Mechanisms of Stromal Cell Activation by the Developing Tumor

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It is well established that the tumor stroma contributes to tumor development by promoting tumor growth and migration from the primary tumor to metastatic sites. One of the components of stromal responsive cells are fibroblasts in the normal surrounding tissue that are reprogrammed by the tumor and are referred to as cancer-activated fibroblasts (CAFs). The mechanisms whereby CAFs promote tumor progression are only beginning to emerge. CAFs have been shown to express high levels of proteolytic enzymes, including matrix metalloproteinases (MMPs) and cathepsins (1-3), which may stimulate tumor dissemination as well as growth. CAFs may secrete chemokines such as monocyte chemotactic protein-1 and cytokines such as interleukin-1. In addition, CAFs secrete stromal-derived factor-1 (SDF-1) which mediates bone marrow-derived endothelial cell recruitment and directly increases tumor proliferation. To investigate the mechanisms of stromal cell reprogramming in human cancers, we established fibroblast cell lines derived from human endometrial cancer specimens and matched normal endometrial tissue. This project was performed in collaboration with the Division of Gynecologic Oncology, University of Virginia. The goal of the project is to characterize the pathways affected in CAFs compared to normal fibroblasts by comparing gene expression profiles, using a microarray approach and protein expression, using mass spectroscopy in cancer and normal specimens. Since the mechanism(s) of fibroblast reprogramming are unknown, we also set out to investigate the role of differential expression of microRNAs in stromal cell activation by tumors and to analyze its possible function in promoting tumor growth and/or metastasis. We accumulated 7 pairs of normal/tumor-derived fibroblasts and determined the purity of cell population by staining with epithelial and fibroblasts markers. We also completed mRNA and microRNA profiling. The preliminary results show that, in agreement with published data, endometrial CAFs express higher levels of MMPs and a number of other proteolytic enzymes. The expression of TGF beta2 is also upregulated several fold. In addition we observed activation of IGF2 and Wnt 5A. Both of these growth factors are highly tumorigenic. Besides the activation of Wnt5A, we found decreased expression of secreted Frizzled related protein (sFRP4), an inhibitor of the Wnt pathway. These data were validated by real-time PCR. We also studied the possible mechanisms of sFRP downregulation in CAFs and found that it is suppressed by methylation that can be reversed by 5-azacytidine treatment. Profiling of microRNA differentially expressed by CAFs and fibroblasts from normal adjacent tissue resulted in identification of several candidates that are now being validated. At present we are preparing for the further validation of the results by matching the microRNAs with their predicted target gene(s) present in our microarray signature. This screen allowed us to identify several putative targets that maybe involved in tumor progression. One such pair is miR31 (reduced in CAFs) and the SATB2 gene (increased in CAFs). We showed that overexpression of miR-31 in fibroblasts derived from cancer samples downregulated expression of endogenous SATB2 mRNA. Similar results were obtained when we used other types of fibroblasts (normal skin or breast fibroblasts).