©2017 Allison L. Isola ALL RIGHTS RESERVED ROLE OF GRM1 IN EXOSOME PRODUCTION AND MELANOMA METASTASIS By ALLISON L. ISOLA A Dissertation submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Toxicology Written under the direction of Dr. Suzie Chen And approved by ______________________________________ ______________________________________ ______________________________________ ______________________________________ ______________________________________ New Brunswick, New Jersey October 2017 ABSTRACT OF THE DISSERTATION Role of GRM1 in Exosome Production and Melanoma Metastasis By Allison L. Isola Dissertation Director: Suzie Chen Exosomes are naturally occurring membrane-bound nanovesicles generated constitutively and released by various cell types, and often in higher quantities by tumor cells. Exosomes have been postulated to facilitate communication between the primary tumor and its local microenvironment, supporting cell invasion and other early events in metastasis. A neuronal receptor, metabotropic glutamate receptor 1 (GRM1), when ectopically expressed in melanocytes, induces in vitro melanocytic transformation and spontaneous malignant melanoma development in vivo in a transgenic mouse model. Earlier studies showed that genetic modulation in GRM1 expression by siRNA or disruption of GRM1-mediated glutamate signaling by pharmacological inhibitors interfering with downstream effectors resulting in a decrease in both cell proliferation in vitro and tumor progression in vivo, suggesting that active GRM1 may participate in melanomagenesis in our system. The overall goal of this dissertation is to determine whether the presence and activation of GRM1 plays a role in exosome formation, and subsequent tumor development and progression. To test this, the first aim utilized in vitro cultured cells in which GRM1 expression ii and function were modulated by pharmacological and genetic means and consequences on exosome production by such manipulations were evaluated in vitro. We also assessed if exosomes derived from GRM1 expressing melanoma cells promote cell growth, migration, invasion as well as colony formation under anchorage-independent growth condition of GRM1 negative cells. Results showed that GRM1 expression in cells, per se, did not modulate exosome quantity, however, modified the qualities and functions of these exosomes. In Aim 2 we used riluzole, a glutamate signaling blockade, in a melanoma prone mouse model (TGS) for the in vivo assessment of exosomal quantity and quality. Daily treatment of TGS mice with riluzole had no detectable effect on the quantity of exosomes in circulation, however riluzole treatment influenced the effects of the circulating exosomes on metastatic behavior of recipient cells. iii Acknowledgements I would like to express my tremendous gratitude to my mentor, Dr. Suzie Chen, who has guided me through my dissertation, and has helped to mold me into a creative scientist and an independent thinker. Her support, guidance and intelligence have been essential to my success as a scientist. Thank you for believing in me. I would like to thank Dr. Lori White for her mentorship and support throughout my entire scientific career as an undergraduate, technician and graduate student. She has been an essential part of my success. I would also like to thank my committee members, and Philip Furmanski, James Goydos and Helmut Zarbl who have given me great advice and insight, contributing to my understanding of cancer biology, and helping my advancement through my studies. Additionally, I want to express my gratitude to Kevinn Eddy, who has been a tremendous help to this project. I hope that I have contributed to your growth as a scientist as much as you have mine. iv Dedication This thesis is dedicated to the memory of my grandmother, Catherine Wedel; her strength and passion I could only dream to have. And to my parents, whose love and support has made this dream possible. v Table of Contents ABSTRACT OF THE DISSERTATION .......................................................................... ii Acknowledgements ............................................................................................................... iv Dedication .................................................................................................................................. v Table of Contents ................................................................................................................... vi List of Tables ........................................................................................................................... ix List of Figures ............................................................................................................................ x Introduction .............................................................................................................................. 1 Melanoma ............................................................................................................................................ 1 Melanocytes ........................................................................................................................................ 2 Melanoma types ................................................................................................................................. 2 Genetic mutations associated with melanoma development ............................................ 3 G-Protein Coupled Receptors ........................................................................................................ 6 Metabotropic Glutamate Receptor 1 (GRM1) .......................................................................... 9 Melanoma Treatments ................................................................................................................. 12 Chemotherapies ........................................................................................................................................... 12 Combinatorial Chemotherapies .......................................................................................................... 13 Immunotherapies ........................................................................................................................................ 14 Riluzole ............................................................................................................................................................. 16 Riluzole Pharmacokinetics ................................................................................................................................. 17 Riluzole and Melanoma ....................................................................................................................................... 18 Metastasis ......................................................................................................................................... 18 Formation of the pre-metastatic niche ............................................................................................. 20 Recruitment of Immune Cells ............................................................................................................... 22 Exosomes .......................................................................................................................................... 23 Composition of Exosomes ...................................................................................................................... 24 Formation of Exosomes ........................................................................................................................... 25 Exosome release ........................................................................................................................................... 27 Exosome uptake ........................................................................................................................................... 29 Exosomes and Cancer ................................................................................................................... 30 Unique Composition of Exosomes ..................................................................................................... 33 Promotion of aggressive behavior in cancer cells by exosomes .......................................... 35 Exosomes manipulate primary tumor microenvironment .................................................... 37 Cancer derived exosomes manipulate the pre-metastatic niche ........................................ 38 Cancer derived exosomes induce resistance to treatment ..................................................... 42 Use of exosomes as biomarkers and treatment of cancer ....................................................... 43 GRM1 and Exosomes ..................................................................................................................... 44 vi Section I: Determine the relationship between GRM1 and its subsequent signal transduction cascade and the production of exosomes. .............................. 48 Aim 1: Rationale .................................................................................................................... 48 Materials and Methods ....................................................................................................... 49 Cell lines ...........................................................................................................................................