CENTROSOME NUMBER HOMEOSTASIS: LESSONS FROM CEP135 ISOFORM DYSREGULATION IN BREAST CANCER By DIVYA GANAPATHI SANKARAN Bachelor of Technology, Anna University, India, 2013 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cancer Biology Program 2019 This thesis for the Doctor of Philosophy degree by Divya Ganapathi Sankaran has been approved for the Cancer Biology Program By Rytis Prekeris, Chair Jeffrey Moore David Bentley Heide Ford Mary Reyland Chad G Pearson, Advisor Date: 05/17/2019 ii Ganapathi Sankaran, Divya (Ph.D., Cancer Biology) Centrosome Number Homeostasis: Lessons from CEP135 Isoform Dysregulation in Breast Cancer Thesis Directed by Associate Professor Chad G. Pearson ABSTRACT The centrosome, comprised of two centrioles surrounded by pericentriolar material, is the cell’s central microtubule organizing center. Centrosome duplication is coupled with the cell cycle such that centrosomes duplicate once in S phase. Loss of such coupling produces supernumerary centrosomes, a condition called centrosome amplification (CA). CA can promote hallmarks of tumorigenesis. In this thesis, I investigate the contribution of centriole overduplication to CA and its consequences on microtubule organization and genomic stability in breast cancer cells. CEP135, a centriole assembly protein, is dysregulated in some breast cancers. We previously identified a short isoform of CEP135, CEP135mini that represses centriole duplication. CEP135mini represses centriole duplication by limiting the localization of essential proteins required for centriole duplication. Interestingly, the relative level of CEP135full to CEP135mini (the CEP135full:mini ratio) is higher in centrosome amplified breast cancer cell lines. Specifically, I demonstrate that inducing expression of CEP135full increases CA. In contrast, elevating CEP135mini reduces centrosome number in breast cancer cells. I find that the CEP135 isoforms in vivo are generated by alternative polyadenylation. A directed genetic mutation near the CEP135mini alternative polyadenylation signal reduces the CEP135full:mini ratio and decreases CA, accordingly. Thus, dysregulation of the CEP135 isoforms can promote CA in breast cancer cells. Furthermore, in order to iii characterize the consequences of CA in microtubule organization and chromosome segregation, we designed a semi-automatic algorithm using machine learning to detect and analyze centrosomes and microtubule organization. Using this algorithm, I find that centrosome amplified breast cancer cells have microtubule organization defects such as increased microtubule density. Moreover, dysregulation of CEP135 isoforms that promotes CA also increases the frequency of multipolar spindles, anaphase-lagging chromosomes, and micronuclei leading to chromosome segregation defects. I conclude that dysregulation of the CEP135 isoforms can promote centriole overduplication and results in chromosome segregation errors in breast cancer cells. The form and content of this abstract are approved. I recommend its publication. Approved: Chad G Pearson iv ACKNOWLEDGEMENTS I am very grateful to Dr. Chad G Pearson for all the life lessons I have acquired from him over the past five years. He has taught me to be focused, to be thoughtful and to never stop questioning. Chad has shown respect for me as a scientist through his personal investment in my learning. I am very thankful for all his hard work and have enjoyed being his student. I especially would like to thank him for giving me those chances to bounce back when I struggled and for space he provided me to dig intellectually deeper, grow and be successful. I would also like to thank my lab members Alexander Stemm-Wolf, Kristin Dahl, Dr. Nick Galati, Dr. Marisa Ruehle, Dr. Brian Bayless, Anthony Junker, and Adam Soh for creating a very collaborative and friendly environment for the last five years. My lab members have believed in me and encouraged me on my hardest days. I have learned many things from all my lab members and they have been my home away from India. I would like to dedicate my thesis to my husband Dr. Bharath Hariharan, my parents Ganapathi Sankaran, Banumathi Ganapathi Sankaran and, my sister Dr. Jayadurga Ganapathi Sankaran. My parents have worked very hard and selflessly for me. Bharath has motivated me relentlessly. They showed me how to work hard, taught me to never look down upon failures and be very courageous throughout this journey. I would also like to acknowledge my-in-laws Rama Hariharan and Hariharan, who have been very kind and supported me throughout my graduate career. Most importantly, I would also like to thank my committee members Dr. Jeffery Moore, Dr. Rytis Prekeris, Dr. David Bentley, Dr. Mary Reyland and Dr. Heide Ford for all the great advice and the Cancer Biology Program for helping me pursue my interests. v TABLE OF CONTENTS CHAPTER CENTROSOME NUMBER HOMEOSTASIS IN DEVELOPMENT AND DISEASE ......... 1 Introduction .................................................................................................................. 1 Centrioles, Pericentriolar Material, and Centrosome Self-Assembly ........................ 2 Centrosome Number Homeostasis and its Loss during Development and Disease ............................................................................................................................... 14 Centrosome Amplification in Cancer .......................................................................... 20 Centrosome Aberrations in Solid and Hematological Tumors ................................ 21 Aggressive Breast Cancer Cells possess Increased Number of Centrioles and Centrosome Amplification ...................................................................................... 25 Causes of Centrosome Amplification ......................................................................... 27 Cell Cycle Aberrations ............................................................................................ 27 Centriole Overduplication ....................................................................................... 29 Consequences of Centrosome Amplification ............................................................. 30 Genome Instability ................................................................................................. 30 Centrosome Amplification in Tumorigenesis .......................................................... 33 Thesis Outline ............................................................................................................ 38 Materials and Methods for Chapter I.......................................................................... 39 Cell Culture ............................................................................................................ 39 Immunofluorescence .............................................................................................. 39 vi Microscopy ............................................................................................................. 40 Centriole and Centrosome Number Counts ........................................................... 40 Statistics and Biological Replicates ........................................................................ 41 CEP135 ISOFORM DYSREGULATION PROMOTES CENTROSOME AMPLIFICATION IN BREAST CANCER CELLS ....................................................................................... 42 Introduction ................................................................................................................ 42 Results ....................................................................................................................... 48 Centriole Overduplication contributes to Centrosome Amplification in Breast Cancer Cells ....................................................................................................................... 48 The CEP135full:mini Ratio is Elevated in Centrosome Amplified Breast Cancer Cells ............................................................................................................................... 52 Elevated CEP135full is Sufficient to Increase Centrosome Amplification in Breast Cancer Cells........................................................................................................... 57 Elevated CEP135mini Limits the Centriolar Levels of Essential Duplication Factors 60 Elevated CEP135mini is Sufficient to Repress Centrosome Frequency in Breast Cancer Cells........................................................................................................... 62 Mutations Affecting CEP135mini Alternative Polyadenylation Reduce the CEP135full:mini Ratio and Centrosome Amplification ................................................ 64 Discussion ................................................................................................................. 71 Centriole Overduplication in Breast Cancer ........................................................... 71 vii CEP135 Isoform Dysregulation Promotes Centriole Overduplication in Breast Cancer ................................................................................................................... 72 CEP135mini in Repression of Centriole Duplication................................................. 73 Alternative Polyadenylation in CEP135 Isoform Regulation ................................... 74 Materials and Methods for Chapter II ........................................................................