UNIVERSITÄTSKLINIKUM HAMBURG-EPPENDORF Zentrum für Experimentelle Medizin Institut für Biochemie und Signaltransduktion Prof. Dr. Aymelt Itzen Role of diaphanous homolog 1 (DIAPH1) in chromosomal instability of colorectal carcinoma cells Dissertation zur Erlangung des Grades eines Doktors der Dr. rer. biol. hum. an der Medizinischen Fakultät der Universität Hamburg. vorgelegt von Shumin Miao aus Ganzhou Hamburg 2020 I Angenommen von der Medizinischen Fakultät der Universität Hamburg am: 18.11.2020 Veröffentlicht mit Genehmigung der Medizinischen Fakultät der Universität Hamburg. Prüfungsausschuss, der/die Vorsitzende: Prof. Dr. Sabine Windhorst Prüfungsausschuss, zweite/r Gutachter/in: PD Dr. Leticia Oliveira-Ferrer II Content 1 Introduction ........................................................................................................................ 1 1.1 Epidemiology and tumorigenesis of colorectal carcinoma .......................................... 1 1.2 Role of dysfunction of spindle MTs in CIN ................................................................ 5 1.3 Microtubule-targeted drugs in CRC treatment considering genomic diversity ......... 10 1.4 Cellular roles of DIAPH1 .......................................................................................... 13 1.5 Aim of the project ...................................................................................................... 16 2 Material ............................................................................................................................ 17 2.1 Plasmids ..................................................................................................................... 17 2.2 Primers ....................................................................................................................... 17 2.3 Antibodies .................................................................................................................. 18 2.4 Media ......................................................................................................................... 19 2.5 Buffers, gels and other solutions ............................................................................... 19 3 Methods ............................................................................................................................ 23 3.1 Biochemical methods ................................................................................................ 23 3.1.1 Cell culture ......................................................................................................... 23 3.1.2 Generation of HCT-116 DIAPH1 stably depleted cells ..................................... 23 3.1.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ..................................... 24 3.1.4 Bradford protein assay ....................................................................................... 24 3.1.5 Western blot analysis ......................................................................................... 25 3.1.6 Cell synchronization with double Thymidine Block .......................................... 26 3.1.7 Cell fixation and immunostaining ...................................................................... 26 3.1.8 Chromosome preparation and staining ............................................................... 28 3.1.9 Micronucleus Test .............................................................................................. 28 3.1.10 Proliferation and MTS assay .............................................................................. 28 3.1.11 Live cell imaging for spindle MT-dynamics ...................................................... 29 III 3.1.12 Measurement of caspase 3/7 activities ............................................................... 30 3.1.13 Paclitaxel treatment of CRC cells ...................................................................... 30 3.1.14 Quantification of mRNA levels for IFNs by RT-qPCR ..................................... 31 3.1.15 Human Cytokine Array (Proteome ProfilerTM Array) ........................................ 32 3.2 Microbiological and molecular biological methods .................................................. 33 3.2.1 Phusion PCR ...................................................................................................... 33 3.2.2 Agarose gel electrophoresis ............................................................................... 34 3.2.3 SLIC reaction ..................................................................................................... 34 3.2.4 KLD reaction ...................................................................................................... 35 3.2.5 Transformation of plasmids into competent E. coli cells ................................... 37 3.2.6 Preparation of XL-1 blue cells for plasmid purification .................................... 37 3.2.7 Recombinant expression of GST-DIAPH1 FL, GST-DIAPH1 FH2 and GST- DIAPH1 dFH2 in bacteria ................................................................................................ 37 3.2.8 Recombinant expression of GFP-DIAPH1 FL, GFP-DIAPH1 FH2 and GFP- DIAPH1 dFH2 in bacteria ................................................................................................ 38 3.2.9 Determination of the binding ratio between DIAPH1 and tubulin .................... 39 3.2.10 MT polymerization assay ................................................................................... 40 3.2.11 MT bundling assay ............................................................................................. 40 3.2.12 MT binding assay ............................................................................................... 41 3.3 Statistical analysis ...................................................................................................... 41 4 Results .............................................................................................................................. 43 4.1 Cellular localization of DIAPH1 ............................................................................... 43 4.2 Depletion of DIAPH1 in colorectal carcinoma cells ................................................. 43 4.3 Effect of DIAPH1 in spindle MT dynamics during metaphase ................................. 44 4.4 Effect of DIAPH1 on MT-polymerization in vitro .................................................... 46 4.5 DIAPH1 enhanced chromosomal stability ................................................................ 48 4.6 Effect of DIAPH1 on cell proliferation and apoptosis .............................................. 52 IV 4.7 Effect of DIAPH1 on chemotherapy treatments of colorectal carcinoma cells ........ 54 4.8 Cytokine expression in control and DIAPH1-depleted cells ..................................... 55 4.9 Effect of DIAPH1 on MT-bundling assay ................................................................. 56 5 Discussion ........................................................................................................................ 57 5.1 DIAPH1 co-localized with spindle MTs and regulated MT- dynamics .................... 58 5.2 Role of DIAPH1 in chromosomal instability ............................................................ 59 5.3 Effect of DIAPH1 on cytokines expression .............................................................. 61 5.4 Potential role of DIAPH1 in colorectal carcinoma treatment .................................... 62 5.5 Limitation .................................................................................................................. 63 5.6 Outlook ...................................................................................................................... 64 5.6.1 Role of DIAPH1 for colorectal carcinoma cells on immune cell response ....... 64 5.6.2 Impact of activated monocytes on the metastatic potential of colorectal carcinoma cells ............................................................................................................................ 65 5.6.3 Effect of pharmacologic DIAPH1 inhibition on the metastatic potential and on chromosome segregation of colorectal carcinoma cells ................................................... 65 6 Conclusion ........................................................................................................................ 65 7 Abstract ............................................................................................................................ 67 8 Zusammenfassung ............................................................................................................ 68 9 Appendix .......................................................................................................................... 69 9.1 Supplementary data ................................................................................................... 69 9.2 List of abbreviations .................................................................................................. 76 9.3 List of figures ............................................................................................................. 82 9.4 List of tables .............................................................................................................. 86 10 Reference .......................................................................................................................... 87 11 Acknowledgements .........................................................................................................