Role of Inflammation in the Development and Progression of Pancreatic Cancer

我们首先通过使用qRT/PCR测量其在来自PDAC病例的切除肿瘤中的表达来评估MIF表达水平是否与患者结果相关（N=66）。发现MIF的较高表达与切除患者的较差存活率相关（p=0.010）。免疫组化结果显示MIF在上皮和间质均有表达。产生MIF过表达稳定的胰腺癌细胞系以研究MIF在胰腺癌中的机制作用。MIF过表达诱导的上皮间质转化（EMT）样的特征与形态学变化，从上皮样外观延长，成纤维细胞间质表型，减少E-钙粘蛋白和增加波形蛋白，mRNA和蛋白质的表达在稳定转染的Capan 2细胞。为了探索MIF诱导EMT相关基因改变的机制，我们测定了miR-200家族成员的表达，该家族成员先前已报道下调E-cadherin的阻遏物Zeb 1。MIF过表达减少miR-200 b，增加Zeb 1和Zeb 2的表达。在MIF过表达细胞中重新表达miR-200 b拯救了E-cadherin，同时降低了Zeb 1和Zeb 2的表达。此外，与对照细胞相比，MIF过表达细胞还显示出增加的细胞增殖、体外测定中的迁移和NOD/SCID小鼠中的肿瘤生长。我们在原位小鼠模型中进一步扩展了我们的研究，以探讨MIF在肿瘤侵袭性中的作用。在Panc 1细胞中，siRNA介导的MIF抑制增加了E-钙粘蛋白，降低了细胞增殖和侵袭。增加的MIF表达降低了Capan 2细胞对吉西他滨（胰腺癌的标准化疗药物）的敏感性。这些发现与MIF促进胰腺癌进展并与患者结局相关的假设一致。基于这些发现，我们建议MIF可能是胰腺癌治疗的候选靶点。在我们对FOX转录因子在胰腺癌中的作用的研究中，我们发现FOXL 1的低表达与PDAC病例切除后的不良预后相关。然后，我们研究了FOXL 1在胰腺癌生长和进展中的作用。FOXL 1的表达增加抑制了Panc 1和MiaPaCa 2胰腺癌细胞系中锚定依赖性和非锚定性细胞生长、肿瘤细胞侵袭，并诱导caspase-3活性和凋亡。TGF-β或HGF诱导的EMT抑制FOXL 1的内源性表达。siRNA抑制FOXL 1增加了Zeb 1和波形蛋白，这是间充质表型的标志物。此外，FOXL 1抑制Zeb 1的转录通过结合到其启动子。FOXL 1是miR-203的预测靶点。我们目前正在研究FOXL 1与miRNA-203的相互作用。在我们的PDAC病例样本集中，miR-203表达增加与不良结局相关。最后，我们正在测试FOXL 1过表达抑制裸鼠肿瘤生长的假设。在我们对转录因子FOX家族的另一个成员FOXO的初步研究中，我们发现，与PDAC病例中相邻的非肿瘤组织相比，肿瘤中表达胞质磷酸化FOXO 3（非活性形式）的细胞数量显著增加，这是通过免疫组织化学染色确定的。这一观察结果正在PDAC病例的独立队列中得到验证。

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.