SYMPLEKIN AND TRANSFORMING ACIDIC COILED-COIL CONTAINING PROTEIN 3 SUPPORT THE CANCER CELL MITOTIC SPINDLE Kathryn M. Cappell A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Pharmacology, School of Medicine. Chapel Hill 2011 Approved by: Advisor: Dr. Angelique Whitehurst Reader: Dr. David Siderovski Reader: Dr. Channing Der Reader: Dr. Pilar Blancafort Reader: Dr. Mohanish Deshmukh ABSTRACT KATHRYN CAPPELL: Symplekin and Transforming Acidic Coiled-Coil Containing Protein 3 Support the Cancer Cell Mitotic Spindle (Under the direction of Dr. Angelique Whitehurst) An increased rate of proliferation in cancer cells, combined with abnormalities in spindle architecture, places tumors under increased mitotic stress. Previously, our laboratory performed a genome-wide paclitaxel chemosensitizer screen to identify genes whose depletion sensitizes non- small cell lung cancer (NSCLC) cells to mitotic stress induced by paclitaxel treatment. This screen uncovered a cohort of genes that are required for viability only in the presence of paclitaxel. Two genes uncovered in this screen were the polyadenylation scaffold symplekin and the gametogenic protein transforming acidic coiled-coil containing protein 3 (TACC3). Herein, we examine the impact of polyadenylation and gametogenesis on the tumor cell mitotic spindle. First, we demonstrate that depletion of SYMPK and other polyadenylation components sensitizes many NSCLC cells, but not normal immortalized lines, to paclitaxel by inducing mitotic errors and leading to abnormal mitotic progression. Second, we demonstrate that multiple gametogenic genes are required for normal microtubule dynamics and mitotic spindle formation in the presence of paclitaxel. Additionally, we show that the gametogenic protein TACC3 is uniquely required for mitosis only in transformed cell lines but not normal immortalized cell lines and that this unique dependency can be targeted in vitro with a small molecule. These studies reveal an unanticipated dependence of the cancer cell mitotic spindle on polyadenylation and gametogenic genes. We propose that, faced with mitotic stress, cancer cells develop conditional dependencies on processes such as polyadenylation that occur in all cells and emergent dependencies on gametogenic genes that are overexpressed in tumor cells. ii ACKNOWLEDGEMENTS First and foremost, I would like to thank my mentor. Angelique Whitehurst has proven to be a fantastic mentor who has guided me on the path towards becoming a scientist. Angelique is always full of energy and excitement to share her many ideas and this has made it a great experience to be her graduate student. She has challenged me where I needed it but at the same time introduced me to the joys of scientific research. I will always be grateful for the time I spent in her laboratory and look forward to watching as her career develops in the future. I would particularly like to thank our laboratory managers Brittany Larson and Moriah Scarbrough who have provided much assistance to my projects and Charlene Ross in the UNC- Animal Core who assisted with mouse studies. I am also thankful to the Department of Pharmacology, the Cancer Cell Biology Training Program and the UNC MD/PhD program. During rough patches of my PhD, these programs have really stood by me and for that I will always be grateful. Dr. Eugene Orringer and Dr. David Siderovski deserve special credit for all the support they have given me personally and for making UNC such a wonderful place to be an MD/PhD student. I would also like to thank my committee, including Dr. Pilar Blancafort, Dr. David Siderovski, Dr. Mohanish Deshmukh and Dr. Channing Der for their insights. Finally, I am grateful to my friends and family for all their support. I am thankful to my friends and labmates who have shared the ups and downs of research with me. I would also like to thank my family and in-laws for their support and for understanding what takes their daughter/sister/aunt so far away from them. Most importantly, I want to thank my husband, Steven Cappell, who is the best half of Team Cappell and without whom this dissertation would probably be formatted completely incorrectly. iii TABLE OF CONTENTS LIST OF TABLES ..........................................................................................................................vi LIST OF FIGURES........................................................................................................................vii LIST OF ABBREVIATIONS AND SYMBOLS..........................................................................viii INTRODUCTION...............................................................................................................1 Hallmarks of cancer and the stress phenotype of cancer cells....................2 Mitotic stress in cancer ...............................................................................4 Paclitaxel and genome-wide screen to identify modulators of chemosensitivity .........................................................................................9 Role of symplekin in polyadenylation and tumorigenesis........................17 The cancer-testis antigens.........................................................................24 Transforming acidic coiled-coil containing protein 3...............................27 Thesis Summary........................................................................................29 SYMPLEKIN IS REQUIRED FOR APPROPRIATE MICROTUBULE FUNCTION AND MITOSIS......................................................................................31 Summary...................................................................................................32 Introduction...............................................................................................33 Results.......................................................................................................34 Discussion.................................................................................................48 Experimental Procedures ..........................................................................51 TACC3 AND MULTIPLE GAMETOGENIC GENES SUPPORT THE CANCER CELL MITOTIC SPINDLE ......................................................................55 Summary...................................................................................................56 Introduction...............................................................................................57 iv Results.......................................................................................................58 Discussion.................................................................................................75 Experimental Procedures ..........................................................................76 CLINICAL RELEVANCE AND FUTURE DIRECTIONS.............................................79 Summary...................................................................................................80 Future directions .......................................................................................81 Clinical implications .................................................................................89 Conclusions...............................................................................................94 REFERENCES...............................................................................................................................95 v LIST OF TABLES Table 1.1 Characteristics of mammalian cleavage and polyadenylation factors............................21 Table 3.1 Characteristics of gametogenic proteins identified in screen.........................................59 vi LIST OF FIGURES Figure 1.1 The spindle assembly checkpoint ...................................................................................5 Figure 1.2 Variation in response of mitotic cells to anti-mitotic therapies ....................................13 Figure 1.3 Protein factors involved in cleavage and polyadenylation and impact of their depletion in two genome-wide screens .........................................................................20 Figure 2.1 SYMPK is required for spindle integrity after exposure to paclitaxel.........................35 Figure 2.2 SYMPK is required for normal mitotic progression in tumor cells..............................37 Figure 2.3 SYMPK is necessary for mitosis in multiple tumor cell lines ......................................39 Figure 2.4 Depletion of SYMPK impairs tumor growth in vivo....................................................41 Figure 2.5 Depletion of SYMPK reduces microtubule stability ....................................................43 Figure 2.6 SYMPK depletion leads to loss of CKAP5 ..................................................................45 Figure 2.7 SYMPK alters CKAP5 levels post-transcriptionally ....................................................47 Figure 2.8 Polyadenylation is required for CKAP5 expression and mitosis..................................49 Figure 3.1 Multiple gametogenic genes sensitize H1155 NSCLC cells to paclitaxel....................62 Figure 3.2 Loss of gametogenic genes impairs formation of the bipolar mitotic spindle ..............64 Figure 3.3 Oncogenic changes alter mitotic properties ..................................................................68 Figure