Prostate cancer is the second most common form of cancer in men. Despite the alarming figures and decades of research, a cure still eludes prostate cancer patients. The morbidity associated with advanced prostate cancer is due to the metastasis of the primary tumor primarily to the bone. Hepsin is a gene that is over-expressed in more than 90% of human prostate cancer patients and thus assumes tremendous relevance in prostate cancer as a therapeutic target in the future. Similarly, matriptase is another gene that is over-expressed in prostate cancer patients. The expression of both genes positively correlates with the aggressiveness of human prostate tumors. Though both genes are known to be over-expressed in human prostate cancer, their mechanism of action is still unknown. Both hepsin and matriptase are within a category of genes known as proteases that have the potential ability to cleave or digest the matrix or nests (called basement membrane) that surrounds the tumor cells in a primary tumor and inhibits cells from escaping and eventually metastasizing. Tumor cells escape the primary tumor by interacting and digesting the basement membrane and travel through the blood stream to reach the bone environment. This study reveals the interaction between extracellular membrane component Ln-332, which is lost in prostate cancer and two type II transmembrane serine proteases, hepsin and matriptase that are consistently over-expressed in human prostate cancer cases. We have found in our study that hepsin and matriptase individually cleave or digest Laminin-332 and that this cleavage facilitates movement of human prostate cells in tissue culture. We found that in the tumor tissues of the prostate of hepsin overexpressing mice there is degradation of Ln-332. Furthermore, hepsin or matriptase overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Also, matriptase over-expression causes increased persistence of LNCap cells on Laminin-332. This cleavage of Ln-332 by hepsin and/or matriptase may play the critical mechanistic role for the spread of human prostate cancer thus assumes tremendous relevance in the field of prostate cancer research in finding a future therapeutic target.

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining the mechanisms by which the bacterium escapes the immune response are clinically important. Our aim was to determine if induction of arginase II (Arg2) by H. pylori modulates host defense. We have demonstrated that macrophage nitric oxide production can kill H. pylori; however, induction of macrophage Arg2 inhibits inducible nitric oxide synthase (iNOS) translation, induces apoptosis, and restricts bacterial killing. After 4 mo infection with H. pylori, we found that enhanced gastritis correlated with decreased bacterial colonization in Arg2/ mice, but not WT mice. We found that iNOS+ macrophages were more abundant in Arg2/ mice, less apoptotic, and had increased nitrotyrosine staining. This correlated with enhanced pro-inflammatory cytokine production. These studies suggest that Arg2 mediates the immune evasion of H. pylori by restricting macrophage function.

Myeloid derived suppressor cells (MDSCs) play an important role in cancer progression, and elucidating the mechanisms involved in the accumulation and function of these cells is important. Preliminary data indicated that Rgs2 may have a role in MDSC production and differentiation. As such, its role in hematopoiesis and myeloid cell differentiation was examined. Overall, hematopoiesis was not affected by loss of Rgs2, and MDSC production and heterogeneity in the spleen were not altered. However, Rgs2 deficiency in MDSCs abrogated the tumor promoting roles of these cells, accompanied by drastically decreased MCP-1 production, and subsequent effects on endothelial cells and leukocytes. Together, these data suggest that Rgs2 affects the tumor promoting functions of MDSCs through mediating MCP-1 production. In targeting the signaling pathway involving Rgs2 in MDSCs, we may be able to promote an anti-tumor response to cancer, enhancing patient outcome.

The interactions between malignant plasma cells and the bone marrow microenvironment are critical for the development of multiple myeloma and the associated osteolytic bone disease. Only recently are studies beginning to elucidate mechanisms by which the altered microenvironment contributes to cancer progression in hematological malignancies. The contributions of the host microenvironment during the initial stages of myeloma development are poorly understood. In the studies presented in this dissertation, we (1) developed a new murine model of myeloma to enable investigation of the host bone marrow microenvironment in myeloma pathogenesis, (2) determined the role of bone marrow stromal cell-derived Dkk1 in myeloma cell establishment and growth, and (3) determined the role of decreased circulating adiponectin in myeloma pathogenesis. Many features of human myeloma bone disease are recapitulated in the well-characterized 5T Radl myeloma model, where transplantation of 5T myeloma cells into recipient mice of the C57Bl/KaLwRij substrain results in propagation of myeloma. Our studies utilized these mice as a tool to provide valuable insights into the development of human myeloma and the associated bone disease. After the initial identification of these factors in the C57Bl/KaLwRij mice, findings in patients, either from the literature or by direct examination of specimens, provide strong support that these changes have clinical relevance. The ultimate goal of this dissertation was to gain a better understanding of the mechanisms that precede myeloma bone disease. Our findings suggest that bone marrow stromal cell-derived Dkk1 and decreases in circulating adiponectin are important in myeloma pathogenesis.

