Role of Immune and Inflammation Mediators in Progression of Pancreatic Cancer

MIF在胰腺癌中的作用：a) MIF在胰腺癌患者中的表达和临床预后我们检验了MIF有助于胰腺癌侵袭性和预测切除病例预后的假设。Kaplan-Meier分析显示，肿瘤中MIF表达较高的患者的生存期明显低于MIF表达较低的患者（P=0.032， log-rank检验）。采用单变量和多变量Cox比例风险分析进一步评价肿瘤中MIF表达及其他临床预后因素与患者预后的关系。单变量Cox分析显示，高MIF表达（HR, 2.21, 95% CI, 1.16-4.22， P=0.016）和高分化等级（HR, 1.86, 95% CI, 1.01-3.45， P=0.048）均与生存率低相关。在这个队列中，我们没有看到肿瘤分期或切除边缘状态与生存率的任何关联。此外，多变量分析显示，MIF与患者生存相关，与肿瘤分级无关（HR, 2.26, 95% CI, 1.17-4.37， P=0.015）。这些数据表明，MIF是PDAC切除患者生存的独立预测因子（Funamizu et. al. Int J Cancer, 2012）。b) MIF在胰腺癌进展中的机制作用我们检验了MIF在胰腺癌中EMT表型获得中起作用的假设。稳定过表达mif的Capan 2和Panc 1胰腺癌细胞系显示E-cadherin表达减少，vimentin mRNA和蛋白表达增加。此外，MIF过表达降低了Capan 2细胞中miR 200b的表达，增加了ZEB1和ZEB2的表达。在MIF过表达细胞中重新表达miR 200b可降低ZEB1、ZEB2和vimentin的表达，增加E-cadherin的表达。我们通过shrna介导的MIF在这些细胞中的下调进一步证实了这些发现。抑制MIF可显著提高miR200b和Ecadherin的表达，同时降低ZEB1和ZEB2的表达。这些数据表明，if诱导的EMT至少部分是通过miR-200/Zeb/Ecadherin轴介导的。c)体内MIF对胰腺肿瘤生长和转移的影响为了进一步阐明MIF在胰腺癌中的作用，我们研究了MIF对肿瘤生长和转移的影响。皮下注射稳定的Capan 2 mif过表达细胞或对照细胞的裸鼠，与对照组相比，mif过表达细胞的肿瘤生长明显增加。获取由Capan 2稳定的mif -转染物和对照组产生的皮下肿瘤，切成约1mm3的块，通过手术植入胰腺原位异种移植物。原位植入后47天，对小鼠实施安乐死。转染mif的肿瘤植入物显示出肿瘤生长和转移的显著增加。远处转移的主要部位包括肝脏、淋巴结、腹膜、肠和脾脏。这些体内研究结果表明，MIF加速了原发性肿瘤的生长和胰腺癌的全身传播，表明其作为候选治疗靶点的潜力。e) mif缺乏对胰腺癌基因工程小鼠模型胰腺癌生长、进展和存活的影响。本研究采用胰腺癌基因工程小鼠模型LSL-KrasG12D、LSL-Trp53R172H/+、Pdx Cre (KPC)，真实再现了人类胰腺ducal腺癌的发生和发展过程。与野生型对照相比，KPC小鼠的胰腺肿瘤表达更高水平的MIF。基于我们早期的研究结果，显示了MIF在胰腺肿瘤生长和进展中的作用，我们正在进一步验证MIF缺乏减少胰腺癌生长和进展并增加胰腺癌基因工程小鼠模型存活的假设。为了验证这一假设，我们建立了mif缺陷KPC胰腺癌小鼠模型。跟踪观察mif缺陷小鼠和野生型同窝KPC小鼠，直至出现死亡迹象。在这项研究中，我们提出了两个具体的问题：1)mif缺乏是否会延长致命PDAC的KPC小鼠的寿命？2) mif缺陷的KPC小鼠是否表现出转移减少？我们的初步研究结果显示，MIF缺失的KPC小鼠比MIF野生型的KPC小鼠存活时间更长（Kaplan-Meier分析，Log-rank检验，P0.01）。此外，MIF缺陷的KPC小鼠显示出远处转移的显著减少。我们目前正在研究MIF促进KPC小鼠胰腺肿瘤进展的分子机制。这项正在进行的遗传研究的初步发现证明了MIF抑制可能具有抗肿瘤作用，并应进一步评估作为胰腺癌治疗干预的潜在策略。几种MIF小分子抑制剂已被开发并成功应用于炎性疾病的动物模型。我们将进一步将小分子mif -抑制剂应用于KPC小鼠胰腺癌模型，评估其潜在的治疗效果。NOS2/NO在胰腺癌中的作用：1)NOS2在胰腺癌患者中的表达与临床预后我们首先通过测定107例手术切除的PDAC患者肿瘤中NOS2表达水平与生存的关系来评估NO在人胰腺癌中的生物学相关性。采用qRT-PCR检测NOS2 mRNA表达，并按中位数分为高（高于中位数）组和低（低于中位数）组。肿瘤中NOS2高表达患者的生存期较NOS2低表达患者差（Kaplan-Meier分析，Log-rank检验，P=0.011）。2)在胰腺癌基因工程小鼠模型（KPC）中，通过NOS2基因缺失检测NOS2/NO在胰腺癌进展中的作用。我们验证了NO促进胰腺癌进展的假设。为了验证这一假设，我们在通过cre重组酶激活突变体kras和p53的胰腺癌基因工程小鼠模型（KPC小鼠）中使用了一种删除NOS2的遗传策略。癌症治疗的临床相关终点之一是生存获益。我们评估了nos2缺乏是否会给患有PDAC的KPC小鼠带来生存优势。产生NOS2缺陷（KPC/NOS2-/-） （N=48）和NOS2野生型KPC （N=53）的窝仔，并进行跟踪，直到小鼠出现与死亡相关的迹象，这些迹象之前被描述为PDAC小鼠模型的死亡前指征。对小鼠实施安乐死，并对每只小鼠进行彻底尸检。我们发现KPC/NOS2-/-小鼠比野生型NOS2的KPC小鼠存活时间更长（Kaplan-Meier分析，Log-rank检验，P0.01）。目前，我们正在研究NOS2/NO信号在KPC小鼠肿瘤发生发展中的机制和功能作用。与来自KPC/NOS2-/-小鼠的肿瘤细胞相比，KPC小鼠分离的原代胰腺肿瘤细胞的增殖、迁移和侵袭能力增强。此外，与KPC/NOS2-/-小鼠胰腺肿瘤相比，cleaved caspase-3检测KPC小鼠胰腺肿瘤细胞凋亡减少，E-cadherin表达水平较低。

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 Capan 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 MIF-inhibitor 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. Primary pancreatic tumor cells isolated from KPC mice showed enhanced proliferation, migration and invasion as compared to tumor cells from KPC/NOS2-/- mice. Furthermore, pancreatic tumors in KPC mice exhibited decrease in apoptosis as determined by cleaved caspase-3 and expressed a lower level of E-cadherin as compared to the pancreatic tumors in KPC/NOS2-/- mice.