Role of Immune and Inflammation Mediators in Progression of Pancreatic Cancer

我们报道了肿瘤中MIF的高表达与PDAC患者的较差生存率相关。此外，我们在另外3个队列中验证了这些发现，包括在公开可用的数据集中。我们假设MIF是PDAC的潜在治疗靶点。为了验证这一假设，我们使用了一种遗传策略，通过删除LSL-KrasG12D/+、LSL-Trp53R172H/+和LSL-Trp53R172H/+中的MIF基因。Pdx-1-Cre （KPC）小鼠，PDAC基因工程小鼠模型，反映了人类疾病的发生、进展、分子和组织病理学特征。Kaplan-Meier分析显示，MIF缺陷型KPC （MKPC）小鼠的存活时间明显长于MIF野生型KPC小鼠（P0.0001）。此外，MKPC小鼠的转移负荷显著降低（P0.01）。KPC和MKPC小鼠的大部分转移都发生在肝脏。此外，与KPC小鼠的原代肿瘤细胞相比，MKPC小鼠的原代肿瘤细胞中E-cadherin的表达增强，vimentin、zeb1和N-cadherin的表达在蛋白和mRNA水平上均降低，表明MKPC肿瘤中EMT的衰减。这些发现提供了体内概念证明，MIF是PDAC的潜在治疗靶点。目前，我们正在使用药理学MIF抑制剂和抗MIF抗体，通过包括KPC小鼠、PDTX和人类胰腺癌类器官在内的几种临床前模型，研究MIF抑制对胰腺癌生长、进展和生存的影响。此外，我们评估了NOS2/NO信号在PDAC患者中的临床相关性，并通过qRT-PCR检测了107例早期切除肿瘤中NOS2的表达。然后根据NOS2表达中值将患者分为NOS2高组和NOS2低组。将肿瘤NOS2表达高于中值的患者定义为NOS2高组，将NOS2表达低于中值的患者定义为NOS2低组。Kaplan-Meier分析显示，与切除肿瘤中NOS2表达较低的患者相比，NOS2表达较高的患者的生存期明显较差（Log-rank检验p=0.012）。此外，单变量（风险比=1.73,95%CI=1.09-2.75, p=0.020）和多变量（风险比=1.75,95%CI=1.10-2.79, p=0.018） cox -回归分析表明，肿瘤中较高的NOS2预示着不良预后。此外，免疫组化染色显示肿瘤胰腺管中NOS2蛋白的表达明显高于非肿瘤胰腺管。这些研究结果表明，NOS2是手术切除的早期PDAC患者的候选预后标志物，NOS2/NO信号可能在胰腺癌进展中发挥作用。为了研究NOS2/NO信号在胰腺癌进展中的作用，我们采用了一种遗传策略，在PDAC的KPC小鼠模型中删除NOS2基因，并产生NOS2缺陷的NKPC小鼠。KPC小鼠的胰腺肿瘤表达高水平的NOS2蛋白，正如预期的那样，在NKPC小鼠的肿瘤中检测不到。Kaplan-Meier分析显示，NKPC小鼠的存活时间明显长于KPC小鼠。与KPC小鼠的肿瘤相比，通过小鼠巨噬细胞标志物F4/80的免疫组织化学分析发现，NKPC肿瘤的巨噬细胞浸润明显减少，趋化因子配体2 (CCL2)，也称为单核细胞趋化蛋白-1 （MCP1）的表达明显降低。此外，qRT/PCR分析显示，与来自KPC小鼠的肿瘤相比，NKPC肿瘤中mir-21的表达显著降低。这些使用遗传策略的研究结果提供了体内概念证明，即靶向NOS2可能具有潜在的治疗益处。此外，NOS2/NO信号可能会增强PDAC中的炎症和miR-21表达。目前，我们正在多个PDAC临床前模型中使用一种小分子NOS2特异性抑制剂对NOS2进行药理抑制，以评估NOS2抑制对PDAC的治疗效果。此外，基于我们观察到NOS2缺陷小鼠胰腺肿瘤中miR-21的低表达，我们正在通过研究NO和miR-21在胰腺癌进展和疾病侵袭中的相互作用来扩展我们对NOS2信号传导的机制作用的研究。我们正在进行的研究建立在我们目前关于MIF和NO在胰腺癌中的作用的发现之上。如前所述，我们最近发现MIF和NOS2的高表达与胰腺癌的低生存率相关。此外，在PDAC基因工程小鼠模型中，基因消融MIF或NOS2均可延长生存期。我们假设MIF和NOS2是PDAC的候选治疗靶点。如上所述，基于基因概念证明，MIF和NOS2可能是PDAC的潜在靶点，我们正在利用KPC小鼠、患者来源的肿瘤异种移植物和患者类器官进行几项临床前研究。这些临床前研究包括单独使用MIF和NOS2或与标准护理药物吉西他滨联合使用MIF和NOS2，以评估它们对胰腺癌生长、进展和生存的影响。我们的主要目标是建立强有力的临床前证据，为在CCR/NCI临床中心开展临床试验提供关键支持。此外，我们还扩展了MIF-和NO介导的信号通路在胰腺癌进展中的调控研究，包括NO和miR-21在胰腺癌进展中的相互作用，以及MIF活性在PDAC患者疾病侵袭性中的遗传调控。在胰腺特异性p53和Kras突变的背景下，NOS2和mir-21双敲除小鼠的产生正在进行这些研究。为了研究MIF的遗传调控，我们重点研究了MIF启动子中存在较多的CATT微卫星重复序列。高于5catt的重复序列会增加MIF的产生和几种炎症性疾病的严重程度。我们正在验证MIF启动子中超过5个CATT重复序列与胰腺癌患者较差的生存率相关的假设。为了验证这一假设，我们与DCEG、NCI合作，对PLCO患者的DNA进行了检测。

