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ELUCIDATING the ROLE of SEMAPHORIN 7A in BREAST CANCER by Ramon Antonio Garcia-Areas a Dissertation Submitted to the Faculty Of

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ELUCIDATING the ROLE of SEMAPHORIN 7A in BREAST CANCER by Ramon Antonio Garcia-Areas a Dissertation Submitted to the Faculty Of ELUCIDATING THE ROLE OF SEMAPHORIN 7A IN BREAST CANCER by Ramon Antonio Garcia-Areas A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, FL December, 2016 Copyright 2016 by Ramon Antonio Garcia-Areas ii ELUCIDATING THE ROLE OF SEMAPHORIN 7 A IN BREAST CANCEl.~ by Ramon Antonio Garcia-Areas 1 This dissertation was prepared under the direction of the candidate's dissertation advisor, Dr. Vijaya lragavarapu-Charyulu, Department of Biomedical Science\ and has been approved by the members of his supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accept~d in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 1 I SUPERVISORY COMMITTEE: ~7 -tt~L Vijaya lrag~ha~h. D . ~a~~visor Michael Lu, Ph 1 Ta Ja Godenschwege, Ph.D ~ .. ~ ...... h . }iana LOpez. Ph.D. ~Q Dec.eMber lo, 2ol6 Date iii ACKNOWLE DGEMENTS I would like to acknowledge my Dissertation advisor, Dr. Vijaya lragavarapu-Charyulu. for her constant support, dedication and generosity. would like to thank Dr. Stephania Libreros for always pushing us to bigger and better things, but most of all, for her friendship. The completion of my dissertation would not have been achieved without the diligence and dedication of: Samantha Amat, Camila Castro-Silva, Nathalia Gazaniga and Michael Simoes. To my collaborators, Dr. Patricia Keating, Dr. Kathy Schilling and Lillian Onwuha­ Ekpete, I extend my deepest gratitude. I would like to thank Dr. James Hartmann for introducing me to the greatest joy, the joy of discovery. I also thank Dr. Michael Lu for selflessly sharing with me his deep knowledge of science through his example of integrity and discipline. I thank Dr. Tanja Godenschwege for teaching me the intricate art of scientific communication. I thank Dr. Mahyar Nouri-Shirazi for his insightful feedback and perspectives. I thank my partner Andy Litvack for his unwavering and loving support. I thank the Biological Sciences Department for supporting my degree, specially to Dr. Evelyn Frazier, the late Dr. John Nambu and especially Geri Mayer for her friendship and guidance. Finally, I thank all the great scientists, teachers, students, friends and family members who may be unmentioned in this dedication, but who's contributions in my life have enriched the privilege ofthis undertaking. iv ABSTRACT Author: Ramon Antonio Garcia-Areas Title: Elucidating the role of Semaphorin 7 A in breast cancer Institution: Florida Atlantic University Thesis Advisor: Dr. Vijaya lragavarapu-Charyulu Degree: Doctor in Philosophy Year: 2016 Solid tumors can hijack many of the same programs used in neurogenesis to enhance tumor growth and metastasis, thereby generating a plethora of neurogenesis-related molecules including semaphorins. Among them, we have identified Semaphorin7A (SEMA7A) in breast cancer. We first used to the DA-3 mammary tumor model to determine the effect of tumor-derived SEMA7A on immune cells. We found that tumor-derived SEMA7A can modulate the production of proangiogenic chemokines CXCL2/MIP-2 and CXCL1 , and pro­ metastatic MMP-9 in macrophages. We next aimed to determine the expression and function ofSEMA7A in mammary tumor cells. We found that SEMA7Ais highly expressed in both metastatic human and murine breast cancer cells. We show that both TGF-f) and hypoxia elicits the production of SEMA7 A in mammary cells. SEMA7A shRNA silencing in 4T1 cells resulted in decreased mesenchymal markers MMP-3, MMP-13, Vimentin and TGF-1). SEMA7Asilenced cells show v increased stiffness with reduced migratory and proliferative potential. In vivo, SEMA?A silenced 4T1 tumor bearing mice showed decreased tumor growth and metastasis. Genetic ablation of host-derived SEMA 7 A synergized to further decrease the growth and metastasis of 4T1 cells. Our findings suggest novel functional roles for SEMA?A in breast cancer and that SEMA?A could be a novel therapeutic target to limit tumor growth and metastasis iv DEDICATION I dedicate my dissertation to my parents, Gloria and Benigno Garcia, who sacrificed much to afford me a world of possibilities with their unconditional love and support. To my mother, who showed the value of hard work and the importance of kindness. To my father, who taught me a love for knowledge and the value of integrity. To my siblings, Maria and Gabriel, who's presence in my life has always fueled my desired to excel. I also dedicate this dissertation to my godparents Francis and Brian Waite who without any obligation gave me the opportunity to fulfill my dream of a higher education in the United States of America. Their boundless generosity towards me is only surpassed by their love and support. To God, for all the good that surfaces in my life through his eternal mercy. ELUCIDATING THE ROLE OF SEMAPHORIN 7A IN BREAST CANCER LIST OF FIGURES ......................................................................................................... xi GENERAL INTRODUCTION ......................................................................................... 