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© 2016 Hillary Stires ALL RIGHTS RESERVED © 2016 Hillary Stires ALL RIGHTS RESERVED ELUCIDATING MECHANISMS THAT DRIVE INCREASED TUMORIGENESIS IN RATS EXPOSED TO ALCOHOL IN UTERO By HILLARY STIRES A dissertation submitted to the Graduate School – New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Endocrinology and Animal Biosciences written under the direction of Dr. Wendie S. Cohick and approved by ______________________________ ______________________________ ______________________________ ______________________________ New Brunswick, New Jersey May 2016 ABSTRACT OF THE DISSERTATION Elucidating Mechanisms That Drive Increased Tumorigenesis in Rats Exposed to Alcohol in Utero By HILLARY STIRES Dissertation Director: Dr. Wendie S. Cohick Alcohol exposure during gestation increases breast cancer risk in offspring. While the mechanisms that underlie this effect are not fully understood, serum estradiol (E2) is increased in these animals during proestrus, suggesting a role for the estrogen axis. To test this hypothesis, it was necessary to develop a stress-free method of hormone administration that could be used in long-term studies of carcinogen exposure to ovariectomized (OVX) rats. Rats were OVX on post- natal day (PND) 40 then treated with daily peroral E2 with or without P4 by adding hormones to peanut butter. On PND 50 rats were injected with nitrosomethylurea (NMU) to induce mammary tumor development. After 26 weeks, there was no difference in tumor incidence suggesting that E2 alone at a normal physiological level can result in tumor development. These results indicate that this may be a useful method to examine the mechanisms of steroid action in mammary tumorigenesis. In addition to changes in circulating E2, local estrogen signaling may be altered in the mammary gland in response to alcohol in utero. To explore this possibility as well as obtain a global view of changes that occur in the mammary gland transcriptome, pregnant Sprague-Dawley rats were treated with alcohol or a control diet during pregnancy. Serum analysis demonstrated an increase in circulating E2 in alcohol-exposed dams during gestation suggesting that alcohol in utero may act as an endocrine disruptor during early development. Mammary glands from PND 2, 10, and 20 offspring were analyzed by RNASeq to look for changes in the estrogen axis. Initial analysis ii with cummeRbund using a low read depth of 25 million reads demonstrated limited differences between alcohol-exposed rats and controls at PND 2 and PND 10. An analysis of additional samples at PND 20 using qRTPCR suggested that heterogeneity of the gland may prevent differences from being observed using the approach taken here. Further analyses of changes in the transcriptome from PND 2 to 10 suggested an increase in stromal cells over time that was corroborated by changes in mammary gland morphology. Comparisons were made with triple negative breast cancer (TNBC), a disease characterized by hormone independent growth similar to this period of mammary gland development. These results suggest that this may be a useful model to study TNBC. Future studies using greater read depth or a larger sample size may uncover differences between alcohol-exposed offspring and controls that were not seen with the present analysis. iii Dedication To my 7th grade science teacher, Mrs. Ambos: for inspiring my passion for science. To my AP Biology teacher, Mr. Gunzelman: for inspiring my passion for biology. To my research advisor at Lehigh, Carrie Garrippa: for inspiring my passion for research. And to my parents: for their unconditional love and support every step along the way. iv Acknowledgments First I would like to thank the Cohick lab for their support during my PhD studies. Thanks to Wendie for letting work in her lab and for always pushing me to be my best. Thanks to Amanda for being my rock in the lab and always being a great listener. Thanks to the women who paved the way before me (Catina and Allyson) and good luck to the women coming after (Mariana, Jenn, and Jen). And a special thank you to my undergrads, Sam, Elena, Rebecca, Ashleigh, and Kathryn for helping me with my studies. Thank you to my committee members for all of your support and insights. Thanks to Troy for meeting with me about our favorite hormone estrogen and giving me invaluable advice about my career. Thank you to Bill Belden for teaching me all there is to know about coding and RNASeq. Your love for science is infectious and I caught the disease whenever we worked together. Thank you to Nick Bello for meeting me about statistics and discussing career advice. Thank you to Dr. Reuhl for looking through slides with me and for using every moment as a teaching opportunity. Thank you to Aparna