These studies examine the consequences of tissue-specific oncogene or a tumor suppressor gene expression in either human prostatic epithelial or stromal cells. This approach allows us to monitor the growth and differentiation status of such grafts in vivo. We utilized retroviral gene transfer to infect initiated epithelial cells (BPH-1 cells-SV40 immortalized human prostatic epithelial cells), benign prostate epithelial cells (PrE3) and primary cultures of normal prostatic fibroblasts (NPF) with either cyclin D1 overexpressing, PTEN shRNA or myr-AKT vectors. The resulting cells were then examined using tissue recombination approaches. Overexpression of cyclin D1 in BPH-1 cells increased their proliferation rate, but did not elicit tumorigenicity in vivo. In contrast overexpression of cyclin D1 in NPF cells that were subsequently recombined with BPH-1 resulted in malignant transformation of the epithelial cells. DNA microarray analysis showed highly concordant profiles of gene expression between Carcinoma Associated Fibroblasts (CAFs) and NPF cells overexpressing cyclin D1 (NPFcyclin D1). Epithelial cells isolated from BPH-1 + NPFcyclin D1 recombinants (BPH-1NPF-cyclin D1) were permanently transformed in vivo, mirroring previously described results using BPH-1 + CAF recombinant-derived cells. Expression of the cell matrix protease cathepsin D was upregulated in NPFcyclin D1, CAFs and BPH-1NPF-cyclin D1 cells. Further work is needed to establish whether this molecule is an important paracrine mediator in this system. A moderate (55%) suppression of PTEN expression elicited a tumorigenic response in BPH-1 cells converting benign epithelial cords into an invasive tumor. Partial suppression of PTEN in benign PrE3 cells caused high-grade PIN. The ability of human prostatic epithelial cells to induce and maintain smooth muscle differentiation in adjacent stroma was found to be negatively correlated to the level of genetic damage sustained by those epithelial cells. This study examined several aspects of the molecular basis for the change in the relationship between stromal and epithelial cells in the transition from benign prostate to prostate cancer and provided new human models for prostate cancer research.

p120 binds and is thought to regulate the cell-cell adhesion molecule E-cadherin. Experiments in a p120-deficient carcinoma cell line suggest a critical role for p120 in stabilizing E-cadherin. p120 downregulation occurs in a wide range of cancers and frequently correlates with E-cadherin loss. Thus I hypothesized that p120 is necessary for maintaining normal cadherin levels and that p120 loss may be a causal event leading to E-cadherin loss in tumors. To test this hypothesis, I knocked down p120 in vitro using siRNA and conditionally knocked out p120 in vivo using MMTV-Cre. My in vitro data demonstrate that p120 is essential for control of E-cadherin stability at the membrane and suggest that without p120, E-cadherin undergoes endocytosis and is degraded. By knocking out p120 in vivo with MMTV-Cre, I had intended to assess the effects of p120 loss on mammary tumorigenesis. Surprisingly, MMTV-Cre-mediated deletion of p120 led to postnatal lethality. Fortunately, MMTV-Cre is also expressed in the salivary gland, which is an excellent model system to study the effects of p120 ablation on glandular morphogenesis and development. As in vitro, p120 loss caused significant downregulation of E-cadherin. Concomitant with p120 loss, I observed premature dilation of salivary ducts and sporadic lack of cell polarity. As p120-null salivary glands developed, ducts extend and branch abnormally and acini do not develop, and indicating that p120 is critical to epithelial morphogenesis. Although these animals did not live long enough to develop cancer, they developed lesions that bore hallmarks of neoplasias including hyperplasia, loss of polarity, loss of cell-cell adhesion, and nuclear crowding. Together, these data reveal an essential role for p120 in regulating E-cadherin function and glandular morphogenesis, and they strongly support my hypothesis that p120 is a tumor modifier and/or suppressor.

