Aus dem Pathologischen Institut der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Thomas Kirchner Significance of heterogeneous WNT and MAPK signaling in colorectal cancer Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Cristina Blaj aus Timișoara 2016 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians- Universität München Betreuer: Prof. Dr. rer. nat. Andreas Jung Zweitgutachter: Prof. Dr. Olivier Giers Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 16.05.2017 Eidesstattliche Versicherung Cristina Blaj Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema „Significance of heterogeneous WNT and MAPK signaling in colorectal cancer“ selbständig verfasst, mich auer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. Ort, Datum: München, 20.05.2017 Unterschrift: Meinen Eltern Table of contents 1. Introduction .................................................................................................................. 7 1.1. Colorectal cancer .............................................................................................................. 7 1.2. The tumor microenvironment ........................................................................................... 9 1.3. Intratumoral heterogeneity ..............................................................................................10 1.4. Intestinal stem cells and colon cancer stem cells ...............................................................11 1.5. Epithelial-mesenchymal transition ...................................................................................15 1.6. The wingless-related integration site pathway ..................................................................17 1.7. The mitogen-activated protein kinase pathway ................................................................19 1.6. Activity-dependent neuroprotector homeobox .................................................................21 Objectives ........................................................................................................................ 23 2. Material ....................................................................................................................... 24 2.1 Chemicals and reagents.....................................................................................................24 2.2. Kits and disposables .........................................................................................................26 2.3. Enzymes ...........................................................................................................................27 2.4. Buffers and solutions ........................................................................................................27 2.5. Laboratory equipment .....................................................................................................29 3. Methods ....................................................................................................................... 30 3.1. Clinical samples and statistical analyses ...........................................................................30 3.2. Gene expression data sets, TCGA data, and GSEA ..........................................................31 3.3. Bacterial cell culture, transformation and plasmid DNA preparation ..............................33 3.4. DNA cloning and sequencing............................................................................................33 3.5. Isolation of RNA and qPCR .............................................................................................34 3.6. Cell and spheroid culture .................................................................................................34 3.7. In vitro treatments ............................................................................................................35 3.8. Lentiviral vectors .............................................................................................................35 3.9. Transient transfections ....................................................................................................37 3.10. Luciferase assays ............................................................................................................37 3.11. Lentiviral transductions and single cell sorting ..............................................................37 3.12. CRISPR/Cas9 genome editing ........................................................................................38 3.13. Gene expression analyses ...............................................................................................38 3.14. Immunoblotting .............................................................................................................39 3.15. Mass spectrometry (MS) ................................................................................................39 3.16. RAS-GTP assays ............................................................................................................40 3.17. Immunohistochemistry, immunofluorescence and imaging ............................................41 3.18. Proliferation, migration and invasion assays ..................................................................43 3.19. Tumor xenografts and in vivo treatments .......................................................................43 4. Results ......................................................................................................................... 45 4.1. Identification of ADNP as a repressor of WNT signaling in colon cancer .........................45 4.1.1. ADNP is overexpressed in colon cancer cells with high WNT signaling activity .................. 45 4.1.2. ADNP is a repressor of WNT signaling in colon cancer ......................................................... 49 4.1.3. ADNP represses malignant traits and tumor growth of colon cancer...................................... 53 4.1.4. Induction of ADNP by sub-narcotic ketamine suppresses tumor growth in vivo .................... 57 4.1.5. High ADNP expression predicts good outcome of colorectal cancer patients ........................ 61 4.2. Analysis of differential MAPK signaling in colorectal cancer ...........................................65 4.2.1. MAPK activity is heterogeneous in colorectal cancer ............................................................. 65 4.2.3. Colorectal cancer cells with high MAPK activity have a distinct phenotype.......................... 67 4.2.5. Lineage tracing reveals a progenitor cell phenotype of colon cancer cells with high MAPK activity ............................................................................................................................................... 72 5. Discussion ................................................................................................................... 76 5.1. ADNP is a therapeutically inducible repressor of WNT signaling in colorectal cancer .....76 5.2. High MAPK activity induces EMT and marks progenitor cells in colorectal cancer ........79 Summary ......................................................................................................................... 81 Zusammenfassung ........................................................................................................... 82 References ....................................................................................................................... 84 Abreviations .................................................................................................................... 95 List of figures .................................................................................................................. 98 List of tables .................................................................................................................... 99 1. Introduction 1.1. Colorectal cancer Cancers figure beside diseases of the circulatory system as the leading cause of death worldwide. With about 14.1 million new cancer cases and 8.2 million deaths worldwide in 2012, cancers represent a massive societal burden affecting both developed and developing countries. While the genetic component and, in some tumor types, inherited genetic susceptibility play a major role in tumor initiation, environmental risk factors like tobacco use, alcoholism, obesity and some chronic infections have a great contribution to the high incidence of cancer (Torre et al., 2015). Cancer is the result of a multistep process transforming normal tissue cells into malignant tumor cells. Most solid human tumors require two to eight sequential genetic alterations to develop over a timeframe of 20 to 30 years (Vogelstein et al., 2013). During malignant transformation, tumor cells acquire several specific capabilities, which have been defined as the hallmarks of cancer. These include malignant traits like proliferation, replicative immortality, angiogenesis, invasion, metastasis, reprogramming