1 Semaphorin7A .......... .................................................................................................... 1 Semaphorin7A and its receptors ................................................................................ 2 Role ofsemaphorin7A in innate immunity ................................................................ 4 Role ofsemaphorin7A in adaptive immunity ...... ...................................................... 6 Semaphorin?A's role in inflammatory diseases ....................................................... 9 Semaphorin7A and Cancer ....................................................................................... 10 CHAPTER 1. SEMAPHORIN7A PROMOTES TUMOR GROWTH AND EXERTS A PRO-ANGIOGENIC EFFECT IN MACROPHAGES OF MAMMARY TUMOR-BEARING MICE ............. ....................... .............................. 13 1.1 lntroduction ....................................................................................................... 13 1 .2 Materials and methods ........................................................... ........................ 15 1.2.1 Mice and cellli nes .................................................................................... 15 1.2.2 Cell cultures ............................................................................................... 16 1.2.3 lmmunoflluorescence ................................................................................ 17 1.2.4 RNA isolation and real-time reverse transcriptase-polymerase chain reaction ......... .... ....................... ....................................................... .......... 17 1.2.5 Flow cytometry studies ............................................................................ 18 1.2.6 Silencing of SEMA 7 A in macrophages .................................................. 18 vii 1.2.7 Silencing of SEMA7 A in DA-3 murine mammary tumor cells ............ 19 1.2.8 Monocyte migration assay ....................................................................... 19 1.2.9 Protein determination ............................................................................... 20 1.2.10 lmmunohistochemistry ............................................................................. 21 1.2.11 Tumor measurements and in vivo imaging for angiogenesis ............ 22 1.2.12 Statistical analysis .................................................................................... 22 1.3 Results ... .. .......................................................................................................... 22 1.3.1 SEMA7A is expressed in DA-3 mammary tumor cells and expression is increased in peritoneal elicited macrophages of DA-3 mammary tumor-bearing mice ......................................................................... 22 1.3.2 Expression of SEMA7 A receptor, ~1 integrin (CD29) is increased in DA-3 mammary tumor cells and macrophages from mammary tumor-bearing mice ......................................................................... 24 1.3.3 Treatment of macrophages with rmS EMA7A induces production of angiogenic CXCL2/MIP-2 ............................................................................. 25 1.3.4 Decreased tumor-derived SEMA7 A results in reduced in vitro macrophage migration and CXCL2/MIP-2 production ................................. 27 1.3.5 Decreased tumor growth in mice bearing SEMA7A silenced mammary tumors ............................................................................................... 29 1.3.6 Peritoneal elicited macrophages from mice bearing SEMA7A KD tumors produce decreased levels of angiogenic molecules ....................... 30 1.4 Discussion ..... .. .. .. .. .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. .. .. .. .. ....... .. .. .. .. .. .. .. .. .. .. .. .. ....... .. .. ... 31 viii CHAPTER 2. SEMAPHORIN 7A PLAYS AN IMPORTANT ROLE IN BREAST CANCER GROWTH AND METASTASIS ........................................... 42 2.1 lntroduction ...................................................... .. ..................... .. ..................... ... 42 2.2 Materials and Methods ................................ ............................... ................... .44 2.2.1 Mice and cell lines ... ........ .... .... .... .... .... ... .... .... .... .... .... .... ....... .... .... .... .... ... 44 2.2.2 RNA
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