The findings presented in this dissertation identify cytosolic phospholipase A2 (cPLA2) as a key component in tumor angiogenesis and the response of vascular endothelial cells to therapeutic doses of ionizing radiation (2-5 Gy). Through extensive experimental analysis, the described results demonstrate that cPLA2 activation leads to increased production of the lipid second messenger, lysophosphatidylcholine (LPC), and the subsequent downstream phosphorylation of the pro-survival kinases Akt and ERK1/2. Furthermore, LPC is frequently converted to lysophosphatidic acid (LPA) by an enzyme known as autotaxin. Once generated, LPA can then initiate its own pro-survival signaling cascades through various G protein-coupled receptors. The result of these collective events is increased tumor vascularization and radioresistance of tumor blood vessels which can significantly enhance tumor progression. In summary, this dissertation project implicates key regulatory roles for cPLA2 and lipid second messengers in 1) the vascular endothelium response to ionizing radiation and 2) tumor blood vessel formation. These data suggest that the inhibition of cPLA2, LPC, and LPA may significantly improve the treatment response of previously resistant tumors including lung cancer and glioblastoma.

Chemokines are a family of small chemotactic cytokines that bind seven transmembrane G protein-coupled receptors. Roles for CXC chemokines and the receptors CXCR2 and CXCR4 in inflammation-mediated tumorigenesis, angiogenesis, and metastasis have established these proteins as anti-cancer therapeutic targets. Therefore, understanding the biology of chemokines and their receptors is crucial for the development of novel therapeutics. We hypothesized that the cellular responses elicited by chemokines are in part regulated through the internalization and intracellular trafficking of chemokine receptors through small GTPases and by novel protein complexes that interact with the cytoplasmic domains of the receptors. We used three approaches to investigate this hypothesis: 1) We investigated the role of the small GTPase RhoB in the intracellular trafficking of CXCR2. Experimental results indicate that the oscillation of the RhoB GTPase activity is essential for appropriate sorting decisions and for directing CXCR2 degradation and recycling, events that are required for optimal chemotaxis. 2) A proteomics approach for the identification of novel ligand-dependent CXC chemokine receptor-interacting proteins was utilized. This approach led to the identification of a number of CXCR2-interacting proteins, including the scaffolding protein IQGAP1 and the actin cytoskeletal modifying protein VASP which may link CXCR2 to major signaling pathways and the actin cytoskeleton. Our studies also describe a direct, phosphorylation-dependent interaction between CXCR2 and VASP and suggest that VASP plays a critical role in CXCR2-mediated leukocyte recruitment in vivo as VASP -/- mice exhibit impaired responses. In addition, a direct interaction of CXCR2 with the amino-terminus of IQGAP1 was identified. CXCL8 stimulation was further shown to regulate IQGAP1 association with the small GTPase Cdc42, which plays a major role in the establishment of cell polarity. 3) The role of the carboxyl-terminus of CXCR4 in breast cancer tumorigenesis was also investigated. We found that the truncated CXCR4 receptor constitutively activates signaling pathways due to enhanced ligand-independent recycling. As a result, this truncated receptor leads to enhanced motility, proliferation, and metastasis of breast cancer cells. Taken together, our data demonstrate the critical importance of the proper intracellular trafficking of CXC chemokine receptors and of its interacting proteins in mediating biological responses elicited by chemokines.

The Eph family of receptor tyrosine kinases has important roles in multiple aspects of cancer development and progression. Substantial advances have been made in understanding the Eph receptor signalling in the pathways that govern fundamental cellular processes in cancer, such as proliferation, survival, migration and invasion, as well as those that regulate intercellular communication during tumorigenesis. Common themes and controversies of Eph-ephrin in cancer biology have been gleaned from intensive research efforts in the field, and continue to be uncovered. As part of them, our current results, presented herein, primarily explore the role of EphA receptor signaling in breast and lung cancer progression, which has not been elucidated systematically and comprehensively. We have now shown the pro-tumorigenic effects of EphA2 receptor in breast cancer by crosstalking with HER2 signaling, and anti-tumorigenic effects of EphA3 receptor in lung cancer by inhibiting the mTOR pathway. Our increased understanding of the genetic, molecular, and biological characteristics of the Eph-ephrin system is essential for the rational development of novel anti-cancer therapies.

