! ""! ACKNOWLEDGEMENTS I dedicate this thesis to my parents who inspired me to become a scientist through invigorating scientific discussions at the dinner table even when I was too young to understand what the hippocampus was. They also prepared me for the ups and downs of science and supported me through all of these experiences. I would like to thank my advisor Dr. Elizabeth Blackburn and my thesis committee members Dr. Eric Verdin, and Dr. Emmanuelle Passegue. Liz created a nurturing and supportive environment for me to explore my own ideas, while at the same time teaching me how to love science, test my questions, and of course provide endless ways to think about telomeres and telomerase. Eric and Emmanuelle both gave specific critical advice about the proper experiments for T cells and both volunteered their lab members for further critical advice. I always felt inspired with a sense of direction after thesis committee meetings. The Blackburn lab is full of smart and dedicated scientists whom I am thankful for their support. Specifically Dr. Shang Li and Dr. Brad Stohr for their stimulating scientific debates and “arguments.” Dr. Jue Lin, Dana Smith, Kyle Lapham, Dr. Tet Matsuguchi, and Kyle Jay for their friendships and discussions about what my data could possibly mean. Dr. Eva Samal for teaching me molecular biology techniques and putting up with my late night lab exercises. Beth Cimini for her expertise with microscopy, FACs, singing, and most of all for being a caring and supportive friend. Finally, I would like to thank Dr. Imke Listerman, my scientific partner for most of the breast cancer experiments. She taught me to enjoy and benefit from a scientific collaboration. She was also wonderful mentor while at the same time treating me as a scientific peer and friend. ! """! I would also like to thank my family and friends who helped me maintain a lab/life balance. My brother Zack has always blindly and enthusiastically supported me without any need for understanding what I do. My sisters Marin, Anne, Kate, and Zaz for supporting their nerdy little sister. My nieces and nephews Lilly, Emmy, Garth, Dante, and Rebecca who always put a smile on my face even when experiments fail. My best friends Karen and Cleya who have made life outside of the lab fun with wine, hikes, and too many good times to list. Finally, I would like to thank my fiancé and partner in adventures, Richard Novak, who is always there to talk science, cook dinner, or just make me smile. ! "#! CONTRIBUTIONS I performed the majority of the experiments in the T cell portion of this thesis with the exception of the following experiments: the Gladstone institute performed the microarray, Dr. Richard Novak performed the microarray analysis, and Beth Cimini performed FACs sorting for GFP + cells. The breast cancer portion of the thesis was a team effort. I performed the telomere length analysis and Dr. Imke Listerman performed telomerase expression and activity data described in this thesis. This data was published in (Listerman, I., Sun, J., Gazzaniga, F.S., Lukas, J.J., Blackburn, E.H. 2013. The major reverse transcriptase-incompetent splice variant of the human telomerase protein inhibits telomerase activity but protects from apoptosis. Cancer Research. May 1;73(9):2817-28). The majority of the breast cancer portion of this thesis was reprinted from (Gazzaniga, F.S., Listerman, I., Blackburn, E.H. 2013. An investigation of the effects of the telomerase core protein TERT on Wnt signaling in human breast cancer cells. Molecular Cell Biology. Epub. Nov 11 2013 doi: 10.1128/MCB.00844-13) in which Listerman and I were co-first authors. I performed the majority of cell culture and the qPCR arrays. Dr. Listerman performed the biochemical and bioinformatic analyses. Both Dr. Listerman and I performed the Wnt reporter assays, stained and imaged cells, and wrote the paper. Pierre de la Grange from Genosplice performed the data analysis correlating telomerase activity and gene expression. ! #! ABSTRACT Non-canonical roles for telomerase in T cells and breast cancer cells Francesca S Gazzaniga Telomerase is a ribonucleoprotein that adds telomeric DNA to the ends of linear chromosomes. It is composed of two core components, the reverse transcriptase component, hTERT, and the RNA template, hTR. In the absence of sufficient telomerase, most differentiated cells experience telomere shortening with each cell division and eventually undergo replicative senescence. Conversely, stem cells and the majority of cancer cells have high telomerase activity, which maintains telomere length allowing them to divide indefinitely. While the role of telomerase in telomere maintenance is clear, recent studies have suggested non-canonical roles for telomerase. The purpose of this thesis is to investigate any non-canonical roles for telomerase in both normal and cancer cells. The first part of this thesis focuses on the role of telomerase in primary human T cells. T cells are one of the few types of differentiated cells that upregulate telomerase activity upon stimulation. While telomerase activity and T cell proliferation correlate, it is unknown if this upregulation is necessary or even quantitatively coupled to T cell survival. Suprisingly, I find that the telomerase RNA, hTR, actually protects CD4+ T cells from apoptosis independent of telomere maintenance or telomerase activity. This is the first report showing a non-telomere role for the telomerase RNA. The second part of this thesis investigates a controversial mechanism for a non-canonical role for hTERT in Wnt signaling. One study published that telomerase activity promotes Wnt/!-catenin signaling in mice and HeLa cells. However, another study was unable to reproduce these findings in mice, but did not investigate these findings in cancer cells. Since ! #"! Wnt signaling is often dysregulated in breast cancer, we sought to determine if we could independently replicate any hTERT -Wnt/!-catenin interactions in breast cancer cells. Using genetic, biochemical, and bioinformatic analyses we do not find any data supporting the hypothesis that hTERT promotes Wnt/!-catenin signaling in breast cancer cells and instead suggest that any published interactions detected are cell line and cell context specific. We and others have shown hTERT can promote proliferation, protect from apoptosis, and promote mitochondrial function independent of telomere maintenance and/or telomerase activity in a wide variety of cell types. This thesis shows that hTERT does not promote these functions through Wnt/!-catenin signaling. Furthermore, I show for the first time that hTR also has a non- telomere and non-telomerase role protecting from apoptosis in CD4+ T cells. While the importance of telomerase for maintaining telomeres is clear, this thesis shows that both of the telomerase core components have non-canonical roles in cell survival in normal and cancer cells. ! #""! TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................iii CONTRIBUTIONS ..................................................................................................................... v ABSTRACT................................................................................................................................. vi TABLE OF CONTENTS .........................................................................................................viii LIST OF FIGURES AND TABLES.......................................................................................... ix CHAPTER 1: T CELLS.............................................................................................................. 1 INTRODUCTION ................................................................................................................................. 1 MATERIALS AND METHODS........................................................................................................ 11 RESULTS............................................................................................................................................. 19 CONCLUSIONS AND DISCUSSION............................................................................................... 46 FUTURE STUDIES............................................................................................................................. 62 CHAPTER 2: BREAST CANCER .......................................................................................... 65 INTRODUCTION ............................................................................................................................... 65 RESULTS............................................................................................................................................. 74 DISCUSSION..................................................................................................................................... 100 FUTURE STUDIES........................................................................................................................... 105 APPENDIX............................................................................................................................... 106 REFERENCES......................................................................................................................... 139 LIBRARY RELEASE FORM ................................................................................................ 148 ! #"""! LIST OF FIGURES AND TABLES CHAPTER