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Rna Helicase Ddx5 Regulates Platelet Derived Growth Factor Receptorand Androgen Receptor (Ar) Expression in Breast Cancer

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Rna Helicase Ddx5 Regulates Platelet Derived Growth Factor Receptorand Androgen Receptor (Ar) Expression in Breast Cancer Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology 5-4-2020 RNA HELICASE DDX5 REGULATES PLATELET DERIVED GROWTH FACTOR RECEPTORAND ANDROGEN RECEPTOR (AR) EXPRESSION IN BREAST CANCER Neha Panchbhai Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Recommended Citation Panchbhai, Neha, "RNA HELICASE DDX5 REGULATES PLATELET DERIVED GROWTH FACTOR RECEPTORAND ANDROGEN RECEPTOR (AR) EXPRESSION IN BREAST CANCER." Dissertation, Georgia State University, 2020. https://scholarworks.gsu.edu/biology_diss/238 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. RNA HELICASE DDX5 REGULATES PLATELET DERIVED GROWTH FACTOR RECEPTOR β AND ANDROGEN RECEPTOR (AR) EXPRESSION IN BREAST CANCER by NEHA ARUN PANCHBHAI Under the Direction of Zhi-Ren Liu, PhD ABSTRACT P68 RNA helicase, a prototypical member of the DEAD box family of RNA helicases is important in many biological processes, including early organ development. However, its aberrant expression contributes to tumor development and progression. In this study, we show that p68 upregulates the transcriptional expression of the growth factor receptor, PDGFRβ P68 knockdown in MDAMB231 and BT549 breast cancer cells significantly decreases mRNA and protein levels of PDGFRβ and EMT markers, resulting in decreased migration. We have previously shown that PDGF-BB induces p68 phosphorylation, resulting in EMT via nuclear translocation of β -catenin. Here, we show that p68 promotes migration in response to PDGF-BB stimulation via upregulation of PDGFRβ in breast cancer cells, suggesting that PDGFRβ is in turn regulated by p68 to maintain a positive feedback loop. Further, our study reveals the association of p68 in androgen receptor (AR) response via PDGFRβ. We demonstrate that p68 and PDGFRβ co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells, highlighting the critical role of p68-PDGFRβ axis in AR regulation INDEX WORDS: RNA helicase, DDx5, Platelet derived growth factor receptor, Breast cancer RNA HELICASE DDX5 REGULATES PLATELET DERIVED GROWTH FACTOR RECEPTOR β AND ANDROGEN RECEPTOR (AR) EXPRESSION IN BREAST CANCER by NEHA ARUN PANCHBHAI A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2020 Copyright by Neha Arun Panchbhai 2020 RNA HELICASE DDX5 REGULATES PLATELET DERIVED GROWTH FACTOR RECEPTOR β AND ANDROGEN RECEPTOR (AR) EXPRESSION IN BREAST CANCER by NEHA ARUN PANCHBHAI Committee Chair: Zhi-Ren Liu Committee: D. D. Merlin Jenny Yang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2020 iv DEDICATION I dedicate this dissertation to my family, friends and teacher, without their great, selfless, emotional spiritual and financial support in every aspect, my pursuit of knowledge for the past five years would have been just a dream. v ACKNOWLEDGEMENTS In the difficult journey called life, it is impossible to succeed without the encouragement and support from various people who come along your way. Without acknowledging them this work will be incomplete. So I would like to express my honest gratitude to them. I would like to thank, Dr. Zhi-Ren Liu, for giving me an opportunity to get me onboard in his lab. His time to time guidance and support paved my way to achieve this point. Despite of numerous failed experiments and hypothesis, his trust in me, gave me the push to hold on. The members of my committee, Dr. Merlin and Dr. Yang, have always put me on the right path through their valuable suggestions. I would like to thank the staff of the biology department, LaTesha Warren, Tameka Hudson, Larialmy Allen, Sonja Young, Debbie Walthall, Mary Karom, who were quick to resolve any issues that I have faced as a graduate student in these years. I would also like to thank Dr. Francisco Cruz and Julie Stowell, who have given me an opportunity to learn the art of teaching by graciously offering me a TA position for many semesters. This project would have been far from real without the support of my fellow graduate students from various laboratories who have helped me in a plethora of issues from laboratory-based experiments to support in my personal life. I extend my thanks to Dr. Liangwei Li, Dr. Yinwei Zhang, Falguni Mishra, Guangda Peng, Hongwei Han and Yang Huang, Chakravarthi Garlapathi, Adani who have helped me in various ways without which I wouldn’t have completed my studies. Special thanks go to Ganesh Satyanarayana, Dr. Ravi Chakra Turaga, and Malvika Sharma, colleagues and my friends. These are the people who stood by me through thick and thins in this 5-year journey. Their intellectual inputs helped me shape this project. Looking at their ability to work hard encouraged me push my limits. Doctoral degree is not just an academic degree, it is a vi package of life and professional lessons. So there are times when you almost feel that you had enough. In these moments Ganesh, Ravi and Malvika were my life support. Thanking them here is beyond my word limits. I would like to extend my thanks to Divya Ravi, Rakesh Balan, Nitika Sharma, Aashlesha Rajankar, and Kanchan Shivankar - my friends / family for holding me together in all possible situations. Their advice in my personal life and their push kept me going in my low points. I would also like to thank Parag Bhure for his support at initial stages of my journey, his lessons helped me grow as a person, step into the new life. Lastly, my family, my father - Arun Panchbhai, mother - Vijaya Panchbhai, brother and sister in law- Swapnil and Roshni Panchbhai who were, are and always will be there, standing strong with immense trust in me, whenever I have to look back for support. I cannot thank them enough for the lessons in my early life which made me what I am today. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................ V LIST OF FIGURES ..................................................................................................................... X LIST OF ABBREVIATIONS .................................................................................................... XI 1. INTRODUCTION ............................................................................................................ 1 1.1. Cancer ........................................................................................................................... 1 1.1.1. Normal cells vs cancer cells .................................................................................... 1 1.2. Breast cancer ................................................................................................................ 3 1.3. Cell migration ............................................................................................................... 5 1.3.1. Metastasis ............................................................... Error! Bookmark not defined. 1.4. Helicases ........................................................................................................................ 7 1.4.1. Super family 1 (SF1) ............................................................................................... 8 1.4.2. Super Family 2 (SF2) .............................................................................................. 9 1.5. RNA helicase p68 ....................................................................................................... 13 1.6. Platelet derived growth factors receptor (PDGFR) ................................................ 16 1.6.1. Platelet derived growth factor receptor beta (PDGFR β) ..................................... 22 1.7. Androgen receptor (AR) ............................................................................................ 24 2. MATERIALS AND METHODS ................................................................................... 28 2.1. Cell Culture ................................................................................................................ 28 2.2. Transfection ................................................................................................................ 28 viii 2.3. DHT – MTT ................................................................................................................ 28 2.4. Immunoblotting .......................................................................................................... 29 2.5. Scratch Assay .............................................................................................................. 29 2.6. Boyden chamber assay ............................................................................................... 29 2.7. Immunohistochemistry .............................................................................................. 30 2.8. Immunofluorescence microscopy ............................................................................. 30 2.9. Real time PCR ............................................................................................................ 31 2.10. Statistical analysis .....................................................................................................
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