TAL1/SCL encodes a basic helix-loop-helix transcription factor and was first identified through its involvement in a chromosomal translocation in T-cell acute lymphoblastic leukemia. The mouse Tal1 cDNA was cloned from a bone marrow (BM) macrophage cDNA library and although Tal1 has critical roles in other hematopoietic lineages, its function in monocyte-macrophage (MM) differentiation has not been studied. <p>Using an ex vivo system of MM differentiation we detected Tal1 expression at all stages of differentiation including activated macrophages. Over-expression of Tal1 in MM precursor cells increased differentiation and proliferation and activated immune response pathways. Gene knockout studies identified severe cell cycle and proliferative defects in Tal1-null cells but had minimal effects on cell survival and only modestly increased terminal differentiation. In conjunction with the cell cycle defect in Tal1-null cells, steady state levels of p16(Ink4a) mRNA were increased and Gata2 mRNA decreased. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA element in a Gata2 enhancer and with three E boxes upstream of p16(Ink4a) at early stages of differentiation. But this association became low to non-existent in more mature cells. ChIP analysis in M1 myelo-monocytic leukemia cells detected Tal1 and E2A association in untreated cells and interleukin-6 (IL-6)-induced differentiation lowered Tal1 association only. In contrast, E2A occupancy in p16(Ink4a) sites was detected in both treated and untreated cells, consistent with relief of Tal1-directed repression of p16(Ink4a) in M1 cells upon differentiation. In summary, these studies have uncovered a critical role for Tal1 in cell cycle regulation during monocytopoiesis and suggest that TAL1 repression of p16(Ink4a) transcription and activation of Gata2 likely contributes. Additionally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null cells that underscores its requirement for DNA binding. Finally, the results in Tal1-over-expressing MM precursors may be relevant to the action of TAL1 in T-lymphoid and, in particular, myeloid leukemias.

The major goal of this thesis is to examine the role of beta1 integrin in kidney development. Our results indicate that beta1 integrin is necessary for ureteric bud branching morphogenesis and maintenance of collecting duct structural integrity. We found that beta1 integrins can regulate branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors. We also demonstrated that â1 integrin expression by podocytes is required for normal development and maintenance of glomerular filtration barrier. Overall, our studies discussed in this thesis proved that beta1 integrin is critical for kidney development. These finding have significantly improved the current knowledge of the role of beta1 integrins and facilitated further in depth investigations.

This research project is to identify FoxA1 interacting partners. In this study, a potential FoxA1 binding partner, Upstream Stimulatory Factor 2 (USF2), has been examined; its DNA binding activity was determined and the function of USF2 was monitored in USF2 knockdown LNCaP cells. FLAG-6His-FoxA1 stable expressing LNCaP cell lines were established. The FoxA1 protein complex from LNCaP tagged-FoxA1 cells was purified and analyzed by Liquid Chromatography Tandem Mass Spectrometry. USF2 has been confirmed to interact with FoxA1 using ImmunoPrecipitation and glutathione-S-transferase pull-down assays. Our study also indicates, as FoxA1, USF2 binds to prostatic specific gene promoters such as probasin promoter, Spermine Binding Protein promoter, and the Prostate Specific Antigen core enhancer. PSA mRNA levels were increased in USF2 knocked down LNCaP cells compared with control LNCaP cells when the LNCaP cells were treated with DHT (10-8 M). Using LC-MS/MS analysis, a total of 16 proteins that appear to interact with Foxa1 have been identified and eight of them have been previously reported as AR interacting proteins. Further work is needed to confirm the binding between FoxA1 and these identified proteins and to perform functional studies of the confirmed FoxA1 binding partners. This study has identified FoxA1 interacting partners which helps unravel the critical transcription factors that control cell type specific gene expression and to further understand the role of FoxA1 during prostate development. This work gives us an insight into the essential mechanism of androgen regulation of the prostate gland and of hormonal regulation of prostate cancer.

STRAP inhibits transforming growth factor-Beta (TGF-Beta) signaling and enhances tumorigenicity. The aim of our current research project was to identify novel TGF-Beta independent functions of STRAP. STRAP acts as a scaffold for the assembly of multi-protein complexes and our study has uncovered two novel but independent functions of STRAP. In the first part, we report, for the first time, that deletion of STRAP from Mouse Embryonic Fibroblasts (MEFs) results in a loss of mesenchymal morphology. These cells lose their spindle shape and exhibit cobloid epithelial morphology. Loss of STRAP leads to upregulation of WT1 that subsequently upregulates E-cadherin leading to the formation of adherens junctions, and sequesters Beta-catenin to the cell membrane and downregulation of the mesenchymal markers like LEF1. Finally, stable expression of STRAP in these cells results in a loss of WT1 and E-cadherin expressions, and a reversal from epithelial to the mesenchymal morphology. In the second part, we validated that STRAP binds with GSK3-Beta, an enzyme that plays multiple roles in a cell, including insulin and Wnt signaling. In a completely novel finding, we observed that STRAP, GSK3-Beta and Axin form a ternary complex. We also, for the first time show that intracellular fragment of Notch3 (ICN3) binds with GSK3-Beta, suggesting that Notch3 may be a novel substrate of GSK3-Beta. We show that STRAP binds ICN3 in a proteasomal inhibition-dependent manner. Further studies revealed that STRAP binds ICN3 through the same ankyrin repeat region. In-vivo ubiquitination studies indicate that STRAP is able to reduce ubiquitination of ICN3, raising a possibility that STRAP may stabilize ICN3 leading to a longer half life in the cells. STRAP and Notch3 are both known to be upregulated in lung cancers and we observed that STRAP and ICN3 are co-overexpressed in 59 % of lung cancers in a tissue microarray study. STRAP shows oncogenic activity and our results from the two independent studies provide additional insights into how STRAP may behave as an oncogene using diverse mechanisms.

