Notch 1 -dependent regulation of cell fate in colorectal cancer Referees: PD Dr. Tobias Dick Prof. Dr. Wilfried Roth http://d-nb.info/1057851272
CONTENTS Summary 1 Zusammenfassung 2 1 INTRODUCTION 3 1.1 Colorectal cancer 3 1.1.1 Pathogenesis 3 1.1.2 Classification 5 1.1.3 Epidemiology and risk factors 6 1.1.4 Diagnosis and therapy 6 1.2 Cell cycle 8 1.2.1 Cyclin-dependent kinase inhibitors 9 1.2.1.1 p27 Kip1 10 1.2.1.2 p21 Cip1 10 1.2.2 Cellular senescence 11 1.3 Apoptosis 12 1.3.1 Caspases 12 1.3.2 Extrinsic apoptotic pathway 14 1.3.3 Intrinsic apoptotic pathway 16 1.3.4 Apoptosis and cancer 18 1.4 Notch signaling 19 1.4.1 Mechanism of Notch signaling 19 I
1.4.2 Notch signaling and cancer 21 Aims of the work 23 2 MATERIALS 24 2.1 Cell lines 24 2.2 Chemicals 24 2.3 Common buffers, solutions and media 25 2.3.1 Buffers for immunoblot analysis 25 2.3.2 Buffer for agarose gel electrophoresis 26 2.3.3 Staining solutions 26 2.3.4 Loading buffers 26 2.3.5 FACS buffer 26 2.3.6 Media for culturing and freezing Escherichia coli 27 2.3.7 Buffers for KCM-competent E. coli Top10 27 2.4 Antibodies 27 2.5 Oligonucleotides 28 2.5.1 sirna 28 2.5.2 PCR primers 29 2.5.3 qrt-pcr primers 29 2.5.4 Sequencing primers 29 2.6 Plasmids 30 2.7 Equipment 30 3 METHODS 31 II
3.1 Cell biology methods 31 3.1.1 Culturing human CRC cell lines 31 3.1.2 Storage of CRC cell lines 31 3.1.3 Transfection of sirna and plasmid DNA 32 3.1.4 Adenoviral transduction 32 3.1.5 Clonogenicity assay 32 3.1.6 Cell counting using trypan blue exclusion 33 3.2 Microbiological methods 33 3.2.1 Generation of KCM-competent E. coli Top10 bacteria 33 3.2.2 Transformation of KCM-competent E. coli Top10 bacteria 33 3.2.3 Transformation of MAX efficiency DH5a competent bacteria 34 3.3 Molecular biology methods 34 3.3.1 Enzymatic restriction 34 3.3.2 Ligation 34 3.3.3 PCR with Phusion polymerase 35 3.3.4 DNA sequencing 35 3.3.5 Isolation and reverse transcription of RNA 35 3.3.6 Quantitative real-time PCR 36 3.3.7 Analyzing qrt-pcr primer efficiency 37 3.4 Biochemical methods 37 3.4.1 PCR primer design 37 3.4.2 Agarose gels 37 3.4.3 Gel extraction 37 III
3.4.4 Preparation of lysates 38 3.4.4.1 Total lysates 38 3.4.4.2 Subcellular fractionation 38 3.4.5 Immunoblot analysis 38 3.4.6 FACS analysis 39 3.4.6.1 Determining cell death using Pl-staining 39 3.4.6.2 Cell cycle analysis 39 3.4.7 Brdll assay 39 3.4.8 Senescence assay 40 3.4.9 Immunohistochemistry 40 3.4.10 Pulse-chase analysis 41 4 RESULTS 42 4.1 Characterization of Notchl-dependent regulation of p27 42 4.1.1 Analysis of NICD1 and p27 levels in CRC cell lines 42 4.1.2 Notchl knockdown causes upregulation of p27 43 4.1.3 Notchl regulates p27 levels post-transcriptionally 43 4.1.4 Notchl controls p27 half-life 44 4.2 Notchl modulates p27 proteasomal degradation by controlling the expression of p27-targeting E3 ubiquitin-protein ligase subunits SKP2, KPC1 and KPC2 45 4.2.1 Knockdown of Notchl induces a decrease of the protein levels of SKP2, KPC1 and KPC2 45 4.2.2 The regulation of SKP2, KPC1 and KPC2 by Notchl occurs at least partially on transcriptional level 46 IV
4.2.3 The regulation of SKP2 and p27 by Notch 1 signaling is additionally demonstrated by shrna-mediated Notchl depletion 48 4.2.4 The knockdown of Notchl leads to increase of p27 levels in the cytoplasm as well as the nucleus 48 4.2.5 The knockdown of SKP2 and KPC1 causes similar upregulation of p27 as Notchl depletion 49 4.2.6 Transient overexpression of SKP2 but not KPC2 decreases p27 levels 50 4.2.7 Notchl-dependent regulation of p27 is probably not mediated by p53... 50 4.3 Role of p27 in Notchl-dependent cell fate decisions 51 4.3.1 p27 knockdown partially rescues the decrease in cellular proliferation mediated by Notchl downregulation 51 4.3.2 p27 knockdown partially rescues the decrease in colony formation mediated by Notchl downregulation 53 4.3.3 p27 knockdown partially rescues the G2/M phase arrest mediated by Notchl downregulation 54 4.3.4 Regulation of p21 by Notchl signaling might contribute to the p27- mediated effect of Notchl on cell fate 54 4.3.5 Overexpression of p27 does not resemble Notchl effect on cell cycle... 55 4.3.6 Clinical relevance of p27 regulation by Notchl 56 4.3.6.1 Low expression of p27 is associated with high proliferative capacity 56 4.3.6.2 Low p27 expression and high Notchl expression are observed at the infiltration zones of colorectal carcinomas 57 4.4 Therapeutic significance of Notchl inhibition 57 4.4.1 Notchl-depleted cells undergo cell senescence 57 4.4.2 Notchl knockdown alone causes CRC cell death 58 V
4.4.3 Notch 1 knockdown enhances the effect of chemotherapy 59 4.4.4 Notchl knockdown enhances the effect of radiotherapy 60 4.4.5 Notchl knockdown enhances the effect of small-molecule inhibitors used in the clinical practice 61 4.4.6 HCT116 cells respond differently to Notchl knockdown 61 4.4.7 Notchl regulation of apoptosis 62 4.4.7.1 Kinetics of activation of apoptosis by Notchl knockdown 62 4.4.7.2 shrna- and sirna-mediated Notchl knockdown causes downregulation of the anti-apoptotic proteins Bcl-X L and Mcl-1 64 5 DISCUSSION 67 5.1 Regulation of cell cycle by Notchl signaling 68 5.2 Regulation of cellular senescence by Notchl signaling 72 5.3 Inhibition of apoptosis by Notchl signaling 74 5.4 Therapeutic significance of Notchl signaling inhibition 76 6 REFERENCES 81 Abbreviations 101 Acknowledgements 107 List of publications 108