DISSERTATION PLURIPOTENCY FACTORS AND MIRNAS IN HUMAN OVARIAN CANCER CELLS AND THEIR SECRETED EXOSOMES REGULATE GENE EXPRESSION AND PHENOTYPE Submitted by Vanessa Ann Enriquez Graduate Degree Program in Cell and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2014 Doctoral Committee: Advisor: Gerrit J. Bouma Co-Advisor: Quinton A. Winger Susan M. Bailey Dawn L. Duval i Copyright by Vanessa Ann Enriquez 2014 All Rights Reserved ii ABSTRACT PLURIPOTENCY FACTORS AND MIRNAS FROM HUMAN OVARIAN CANCER CELLS AND THEIR SECRETED EXOSOMES REGULATE GENE EXPRESSION AND PHENOTYPE Ovarian cancer is the fifth most deadly cancer among women in the United States and the most lethal gynecological malignancy in the world. Recent studies reveal that human tumor cells release cell-secreted vesicles called exosomes. Exosomes are endosome-derived vesicles containing bioactive materials, including RNAs and miRNAs that can be detected in body fluids. Importantly, pluripotency factor LIN28, a regulator of let-7 miRNAs, is present in ovarian cancer cells. High LIN28A and low let-7 miRNA levels are associated with aggressive IGROV1 human ovarian cancer cells (Piskounova et al., 2011) so we compared this to low LIN28A and high let-7 miRNA levels in less aggressive OV420 human ovarian cancer cells. Moreover, let-7b, let-7c, let-7g , and let- 7i miRNA signatures were also present in their secreted exosomes. We hypothesized that ovarian cancer cell-secreted exosomes are taken up by target cells and induce changes in gene expression and cell behavior. Our data revealed that IGROV1 secreted exosomes taken up by HEK293 cells lead to significantly higher LIN28A mRNA levels, but not LIN28A protein levels and did not significantly change LIN28B mRNA or protein levels. However, IGROV1 exosome exposure to HEK293 cells did significantly increase invasion and migration. In addition, various genes involved in epithelial-mesenchymal transition, including TIMP1 (25-fold higher), NOTCH1 (11-fold-higher), SNAI1 (SNAIL) ii (7-fold higher), CDH1 (6-fold higher), (MMP9 (4-fold higher), and ZEB1 (3-fold higher), were significantly higher in HEK293 cells following uptake of IGROV1 secreted exosomes. We also postulated that ovarian cancer cell-secreted exosomes contain a distinct RNA signature capable of inducing phenotypic changes in target cells, as well as distinguishing aggressive, advanced ovarian exosomes from less aggressive ovarian exosomes. Since IGROV1 cells are a more aggressive epithelial ovarian cancer cell line, while OV420 cells are a less aggressive cell line, we performed RNAseq transcriptome analysis on exosomes from IGROV1 cells and exosomes from OV420 cells. The results yielded 312 differentially expressed RNAs. Future studies will allow us to detect the RNAs present in exosomes from urine in individuals with early stage versus late stage ovarian cancer. This may enable investigators to distinguish a poor prognosis group from a good prognosis group leading to a potential biomarker for ovarian cancer detection. Data from this study is important for elucidating the role ovarian cancer cell-secreted exosomes have on early metastasis and tumor progression, an area in ovarian cancer biology in critical need of advancement. iii ACKNOWLEDGEMENTS “If I have seen farther than others, it is because I stood on the shoulders of giants.” Sir Isaac Newton First and foremost, I would like to thank my advisor Dr. Gerrit J. Bouma for his constant guidance, patience, and mentorship. I remember meeting you at an ARBL tour (before I had even picked a lab) and thought to myself, “Wow, he seems so down to earth and smart. If I ever get to work with this person, I would be not only be gaining and extremely accomplished education, but I would be exceptionally fortunate to have such an incredible mentor.” I never would have thought that I would get the chance to work with you and feel very blessed to have such an unbelievable advisor. Jerry, you have shown me how to be the scientist I am today and I can only hope to one day inspire others in the lessons I’ve learned from you as I continue discovering my role in the scientific community. I would also like to thank my co-advisor Dr. Quinton Winger for sharing his passion for placentation development with me and allowing me to gain some experience in this field. Also, I would like to thank you for showing me the importance for experimental design and always willing to speak with me about interpreting my data. To Drs. Dawn Duval and Susan Bailey, I would especially like to thank you for not only being wonderful insightful committee