Extracellular Redox Signaling in mammary tumor-fibroblast interactions

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DESCRIPTION (provided by applicant): There is a fundamental gap in knowledge of the role of oxidative stress in the extracellular environment in the etiology of breast cancer. Our long term goal is to elucidate the role of extracellular redox signaling in promoting mammary tumor-fibroblast interactions. Our preliminary data suggest that loss of an extracellular antioxidant enzyme, extracellular superoxide dismutase (EcSOD) is a frequent event in clinical breast cancer tissue and there is an inverse correlation between its expression and clinical stage. We also observed that re- expression of EcSOD in c-Met expressing breast cancer cells strongly attenuated oncogenic stimulation by mammary fibroblasts that overexpress the c-Met ligand, HGF. These findings led us to our hypothesis that loss of EcSOD expression contributes to breast cancer progression and metastasis through oxidative-mediated tumor-fibroblast interactions, via activating the HGF/c-Met axis. The objectives of this study are to identify the mechanisms involved in deregulating the expression of EcSOD in breast cancers and the consequences of the loss of EcSOD expression (and the resulting increase in extracellular oxidative stress) in promoting HGF/c- Met-mediated oncogenesis. We will first determine the involvement of aberrant DNA promoter methylation in regulating EcSOD gene silencing in clinical breast cancer tissues. Next, we will test a novel idea that oxidative tumor microenvironment is one of the key factors that promotes HGF/c-Met signaling. It has been well established that Hepatocyte growth factor (HGF) is often overexpressed and secreted by cancer-associated fibroblasts. This paracrine factor activates its receptor, c-Met on the surface of cancer epithelial cells. In the second aim, we will determine whether loss of EcSOD promotes tumorigenic transformation of normal mammary epithelial cells by HGF-overexpressing mammary fibroblasts. Conversely, we will evaluate whether re-expression of EcSOD in breast cancer cells will attenuate oncogenic stimulation induced by fibroblast- derived HGF. We will further examine whether NADPH oxidase-generated oxidative stress on the plasma membrane is involved in HGF/c-Met activation. We will utilize a 3-dimensional co-culture model that includes both the cancer epithelial cells and their oncogenic 'partner', fibroblasts. In addition t determining the tumorigenic stimulation of fibroblasts on cancer epithelial cells, we will also examine how cancer cells reciprocally alter the phenotype of fibroblasts. We will next investigate the effects of EcSOD on tumor growth and metastasis of c-Met expressing breast cancer cells using a syngeneic mouse model as well as using an orthotopic tumor-fibroblast model. Overall, our multi-pronged study will utilize overexpression, knock-down, inactive mutant protein expression, pharmaceutical SOD mimetic and chemical inhibition approaches to validate the role of extracellular oxidative stress in promoting progression and metastasis of breast cancer. Positive outcome will help in expanding our understanding of the role of extracellular oxidative stress in fueling cancer epithelial-fibroblast interactions, towards development of novel therapeutic interventions.