Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by M.Sc. Emre Sofyali born in: Edirne, Turkey Oral examination: 09.09.2019 GLYATL1 PROMOTES ENDOCRINE THERAPY RESISTANCE IN BREAST CANCER Referees: Prof. Dr. Stefan Wiemann PD Dr. rer. nat. Karin Müller-Decker Declaration of Authorship I hereby declare that the work presented in my dissertation was carried out between August 2015 and July 2019 under the supervision of Prof. Dr. Stefan Wiemann in the group of Molecular Genome Analysis at the German Cancer Research Center (DKFZ, Heidelberg, Germany). If not stated differently and referenced within the text, the data described in my dissertation is original, has been gathered by myself and has not yet been presented as a part of a university examination. All main sources, as well as the work of joint cooperation have been referenced appropriately. I, as author, hereby declare no potential conflict of interest. Heidelberg, Emre Sofyali Table of Contents Summary ......................................................................................................................................... 1 Zusammenfassung ........................................................................................................................... 3 1. Introduction ............................................................................................................................. 5 1.1 Breast Cancer ................................................................................................................... 5 1.1.1 Molecular Subtypes of Breast Cancer .......................................................................... 5 1.1.2 Endocrine Therapy ...................................................................................................... 8 1.2 Epigenetics ..................................................................................................................... 14 1.2.1 DNA methylation ....................................................................................................... 14 1.2.2 Histone modifications ................................................................................................. 16 1.3 CRISPR/dCas9-mediated Epigenetic Editing ................................................................ 21 2. Aims ....................................................................................................................................... 25 3. Materials and Methods .......................................................................................................... 27 3.1 Materials ......................................................................................................................... 27 3.1.1 Instruments ................................................................................................................. 27 3.1.2 Chemicals and Reagents ............................................................................................. 28 3.1.3 Assay Kits ................................................................................................................... 30 3.1.4 Consumables ............................................................................................................... 31 3.1.5 Software ...................................................................................................................... 32 3.1.6 Database ...................................................................................................................... 33 3.1.7 Buffers and solutions .................................................................................................. 33 3.1.8 Antibodies ................................................................................................................... 34 3.1.9 siRNAs........................................................................................................................ 34 3.1.10 Primers .................................................................................................................... 35 3.2 Methods .......................................................................................................................... 36 3.2.1 Cell Culture and Growth Conditions .......................................................................... 36 3.2.2 siRNA Transfections .................................................................................................. 38 3.2.3 Analysis of RNA expression ...................................................................................... 39 3.2.4 Analysis of protein expression ................................................................................... 41 3.2.5 Functional assays ........................................................................................................ 44 3.2.6 Determination of methylation changes via Illumina EPIC 850k array ...................... 47 3.2.7 Assay for Transposase Accessible Chromatin with High Throughput Sequencing (ATAC-Seq) .......................................................................................................................... 48 3.2.8 CRISPR/dCas9-mediated targeted epigenetic editing experiments ........................... 48 3.2.9 Dataset Analysis ......................................................................................................... 55 3.2.10 Statistical Analysis .................................................................................................. 56 3.2.11 Graphical Illustrations ............................................................................................. 56 4. Results .................................................................................................................................... 57 4.1 Part I: Elucidating the Role of GLYATL1 in Endocrine Therapy Resistant Breast Cancer... 57 4.1.1 Establishment and characterization of endocrine therapy resistant breast cancer cell lines .................................................................................................................................... 57 4.1.2 Profiling of resistant cell line repertoire ..................................................................... 60 4.1.3 Clinical Significance of GLYATL1 ............................................................................. 64 4.1.4 GLYATL1 as a novel gene in endocrine therapy resistance context ........................... 67 4.1.4.1 Effect of GLYATL1 knockdown on cell viability ................................................... 67 4.1.4.2 Effect of GLYATL1 overexpression on cell proliferation ..................................... 69 4.1.4.3 Effect of GLYATL1 overexpression on cell cycle ................................................. 72 4.1.4.4 Effect of GLYATL1 overexpression on apoptosis ................................................. 73 4.1.5 Investigating the role of GLYATL1 as a potential HAT............................................ 74 4.1.6 Regulation of GLYATL1 ............................................................................................. 81 4.1.6.1 Epigenetic Regulation ................................................................................................. 81 4.1.6.2 Regulation by HER2 ............................................................................................... 85 4.2 Part II: Establishing CRISPR/dCas9-mediated Epigenetic Editing Methodology to Modulate the Epigenetic Landscape of Endocrine Therapy Resistant Breast Cancer ................................. 89 4.2.1 CRISPR/dCas9-mediated targeted epigenetic editing ................................................ 89 5. Discussion ............................................................................................................................ 105 5.1 Validation of in vitro models ....................................................................................... 105 5.2 GLYATL1 depletion leads to partial re-sensitization to endocrine therapy conditions 106 5.3 GLYATL1 overexpression confers endocrine therapy resistance ............................... 107 5.4 GLYATL1 overexpression counteracts induction of cell cycle arrest and apoptosis in endocrine therapy-sensitive cells ............................................................................................ 107 5.5 GLYATL1 is involved in histone acetylation .............................................................. 108 5.6 GLYATL1 expression correlates with poor prognosis in breast cancer patients .......... 111 5.7 GLYATL1 expression is regulated by HER2 and luminal transcription factors ........... 112 5.8 GLYATL1 is regulated by methylation ......................................................................... 113 5.9 GLYATL1 as a link between metabolism and epigenetics .......................................... 115 5.10 CRISPR/dCas9-mediated epigenetic editing is effective in fine tuning target genes .. 116 5.11 Wnt signaling pathway: BAMBI –CD44 axis ............................................................... 119 5.12 Conclusions and Outlook ............................................................................................. 123 References ................................................................................................................................... 125 Abbreviations .............................................................................................................................