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Analysis of the Role of Two Autophagy Pathway Related Genes, Becn1 and Tsc1, in Murine Mammary Gland Development and Differentiation

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University of Kentucky UKnowledge Theses and Dissertations--Biology Biology 2014 ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION Amber N. Hale University of Kentukcky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Hale, Amber N., "ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION" (2014). Theses and Dissertations--Biology. 18. https://uknowledge.uky.edu/biology_etds/18 This Doctoral Dissertation is brought to you for free and open access by the Biology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Biology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Amber N. Hale, Student Dr. Edmund Rucker, Major Professor Dr. David Westneat, Director of Graduate Studies ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION __________________________ DISSERTATION __________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Amber Nicolle Hale Lexington, Kentucky Co-Directors: Dr. Edmund Rucker, Associate Professor of Biology and Dr. Vincent Cassone, Professor of Biology Lexington, Kentucky 2014 Copyright © Amber Nicolle Hale 2014 ABSTRACT OF DISSERTATION ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION The mammary gland is a dynamic organ that undergoes the majority of its development in the postnatal period in four stages; mature virgin, pregnancy, lactation, and involution. Every stage relies on tightly regulated cellular proliferation, programmed cell death, and tissue remodeling mechanisms. Misregulation of autophagy, an intracellular catabolic process to maintain energy stores, has long been associated with mammary tumorigenesis and other pathologies. We hypothesize that appropriate regulation and execution of autophagy are necessary for proper development of the mammary ductal tree and maintenance of the secretory epithelia during late pregnancy and lactation. To test this hypothesis we examined the role of two genes during development of the mammary gland. Beclin1 (Becn1) is an essential autophagy gene. Since the Becn1 knockout model is embryonic lethal, we have generated a Becn1 conditional knockout (cKO). We used two discrete mammary gland-specific Cre transgenic lines to interrogate the role of BECN1 during development. We report that MMTV-CreD; Becn1fl/fl mice have a hyper- branching phenotype and WAP-Cre; Becn1fl/- mice are unable to sustain a lactation phase. Becn1 mutants exhibit abnormal glandular morphology during pregnancy and after parturition. Moreover, when autophagy is chemically inhibited in vitro, mammary epithelial cells have an increased mean number of lipid droplets per cell. MTOR inhibits autophagy upstream of BECN1; we looked higher in the regulatory pathway for regulatory candidates. It has been well characterized that Tuberous sclerosis complex 1 (TSC1), in a heterodimer with its primary binding partner TSC2, inhibits MTOR signaling via inhibition of RHEB. Using the Tsc1 floxed model we generated a mammary gland specific Tsc1 cKO and found that these mice phenocopy the Becn1 cKO mice, including a gross lactation failure. Tsc1 cKO glands have altered morphology, retained lipid droplets in secretory epithelia, and an overall increase in MTOR signaling. We show that TSC1 and BECN1 are interacting partners, and that the interaction is nutrient responsive. These results suggest that Becn1 and Tsc1 are necessary for proper mammary gland development and differentiation. Furthermore, we have demonstrated a novel murine protein-protein interaction and an important link between regulation of MTOR pathway and regulation of autophagy in a developmental context. KEYWORDS: Autophagy, Bax, Beclin1, mammary gland, Tuberous Sclerosis Complex 1 Amber Nicolle Hale Student’s Signature March 13, 2014 Date ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION By Amber Nicolle Hale Dr. Edmund Rucker Co-Director of Dissertation Dr. Vincent Cassone Co-Director of Dissertation Dr. David Westneat Director of Graduate Studies March 13, 2014 Date This work is dedicated to my father, Joseph Hale, who will always be with me. Acknowledgements I first want to thank my mentor Dr. Edmund Rucker. He is the one who encouraged me to pursue research and has been the driving force behind the projects that are presented here. He has been an unwavering supporter and has helped me become a well-rounded scientist. Many thanks go to my lab mates Dan Ledbetter and Tom Gawriluk, who are collaborators and friends. My committee, Dr. Vincent Cassone, Dr. Philip Bonner, and Dr. Vivek Rangnekar has been an asset. I would like to recognize Dr. Kay-Uwe Wagner who has been a collaborator and scientific influence. The Forum in Reproductive Sciences and Women’s Health has been an avenue for me to interact with other reproductive biologists and gain exposure to a broader perspective on women’s health issues. I want to extend my thanks to Dr. Tom Curry, Dr. Michael Kilgore, and Dr. Misung Jo who have been supporters. Dr. Morris Grubbs was instrumental in my effort to earn the Graduate Certificate in College Teaching and Learning. My thanks go to the faculty, staff, and fellow graduate students of the Department of Biology. Specifically, Dr. Brian Rymond, Dr. Kellum, Beverly Taulbee, and Jacqueline Burke have been particularly helpful. I have had the opportunity to mentor many undergraduate students. They have been wonderful to teach, have aided in projects, and had projects of their own. I am proud of all they have and will accomplish. I want to acknowledge and thank my immediate and extended family who are incredibly supportive. My sincerest thanks go to David Keesling, who is my partner, rock, and source of sanity. You make every day a happy one. iii Table of Contents Acknowledgements ............................................................................................................. iii List of Tables ....................................................................................................................... ix List of Figures ....................................................................................................................... x Chapter One: Autophagy: Regulation and Role in Development ....................................... 1 Overview ......................................................................................................................... 1 Core machinery involved in autophagosome formation ................................................ 2 Sources of Autophagosomal Membranes ....................................................................... 4 Cellular Stress Responses ................................................................................................ 6 Types of Autophagy ....................................................................................................... 14 Aggrephagy ................................................................................................................ 14 Allophagy, crinophagy, and zymophagy .................................................................... 14 Exophagy .................................................................................................................... 15 Heterophagy and endosomal microautophagy ......................................................... 16 Immunophagy ............................................................................................................ 17 Lipophagy
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  • Beclin-1 Expression Is Retained in High-Grade Serous Ovarian Cancer Yet Is Not Essential for Autophagy Induction in Vitro Rohann J

    Beclin-1 Expression Is Retained in High-Grade Serous Ovarian Cancer Yet Is Not Essential for Autophagy Induction in Vitro Rohann J

    Western University Scholarship@Western Biochemistry Publications Biochemistry Department 8-2015 Beclin-1 Expression is Retained in High-Grade Serous Ovarian Cancer yet is Not Essential for Autophagy Induction In Vitro Rohann J. M. Correa Western University Yudith Ramos Valdes London Regional Cancer Program Trevor G. Shepherd Western University Gabriel E. DiMattia Western University, [email protected] Follow this and additional works at: https://ir.lib.uwo.ca/biochempub Part of the Biochemistry Commons Citation of this paper: Correa, Rohann J. M.; Valdes, Yudith Ramos; Shepherd, Trevor G.; and DiMattia, Gabriel E., "Beclin-1 Expression is Retained in High- Grade Serous Ovarian Cancer yet is Not Essential for Autophagy Induction In Vitro" (2015). Biochemistry Publications. 166. https://ir.lib.uwo.ca/biochempub/166 Correa et al. Journal of Ovarian Research (2015) 8:52 DOI 10.1186/s13048-015-0182-y RESEARCH Open Access Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro Rohann J. M. Correa1,2, Yudith Ramos Valdes1, Trevor G. Shepherd1,3,4,5† and Gabriel E. DiMattia1,2,3,4,6*† Abstract Background: Autophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation.