Role of Inflammation in the Development and Progression of Pancreatic Cancer

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We first evaluated if the MIF expression level is associated with patients outcome by measuring its expression using qRT/PCR in resected tumors from PDAC cases (N=66). A higher expression of MIF was found to be associated with poorer survival in resected patients (p=0.010). Immunohistochemical analysis of tumors indicated that MIF is expressed both in epithelium and stroma. MIF-overexpressing stable pancreatic cancer cell lines were generated to investigate the mechanistic role of MIF in pancreatic cancer. MIF-overexpression induced epithelial-mesenchymal-transition (EMT)-like characteristics with morphological changes from an epithelial-like appearance to elongated, fibroblastic mesenchymal phenotype, a decreased E-cadherin and an increased vimentin, mRNA and protein expression in stably transfected Capan 2 cells. To explore the mechanism of MIF-induced alterations in EMT-related genes, we determined the expression of miR-200 family members, which has been earlier reported to down-regulate Zeb1, a repressor of E-cadherin. MIF overexpression reduced miR-200b and increased both Zeb1 and Zeb2 expression. Re-expression of miR-200b in MIF overexpressing cells rescued E-cadherin with a decrease in Zeb1 and Zeb2 expression. Furthermore, MIF overexpressing cells also showed an increased cell proliferation, migration in in vitro assay and tumor growth in NOD/SCID mice as compared to control cells. We have further extended our investigation in orthotopic mouse model to explore the role of MIF in tumor aggressiveness. siRNA-mediated inhibition of MIF in Panc1 cells increased E-cadherin and decreased cell proliferation and invasion. An increased MIF expression reduced the sensitivity of Capan 2 cells to Gemcitabine, a standard chemotherapeutic drug for pancreatic cancer. These findings are consistent with the hypothesis that MIF contributes to the pancreatic cancer progression and is associated with patients outcome. Based on these findings, we propose that MIF may be a candidate target for pancreatic cancer therapy. In our investigation of the role of FOX transcription factors in pancreatic cancer, we found that a lower expression of FOXL1 is associated with poor outcome in PDAC cases following resection. We then investigated the role of FOXL1 in the growth and progression of pancreatic cancer. Increased expression of FOXL1 inhibited anchorage dependent and independent cell growth, tumor cell invasion, and induced caspase-3 activity and apoptosis in Panc1 and MiaPaCa2 pancreatic cancer cell lines. TGF-beta or HGF-induced EMT inhibited endogenous expression of FOXL1. siRNA inhibition of FOXL1 increased Zeb1 and vimentin, which are markers for mesenchymal phenotype. Furthermore, FOXL1 suppressed Zeb1 transcription by binding to its promoter. FOXL1 is a predicted target of miR-203. We are currently investigating the interaction of FOXL1 with miRNA-203. An increased miR-203 expression is associated with poor outcome in our sample set of PDAC cases. Lastly, we are testing the hypothesis that FOXL1 over-expression inhibits tumor growth in nude mice. In our initial investigation of FOXO, another member of FOX family of transcription factor, we found that a significantly higher number of cells expressed cytoplasmic phosphorylated-FOXO3 (inactive form) in tumors as compared to the adjacent nontumor tissue in PDAC cases as determined by immunohistochemical staining. This observation is being validated in an independent cohort of PDAC cases.