Role of stathmin 1 in colorectal cancer metastasis and chemo resistance

ROLE OF STATHMIN-1 IN COLORECTAL CANCER METASTASIS AND CHEMO-RESISTANCE WU WEI B Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Wu Wei 23rd January 2014 i Acknowledgement “All that is valuable in human society depends upon the opportunity for development accorded the individual.” Albert Einstein It was my utmost privilege to receive the mentorship of three great scientists As I reflect in the final lap upon this journey, these opportunities appear even more valuable than ever I am immensely grateful for the opportunity to work under A/P Maxey Chung Ching Ming, who saw faith in an enthusiastic but otherwise mediocre student Without his encouragement and dedicated care, this amazing four-year adventure would never have been any more than the desire to explore I also thank Dr David Balasundaram for captivating me with his passion for science almost a decade ago Till this day, I remember the shine in his eyes at that eureka moment we shared I am also indebted to A/P Alan G Porter who provided the best environment to learn experimental techniques, and for trusting that a third-year undergraduate just picking up cancer biology was worth his time I am also blessed to have supportive thesis advisors who were genuinely concerned, as well as efficient department staff whom I trouble frequently, but still remain friendly This voyage was never lonely, for I had labmates who shared the joy of making discoveries and never failed to spur me on Thank you all This thesis is dedicated to my parents, who held the conviction that I was meant to pursue this path, and bestowed upon me the fortitude to take on this challenge With your love, I made it ii Table of Contents Acknowledgement .ii List of figures vii List of Tables x Summary xii Abbreviations xiii Chapter Introduction 1.1 Colon cancer 1.1.1 1.1.2 Diagnosis and staging 1.1.3 CRC survival 1.1.4 1.2 Colorectal carcinoma CRC treatment Stathmin-1 1.2.1 The Stathmin family 1.2.2 STMN1 in microtubule regulation 10 1.2.3 STMN1 in cell cycle regulation 12 1.2.4 STMN1 in cancer 14 Chapter Objective of study 15 2.1 Motivation of study 16 2.1.1 2.1.2 2.2 STMN1 up-regulation in metastatic CRC 16 Knowledge gaps and experimental aims 20 Workflow 22 Chapter Results 25 3.1 Stable STMN1 knockdown and over-expression 26 3.1.1 3.1.2 STMN1 over-expression 30 3.1.3 3.2 STMN1 knockdown 28 Summary 31 STMN1 expression is required for metastatic processes in vitro 33 iii 3.2.1 3.2.2 Invasion 36 3.2.3 Adhesion 37 3.2.4 Colony formation 38 3.2.5 Growth 39 3.2.6 3.3 Migration 34 Summary 40 STMN1 silencing regulates the metastatic proteome 41 3.3.1 3.3.2 Metastatic balance 44 3.3.3 Cell junctions and intracellular architecture 46 3.3.4 Apoptotic defense 48 3.3.5 Validation 50 3.3.6 3.4 iTRAQTM Summary statistics 42 Summary 54 STMN1 silencing enhances cellular anchorage and intracellular rigidity 55 3.4.1 3.4.2 Desmosomes and intermediate filaments 57 3.4.3 3.5 Hemidesmosomes 56 Summary 58 STMN1 silencing promotes 5-Fluorouracil sensitivity 59 3.5.1 3.5.2 5-Fluorouracil sensitisation 62 3.5.3 Caspase-dependent apoptosis 64 3.5.4 Caspase activity 66 3.5.5 3.6 General cytotoxicity 60 Summary 68 STMN1 silencing regulates transcript abundance 69 3.6.1 3.6.2 Quality control 72 3.6.3 CRC progression and cytoskeletal remodelling 74 3.6.4 Metastatic and EMT transcriptional profile 76 3.6.5 Validation 79 3.6.6 3.7 p38 phosphorylation 70 Summary 80 Regulation of STMN1 function 81 3.7.1 STMN1 interactions 82 3.7.2 Fibronectin stimulation 86 3.7.3 p53 dependence 87 3.7.4 STMN1 phosphorylation 90 3.7.5 S25/38 phosphorylation in metastatic processes 92 iv 3.7.6 Summary 94 Chapter Discussion 95 4.1 Experimental strategy 96 4.2 STMN1 expression drives in vitro metastatic phenotype 97 4.3 Molecular benefits of STMN1 silencing 99 4.4 STMN1 interactions and S25/38 phosphorylation determine pro-metastatic activity 101 4.5 STMN1 silencing regulates metastatic networks 103 4.6 STMN1 silencing: a potential therapy against metastatic CRC 104 Chapter Conclusion and future work 107 Chapter Materials and methods 110 6.1 Cell lines and constructs 112 6.1.1 6.1.2 Preparation of whole cell lysate 112 6.1.3 STMN1 KD and OE constructs 113 6.1.4 Mutagenesis 113 6.1.5 6.2 HCT116 and E1 cell lines 112 Transfection 114 Cell-based assays 115 6.2.1 6.2.2 Wound healing 115 6.2.3 Transwell migration 116 6.2.4 Matrigel invasion 117 6.2.5 Cell adhesion 117 6.2.6 6.3 Proliferation 115 Anchorage-independent