Understanding how cells maintain genome integrity is necessary to gain insight into the pathology of cancer and to identify therapeutic targets and biomarkers. The DNA damage response kinase ATR is essential for the maintenance of the cellular genome. In this dissertation, I hypothesize that ATR is phosphorylated in response to DNA damage. I also characterize a novel ATR inhibitor and explore its efficacy as an anti-cancer agent. I discover that ATR is phosphorylated on T1989 in response to DNA damage and marks an activated kinase. T1989 phosphorylation is not critical for ATR function. As a proximal marker for ATR activity, T1989 phosphorylation has significant potential as a biomarker. I also identify a novel regulatory region in ATR that separates the essential function of ATR from its G2 checkpoint activity. Finally, I demonstrate that an ATR inhibitor sensitizes cells to replication stress, suggesting ATR inhibition may be a good therapeutic strategy to pursue.

Synovial sarcoma (SS) is a rare but aggressive malignancy that is typically diagnosed in adolescents and young adults. These tumors are characterized by the presence of a specific chromosomal translocation t(X;18)(p11.2;q11.2) resulting in the fusion of the SYT gene from chromosome 18 with one of the SSX genes located on the X chromosome. The wild-type SYT and SSX proteins function as a transcriptional co-activator and transcriptional co-repressor, respectively, thus it is believed that the tumorigenic activity of the resultant SYT-SSX fusion protein is based on transcriptional deregulation. Moreover, it has been hypothesized that SS arises from an undifferentiated cell type. Here we show that SYT-SSX2 expression in mesenchymal stem and progenitor cells results in the aberrant activation of a neural program through the direct targeting of genes involved in the differentiation and function of this lineage. In addition, this phenotype is dependent on signaling through Fgfr2, a gene that is also directly occupied and upregulated by the chimeric protein. Genome-wide binding and expression studies included in this work reveal that SYT-SSX2 may reverse the transcriptional activity, whether silent or expressed, of its target genes. These analyses also demonstrate that SYT-SSX2 is primarily recruited to genes regulated by Polycomb repressive complexes and antagonizes their silencing function. At the molecular level this may be mediated by the opposition of the ubiquitin E3-ligase activity of Ring1b, a core component of Polycomb Repressive Complex 1, by SYT-SSX2 and/or the proteins with which it interacts. Overall, the data presented here indicate that by altering lineage-specific factors, signaling pathways, and epigenetic regulators, SYT-SSX2 mediates transformation through aberrant cellular reprogramming.

<p>Nuclear factor-êB (NF-êB) inducing kinase (NIK) is a MAP3K that regulates activation of NF-êB. NIK is often over-expressed in tumor cells, including melanoma, but the significance of this in melanoma progression remains unclear. Tissue microarray analysis of NIK expression reveals that dysplastic nevi (n=22), primary (n=15) and metastatic melanoma (n=13) lesions showed a statistically significant elevation in NIK expression when compared to benign nevi (n=30). Depletion of NIK using shRNA in melanoma cell lines decreased proliferation, increased apoptosis, delayed cell cycle progression, and reduced tumor growth in a mouse xenograft model. Consistent with the previous studies, NIK deficiency reduced activation of the non-canonical NF-êB pathway, while canonical NF-êB activation remained intact. NIK depletion also reduced expression of genes that contribute to tumor growth, including CXCR4, c-MYC and c-MET, as well as pro-survival factors BCL2 and survivin. These changes in gene expression are not fully explained by the attenuation of the non-canonical NF-êB pathway. Shown here for the first time is the demonstration that NIK depletion decreases â-catenin mediated transcription to down-regulate expression of survivin as well as other â-catenin regulated genes including c-MYC, c-MET and CCND2. These data indicate NIK mediates both â-catenin and NF-êB regulated transcription to modulate melanoma survival and growth. Thus, NIK may be a promising therapeutic target for melanoma. In addition, novel NIK-interacting proteins were identified by using mass spectrometry analysis. Some of these proteins include heat shock protein 90 (Hsp90), ribosomal protein S3 (RPS3) and DEAD box polypeptide 5 (DDX5). <p>Inhibitor of IêB kinase â (IKKâ), another kinase of NF-êB pathway, also contributes to melanoma growth. Systemic inhibition of this kinase in a melanoma xenograft model was characterized using a pharmacological IKKâ inhibitor, BMS-345541. Results show that inhibition of IKKâ alters the host immune cell composition and the composition of leukocytes infiltrating tumor, suggesting that systemic NF-êB inhibition should be evaluated more carefully before going forward as a cancer therapy. <p>In this research, I identified NIK as a new potential target for melanoma growth, revealed important new biological functions of NIK, and unraveled key effects of IKKâ inhibition on the tumor microenvironment.