The oncogenic PIM1 kinase has been implicated as a cofactor for the oncogene c-MYC in prostate carcinogenesis. In this study, we show that co-expression of c-MYC and PIM1 is associated with high Gleason grade in prostate cancer. Using a tissue recombination model coupled with lentiviral-mediated gene transfer we show that Pim1 is weakly oncogenic in naive adult mouse prostatic epithelium. However, it cooperates dramatically with c-MYC to induce prostate cancer within 6 weeks. Importantly, c-MYC and Pim1 synergy is critically dependent on Pim1 kinase activity. c-MYC/Pim1 expressing tumors showed increased phosphorylation of MYC on serine 62. Expression of a phosphomimetic c-MYC S62D mutant resulted in higher rates of proliferation compared to that of wild type c-MYC. However, c-MYC S62D did not result in tumors like c-MYC/Pim1 grafts, indicating that Pim1 cooperativity with c-MYC involves additional mechanisms other than enhancement of c-MYC activity by S62 phosphorylation. In addition, c-MYC/Pim1-induced prostate carcinomas demonstrate evidence of neuroendocrine (NE) differentiation. Additional studies, including the identification of tumor cells co-expressing androgen receptor and NE cell markers synaptophysin and Ascl1 suggest that NE tumors arise from adenocarcinoma cells through transdifferentiation. These results directly demonstrate functional cooperation between c-MYC and Pim1 in prostate tumorigenesis in vivo and support therapeutic strategies for targeting PIM1 in prostate cancer. Since Pim1 deficiency is well tolerated in vivo, it has been proposed that Pim1 inhibition may offer an attractive option to impede prostate cancer progression. In the current study, we used small hairpin interfering RNA (shRNA) directed against Pim1 to determine the anti-tumor effects in prostate cancer cell lines. In mouse c-MYC/Pim1 prostate tumor-derived cells and human prostate cancer cells, Pim1 knockdown markedly decreased cellular proliferation, survival, and tumorigenicity. Further studies indicate that in prostate cancer cells, Pim1 is required to maintain ERK/MAPK signaling pathway activation. Thus, Pim1 is necessary to maintain tumorigenicity, and may represent an efficient target for prostate cancer therapy. (Note: Human MYC and mouse Pim1 are cloned in lentiviral constructs and used for this study)

Overexpression of activated oncogenes such as Ras(V12) in primary cells induces senescence rather than transformation, thus suppressing tumorigenesis. C/EBPbeta is required for Ras(V12)-induced senescence in mouse embryonic fibroblasts. C/EBPbeta is a transcription factor in which three protein isoforms exist due to alternative translation at three in-frame methionines. Upregulation of IL6 by C/EBPbeta is necessary for oncogene-induced senescence (OIS), but the specific isoform of C/EBPbeta has not been investigated. We show that the C/EBPbeta1 isoform upregulates IL6 and induces senescence when introduced into normal fibroblasts. In contrast to normal fibroblasts, overexpression of Ras(V12) in MCF10A cells, an immortalized mammary epithelial cell line, leads to transformation and degradation of C/EBPbeta1, suggesting a mechanism exists to bypass OIS in these cells. When MCF10A-Ras cells are forced to express C/EBPbeta1, these cells degrade p52-C/EBPbeta1. Treatment of MCF10A-Ras-C/EBPbeta1 cells with the cdk inhibitor roscovitine stabilizes p52-C/EBPbeta1. Cdk2 phosphorylates C/EBPbeta1 on Thr235. Mutation of Thr235 of alanine in C/EBPbeta1 restores stability of p52-C/EBPbeta1 expression in MCF10A-Ras cells, indicating that phosphorylation of C/EBPbeta1 on Thr235 by cdk2 is leading to degradation of p52-C/EBPbeta1 in the MCF10A-Ras cells. C/EBPbeta1 is also regulated by Ras in breast cancer cells by the post-translational modification SUMO-2/3. Although p52-C/EBPbeta1 is not detected in anti-C/EBPbeta1 immunoblots of breast cancer cells, higher molecular weight bands are frequently observed. Exogenously expressed C/EBPbeta1 is sumoylated in breast cancer cells, and the higher molecular weight bands observed in anti-C/EBPbeta1 immunoblots of breast cancer cell lines is sumoylated C/EBPbeta1. Phosphorylation oftentimes enhances sumoylation, and phosphorylation cascades, including the Ras pathway, are activated in breast cancer cells. C/EBPbeta1 can be phosphorylated on Thr235 by Erk-2, a downstream effector of Ras. Phosphorylation of purified C/EBPbeta1 by Erk-2 enhances sumoylation, in vitro. Additionally, sumoylated C/EBPbeta1 is phosphorylated on Thr235 and the C/EBPbeta1T235A mutant shows a 3-fold decrease in sumoylation as compared to wild type C/EBPbeta1. Taken together, degradation or sumoylation of C/EBPbeta1 by Ras activation may represent mechanisms to bypass OIS.