The EphA2 receptor belongs to the recently cloned Eph family of receptor tyrosine kinase (RTK). High levels of EphA2 RTK have been detected in 60-90% of human breast cancer specimens, both in breast cancer cells and in tumor vascular endothelial cells. However, the mechanisms by which EphA2 promotes breast cancer malignancy are not completely clear. In my dissertation work, I first investigated the effect of EphA2 overexpression on tumor cells. I found that increased EphA2 expression in normal epithelial cells contributes to destabilization of cell-cell adhesion, an early step towards tumor cell malignancy; whereas high levels of EphA2 expression in tumor cells increase tumor cell motility and lung metastasis. In addition to regulating tumor cell malignancy, EphA2 also promotes tumor angiogenesis. In order to dissect EphA2 signaling in vascular endothelial cells, I mapped phosphorylated tyrosine residues on the EphA2 receptor, identified interacting proteins that bind to these sites, and tested the effects of a series of phosphorylation-defective EphA2 mutants on angiogenesis. These studies established a critical role for tyrosine phosphorylation of EphA2 in tumor angiogenesis. Taken together, my thesis work demonstrated that EphA2 regulates tumor progression by promoting both tumor cell malignancy and tumor angiogenesis.

The transforming growth factor beta (TGF-â) pathway significantly regulates mammary development and tumorigenesis. In human cancer, its signaling pathways are often modified or lost during tumor progression. Prior to initiation and early during progression TGF-â acts upon the epithelium as a tumor suppressor, however at later stages it is often a tumor promoter. This dual role has been previously described as the TGF-â paradox. Importantly, our current studies involving stromal-epithelial and host-tumor interactions, including those presented in this dissertation, are beginning to provide an explanation and mechanisms for the dual nature of TGF-â signaling during tumor progression. The results reported herein, primarily explore the mammary carcinoma cell response to TGF-â signaling and the subsequent impact on the adjacent tumor microenvironment. Notably, we have been able to demonstrate, using two independent parallel model systems, that loss of carcinoma cell specific TGF-â response leads to increased extravascular pulmonary metastasis. In addition, we have shown that the loss of the carcinoma cell specific TGF-â response in primary mammary tumors can result in enhanced carcinoma cell survival, increased adjacent smooth muscle actin positive stroma, increased tumor cell heterogeneity, inflammatory gene expression and inflammation involving bone marrow derived myeloid cells that are known to promote tumor progression and metastasis. To provide mechanisms for these observations, we have performed gene profiling studies with intact or TGF-â signaling deficient carcinoma cells. Importantly, the gene expression profiling results demonstrated regulation of known inflammatory genes by TGF-â and also demonstrated a significant link between the loss of carcinoma cell specific TGF-â signaling and increased risk for relapse in human breast cancer. Together, our results and the current literature, strongly suggest that gain or loss of signaling through the TGF-â pathway in carcinoma cells can promote tumor progression to metastasis though distinct mechanisms in vivo. In addition, if a signature indicative of carcinoma cell TGF-â signaling deficiency is detected in human breast cancer, our results may suggest that the patient should be treated more aggressively to reduce the risk of subsequent relapse and mortality.

Gastric adenocarcinoma is strongly associated with the presence of H. pylori, and both microbial and host factors influence the risk for carcinogenesis. A novel role for H. pylori in the stimulation of the p120ctn/Kaiso signaling axis leading to relief of Kaiso-mediated repression of mmp-7 transcription in vitro was identified. Increased expression of MMP-7 in response to H. pylori infection may alter a number of processes involved in carcinogenesis, including the inflammatory response, proliferation, and apoptosis. Bacterial challenge of mice deficient in MMP-7 resulted in enhanced inflammation and cellular turnover when compared to wild type mice, suggesting that MMP-7 may serve a protective role within H. pylori-infected gastric mucosa. Taken together, these data indicate that H. pylori stimulates increased gene transcription through dysregulation of a number of signaling pathways and that upregulation of these host effectors mediates the development of malignant lesions.

FUNCTIONAL STUDIES ON INTERLEUKIN 24 IN VITRO AND IN VIVO MIAO HE Thesis under the direction of Professor Peng Liang Among IL-20 family of cytokines, IL-19, IL-20 and IL-24 can each activate IL20R1/IL-20R2 heterodimeric receptor in vitro; whereas IL-20 and IL-24 can also signal through IL22R1/IL-20R2. The significant receptor sharing among these cytokines based on in vitro biochemical assays with cultured cells thus has raised questions about whether the three cytokines have redundant biological functions in vivo or they may use the same receptors for different biological end points in a tissue specific or temporally regulated manner. However, until now only IL-20 has been proven to play an important role in vivo in the epidermis where both receptors are expressed. In this study, we created IL-24 transgenic mice targeted to the skin. IL-24 transgenic mice exhibit many phenotypes similar to that of IL-20, including neonatal lethality, epidermal hyperplasia and abnormality in keratinocyte differentiation, other than infiltrating macrophages. Moreover, homodimeric IL-20R2 soluble receptor can bind to both IL-20 and IL-24 in vitro with high affinity and potently blocks their ability to bind to cell surface receptors. These results support a redundant role for IL-20 and IL-24 in epidermal functions and a potential therapeutic use of soluble IL-20R2-Fc in autoimmune diseases such as psoriasis where both IL-20 and IL-24 levels are elevated.

