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. We established a coculture system with human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts in order to determine the effects of crosstalk between the stromal and tumor compartments. Two types of homotypic cultures, with fibroblasts and breast cancer cells respectively, were established and compared to a coculture of both cell types. We were able to show that medium conditioned by cocultures of increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. We were also able to demonstate that co-cultures of tumor cells and fibroblasts influence migratory patterns of both partners. A manuscript on these results, "Transient tumor-fibroblast interactions durably increase tumor cell malignancy by a TGF-beta mediated mechanism" was published in PLoS ONE. To further investigate the mechanisms of stromal cell reprogramming in human cancers, we also established fibroblast cell lines derived from human endometrial cancer specimens and patient matched normal endometrial tissue. Specimens for this project were contributed by collaborators in 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. We also observed activation of IGF2 and Wnt 5A. Both of these growth factors are highly tumorigenic. In addition to 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. We identified an 11 microRNA signature of endometrial cancer CAFs and showed that miR-31 represses cancer cells migration and invasion. We identified SATB2 chromatin remodeling protein as a direct target of miR-31. Manipulation of SATB2 protein levels in fibroblasts suggest its function in inducing cell motility. These results were confirmed in mouse tail vein injection experiments. CAFs stimulate tumor cell growth and SATB2 overexpressing fibroblasts may increase the metastatic potential of breast cancer cells when injeected into mice. We have also identified WNT10B as a target of mir-148a. A manuscript on the mir31 - SATB2 interactions has been submitted to PLoS genetics.