VAPB (VAMP-associated protein B) is an endoplasmic reticulum protein that regulates multiple biological functions. VAPB protein expression is elevated in human breast cancers and correlates with poor prognosis. Despite aberrant expression of VAPB in breast cancer, its function in tumor cells is poorly understood. In this dissertation we demonstrate the causal role between VAPB and breast cancer, specifically in three areas (1) cell growth (2) invasion and (3) tumor growth in vivo. Additionally, we provide mechanistic evidence that VAPB regulates breast tumor cell proliferation and invasion through enhanced ATK activity and vesicle mediated protein secretion.

Inactivating mutations in TGF-©¬ receptors and Smad signal transducers that contribute to resistance to TGF-©¬, are associated with only very small number of NSCLC. The Smad dependent pathway is involved in the tumor suppressive functions of TGF-¥â. Epidemiological studies have demonstrated that most cases of lung cancer (85-90%) are directly attributable to cigarette smoking. However, nothing is known how smoking is involved in inhibiting tumor suppressor functions of TGF-©¬ and whether or how the chemo resistance of platinum-based drugs seen in lung cancer patients who are smokers is dependent on this pathway. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated (300 days) with cigarette smoke condensate (CSC) and Dimethyl sulphoxide (DMSO, as a control) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in decreased Smad-mediated TGF-¥â signaling due to reduced expression of Smad3 both in the protein and mRNA level. The decrease in Smad3 is due to histone deacetylation. Long-term CSC treatment reduced apoptosis, increased cell viability, decreased TGF-¥â-mediated growth inhibition, and enhanced tumorigenicity both in vitro and in vivo. Re-expression of Smad3 in the long-term CSC treated cells reversed the cancerous phenotypes observed due to long-term CSC treatment. Looking further into public databases revealed that the expression of Smad3 is lower in lung tumors of current smokers compared to that observed in never-smokers. The long-term CSC treatment also rendered the cells resistant to platinum-based chemotherapy; by up regulating Bcl2. Blocking the effect of Bcl2 both by small molecule inhibitor ABT-737 and siRNA approaches re-sensitized the cells to platinum-based chemotherapy. The re-expression of exogenous Smad3 in the long-term CSC treated cells, decreased the Bcl2 levels and re-sensitized the cells to platinum-based chemotherapy. Thus, Smad3 controls the expression of Bcl2 and sensitivity to platinum based drugs in our model system. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity and makes the cells resistant to platinum-based chemotherapy by abrogating apoptosis, partly by reducing expression of Smad3.

C/EBPbeta is a member of a family of basic-leucine zipper transcription factors. It has been shown to be a key regulator of growth and differentiation in the mammary gland. There are three different protein isoforms of C/EBPbeta. C/EBPbeta-1 and -2 are transactivators, and differ by just 23 N-terminal amino acids present in beta-1 only. C/EBPbeta-3 (LIP) lacks the transactivation domain and represses transcription. Overexpression of LIP is incompatible with cell proliferation and induces cell death in breast cancer cell lines. LIP expression stimulates autophagy, an evolutionarily conserved cellular process responsible for self-cannabalization through a lysosomal degradation pathway. Interestingly, I find that LIP expression not only leads to self-cannibalization in the MDA-MB-468 breast cancer cell line, but cell cycle profiling reveals a dramatic increase in DNA content in LIP expressing cells. I present data that the induction of autophagy appears to accompany or possibly follow the cannibalization or engulfment of neighboring cells by the LIP expressing cells. LIP expression was found to upregulate HSPA1A transcripts and concomitantly leads to increases in HSP70 protein levels in exosomes. Exosomes are 30- to 100-nm lipid bilayer vesicles that function to promote intercellular communication. LIP-derived exosomes appear to play a role in marking target cells for engulfment. Finally, I present data to show that LIP-induced cell engulfment may play a physiological role during involution of the mammary gland.

