Implications of BRCA1 mutations in basal-like breast cancer development and treatment Yuexi Gu Institute for Molecular Medicine Finland (FIMM) Division of Genetics Department of Biosciences Faculty of Biological and Environmental Sciences Integrative Life Science Doctoral Program (ILS) University of Helsinki Academic dissertation To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences, University of Helsinki, in Biomedicum Helsinki 1 (Haartmaninkatu 8), Lecture Hall 2, on June 16, 2015, at 12:00 noon. Helsinki 2015 Supervisor Sergey Kuznetsov, Ph.D. Institute for Molecular Medicine Finland (FIMM) University of Helsinki Thesis advisory committee Professor Marikki Laiho, M.D., Ph.D. Docent Juha Klefström, Ph.D. The Johns Hopkins University School of Medicine University of Helsinki Pre-examiners Krister Wennerberg, Ph.D. Daniel Abankwa, Ph.D. Institute for Molecular Medicine Finland (FIMM) Turku Centre for Biotechnology University of Helsinki Åbo Akademi University Opponent Professor Claus Storgaard Sørensen, Ph.D. Biotech Research & Innovation Centre University of Copenhagen Custos Professor Minna Nyström Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Published in Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis ISBN 978-951-51-1195-1 (paperback) ISBN 978-951-51-1196-8 (PDF) ISSN 2342-3161 (Print) ISSN 2342-317X (Online) Cover layout by Anita Tienhaara Cover image: Breast cancer awareness ribbon woman head composite. Hansaprint Oy, Vantaa, Finland 2015 If you don’t know your future, then go for it. Table of contents List of original publications .................................................................................. 6 Abbreviations ......................................................................................................... 7 Summary ................................................................................................................ 9 Introduction ......................................................................................................... 11 1. Review of the literature ................................................................................. 13 1.1 Molecular basis of breast cancer ......................................................................... 13 1.1.1 Breast cancer as a major healthcare problem ................................................. 13 1.1.2 Different breast cancer subtypes inform on their heterogeneous biology ...... 14 1.1.3 Inherited breast cancer predisposition is often associated with DNA repair genes ............................................................................................................... 17 1.2 DNA damage response and repair ....................................................................... 19 1.2.1 Cancer cells often experience elevated DNA damage .................................... 19 1.2.2 Regulation of DNA damage response and repair ........................................... 20 1.2.3 Targeting DNA damage repair pathways for the treatment of cancer ............ 27 1.3 BRCA1 as a cancer susceptibility gene ............................................................... 31 1.3.1 Functional domains of BRCA1 protein .......................................................... 31 1.3.2 BRCA1 is an E3 ubiquitin ligase .................................................................... 32 1.3.3 Functions of BRCA1 in DNA damage response and repair ........................... 34 1.3.4 BRCA1 regulates cell cycle checkpoints ....................................................... 37 1.3.5 The role of BRCA1 in development and stem cell regulation........................ 41 1.3.6 Therapeutic implications of BRCA1 mutations for cancer treatment ............ 42 2. Aims of the study ........................................................................................... 45 3. Materials and methods .................................................................................. 46 4. Results and discussion ................................................................................... 50 4.1 Heterogeneity of BRCA1-mutant breast cancer cell lines ................................. 50 4.1.1 BRCA1-mutant cells are resistant to DNA-damaging agents ........................ 50 4.1.2 Characterization of homologous recombination efficiency in BRCA1-mutant cells ................................................................................................................. 52 4.1.3 High-throughput chemical compound screen in BRCA1-mutant cell lines ... 53 4.2 Loss of BRCA1 sensitizes cells to proteasome inhibitors in a DNA repair- independent manner ............................................................................................. 