CANCER BIOLOGY MOUSE MODELS OF PROSTATE CANCER PROGRESSION AND BONE METASTASIS Srinivas Rao Nandana Dissertation under the direction of Robert J. Matusik, PhD Prostate cancer is the second leading cause of deaths due to cancer in men in the United States. The American Cancer Society has projected that 192,280 new cases and 27,360 deaths will occur in the year 2009. Mostly, men over the age of 50 are afflicted by the disease and more than 70% of the men diagnosed with prostate cancer are over the age of 65. Most of the patients who suffer from prostate cancer do not die due to the tumor at the primary site but rather due to complications when the tumor has spread to the bone. It is estimated that each year, about 350,000 people die of bone metastasis in the United States. The causative tumor becomes incurable once the bone has been invaded and only 25% of prostate cancer patients are able to live 5 years subsequent to their diagnosis of bone metastasis. The interaction between prostate cancer cells and the bone creates a vicious cycle of bone formation and bone destruction thereby destabilizing the inherently delicate homeostasis within the bone. In prostate cancer, this process leads to metastatic lesions that are predominantly osteoblastic. However within the background of bone formation, several groups have reported that bone resorption is an integral part of prostate cancer bone lesions. Therefore, early detection and treatment before the tumor metastasizes is critical for the survival of the patient. Creating and characterizing mouse models that better mimic the progression in human prostate cancer is a powerful tool to study and delineate the various steps in tumor progression. Most of the currently available mouse models of prostate cancer successfully recapitulate the early steps of tumor progression in the primary site but fail to metastasize to other organs especially to the bone. Amongst the existing ones, transgenic models that are created by dysregulating a gene that is widely known to be altered in human prostate cancer are considered to be more reflective of the human disease. This is in contrast to transgenic mouse models that overexpress the small t and large T antigens that despite the phenotype they produce, are considered to have little physiological significance. This first part of the thesis describes the creation and characterization of the hepsin/myc bigenic model that develops adenocarcinoma in the primary site. The second part of the thesis describes a model that delineates the interactions between prostate cancer cells and the bone microenvironment. This is approached by dysregulating the T-box transcription factor Tbx2 in PC3 human prostate cancer cells and looking at the effect of this dysregulation on the interaction of the cells with the bone microenvironment. Approved______________________________________Date_________

C/EBPbeta is essential for mammary gland growth and development and has been associated with poor prognosis in breast cancer. Overexpression of C/EBPbeta2 in MCF10A cells, a model of normal mammary epithelial cells, results in a variety of cancer phenotypes including EMT and ErbB independence. IL1B is dramatically upregulated in MCF10A-C/EBPbeta2 cells but there is little, if any, processing to the mature 17 kD form. Although proIL1B has previously been considered to be biologically inactive, we demonstrate proIL1B is not only localized to the nucleus, but is also tightly associated with the chromatin. We show that proIL1B is bound at specific locations in the genome and is positioned in such a way to play a role in the cancer phenotypes observed in MCF10A-C/EBPbeta2 cells. Given that MCF10A cells overexpressing C/EBPbeta-2 are no longer dependent on ErbB signaling for survival, we sought to investigate whether aberrant C/EBPbeta-2 expression could contribute to the resistance of some breast cancers to ErbB targeted therapies, such as trastuzumab. C/EBPbeta2 was retrovirally introduced into two ErbB2 overexpressing cell lines, BT474 and HCC1954, which are sensitive to trastuzumab. These cells were then assayed for trastuzumab sensitivity and it was found that C/EBPbeta2 confers resistance to trastuzumab in both cell lines albeit by different mechanisms. In BT474 cells overexpression of C/EBPbeta2 was able to mediate resistance by the loss of ErbB2 and stem cell-like gene expression. C/EBPbeta2 overexpression in HCC1954 cells resulted in upregulation of proIL1B. The proIL1B in HCC1954-C/EBPbeta-2 cells was localized to the nucleus. Moreover, nuclear IL1B is detected in some human breast tumor samples. This study demonstrates the presence of nuclear proIL1B in transformed mammary epithelial cells providing the first evidence that IL1B may be a dual function cytokine with distinct secreted and nuclear functions.

The major goal of this thesis is to investigate the role of MT1-MMP in kidney development. Our results demonstrate that loss of MT1-MMP leads to a renal phenotype characterized by a moderate decrease in ureteric bud branching morphogenesis and a severe proliferation defect. This phenotype is independent of the MT1-MMP target, MMP-2. The kidneys of MT1-MMP-null mice have increased deposition of the major components of renal basement membranes: collagen IV, laminins, perlecan, and nidogen. Utilizing in vitro systems we demonstrate that MTI-MMP proteolytic activity is required for renal tubule cells to proliferate in three dimensional matrices and to migrate on the principal kidney basement membranes components. Together, these data suggest an important role for MT1-MMP in kidney development, which is mediated by its ability to regulate cell proliferation and migration by proteolytically cleaving kidney basement membrane extracellular matrix components. These finding have significantly improved the current knowledge of the role of MMPs renal development.