Zama for your help and advice about histology. Thank you to my amazing friends in general biology. I found my passion for teaching science through the opportunity to start and develop the BRL program. Thank you to Gregg for seeing something in me that I sometimes was unable to see myself. Thank you to Monica and Christina for helping make the program a success and for continuing to develop it in my absence. Thanks to Diana and David for all of your hard work, dedication, and support while we were building the program. I wish you all the best in the future. Thank you to mah ladies, my grad student family. It is really amazing how quickly you can become so attached and close with people in the same position as you. You all have been (and will continue to be) the best support system. Dr. Jess Verpeut, you have been the best shoulder to cry on, the best cheerleader, and the best friend a girl could ask for. v Thank you to my biological family and friends that have become family. My parents and siblings (SILS and Lucy too!) have been so incredibly supportive throughout all of my years of schooling. Thanks to Gram and Pod for always supporting me… now we have two PhDs in the family! Thank you to my aunts, uncles, and all of my cousins, especially Rachel for always listening. Thank you to Nicole (and Danny) for living with me and always supporting me even though I was probably a terror 90% percent of the time. Thanks to Caitlin O’Neill, RD for being such a supportive friend (and Ed!). Thanks to all of my other friends from Lehigh, my Jersey City girls (the boobtastics), and my friends from home for all of your love and support. I never would have been able to do this without you! vi Table of Contents Abstract…………………………………………………………………………...………………ii Dedication…………………………………………………………………………...……………iv Acknowledgements…………………………………………………….….…………...…………v Table of Contents………………………………………………………..…………………....…vii List of Tables……………………………………………………………………………….…….ix List of Figures……………………………………………………………………………………..x List of Abbreviations…………………………………………………………………………….xii Chapter 1: Review of the Literature Introduction………………………………………………………………………………..1 Mammary Gland Development……………………………………………………………3 Overview of Morphological Changes during Mammary Gland Development……..3 Species Comparisons in the Mammary Gland……………………………………...5 Ovarian Hormones in Mammary Gland Development…………………………………….5 Role of Ovarian Hormones in Prepubertal Mammary Gland Development………6 Role of Ovarian Hormones in Pubertal Development of the Mammary Gland……7 Changes in Ovarian Hormones During the Adult Estrous Cycle………………….8 Paracrine Mediators of E2 and P4…………………………………………………9 E2 and P4 in Breast Cancer……………………………………………………………….10 Evidence from Epidemiological Studies………………………………………...11 Evidence from Animal Models…………………………………………………..11 Importance of Hormone Concentration………………………………………….12 Mechanisms of How Estrogens Increase Breast Cancer Risk……………………13 Breast Cancer Heterogeneity……………………………………………………………..14 Endocrine Disruptors and Breast Cancer…………………………………………………16 Objectives………………………………………………..……………………………………...20 vii Chapter 2: Peroral Estradiol is Sufficient to Induce Carcinogen-Induced Mammary Tumorigenesis in Ovariectomized Rats without Progesterone Abstract………………..…………………………………………………………………22 Introduction…………...………………………………………………………………….23 Materials and Methods…………………………………………………………………...25 Results……………………………………………………………………………………31 Discussion………………………………………………………………………………..35 Chapter 3: Effect of Alcohol Exposure in Utero On the Mammary Transcriptome in Early Development Abstract…………………………………………………………………………………..47 Introduction…………………………………………………………………………....…48 Materials and Methods…………………………………………………………………...51 Results……………………………………………………………………………………54 Discussion………………………………………………………………………………..59 Chapter 4: Prepubertal Mammary Gland Development as a Model for Hormone Independent Breast Cancer Abstract…………………………………………………………………………………..75 Introduction………………………………………………………………………………76 Materials and Methods…………………………………………………………………...79 Results……………………………………………………………………………………80 Discussion………………………………………………………………………………..86 Conclusions and Future Directions………………………………….....…………………..100 References……………………………...……………………………………………………….107 Appendix…………………..…………………………………………………………………....130 viii List of Tables Chapter 3 Table 1. Accession numbers and primer sequences for genes analyzed by qRTPCR…...…………63 Table 2. Alcohol levels are elevated for at least 6 hours following lights off………………...……64 Table 3. Concentration and RNA integrity values for samples analyzed……………………….....65 by RNASeq prior to and after shipping. Table 4. Four genes were selected to complete qRTPCR troubleshooting……………..…….……66 Table 5. Expression of genes representing
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