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    THE ROLE OF CXCL12 AND CXCL14 CHEMOKINES IN THE REGULATION OF BREAST CANCER CELL GROWTH AND INVASION

    Scientific Abstract:
    THE ROLE OF CXCL12 AND CXCL14 CHEMOKINES IN THE REGULATION OF BREAST CANCER CELL GROWTH AND INVASION Background: Breast cancer is a leading cause of cancer related death in women of the western world. Last year in the United States alone over 180,000 new cases were diagnosed and over 40,000 women died from breast cancer. Thus, improved molecular based breast cancer preventative and therapeutic strategies are needed. To identify genes implicated in breast cancer, we have analyzed the gene expression profiles of normal mammary epithelial cells and breast carcinomas of different stages using SAGE (Serial Analysis of Gene Expression). In addition, we have recently generated SAGE libraries from purified luminal epithelial, myoepithelial, endothelial cells, infiltrating leukocytes, and fibroblasts from normal and DCIS (ductal carcinoma in situ) breast tissue. Based on these SAGE data and follow up studies using mRNA in situ hybridization and immunohistochemistry, we determined that dramatic gene expression changes occur not only in the epithelial (cancer) cells, but in various stromal cells as well. Many of the genes up-regulated in tumor epithelial or stromal cells encode secreted proteins and receptors implicating abnormal autocrine/paracrine interactions in breast tumorigenesis. Two of the genes specifically up-regulated in DCIS myofibroblasts and myoepithelial cells encode the CXCL12 and CXCL14 chemokines. CXCL12 has been suggested to play a role in breast cancer metastasis, however, its overexpression in DCIS myofibroblasts suggests that it must have additional functions in breast cancer. CXCL14 is a relatively uncharacterized chemokine and its role in breast cancer is unclear. CXCR4, the receptor of CXCL12, and a not yet identified putative receptor for CXCL14 are both present on cancer epithelial cells, suggesting that these chemokines mediate paracrine interactions between cancer epithelial and myoepithelial cells. Objectives/Hypothesis: Based on our preliminary data, we hypothesize that the expression of CXCL12 and CXCL14 in DCIS myoepithelial cells and myofibroblasts may play a role in the regulation of cancer cell proliferation, survival, and invasion. The goal of this study is to characterize the role of CXCL12 and CXCL14 in the regulation of breast cancer cell growth, survival, migration and invasion. Specific aims: (1) To determine the effect of CXCL12 and CXCL14 overexpression on breast cancer cell growth, survival, migration, and invasion. (2) To determine the effect of lack of CXCL12 and CXCL14 on breast cancer cell growth, survival, migration, and invasion. (3) To identify and characterize the CXCL14 signaling pathway in breast cancer cells. Study design: In this proposal we will determine the effect of transient and stable overexpression (aim 1) and elimination (aim 2) of CXCL12 and CXCL14 on breast cancer cell proliferation, survival, migration, and invasion using various in vitro conditions. Similar experiments will be performed using recombinant CXCL12 and CXCL14 proteins to determine if the observed effects are concentration dependent. To investigate the effect of CXCL12 and CXCL14 expression or elimination on tumorigenicity, we will generate xenografts in mouse mammary fat pads by injecting breast cancer cells expressing or lacking CXCL12 or CXCL14. The receptor and signaling pathway mediating the actions of CXCL14 will be identified and characterized in Aim 3 using a candidate gene testing approach and appropriate antibodies and pharmacological agents. Potential Outcomes and Benefits of the Research: Despite significant improvements in breast cancer diagnosis and treatment, advanced stage breast tumors are still inevitably fatal. Therefore, the identification of new therapeutic targets for the inhibition of tumor progression and treatment of invasive breast tumors are of utmost importance. Targeting aberrant autocrine/paracrine interactions essential for tumor growth are particularly promising therapeutic approaches. The goal of this project is to characterize the role of the CXCL12 and CXCL14 chemokines in breast tumorigenesis. The completion of this project will not only help us understand the role of chemokines in breast cancer, but the therapeutic targeting of CXCL12 and CXCL 14 may provide new molecular targets for breast cancer treatment.

    Lay Abstract:
    Breast cancer is a leading cause of cancer related death in women of the western world. In the past year in the United States alone over 180,000 new cases were diagnosed and over 40,000 women died from breast cancer. The breast cancer mortality rate of patients diagnosed with an early stage tumor (such as DCIS-ductal carcinoma in situ) that has not yet invaded the surrounding tissue is nearly zero, while that of tumors that have already spread into other parts of the body is inevitably fatal. Our relative inability to effectively treat advanced stage tumors is due to the lack of targeted therapy specific for the cancer cells as well as due to the inherent genetic instability of advanced stage tumors that enables them to develop resistance to cancer therapeutic drugs. Characterization of breast tumors at the molecular level will likely lead to improved understanding of the genes and biochemical pathways necessary for the initiation, progression, and maintenance of breast cancer. Specific targeting of these pathways will lead to better and less toxic cancer treatment. However, overcoming acquired resistance that occurs during cancer treatment is still a major problem. Emerging evidence suggests that abnormalities occur not only in breast cancer cells, but in the surrounding stromal cells as well. In this sense, a tumor is an abnormal organ with its own blood vessels, inflammatory, and supportive cells, and effective communication among these cells is essential for the maintenance and growth of the tumor. Unlike cancer cells, stromal cells are thought to be genetically stable, and therefore are less likely to be able to develop resistance to cancer therapy. However, very little is known about the role of epithelial/stromal cell interactions in normal breast tissue and the abnormalities of these interactions in breast tumors. In order to identify gene expression changes that occur in specific cell types, we have recently purified each cell type that composes normal and DCIS breast tissue and analyzed their gene expression pattern using SAGE (Serial Analysis of Gene Expression). Based on our SAGE data and subsequent mRNA in situ hybridization studies, we identified the CXCL12 and CXCL14 chemokines and genes specifically overexpressed in DCIS myoepithelial cells and in myofibroblasts of in situ and invasive carcinomas. The goal of this project is to characterize the role of the CXCL12 and CXCL14 chemokines in breast cancer cell growth. In the first aim we will determine if the expression or exogenous addition of CXCL12 and CXCL14 to breast cancer cells influences their proliferation, survival, migration, and invasion using various in vitro culture conditions. The second aim will address the effect of elimination of CXCL12 and CXCL14 in breast cancer cells that endogenously express these chemokines. The in vivo tumorigenicity of breast cancer cells engineered in Aims 1 and 2 will tested by performing xenograft experiments in nude mice. Finally in the third aim we propose to identify and characterize the signaling pathway mediating CXCL14 responses. Currently the receptor and the signaling pathway activated by this chemokine are unknown. Contrary to the well characterized role of chemokines in the regulation of immune function, their role in breast cancer is poorly defined. Thus, this project is highly innovative and significant since it addresses one of the least understood areas of breast tumorigenesis. In addition, the successful completion of this project could be the basis of future clinical trials that could therapeutically target the CXCL12 and CXCL14 signaling pathways and interrupt potentially crucial autocrine/paracrine regulatory loops in breast cancer.