54 4.2.1 Proteasome inhibitors selectively kill BRCA1- but not BRCA2-depleted cells ........................................................................................................................ 54 4.2.2 Sensitization of BRCA1-depleted cells to proteasome inhibitors does not involve DNA damage response ...................................................................... 55 4.2.3 Sensitization of BRCA1-depleted cells to proteasome inhibitors involves uncontrolled cell division caused by deregulated cell cycle checkpoints ...... 55 4.2.4 53BP1 depletion inhibits sensitization of BRCA1-deficient cells to proteasome inhibitors ........................................................................................................ 57 4.3 Loss of BRCA1 promotes basal-like differentiation by sustaining ΔNp63 activity .................................................................................................................... 58 4.3.1 Loss of BRCA1 promotes basal-like differentiation ...................................... 58 4.3.2 BRCA1 antagonizes the activation of ΔNp63 ................................................ 59 5. Concluding remarks....................................................................................... 61 Acknowledgements ............................................................................................... 62 References ............................................................................................................. 63 List of original publications List of original publications The thesis is based on the following articles referred to in the text by their Roman numerals: I. Gu Y, Bouwman P, Greco D, Saarela J, Yadav B, Jonkers J, and Kuznetsov SG (2014) Suppression of BRCA1 sensitizes cells to proteasome inhibitors. Cell Death Dis 18;5:e1580. II. Munne PM, Gu Y, Tumiati M, Gao P, Koopal S, Uusivirta S, Sawicki J, Wei G, and Kuznetsov SG (2014) TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli. Sci Rep 4:4663. III. Yuexi Gu, Kristiina Väänänen, Jani Saarela, Anna Sokolenko, John Martens, Evgeny Imyanitov, and Sergey Kuznetsov. BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6- thioguanine. Manuscript. The author’s contribution to each publication: I. Y.G. conceived the idea of carrying out a high-throughput chemical compound screen on a pair of BRCA1- and control-knockdown MDA-MB-231 cells, which is originally a basal-like breast cancer cell line expressing wildtype BRCA1. Y.G. designed and perfomed most of the experiments, analyzed the data, and wrote the manuscript together with S.K. II. Y.G. helped to perform several experiments of Western blotting and reverse transcription–quantative PCR (RT-qPCR) that are related to BRCA1. III. Y.G. performed most of the experiments under the supervision of S.K., analyzed the data and helped to write the manuscript. 6 Abbreviations Abbreviations 53BP1 p53-binding protein 1 ATM ataxia telangiectasia mutated ATR ataxia telangiectasia mutated and Rad3-related protein ATRIP ATR-interacting protein BARD1 BRCA1-associated RING domain protein 1 BER base excision repair BLM Bloom syndrome protein BRCA1 breast cancer 1, early onset BRCA2 breast cancer 1, early onset BRCC36 BRCA1/BRCA2-containing complex, subunit 36 BRCC45 BRCA1/BRCA2-containing complex, subunit 45 BRCT BRCA1 C terminus CHK1 checkpoint kinase 1 CHK2 checkpoint kinase 2 CtBP C-terminal-binding protein CtIP CtBP-interacting protein DNA2 DNA replication helicase/nuclease 2 DNA-PKcs DNA-dependent protein kinase, catalytic subunit DSB double-strand break EGFR epidermal growth factor receptor EME1 crossover junction endonuclease EMT epithelial-to-mesenchymal transition ER estrogen receptor ERCC1 excision repair cross-complementation group 1 EXO1 exonuclease 1 H2AX H2A histone family, member X HER2 human epidermal growth factor receptor 2 (ERBB2) HNPCC hereditary nonpolyposis colorectal cancer HR homologous recombination LOH loss of heterozygosity MAPK mitogen-activated protein kinase MDC1 mediator of DNA damage checkpoint 1 MEK mitogen-activated protein kinase kinase (MAPKK, MAP2K) MGMT methylguanine methyltransferase mMEC mouse mammary epithelial cell MMEJ microhomology-mediated end joining MMR mismatch repair MNNG N-methyl-N'-nitro-N-nitrosoguanidine MRN MRE11-RAD50-NBS1 7 Abbreviations MSI microsatellite instability MUS8 ubiquitin-conjugating enzyme E2 2 NER nucleotide excision repair NHEJ non-homologous end joining NLS nuclear localization signal/sequence OGG1 8-oxoguanine DNA glycosylase PALB2 partner and localizer of BRCA2 PARP poly (ADP-ribose) polymerase