colony formation 118 Proteome profiling 119 6.3.1 iTRAQTM: labeling chemistry 119 6.3.2 iTRAQTM: sample preparation 120 6.3.3 iTRAQTM: 2D LC-MS/MS 121 6.3.4 iTRAQTM: protein and peptide identification 122 6.3.5 iTRAQTM: data analysis 123 6.3.6 SWATHTM MS: label-free technology 124 6.3.7 SWATHTM MS: sample preparation and analysis 125 6.3.8 SWATHTM MS: protein identification and quantitation 126 v 6.4 Transcript analysis 127 6.4.1 6.4.2 6.5 RNA extraction and quantification 127 qPCR array 127 Molecular methods 128 6.5.1 6.5.2 2D western blotting 129 6.5.3 Dephosphorylation 130 6.5.4 Immuno-fluorescence 131 6.5.5 Cytotoxicity 132 6.5.6 Flow cytometry 132 6.5.7 Caspase inhibition 133 6.5.8 6.6 1D western blotting 128 Caspase activity 133 Data representation 135 6.6.1 Graphs and data visualisation 135 6.6.2 Images 135 6.6.3 Statistical analyses 135 Appendix I: Proteins regulated by STMN1 silencing (iTRAQTM) 136 Appendix II: Regulated proteins validated by SWATHTM 142 Appendix III: Transcripts regulated by STMN1 silencing (qPCR) 143 Publications 144 Conference presentations 145 Awards 146 Bibliography 148 vi List of Figures Chapter Introduction Figure 1-1: Survival of primary and metastatic CRC Figure 1-2: Stathmin family multiple sequence alignment Figure 1-3: STMN1 regulates MT and mitotic spindle dynamics during cell cycle 12 Chapter Objective of study Figure 2-1: STMN1 is significantly up-regulated in hepato-metastatic cell line E1 17 Figure 2-2: STMN1 expression increases with CRC progression 18 Figure 2-3: STMN1 expression indicates CRC prognosis 19 Figure 2-4: Experimental workflow 23 Chapter Results Figure 3-1: Representative colony amplified from a single stably-transfected cell 28 Figure 3-2: Morphology of STMN1 KD and SC cells 29 Figure 3-3: Morphology of STMN1 OE and vector control cells 30 Figure 3-4: Panel of stable STMN1 KD and OE cell lines 31 Figure 3-5: STMN1 expression is required for efficient wound healing 34 Figure 3-6: STMN1 expression promotes cell migration 35 Figure 3-7: STMN1 expression promotes matrix invasion 36 Figure 3-8: STMN1 expression inhibits cell adhesion 37 Figure 3-9: STMN1 expression promotes anchorage-independent growth 38 Figure 3-10: STMN1 expression confers no proliferative advantage 39 Figure 3-11: Functional classification of targets regulated by STMN1 silencing 43 Figure 3-12: Hemidesmosomes 46 vii Figure 3-13: Desmosomes 47 Figure 3-14: iTRAQTM validation by western blotting 50 Figure 3-15: iTRAQTM validation by immuno-fluorescence 51 Figure 3-16: STMN1 silencing strengthens hemidesmosomes 56 Figure 3-17: STMN1 silencing increases intracellular rigidity 57 Figure 3-18: STMN1 silencing promotes sensitivity to MT inhibitors and 5FU 60 Figure 3-19: Microtubule inhibition and 5FU treatment decrease STMN1 level 61 Figure 3-20: STMN1 silencing amplifies 5FU-dependent apoptosis 63 Figure 3-21: 5FU-induced apoptosis is caspase-dependent 64 Figure 3-22: 5FU sensitisation in STMN1 KD cells depends on Caspases and 65 Figure 3-23: Caspase activity amplifies 5FU sensitivity 66 Figure 3-24: Caspase activation and cleavage of Lamin A 67 Figure 3-25: Model of STMN1 silencing induced 5FU sensitisation 68 Figure 3-26: p38 phosphorylation 70 Figure 3-27: qPCR reproducibility 73 Figure 3-28: STMN1 KD inhibits CRC progression and cytoskeletal remodelling 74 Figure 3-29: STMN1 KD reverses metastatic and EMT transcriptional profile 77 Figure 3-30: qPCR validation by western blotting 79 Figure 3-31: STMN1 is enriched by immuno-precipitation 82 Figure 3-32: 2D separation of STMN1 IP eluate 83 Figure 3-33: STMN1 potentially interacts with RhoGAP8 84 Figure 3-34: STMN1 may regulate G protein signaling 85 Figure 3-35: Fibronectin induces STMN1 expression 86 Figure 3-36: STMN1 silencing perturbs p53 transcirptional network 87 Figure 3-37: Stable STMN1 silencing in HCT116 p53-/- cells 88 Figure 3-38: Functional p53 not required to achieve efficacy of STMN1 silencing 88 Figure 3-39: STMN1 is phosphorylated at S16, 25, 38 and 63 in CRC cells 90 Figure 3-40: Rescue of STMN1 KD by phosphorylation defective mutants 92 viii Figure 3-41: STMN1 pro-metastatic 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Stathmin- 1 1. 2 .1 The Stathmin family 1. 2.2 STMN1 in microtubule regulation 10 1. 2.3 STMN1 in cell cycle regulation 12 1. 2.4 STMN1 in cancer 14 ... Conclusion and future work 10 7 Chapter Materials and methods 11 0 6 .1 Cell lines and constructs 11 2 6 .1. 1 6 .1. 2 Preparation of whole cell lysate 11 2 6 .1. 3 STMN1 KD and