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Wnt-Driven O-Glycosylation by LARGE2 in Human Colon and Colorectal Cancer

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Wnt-Driven O-Glycosylation by LARGE2 in Human Colon and Colorectal Cancer Aus dem Pathologischen Institut der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Thomas Kirchner in der DKTK Arbeitsgruppe „Oncogenic Signaling Pathways in Colorectal and Pancreatic Cancer“ Leiter: Dr.rer.nat. Peter Jung Dissertation zum Erwerb des Doctor of Philosophy (Ph.D.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Wnt-driven O-glycosylation by LARGE2 in human colon and colorectal cancer vorgelegt von Vanessa Dietinger aus Ansbach, Deutschland am 18.06.2020 Gedruckt mit der Genehmigung der Medizinischen Fakultät der Ludwig- Maximilans-Universität München First supervisor: Prof. Dr. rer. nat. Heiko Hermeking Second supervisor: Dr. rer. nat. Peter Jung Dean: Prof. Dr. med. Dent. Reinhard Hickel Day of oral defense: 24.11.2020 II “I was taught that the way of progress was neither swift nor easy” Marie Curie To everyone who supported me III Affidavit Affidavit Pathologisches Institut der LMU Dietinger, Vanessa Thalkirchner Str. 36 80337 München Deutschland I hereby declare, that the submitted thesis entitled „Wnt-driven O-glycosylation by LARGE2 in human colon and colorectal cancer “ is my own work. I have only used the sources indicated and have not made unauthorized use of services of a third party. Where the work of others has been quoted or reproduced, the source is always given. I further declare that the submitted thesis or parts thereof have not been presented as part of an examination degree to any other university. Munich, 30.11.2020 Vanessa Dietinger IV Confirmation of congruency Confirmation of congruency between printed and electronic version of the doctoral thesis Pathologisches Institut der LMU Dietinger, Vanessa Thalkirchner Str. 36 80337 München Deutschland I hereby declare that the electronic version of the submitted thesis, entitled “Wnt-driven O-glycosylation by LARGE2 in human colon and colorectal cancer” Is congruent with the printed version both in content and format. Munich, 30.11.2020 Vanessa Dietinger V Publications Publications Parts of this thesis have been published in the following article: Dietinger, V.*, García de Durango, C.R.*, Wiechmann, S., Boos, S., Michl, M., Neumann, J., Hermeking, H., Küster, B., Jung, P.: Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer. Cell communication and signaling, 2020. * Equal contribution VI Table of contents Table of contents Affidavit ...................................................................................................................... IV Confirmation of congruency between printed and electronic version of the doctoral thesis ...................................................................................................................V Publications ...................................................................................................................... VI Abbreviations ................................................................................................................... XII 1. Introduction .................................................................................................. 1 1.1. The human intestine ..................................................................................... 1 1.2. Canonical Wnt signaling pathway ............................................................... 2 1.3. Incidence of colorectal cancer .................................................................... 5 1.4. Genetic alterations leading to colorectal cancer ....................................... 7 1.5. Aberrant activation of Wnt signaling and the role of APC ...................... 10 1.6. Cellular mechanisms of glycosylation ..................................................... 14 1.7. Dystrophin-associated glycoprotein 1 (DAG1) ........................................ 16 2. Aim of the study ......................................................................................... 21 3. Materials ...................................................................................................... 22 3.1. Chemicals and reagents ............................................................................ 22 3.2. Enzymes ...................................................................................................... 27 3.3. Kits ............................................................................................................... 28 3.4. Antibodies ................................................................................................... 30 3.5. DNA constructs and oligonucleotides ...................................................... 31 3.5.1. Plasmids ....................................................................................................... 31 3.5.2. Taqman Probes ............................................................................................ 32 3.5.3. Primer ........................................................................................................... 33 3.5.3.1. qPCR ............................................................................................................ 33 3.5.3.2. ChIP PCR ..................................................................................................... 34 VII Table of contents 3.5.3.3. Genotyping ................................................................................................... 35 3.5.3.4. In situ hybridization ....................................................................................... 35 3.5.3.5. Cloning and PCR .......................................................................................... 35 3.6. Buffers and solutions ................................................................................. 37 3.7. Laboratory equipment ................................................................................ 42 4. Methods....................................................................................................... 43 4.1. Generation of DNA constructs .................................................................. 43 4.2. Bacterial cell culture .................................................................................. 44 4.3. Mammalian cell culture .............................................................................. 44 4.3.1. 2D human cell lines ...................................................................................... 44 4.3.2. 3D culture ..................................................................................................... 45 4.3.2.1. Organoid culture from human colonic mucosa ............................................. 45 4.3.2.2. Organoid culture from human tumor samples .............................................. 45 4.3.3. Transduction of cell lines and organoids ...................................................... 46 4.3.4. Transfection of oligonucleotides and vector constructs ................................ 46 4.4. Luciferase Assay ........................................................................................ 47 4.5. Mutation detection ...................................................................................... 47 4.6. Chromatin-Immunoprecipitation (ChIP) ................................................... 47 4.7. Single cell sorting and genotyping ........................................................... 48 4.8. Immunolabeling of human cells for FACS analysis ................................ 48 4.9. RNA Isolation and transcription ................................................................ 49 4.10. Protein analysis .......................................................................................... 49 4.10.1. Preparation of protein lysates ....................................................................... 49 4.10.2. Precipitation of Glycoproteins ....................................................................... 50 4.10.3. Western Blot analysis ................................................................................... 50 4.10.4. Laminin Overlay Assay ................................................................................. 50 4.10.5. LC-MS/MS .................................................................................................... 51 VIII Table of contents 4.10.6. Peptide quantification ................................................................................... 52 4.11. Migration Analysis ...................................................................................... 52 4.12. Cell adhesion Analysis .............................................................................. 53 4.13. In situ hybridization .................................................................................... 53 4.14. RNAscope® ................................................................................................. 54 4.15. Immunohistochemistry staining ............................................................... 54 4.16. RNA Sequencing (Next generation sequencing) ..................................... 55 4.17. Gene expression data and gene set enrichment analysis ...................... 56 4.18. Statistics ..................................................................................................... 56 4.19. Clinical samples ......................................................................................... 57 5. Results ........................................................................................................ 59 5.1. Silencing of APC in HT-29 cells activates Wnt target
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