Drug Metabolism Determines Resistance of Colorectal Cancer to Resorcinol-Based HSP90 Inhibitors Dissertation for the award of the degree “Doctor rerum naturalium” (Dr. rer. nat.) in the “Biology of Cells” Program at the Georg August University Göttingen, Faculty of Biology submitted by Hannes Landmann born in Hannover, Germany Göttingen, August 2014 Thesis Committee Prof. Dr. Matthias Dobbelstein Institute for Molecular Oncology, Faculty of Medicine Prof. Dr. Heidi Hahn Tumor Genetics, Department of Human Genetics, Faculty of Medicine Prof. Dr. Dieter Kube Department for Hematology and Oncology, Faculty of Medicine Members of the Examination Board Referee: Prof. Dr. Matthias Dobbelstein, Institute for Molecular Oncology 2nd Referee: Prof. Dr. Heidi Hahn, Tumor Genetics, Department of Human Genetics Further members of the Examination Board Prof. Dr. Dieter Kube Department for Hematology and Oncology, Faculty of Medicine PD Dr. Halyna Shcherbata Research Group Gene Expression and Signaling, Max Planck Institute for Biophysical Chemistry Prof. Dr. Ralph Kehlenbach Department of Molecular Biology, Faculty of Biochemistry PD Dr. Wilfried Kramer Department for Molecular Genetics, Institute for Microbiology and Genetics Date of oral examination: 19th of September 2014 AFFIDAVIT Herewith I declare that I prepared the PhD thesis “Drug Metabolism Determines Resistance of Colorectal Cancer to Resorcinol-Based HSP90 Inhibitors” on my own with no other aids and sources than quoted. _____________________ Hannes Landmann, Göttingen in August 2014 The findings in this work were accepted for publication in the open access journal Cell Death and Disease: Landmann, H., Proia, D.A., He, S., Ogawa, L.S., Kramer, F., Beißbarth, T., Grade, M., Gaedcke, J., Ghadimi, M., Moll, U. & Dobbelstein, M. UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib. Cell Death and Disease (accepted for publication in July 2014). Table of Contents Table of Contents ............................................................................................................... i List of Figures................................................................................................................... iv List of Tables ..................................................................................................................... v Abbreviations ................................................................................................................... vi Abstract.............................................................................................................................. 1 1. Introduction ........................................................................................................... 3 1.1 Global Perspective on Cancer Treatment .......................................................... 3 1.2 Colorectal Cancer .............................................................................................. 3 1.3 The Chaperone Protein HSP90 ......................................................................... 4 1.4 HSP90 Clients Involved in Cancer.................................................................... 6 1.5 Inhibition of HSP90 as a Cancer Therapy Approach ........................................ 7 1.5.1 The First Generation of HSP90 Inhibitors: Derivatives of Geldanamycin . 8 1.5.2 The Second Generation of HSP90 Inhibitors: Small Synthetic Molecules . 9 1.5.3 Rationale for the Clinical Use of HSP90 Inhibitors .................................. 11 1.5.4 Potential Risks Linked to the Clinical Use of HSP90 Inhibitors .............. 11 1.5.5 Varying Pharmacology of Different HSP90 Inhibitors ............................. 12 1.6 Enzymes of the Drug Metabolism ................................................................... 14 1.6.1 Enzymes from the Phase I of the Drug Metabolism ................................. 14 1.6.2 Enzymes from the Phase II of the Drug Metabolism ................................ 15 1.6.3 The UDP-Glucuronosyltransferases and Their Role in Drug Metabolism16 1.7 Scope of the Thesis ......................................................................................... 19 2. Materials and Methods ........................................................................................ 20 2.1 Materials .......................................................................................................... 20 2.1.1 Technical Devices ..................................................................................... 20 2.1.2 Consumables ............................................................................................. 21 2.1.3 Chemicals and Reagents............................................................................ 22 2.1.4 Eukaryotic Cell Culture ............................................................................. 26 2.1.5 Bacteria...................................................................................................... 27 2.1.6 Bacteria Culture Medium .......................................................................... 27 2.1.7 Oligonucleotides and plasmids.................................................................. 27 2.1.8 Software .................................................................................................... 28 i 2.2 Methods ........................................................................................................... 29 2.2.1 Cell Biology .............................................................................................. 29 2.2.2 Molecular Biology..................................................................................... 33 2.2.3 Protein biochemistry ................................................................................. 38 2.2.4 Statistical Analysis .................................................................................... 40 3. Results ................................................................................................................. 42 3.1 Expression of UGT1A Correlates with Resistance to Ganetespib Treatment 42 3.2 UGT1A Knockdown Sensitizes Resistant CRC Cell Lines to Ganetespib ..... 44 3.3 Overexpression of UGT1A10 Renders Sensitive Cells Resistant to Ganetespib Treatment ....................................................................................... 46 3.4 Verification of Ganetespib Glucuronidation by LC-MS/MS .......................... 49 3.5 UGT1A Expression Levels do not Correlate to Susceptibility to the First Generation Inhibitor 17-AAG ........................................................................... 52 3.6 Susceptibility of Ganetespib-Resistant and -Sensitive Cell Lines to Second Generation HSP90 Inhibitors ............................................................................ 53 4. Discussion ........................................................................................................... 55 4.1 UGT1A Renders CRC Cells Resistant to Ganetespib ..................................... 55 4.2 UGT1A and the Resistance to Other Anti-Cancer Drugs ............................... 56 4.3 Implications for the Different Classes of HSP90 Inhibitors ........................... 57 4.4 Role of UGT1A Glucuronidation in Other Cancer Types .............................. 58 4.5 Other Factors that Might Influence the Efficacy of Glucuronidation by UGT1A ............................................................................................................. 59 4.5.1 Transcriptional Regulation ........................................................................ 59 4.5.2 Factors Regulating the Activity of UGT1A .............................................. 61 4.5.3 Potential Influence of Co-Substrate Supply .............................................. 62 4.5.4 Potential Influence of Metabolite Transport ............................................. 62 4.6 UGT1A Isoforms in Ganetespib Resistance ................................................... 63 4.7 Strategies to Circumvent Ganetespib Resistance by Glucuronidation ............ 64 4.8 Implications for the Clinical Use of Resorcinolic HSP90 Inhibitors .............. 65 4.8.1 Mouse Models for in vivo Studies of Resorcinolic Hsp90 Inhibitors ....... 66 4.8.2 Clinical Studies ......................................................................................... 67 4.9 UGT1A as a Drug-Related Biomarker for Ganetespib Treatment .................. 68 4.9.1 Clinical Assessment of UGT1A mRNA Levels ........................................ 69 4.9.2 Standardization of the mRNA Evaluation Process ................................... 70 ii 4.10 Concluding Remarks ....................................................................................... 71 5. Literature ............................................................................................................. 72 6. Appendix ............................................................................................................. 82 6.1 Gene List from the Statistical Analysis ........................................................... 82 Acknowledgement ........................................................................................................... 85 iii List of Figures Figure 1.1: The HSP90 chaperoning cycle ................................................................................ 6 Figure 1.2: Chemical structures of geldanamycin derivatives. .................................................