Exosomes Derived From Colon Cancer Cells Promote Tumor Progression via the Activation of Fibroblasts in Tumor Microenvironment

Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-to-mesenchymal transition (EMT) and functions as a tumor suppressor in colorectal cancer (CRC).Through a yeast two-hybrid screen, BVES was shown to interact with breast cancer anti-estrogen resistance 3 (BCAR3).BCAR3 was originally identified via its upregulation in tamoxifen resistant breast cancer cell lines and functions as an adapter protein, recruiting p130Cas to focal adhesions.We first sought to define the role of BCAR3 in CRC to aid in determining the functional consequence of the BVES-BCAR3 interaction.We began by analyzing the combined Moffitt Cancer Center/Vanderbilt University Medical Center CRC expression array data set consisting of normal, adenomatous, and 250 tumor samples organized by tumor stage.BCAR3 was downregulated in adenomas (p = 0.01075) and further decreased at all CRC stages (p = 4.527e-07).Analysis of TCGA RNASeq data confirmed reductions at all stages (p < 2.2e-16).BCAR3 expression by qRT-PCR was also reduced by three-fold (p < 0.001) in two out of three paired normal and tumor samples acquired from the Cooperative Human Tissue Network (CHTN).Not surprisingly, heterogeneity also existed in BCAR3 protein levels in a panel of CRC cell lines.In Caco2 cells (which express low levels of BCAR3), stable overexpression of BCAR3 impaired migration through uncoated transwells, and reduced both cellular proliferation, as measured by cell number, and clonogenic growth capacity.Stable lentiviral expression of BCAR3 in Caco2 cells impaired migration on uncoated substrates (33.9 ± 1.12 vs. 51.0 ± 4.1 percent wound closure at 48 hours, p = 0.0003) but not on collagen coated or fibronectin coated surfaces as assayed using the magnetically attachable stencils (MAts) assay.shRNA knockdown of BCAR3 and Crispr-Cas9 mediated knockout of BCAR3 in HCT116 cells impaired transwell migration but did not alter proliferation.Additionally, intestinal enteroid 3D cultures from Bcar3 -/- mice had increased branching (4.17 ± 0.32 vs. 2.90 ± 0.33 average branches per day five enteroid, p < 0.01) and have at least 1.4-fold increases in expression of the Wnt target genes Lgr5, Axin2, and Myc (p < 0.01), suggestive of perturbations in the crypt/stem cell compartments.Together, these results implicate BCAR3 in stem cell dynamics, cell migration, and in overall epithelial homeostasis; all processes relevant in the pathogenesis of CRC.