Tu2002 Characterization of Myeloid - Derived Suppressor Cell Populations in Localized Rectal Adenocarcinoma Patients

Introduction: Stromal cells and transforming colonocytes contribute to colon cancer development.EGF receptors on both stromal cells and colonocytes are required for cancer growth.ADAM17, a metalloprotease that regulates release of EGFR ligands, is also expressed on both cell types and upregulated in colon cancer.Bioinformatic analysis suggests that miR-145, miR-148a and miR-152 can target ADAM17, whereas miR-143, polycistronic with miR-145, targets EGFR effector K-Ras.K-Ras is also increased in colon cancer.The goal of this study was to assess the expression of these miRNAs in stromal cells and colonocytes in normal colon and colon cancers.We also examined the ability of these miRNAs to directly regulate ADAM17 in colon cancer cells and colonic fibroblasts.Methods: Colonoscopic biopsies of normal colon (n=9), colon cancers (n=3) and adjacent mucosa (n=3) were obtained.Colonocytes and stromal cells were isolated by incubation in EDTA followed by mechanical dissociation and differential centrifugation.Cell purities were assessed by Western blotting using colonocyte marker cytokeratin 20 (CK-20) and stromal cell marker vimentin (VIM).Proteins and RNA were purified using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen).miRNAs were quantified by real time PCR using Taqman assays.cDNAs encoding luciferase fused to wildtype or mutant ADAM17-3'UTR were transfected into HCT116 colon cancer cells or CCD-18Co colonic fibroblasts to assess miRNA regulation of ADAM17.Results: Stromal cells and colonocytes showed no cross contamination as assessed by CK20 and VIM reactivity, respectively.Table 1 summarizes miRNA expression levels.Note that miR-143 (44.3-fold), miR-145 (4.3-fold) and miR-152 (2.3-fold) are significantly more abundant in stromal cells than in colonocytes from normal samples, whereas miR-148a (10fold) is significantly greater in colonocytes than stromal cells.miR-143, miR-148a and miR-152 were more than 75% downregulated in tumor stroma, compared to adjacent stroma.miR-143, miR-145 and miR-148a were decreased more than 60% in stroma adjacent to tumors compared to stroma in normal samples.Studies are in progress to assess ADAM17 and K-Ras in cancer stroma and malignant colonocytes.In cell culture, miR-145, miR-148a and miR-152 transfected into colon cancer cells or colonic fibroblasts significantly decreased luciferase regulated by wild type but not mutant ADAM17-3'UTR (mutations predicted to abolish interactions with specific miRNAs).Conclusions: miRNAs predicted to regulate ADAM17 and K-Ras are differentially expressed in stromal and epithelial cells and downregulated in colon cancer.These miRNA differences in stromal and epithelial cells could play significant roles in determining cell-specific expression of their important targets, including ADAM17 and K-Ras in colon cancer.