Role of MIF in Pancreatic Cancer: a) MIF Expression and Clinical Outcome in Patients with Pancreatic Cancer We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts outcome in resected cases. Kaplan-Meier analysis showed that patients with a higher MIF expression in tumors had a significantly poorer survival when compared with patients who had a lower MIF expression (P=0.032, log-rank test). Univariable and multivariable Cox proportional hazard analysis was used to further evaluate the association of MIF expression in tumors and other clinical prognostic factors with patient outcome. Univariable Cox analysis showed that a high MIF expression (HR, 2.21, 95% CI, 1.16-4.22, P=0.016) and a high differentiation grade (HR, 1.86, 95% CI, 1.01-3.45, P=0.048) were each associated with poor survival. We did not see any association of tumor stage or resection margin status with survival in this cohort. Furthermore, multivariable analysis showed that MIF was associated with patients survival independent of tumor grade (HR, 2.26, 95% CI, 1.17-4.37, P=0.015). These data indicated that MIF is an independent predictor of survival in resected PDAC patients (Funamizu et. al. Int J Cancer, 2012). b) Mechanistic Role of MIF in the Progression of Pancreatic Cancer We tested the hypothesis that MIF plays a role in the acquisition of EMT phenotype in pancreatic cancer. Stably MIF-overexpressing, Capan 2 and Panc 1 pancreatic cancer cell lines showed a decrease in E-cadherin and an increase in vimentin mRNA and protein expression. Furthermore, MIF over-expression reduced miR 200b and increased ZEB1 and ZEB2 expression in Capan 2 cells. Reexpressing miR 200b in MIF overexpressing cells reduced ZEB1, ZEB2 and vimentin expression and increased the expression of E-cadherin. We further confirmed these findings by shRNA-mediated knockdown of MIF in these cells. Knocking down MIF significantly increased the expression of miR200b and Ecadherin, while decreasing both ZEB1 and ZEB2. These data indicated that MIF-induced EMT is mediated, at least in part, through miR-200/Zeb/Ecadherin axis. c) Effect of MIF on Pancreatic Tumor Growth and Metastasis In Vivo To further elucidate the role of MIF in pancreatic cancer, we investigated the effect of MIF on tumor growth and metastasis. Subcutaneous injection of stable Capon 2 MIF-overexpressing or control cells in nude mice showed a significant increase in tumor growth with MIF-overexpressing cells as compared with control. Subcutaneous tumors produced by Capan 2 stable MIF-transfectants and controls were harvested and cut into pieces of approximately 1 mm3 for pancreas orthotopic xenograft by surgical implantation. Forty-seven days following the orthotopic implantation, mice were euthanized. Tumor implants from MIF-transfectants showed a significant increase in tumor growth and metastasis. The principal sites of distant metastasis included liver, lymph nodes, peritoneum, intestine and spleen. These in vivo findings showed that MIF accelerates primary tumor growth and systemic dissemination of pancreatic cancer indicating its potential as a candidate therapeutic target. e) Effect of MIF-deficiency on pancreatic cancer growth, progression and survival in genetically engineered mouse model of pancreatic cancer In this study, we are using a genetically engineered mouse model, LSL-KrasG12D,LSL-Trp53R172H/+,Pdx Cre (KPC), of pancreatic cancer, which faithfully recapitulates the development and progression of human pancreatic ducal adenocarcinoma. Pancreatic tumors in KPC mice express a higher level of MIF as compared to the pancreas from wild-type controls. Based on our earlier findings, showing a role of MIF in pancreatic tumor growth and progression, we are further testing the hypothesis that MIF-deficiency reduces the growth and progression of pancreatic cancer and increases survival in genetically engineered mouse model of pancreatic cancer. To test this hypothesis, we have generated MIF-deficient KPC pancreatic cancer mouse model. MIF-deficient and wildtype littermate KPC mice were followed till the signs of moribundity appear. In this study we are asking two specific questions: 1) Does MIF-deficiency enhance the life span of KPC mice with lethal PDAC? and 2) Does MIF-deficient KPC mice show reduced metastasis? Our initial finding showed that MIF-deficient KPC mice survive longer as compared to KPC mice with wild type MIF (Kaplan-Meier analysis, Log-rank test, P0.01). Furthermore, MIF deficient KPC mice showed a significant reduction in distant metastasis. We are currently investigating the molecular mechanism by which MIF contributes to the pancreatic tumor progression in KPC mice. The initial findings from this ongoing genetic study provide proof of concept that MIF inhibition may have anti-tumorigenic effect and should be further evaluated as a potential strategy for therapeutic intervention in pancreatic cancer. Several small molecule inhibitors of MIF have been developed and successfully used in animal models of inflammatory diseases. We will further use small molecule inhibitor of MIF in the KPC mouse model of pancreatic cancer to evaluate its potential therapeutic effect. Role of NOS2/NO in Pancreatic Cancer: 1) NOS2 Expression and Clinical outcome in Patients with Pancreatic Cancer We first assessed the biological relevance of NO in human pancreatic cancer by determining the association of NOS2 expression level in tumor and survival in 107 surgically resected patients with PDAC. NOS2 mRNA expression was determined by qRT-PCR and dichotomized by median value into high (above median) and low (below median) groups. The patients with a higher NOS2 expression in tumors showed poorer survival as compared to the patients with lower NOS2 expression level (Kaplan-Meier analysis, Log-rank test, P=0.011). 2) Examining the role of NOS2/NO in pancreatic cancer progression by genetic deletion of NOS2 in a genetically engineered mouse model (KPC) of pancreatic cancer. We tested the hypothesis that NO enhances pancreatic cancer progression. To test this hypothesis, we used a genetic strategy of deleting NOS2 in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas-specific activation of mutant-KRAS and p53 through cre recombinase. One of the clinically relevant endpoints in cancer management is the survival benefit. We evaluated if NOS2-deficiency confers any survival advantage in KPC mice with PDAC. NOS2-deficient (KPC/NOS2-/-) (N=48) and NOS2-wild type KPC (N=53) littermates were generated and followed till the mice showed signs related to moribundity, previously described as indications preceding death in this mouse model of PDAC. Mice were euthanized and a complete necropsy was performed on each mouse. We found that KPC/NOS2-/- mice showed a longer survival time as compared to the littermate KPC mice with wild-type NOS2 (Kaplan-Meier analysis, Log-rank test, P0.01). Currently, we are investigating the mechanistic and functional role of NOS2/NO signaling in tumor development and progression in KPC mice.
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