed its rather tumor suppressive role, leading to the hypothesis on dual and context dependent role in breast cancer development. Third, large- scale total proteome analysis together with the analysis of protein-protein interaction network, gene set enrichment analysis (GSEA) and functional analysis of migration and invasion of MCF7 breast cancer cell line demonstrated the association of elevated PDLIM2, RNF25 and TRAF3IP2 protein levels with cytoskeletal changes, increased proliferation, migration, invasion, and tumor progression. The work contributes to understanding the molecular network involved in luminal-A breast cancer metastasis and its impact on cancer cell phenotype. Acknowledgements I would like to thank my thesis supervisor, Pavel Bouchal for his time, guidance and continuous support. I would like to express my gratitude to him for providing suggestions and commentaries at every step during my PhD studies, while also giving me the freedom to design my own experiments and realize my own ideas. My work could not have been possible without the help and support of former members of Regional Centre for Applied Molecular Oncology. Especially, I would like to thank Monika D. and Iva P., for all discussions we had. I am also thankful to Jakub F., for his valuable technical support with MS experiments. My endless thanks belong to my family and friends. They always stand on my side, helped me, supported me and inspired me every day. Without them I would not become the person that I am today. Pledge I hereby declare that I have worked on this thesis independently, using primary and secondary sources listed in the bibliography, under the supervision of Dr. Pavel Bouchal. In Brno ........................ Josef Maryáš List of selected original publications with dissertant’s contributions The dissertation thesis is based on one peer-reviewed review paper (Review paper 1), two original peer-reviewed papers in journals with impact factor (Research papers 1 - 2), one manuscript in peer-review process published on preprint server (Research paper 3) and one manuscript in preparation (Research paper 4). The dissertant‟s contribution to each paper is described in commentaries below. Publications are listed in Appendices 1 to 5. Review paper 1 (Appendix1): Maryas J, Faktor J, Dvorakova M, Struharova I, Grell P, Bouchal P. Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges. Proteomics. 2014;14(4-5):426-40. (IF2014 = 3.807, Q1 in Biochemical Research Methods, Q1 in Biochemistry and Molecular Biology). https://onlinelibrary.wiley.com/doi/epdf/10.1002/pmic.201300264 Estimated personal contribution: 30 % JM contributed to this work by summarizing the biological basis of cancer metastasis and functional proteomics studies in pro-metastatic mechanisms. Research paper 1 (Appendix 2): Bouchal P, Dvorakova M, Roumeliotis T, Bortlicek Z, Ihnatova I, Prochazkova I, Ho J.T.C, Maryas J, Imrichova H, Budinska E, et al. Combined Proteomics and Transcriptomics Identifies Carboxypeptidase B1 and Nuclear Factor κB (NF- κB) Associated Proteins as Putative Biomarkers of Metastasis in Low Grade Breast Cancer. Mol. Cell. Proteomics MCP. 2015;14:1814-30. (IF2015 = 5.912, Q1/92.857 percentile in Biochemical Research Methods). https://www.mcponline.org/content/mcprot/14/7/1814.full.pdf Estimated personal contribution: 10 % JM‟s contribution to this work consisted of antibodies specificity validation and dilutions optimization using western blot, and preparation of materials for publication. Research paper 2 (Appendix 3): Maryas J, Faktor J, Skladal P, Bouchal P. Pull-down assay on streptavidin beads and surface plasmon resonance chips for SWATH mass spectrometry based interactomics. Cancer Genomics and Proteomics. 2018;15(5):395-404. (IF2018 = 3.147, Q2 in Oncology, Q2 in Genetics and Heredity). http://cgp.iiarjournals.org/content/15/5/395.full.pdf+html Estimated personal contribution: 70 % JM designed and performed majority of experiments (cell cultivation, transfection, cell lysis and sample preparation for Methods 1-4 and pull-down for Methods 1-3), analyzed potential interactors and drafted the manuscript. Research paper 3 (Appendix 4): Maryas J, Pribyl J, Bouchalova P, Skladal P, Bouchal