Role of ABL Family Kinases in Breast Cancer by Jun Wang Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ Ann Marie Pendergast, Supervisor ___________________________ Donald McDonnell ___________________________ Christopher Counter ___________________________ Gerard Blobe ___________________________ Xiao-Fan Wang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2016 i v ABSTRACT Role of ABL Family Kinases in Breast Cancer by Jun Wang Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ Ann Marie Pendergast, Supervisor ___________________________ Donald McDonnell ___________________________ Christopher Counter ___________________________ Gerard Blobe ___________________________ Xiao-Fan Wang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2016 i v Copyright by Jun Wang 2016 Abstract The ABL family of non-receptor tyrosine kinases, ABL1 (also known as c-ABL) and ABL2 (also known as Arg), links diverse extracellular stimuli to signaling pathways that control cell growth, survival, adhesion, migration and invasion. ABL tyrosine kinases play an oncogenic role in human leukemias. However, the role of ABL kinases in solid tumors including breast cancer progression and metastasis is just emerging. To evaluate whether ABL family kinases are involved in breast cancer development and metastasis, we first analyzed genomic data from large-scale screen of breast cancer patients. We found that ABL kinases are up-regulated in invasive breast cancer patients and high expression of ABL kinases correlates with poor prognosis and early metastasis. Using xenograft mouse models combined with genetic and pharmacological approaches, we demonstrated that ABL kinases are required for regulating breast cancer progression and metastasis to the bone. Using next generation sequencing and bioinformatics analysis, we uncovered a critical role for ABL kinases in promoting multiple oncogenic pathways including TAZ and STAT5 signaling networks and the epithelial to mesenchymal transition (EMT). These findings revealed a role for ABL kinases in regulating breast cancer tumorigenesis and bone metastasis and provide a rationale for targeting breast tumors with ABL-specific inhibitors. iv Contents Abstract .......................................................................................................................................... iv List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................ x List of Abbreviations ................................................................................................................. xiii 1. Introduction ............................................................................................................................... 1 1.1 Breast cancer ...................................................................................................................... 1 1.1.1 Breast cancer overview ............................................................................................... 1 1.1.2 Breast cancer bone metastasis .................................................................................... 2 1.2 The ABL family of nonreceptor tyrosine kinases ......................................................... 4 1.2.1 ABL kinases in normal cell biology .......................................................................... 6 1.2.1.1 Structure, modular domains, and enzymatic regulation of the ABL kinases ............................................................................................................................................. 7 1.2.1.2 ABL kinases regulate cytoskeletal dynamics required for cellular protrusions, cell adhesion, polarity, and migration .................................................. 12 1.2.1.3 ABL-dependent regulation of membrane and organelle trafficking .......... 16 1.2.1.4 ABL-dependent regulation of cell proliferation and survival pathways in normal cells ..................................................................................................................... 18 1.2.1.5 Physiological roles of mammalian ABL kinases: insights from knockdown mice .................................................................................................................................. 21 1.2.2 ABL kinases in cancer biology ................................................................................. 22 1.2.2.1 ABL activation in leukemias and development of targeted therapies ....... 22 1.2.2.2 ABL kinases activation in solid tumors .......................................................... 26 v 1.2.2.3 ABL-dependent regulation of cancer cell proliferation ................................ 28 1.2.2.4 ABL-mediated metabolism and oxidative stress in cancer .......................... 29 1.2.2.5 Role of ABL kinases in cancer cell invasion and metastasis ........................ 30 1.2.2.6 Role for ABL kinases in cancer drug resistance ............................................. 33 2. Materials & Methods .............................................................................................................. 37 2.1 Cell Culture ..................................................................................................................... 37 2.2 Antibodies ........................................................................................................................ 38 2.3 Tumor Xenografts and Analysis ................................................................................... 38 2.4 Bone µCT Analysis ......................................................................................................... 40 2.5 In Vitro Osteoclastogenesis Assay ............................................................................... 40 2.6 RNAseq Analysis ............................................................................................................ 40 2.7 Viral Transduction .......................................................................................................... 41 2.8 Immunoblotting .............................................................................................................. 42 2.9 Real-Time RT-PCR .......................................................................................................... 42 2.10 Histological Analysis ................................................................................................... 43 2.11 TUNEL Staining ............................................................................................................ 43 2.12 ELISA and Cytokine Array ......................................................................................... 44 2.13 Invasion Assay .............................................................................................................. 44 2.14 In Vitro Cell Growth Assays ....................................................................................... 44 2.15 CHIP-QPCR Analysis .................................................................................................. 45 2.16 Mutagenesis ................................................................................................................... 45 2.17 Soft Agar Colony Formation ....................................................................................... 46 vi 2.18 Mammosphere Formation ........................................................................................... 46 2.19 Limiting Dilution Analysis .......................................................................................... 46 2.20 Flow Cytometry ............................................................................................................ 47 2.21 Statistical Analysis ........................................................................................................ 47 3. ABL kinases are required for breast cancer bone metastasis ............................................ 48 3.1 Introduction ..................................................................................................................... 48 3.2 Results .............................................................................................................................. 49 3.2.1 Increased expression of ABL kinase-encoding genes correlates with breast cancer metastasis ................................................................................................................ 49 3.2.2 ABL family protein kinases are required for bone metastasis ............................ 50 3.2.3 Allosteric inhibition of the ABL kinases impairs breast cancer bone metastasis ............................................................................................................................................... 57 3.2.4 ABL kinases are required for tumor cell survival and tumor-induced osteolysis in the bone microenvironment ........................................................................................