This project was conducted to further elucidate the role of matrix metalloproteinase 7 (MMP7) in the pathogenesis of the carcinogenic bacteria Helicobacter pylori. Specifically, in this thesis I discuss the role of MMP7 in H. pylori-induced inflammation and injury. Briefly, we performed histological and molecular biological analysis of stomach tissue from wild-type and MMP7-/- mice that had been challenged with H. pylori. We also performed molecular biological analyses of macrophages extracted from the stomachs or derived from bone marrow of wild-type and MMP7-/- mice. We found that loss of MMP7 results in increased production of inflammatory cytokines, and that MMP-/- macrophages display enhanced M1 macrophage phenotypes. In this research we discovered an explanation for the increased inflammation and injury seen in MMP7-/- mice and thus, a potential role for MMP7 in protecting wild-type animals from H. pylori- induced inflammation.

Transforming growth factor-beta (TGF-β) has a dual role during tumor progression initially as a suppressor and then as a promoter. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet, the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. In this dissertation, we hypothesized that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and metastasis. Fluorescently-labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT TGF-β receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. Intravital microscopy of xenografts revealed that fibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by α-SMA and vimentin expression, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors exhibited a two-fold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared to TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Downregulation of Tmeff1 was correlated with disease progression of TGF-β-insensitive mammary cancer. Our findings concerning TGF-β signaling in stromal-epithelial interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment. In the second part of this dissertation, the timing of TGF-β signaling in relation to tumor progression was investigated through the creation of an inducible dominant-negative TβRII (dnTβRII) mouse model (MMTV-PyVmT;MMTV-rtta;dnTβRII). We hypothesized that attenuation of TGF-β signaling prior to tumorigenesis leads to increased metastasis. Results indicated that animals with attenuated TGF-β signaling prior to tumor palpation exhibited increased tumor latency and enhanced metastasis of dnTβRII-expressing carcinoma cells. These animals had an increased MDSC tumor population, as well as increased carcinoma cell secretion of MCP-1/CCL2. Our inducible dnTβRII model has therapeutic implications in determining the necessary timing of therapeutic inhibition of TGF-β signaling during cancer progression.

These studies tested the hypothesis that cell-extracellular matrix (ECM) interactions promote the maturation of invadopodia to fully functional structures. I demonstrate that invadopodia-associated ECM degradation is modulated by substrate stiffness and density. Overexpression of the mechanosensing proteins FAK and p130Cas can promote the invadopodial response to stiffness. In addition, I investigated the role of integrins as promoters of invadopodia formation and function. Adhesion proteins were found to distinctly localize in ring-like structures around invadopodia. Blocking RGD-binding integrin attachment to the ECM or knockdown of integrin-linked kinase specifically affected cellular ECM degradation by reducing MT1-MMP recruitment to invadopodia. This process apparently involves downstream recruitment of the scaffold protein IQGAP. These data support a model in which cell-ECM interactions specifically promote the maturation stage of invadopodia to promote matrix degradation.

Dysregulation of the Notch signaling pathway plays an important role in lung cancer pathobiology. The Notch3 receptor is overexpressed in ∼40% of resected non-small cell lung cancers, and its suppression results in loss of the malignant phenotype both in vitro and in vivo. In this dissertation, I have identified novel ligand binding regions in the Notch3 receptor using a high throughput system and a Notch3 peptide library that spans the extrcellular domain. This knowledge allowed me to generated Fc-fusion proteins and neutralizing monoclonal antibodies to target these regions in the Notch3 receptor thereby interfering with signaling by blocking the interaction of the receptor with its ligands. In addition, I explored the roles of Jagged1, a Notch ligand, in lung cancer. I demonstrate that Jagged1 has the paradoxical roles of increasing cell growth and suppressing migration. Interestingly, this appears to be through a noncanonical, CSL-independent mechanism. The findings of these studies not only give novel insights into Notch3 signaling but also establish a foundation on which targeted therapies can be developed.