Prl-3 Is an Oncogenic Driver in Triple-Negative Breast Cancers And
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PRL-3 IS AN ONCOGENIC DRIVER IN TRIPLE-NEGATIVE BREAST CANCERS AND A MEDIATOR OF THE NOVEL ANTICANCER COMPOUND AMPI-109 by HAMID H. GARI B.S., Colorado State University, 2009 M.Phil., University of Cambridge, 2010 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cancer Biology Program 2016 This thesis for the Doctor of Philosophy degree by Hamid H. Gari has been approved for the Cancer Biology Program by James DeGregori, Chair M. Scott Lucia Heide Ford Jennifer Diamond Bolin Liu James Lambert, Advisor Date: 05/20/2016 ii Gari, Hamid H. (Ph.D., Cancer Biology) PRL-3 is an Oncogenic Driver in Triple-Negative Breast Cancers and a Mediator of the Novel Anticancer Compound AMPI-109 Thesis directed by Associate Research Professor James Lambert ABSTRACT Triple-negative breast cancers (TNBCs) are among the most aggressive and heterogeneous cancers characterized by a high propensity to invade, metastasize and relapse. AMPI-109 is a novel anticancer compound that is selectively efficacious in inhibiting proliferation and in inducing apoptosis of multiple TNBC subtype cell lines as assessed by activation of pro-apoptotic caspases-3 and 7, PARP cleavage and nucleosomal DNA fragmentation. Because AMPI-109 has little to no effect on growth in the majority of non- TNBC cell lines, we utilized AMPI-109 in a genome-wide shRNA screen to investigate the utility of AMPI-109 as a tool in helping to identify molecular alterations unique to TNBC. Our screen identified the phosphatase, PRL-3, as a putative intracellular target of AMPI-109 and an important driver of TNBC growth, migration and invasion. Through stable lentiviral knock downs and transfection with catalytically impaired PRL-3 in TNBC cells, we show that loss of PRL-3 expression, or functionality, leads to substantial growth inhibition. Mechanistically, we discovered that PRL-3 expression in TNBCs is transcriptionally regulated by the oncogenic NF-ĸB pathway and that PRL-3 ablation elicits a TNF-R1 feedback loop that results in TNBC cell cycle arrest and senescence followed by caspase-8 mediated apoptosis. Additionally, AMPI-109 treatment, downregulation of PRL-3 expression or impairment of PRL-3 activity reduced TNBC cell migration and invasion by inactivating Src iii and ERK signaling and downregulating downstream RhoA and Rac1/2/3 GTPase protein levels. This coincided with altered filamentous actin structures necessary for cell migration and invasion. Conversely, overexpression of PRL-3 promoted TNBC cell migration and invasion and promoted upregulation of a key matrix metalloproteinase, MMP-10, which resulted in increased adherence to, and degradation of, the major basement membrane substrate, laminin. Histological evaluation of human breast cancers revealed PRL- 3 expression was significantly associated with the TNBC subtype and correlated positively with the presence of regional and distant metastases, as well as poor relapse free survival. This study is proof-of-concept that AMPI-109, a viable pre-clinical investigational therapeutic candidate for TNBCs, can be used as a molecular tool to uncover unique drivers of disease progression, such as PRL-3, which we show promotes oncogenic phenotypes in TNBC cells. The form and content of this abstract are approved. I recommend its publication. Approved: James Lambert iv I dedicate this work to my family, my wife Janie and daughter Layan, who have supported me fully throughout the journey. Without them, my Ph.D. would not have been possible. In particular, I dedicate this work to my father who continues to lovingly challenge and encourage me to humbly become more than I thought I was capable of. v ACKNOWLEDGEMENTS I would like to thank Dr. Mary Reyland of the Cancer Biology Program and Dr. Ann Thor and the research faculty of the Department of Pathology under the direction of Dr. Steve Anderson for welcoming me into the cancer research community and allowing me to pursue my doctorate degree. I owe a deep gratitude to my friend and mentor, Dr. James Lambert, for advising me every step of the way and giving me unconditional freedom to explore this thesis project. I’m also immensely grateful to have been welcomed as a colleague and as a friend by Drs. Scott Lucia and Steve Nordeen for further inspiring me to challenge the status quo in every facet of this research project and the thoughtful discussions on developing new scientific concepts. A special thank you is deserved for the members of my thesis committee, and my Chair Dr. James DeGregori, who graciously donated their time and intellect despite their immense pre-existing commitments, to help me mature this important body of research. This work was partially funded by a research grant from the Colorado Cancer League and gift funds from David Paradice of Paradice Investment Management, to whom we’re indebted. Finally, I would like to thank the late King Abdullah bin Abdul-Aziz, the Ministry of Higher Education and the Saudi Arabian Cultural Mission in the USA for their generosity in funding my graduate studies and encouraging the Fellows to push for advancements in science as Ambassadors for the Kingdom of Saudi Arabia. vi TABLE OF CONTENTS CHAPTER I. INTRODUCTION .........................................................................................................1 Molecular heterogeneity of breast cancer ......................................................................1 Therapeutic approaches to combating TNBC ................................................................2 AMPI-109: Development history and mechanism of action in TNBC ..........................4 AMPI-109 impairs TNBC cell proliferation and induces apoptosis .................5 Ability of AMPI-109 to block TNBC growth is independent of the VDR ........8 Functional genomic screen identifies PRL-3 as a modifier of AMPI-109 action ..................................................................................................................9 PTPs as potential molecular targets in cancer ..............................................................13 The PTP4A family of phosphatases .............................................................................13 The role of PRL-3 in cancer .........................................................................................14 Expression ........................................................................................................14 Substrates and control of metastatic signaling pathways .................................15 Transcriptional regulation ................................................................................16 NF-ĸB in cancer development and metastasis ............................................................ 16 Hypothesis and aims ....................................................................................................18 II. AMPI-109 TREATMENT AND PRL-3 ABLATION INHIBIT TNBC GROWTH, MIGRATION AND INVASION .................................................................................19 Introduction ..................................................................................................................19 Materials and methods .................................................................................................20 Materials ..........................................................................................................20 Cell culture, immunoblot analysis and transfection .........................................21 vii In silico docking analysis .................................................................................21 Cellular proliferation, migration and invasion assays......................................2 1 Lentivirus .........................................................................................................22 In vitro enzymatic assays .................................................................................22 Site-directed mutagenesis ................................................................................23 Apoptosis assays ..............................................................................................23 Production of wild type and C104A FLAG-tagged recombinant PRL-3 ........23 Immunohistochemistry ....................................................................................24 Microarray and statistical analysis ...................................................................24 Results ..........................................................................................................................25 AMPI-109 binds PRL-3 active site in silico and impairs its catalytic activity in vitro ..................................................................................................................25 PRL-3 knock down and expression of catalytically impaired PRL-3 inhibits TNBC cell growth and confers partial resistance to AMPI-109 ......................29 PRL-3 modulation and AMPI-109 treatment inhibit TNBC cell migration and invasion ............................................................................................................32 PRL-3 expression positively associates with the TNBC subtype, regional and distant metastases .............................................................................................34 Discussion ....................................................................................................................37 III. PRL-3 PARTICIPATES IN