p120 catenin (p120) binds and stabilizes classical cadherins, making it a critical regulator of cell-cell adhesion. Here, we report an efficient technique (designated ReCLIP for Reversible Cross-Link Immuno-Precipitation) for identifying novel p120 binding partners that provides evidence of an interaction between p120 and the RhoA substrate p160 Rho Kinase (ROCK1). Briefly, proteins are covalently crosslinked in situ using thiol-cleavable DSP (Dithiobis[succinimidyl propionate) or DTME (Dithio-bismaleimidoethane) chemistries and then recovered by immunoprecipitating p120. Binding partners are then selectively eluted and identified by single-dimension liquid-chromatography tandem mass spectrometry. Crosslinking dramatically improved the efficiency of p120 co-immunoprecipitation with other members of the cadherin complex and revealed several new putative binding partners. Interestingly, one of these was the RhoA substrate p160 Rho Kinase (ROCK1). Using immunofluorescence and immunoprecipitation-based analyses, we showed that a fraction of ROCK1 associates with E-cadherin-bound p120 at cell-cell junctions. ROCK1 depletion by shRNAt led to disorganization of the adherens junction, but did not effect p120 or E-cadherin expression levels. Instead, disruption of the cadherin complex appeared to be due to a disruption in junctional actin. Using an in vitro kinase assay and phospho-specific p120 antibodies, we demonstrated that ROCK1 phosphorylates p120 on serine 268. These data reveal that ROCK1 can phosphorylate and interact with p120, and implies a role for this interaction in regulating cadherin stability.

Cancer targeted imaging plays an important role in cancer diagnosis. It allows us to rapidly evaluate tumor responses to certain treatments. Peptide-based imaging probes have been widely used in cancer targeted imaging. With the development of phage display technology, a great number of peptides have been identified specifically targeting to tumor cells in vitro and in vivo. However, due to peptides small size and relatively low binding affinity, it is difficult to identify the target of peptides. This thesis research is focusing on: i). identifying the target of specific peptide HVGGSSV, which bind to radiation responsive tumors, by utilizing several affinity purification techniques especially the phage display screening; and validating the target as a marker for evaluation of radiation treatment; ii). exploring the biological functions of the targeting protein.

Eph receptor tyrosine kinases are membrane bound receptors often expressed by normal epithelial cells but are frequently overexpressed in many human cancers. Of the many Eph receptors, EphA2 is present at low levels in normal mammary tissue but highly expressed in breast cancer and correlative with a poor patient prognosis. The focus of this thesis is to understand the endogenous role of EphA2 in mammary gland development and how overexpression can contribute to the poor prognosis through metastasis often seen with breast cancers overexpressing EphA2. Using EphA2-deficient animals, this thesis work demonstrates for the first time that EphA2 receptor function is required for mammary epithelial growth and branching morphogenesis. Loss of EphA2 decreased penetration of mammary epithelium into fat pad, reduced epithelial proliferation, and inhibited epithelial branching. These defects appear to be intrinsic to loss of EphA2 in epithelium, as transplantation of EphA2-deficient mammary tissue into wild-type recipient stroma recapitulated these defects. In addition, HGF-induced mammary epithelial branching morphogenesis was significantly reduced in EphA2-deficient cells relative to wild-type cells, which correlated with elevated basal RhoA activity. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development, functioning downstream of HGF to regulate branching through inhibition of RhoA. Breast cancer metastasis to bone is a major cause of morbidity and mortality in patients. Analysis of human breast-to-bone metastasis samples revealed EphA2 positive staining on tumor cells in close proximity to osteoclast at the tumor-bone interface. To define the role of EphA2 in tumor cell-host bone cell interactions, mouse tibias were injected with osteolytic breast tumor cells lacking EphA2 activity. Our data showed that inhibition of EphA2 activity significantly decreased tumor-induced osteolysis compared to controls. Further in vitro analysis revealed that blocking EphA2 function resulted in defective precursor maturation into functional osteoclasts. A human antibody targeted against EphA2 decreased breast tumor induced osteolysis in vivo. Our studies indicate the selective inhibition of EphA2 at the tumor-bone interface may be a benefit for the treatment of breast-to-bone metastases

The serine threonine kinase receptor-associated protein (STRAP) is a WD40 domain protein that regulates a wide array of biological processes. Previous reports suggest that STRAP possesses oncogenic characteristics and regulates signaling pathways associated with tumorigenesis. The aim of this dissertation was to investigate the functional role of STRAP in cellular proliferation and motility. I have shown that STRAP expression in fibroblasts promotes expression of the c-Jun proto-oncogene by inhibiting ubiquitin-mediated proteolysis. Stabilization of c-Jun was associated with increased expression of the AP-1 target gene, cyclin D1, and increased proliferation. These findings may suggest that STRAP-mediated AP-1 signaling can support tumor growth through cell autonomous and paracrine signaling. With respect to cellular motility, I have shown that STRAP appears to regulate persistent chemotaxis and tail formation during motility. Loss of STRAP expression was associated with increased cellular adhesion as well as alterations in the size and distribution of focal adhesion kinase (FAK) complexes. The results of this study suggest that STRAP promotes efficient chemotaxis through modulation of directionality and adhesion dynamics. Taken together, these studies provide insight into the potential mechanisms by which STRAP can promote tumor growth and metastasis.