Breast to bone metastasis is a common event during breast cancer progression. The resultant lesions often cause extensive bone destruction that results in a number of complications including intense pain that dramatically affects the patients quality of life and leads to increased morbidity. Understanding the mechanisms through which breast cancer cells destroy bone can ultimately lead to the generation of new therapies that will successfully battle the disease. In the osteolytic tumor bone environment, activation of osteoclasts, the cells responsible for bone resorption, is critically dependent on proper signals derived from osteoblasts, the cells responsible for bone synthesis. Matrix metalloproteinases (MMPs) are a family of proteinases that have been implicated in mediating cell-cell communication in the tumor bone environment. Interestingly, in human breast-to-bone metastases samples, MMP-2 has been shown to be expressed by osteoblasts and osteocytes and MMP-7 and MMP-9 were found be localized to bone resorbing osteoclasts. In a bid to define the roles of host derived MMP-2, MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibia of MMP-2, MMP-7 and MMP-9 null mice were injected with osteolytic luciferase tagged mammary tumor cell lines. Our studies demonstrated that osteoblast-derived MMP-2 impacts mammary tumor survival in the bone microenvironment by mediating the release of active TGF-β via the processing of a novel MMP-2 substrate, LTBP-3. Furthermore, our results showed that osteoclast-derived MMP-7 but not MMP-9 affect mammary tumor growth in the bone via the solubilization of RANKL that in turn induces osteoclastic resorption of the bone. Thus, this dissertation demonstrated that MMP-2 and MMP-7 support the vicious cycle of bone metastasis via two distinct mechanisms affecting two of the key components of the process, osteoblasts and osteoclasts. Therefore, these two MMPs and their substrates would be attractive targets for future drug therapies to treat patients suffering from breast cancer-induced lytic bone lesions.

Genetic heterogeneity is a key factor involved in tumor progression, and it is increasingly recognized that heterogeneity of phenotypic traits exists among individual cancer cells. In this dissertation, I investigate the heterogeneity of cancer cell motility via video-microscopic analysis by: 1) quantitating heterogeneity of cell motility, 2) exploring the differences in cell motility between non-tumorigenic and cancer cell lines, and 3) developing novel tools and metrics for the study of dynamic characteristics of cancer cells. To this end, over 7,300 cells were tracked and analyzed, from a variety of cell lines, in an assortment of microenvironmental conditions. The work presented in this dissertation provides important additions to the study of single-cell variability, and provides robust tools for future investigations in this field.

Gastric adenocarcinoma is strongly associated with the presence of H. pylori. Microbial factors of H. pylori and host responses induced by the interactions of H. pylori with gastric epithelial cells play important roles in the development of disease. PI3K and β-catenin/p120 are multifunctional host proteins that coordinate carcinogenic epithelial responses when aberrantly activated, such as in malignant gastric lesions. We demonstrate that H. pylori infection results in upregulation of PI3K-AKT signaling, through stimulation of EGFR. Activation of this pathway reduces rates of epithelial cell death induced by H. pylori and promotes resistance to apoptosis. We also demonstrate that H. pylori infection induces additional host signaling pathways to potentiate a proliferative response in gastric epithelial cells. Specifically, PPARδ, a target of β-catenin transcriptional activation, contributes to increased rates of gastric epithelial cell proliferation in response to H. pylori infection. Based on these findings we hypothesize that an anti-apoptotic response in the presence of increased proliferation increases the risk of retaining mutagenized gastric epithelial cells in the presence of H. pylori induced gastritis. Taken together, these studies have identified effectors that directly mediate host responses related to carcinogenesis. Molecular delineation of such pathways activated by host-microbial interactions will improve our understanding of H. pylori-induced carcinogenesis, allowing for targeted therapies to high-risk individuals, as well as provide insight into other malignancies that arise within the context of pathogen-induced inflammation.

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.