members, but for allowing me to work in your labs during my rotations when I was trying to find my “lab home”. I not only learned valuable iv techniques from you and your students, but also was able to gain personal mentorship from you both and how to function as a woman in science. Your advice was invaluable and I will treasure your teachings for the rest of my life. I am thankful for the McNair Scholars program that introduced me to the idea that I could pursue a higher education degree. This program sparked my interest in research and allowed me to work with my first mentor, Dr. Jodi Huggenvik who guided me toward a passion for scientific research. I am also incredibly thankful to the future Dr. Aspen D’Costa who was my nurse, friend, and supporter during my undergraduate degree. You will always be my lifelong friend! Mrs. Margaret Cech is also a person I would like to acknowledge, as she became an outside personal mentor who was my confidant. I would also like to thank the funding sources that have allowed me to continue my PhD dissertation work here at Colorado State University (CSU). I am thankful for the Bridge to the Doctorate Fellowship (Award #: 0603176) for funding my first two years in graduate school, the CSU CVMBS CRC grant, CSU Cancer Supercluster 2011, and the ACS-IRG #57-001-50 from the American Cancer Society. I would like to extend my humblest appreciation toward my friends at ARBL, the place that was my home so for many years: Dr. Jillian Delehoy (formerly Guttormsen) whom was my close colleague during the first couple years of my PhD for showing me how to run a successful lab, Dr. Juliano C. da Silveria for showing me the ropes in the lab and always willing to talk about experiments and science, Dr. Dilka Murtazina for helping me through frustrating days and always being a listening ear and Brenda Martin for always v reassuring me that I could finish and do well in my future endeavors. I would like to extend a special thank you to the future Dr. Ellie Cleys, Lisa Nulton, and future Dr. Kathleen Eddy for being great lab mates and friends. You girls got me through days when I felt hopeless and alone. I could always depend on your to be a shoulder to cry on, sharing a sarcastic remark with me to make me smile, always reminding me “we can get through this”, celebrating the times when lab results worked or (more often then I would like to admit) telling me “you’ll figure it out” when my results didn’t work. I will always remember our days in the office/lab where you listened to me vent, gave me great advice and how you were always there to bounce ideas off. I am forever grateful for your friendship. Lastly, it is with the deepest gratitude that I thank my entire familia! I can’t even begin to thank you for all that you’ve done and continue to do. You have all given me the strength to keep pushing forward, love that carried me through my toughest hours, and support and prayers to keep on fighting. You have believed in me accomplishing something great since I was a young girl. Mom and Dad, you have sacrificed so much to give your children an education. You always told me, “the key to the world lies in a degree” and I guess I took that literally. There were days when I felt broken down and your kind words lifted me up and reminded me that no matter what happened, you were proud of me. You both are an inspiration to me. God bless you for having the strength to put up with me and having the unwavering love and support, which allowed me to follow my dreams! In a million years with a million words I would never be able to express my gratitude to you; just know, that I love you from the depths of my heart and soul and am vi forever grateful you are my parents! To my siblings, you have each played a significant part in helping me reach my goals. Victor, I remember how proud you looked when you saw me graduate with my Bachelors. You told me to fight for my dreams and I haven’t given up yet, nor will I ever. I love you brother! Jessica, you were always around to listen my frustrations and give me hope and encouragement and for that I will be forever indebted to you. Your drive and resilience is inspirational and I continue to strive for the best because I see you doing this everyday. I love you little sister and know you will go on and accomplish great things! Christopher, my little big brother, you saved my life when you moved to Colorado. You took care of me when I didn’t take care of myself. I remember when you would bring me lunch, sit with me, and watch me eat because you knew I was too busy and would “forget”.