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STRUCTURE AND FUNCTION OF HEDGEHOG ACYLTRANSFERASE IN NORMAL AND CANCER CELLS by Armine Matevossian A Dissertation Presented to the Faculty of the Louis V.Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY May, 2015 ___________________________ _________________________ Marilyn D. Resh, PhD Date Dissertation Mentor Copyright by Armine Matevossian 2015 © DEDICATION To my parents, Azniv and Achot, for showing me how to lead a meaningful and joyous life. To my siblings, Ara and Anouch, for always being by my side in this journey. iii ABSTRACT Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. The Shh signaling pathway has been implicated in the progression of breast cancer. To determine the functional significance of Hhat expression in breast cancer, we used a panel of estrogen receptor (ER) positive and negative cell lines. Here we show that Hhat is a novel target for inhibition of ER positive, HER2 amplified, and tamoxifen resistant breast cancer cell growth. Depletion of Hhat with lentiviral shRNA decreased both anchorage-dependent and anchorage-independent proliferation of ER positive, but not triple negative, breast cancer cells. Treatment with RU-SKI 43, a small molecule inhibitor of Hhat recently identified by our group, also reduced ER positive cell proliferation. Overexpression of Hhat in ER positive cells not only rescued the growth defect in the presence of RU-SKI 43 but also resulted in increased cell proliferation in the absence of drug. Furthermore, depletion or inhibition of Hhat also reduced proliferation of HER2 amplified as well as tamoxifen resistant cells. Moreover, Hhat regulated the proliferation of both Shh responsive and non-responsive ER positive cells, suggesting a Shh independent function for Hhat. Together, these data suggest that Hhat plays a critical role in ER positive, HER2 amplified, and hormone resistant breast cancer proliferation and highlights the potential promise of Hhat inhibitors for therapeutic benefit. To enhance our understanding of Hhat structure and function, we also conducted a comprehensive analysis of its transmembrane topology. Bioinformatics analysis of iv transmembrane domains within human Hhat using ten different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains ten transmembrane domains and two re-entrant loops. The invariant His and highly conserved Asp residues within the membrane bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C- terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O- acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors. v BIOGRAPHICAL SKETCH Armine Matevossian was born in Yerevan, Armenia and moved to Chelmsford, MA at the age of ten. She attended University of Massachusetts in Amherst where she received a Bachelor of Science degree with high honors in Biochemistry and Molecular Chemistry. She entered the Gerstner Sloan Kettering Graduate School of Biomedical Sciences in 2008 and later joined the laboratory of Dr. Marilyn Resh. During her graduate career, Armine examined the function of Hhat in breast cancer and elucidated the topological organization of Hhat. vi ACKNOWLEDGEMENTS I am extremely lucky to be surrounded by exceptionally talented and supportive individuals who have contributed to both my professional and personal development. First and foremost, I would like to thank Dr. Marilyn Resh for being an extraordinary mentor. Without your knowledge, guidance, and immense enthusiasm, these projects would not have been possible. I am extremely grateful for the opportunity to conduct research in your laboratory. I would also like to thank all current and past members of the Resh laboratory for their insightful discussions and input over the last few years. Special thanks to Raisa Louft-Nisenbaum for her extraordinary lab management and daily technical assistance; Debra Alston for administrative help and cheerful attitude; Rayshonda Hardy for patiently showing me how to work with radioiodinated palmitate and consistently having a sunny disposition; Ellie Petrova for initiating the studies with Hhat inhibitors and providing critical analysis of my work; Jessica Rios-Esteves, a labmate and a true friend, for the countless conversations and advice on all aspects of life. It was an absolute pleasure coming to lab every day. I would also like to thank my thesis committee: Dr. Stephen Long and Dr. Xuejun Xiang, for their continued support and scientific insight. In addition, I would like to thank Dr. Lisa Denzin, Dr. Derek Sant’Angelo, Dr. Jayanta Chaudhuri, and all the members of their laboratories for their assistance in my first two years of graduate school. Thanks to everyone who contributed to the completion of this work. Thanks to the Genomic Core Facility for sequencing a seemingly endless array of constructs, the vii Molecular Cytology Core Facility for providing support with confocal imaging microscopy, and all the members of the Jiang and Haynes laboratories for their scientific advice. I would like to thank all members of the Gerstner Sloan Kettering Graduate School of Biomedical Sciences for their tireless support of all graduate students. Thank you to Iwona Abramek, Maria Torres, and Ivan Gerena for your amazing support throughout these years. I would especially like to thank Dr. Ken Marians for his hands-on approach as dean of the graduate program. I truly appreciate your dedication, support, and willingness to listen to students. This journey would not be the same without all of you. I would like to thank my friends who have made these years truly remarkable. To Bethany Reis for your endless patience in listening to me talk about work. To the friends I made in graduate school – Neha Bhagwat, Jessica Rios-Esteves, Berenice Ortiz, and Moriah Nissan – for your constant cheer and support. I am grateful to have such smart and talented friends. Finally, I would like to thank my family for providing unwavering support and lots of laughter. To my love, David Pedersen, for all the cheerful motivation and encouragement. To my sister, Anouch Matevossian, for the priceless (and lengthy) phone conversations. To my brother, Ara Matevossian, for always protecting and challenging me. To my parents, Azniv and Achot Matevossian, for showing me the benefit of hard work, the value of education, and the significance of a loving family. I owe all my accomplishments to you. viii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF ABBREVIATIONS ........................................................................................... xv CHAPTER ONE: Introduction ........................................................................................... 1 The Hedgehog signaling pathway ................................................................................... 3 Non-canonical Hedgehog signaling and crosstalk with other pathways ....................... 12 Ptch independent functions of Hh ............................................................................. 12 Ptch dependent, Smo independent functions of Hh signaling ................................... 13 Gli independent functions of Hh signaling ................................................................ 15 Hedgehog ligand maturation, secretion, and transport .................................................. 17 Hedgehog acyltransferase ............................................................................................. 24 Protein palmitoylation ............................................................................................... 24 Membrane bound O-acyltransferases ........................................................................ 27 Hedgehog acyltransferase .......................................................................................... 29 Hedgehog signaling in cancer development and maintenance...................................... 32 Ligand independent activation of Hh signaling ......................................................... 33 Ligand dependent activation of Hh signaling ............................................................ 34 Hh signaling as a restraint on tumor growth .............................................................
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