We reported that a higher expression of MIF in tumors is associated with poorer survival in PDAC patients. Furthermore, we validated these findings in 3 additional cohorts, including in publicly available data sets. We hypothesized that MIF is a potential therapeutic target in PDAC. To test this hypothesis, we used a genetic strategy by deleting MIF gene in LSL-KrasG12D/+;LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice, a genetically engineered mouse model of PDAC, which mirrors the development, progression, and molecular and histopathological characteristics of human disease. Kaplan-Meier analysis showed that MIF-deficient KPC (MKPC) mice survived significantly longer than that of KPC mice with wild-type MIF (P0.0001). Additionally, MKPC mice showed a significant reduction in metastatic burden (P0.01). Most of the metastasis occurred in liver of both KPC and MKPC mice. Furthermore, primary tumor cells isolated from MKPC mice showed an enhanced expression of E-cadherin and reduced expression of vimentin, zeb1 and N-cadherin at both the protein and mRNA levels, as compared with primary tumor cells from KPC mice suggesting an attenuation of EMT in MKPC tumors. These findings provided in vivo proof-of-concept that MIF is a potential therapeutic target in PDAC. We are currently using pharmacological MIF inhibitors and anti-MIF antibodies to examine the effect of MIF inhibition on pancreatic cancer growth, progression and survival using several pre-clinical models including KPC mice, PDTX and human pancreatic cancer organoids. Additionally, we assessed the clinical relevance of NOS2/NO signaling in the patients with PDAC and examined the NOS2 expression by qRT-PCR in tumors from 107 early stage, resected cases. Patients were then divided into NOS2-high and NOS2-low groups based on the median value of NOS2 expression. Patients with the tumor NOS2 expression above the median value were defined as NOS2-high group, and the patients with the NOS2 expression lower than the median value constituted NOS2-low group. Kaplan-Meier analysis showed that patients with a higher NOS2 had significantly poorer survival as compared to the patients with lower NOS2 expression in resected tumors (Log-rank test p=0.012). Furthermore, a higher NOS2 in tumors predicted poor prognosis by both univariable (hazard ratio=1.73, 95%CI=1.09-2.75, p=0.020) and multivariable (hazard ratio=1.75, 95%CI=1.10-2.79, p=0.018) Cox-regression analyses. Additionally, immunohistochemical staining showed a significantly higher NOS2 protein expression in tumors as compared to nontumor pancreatic ducts. These findings showed that NOS2 is a candidate prognostic marker in early stage PDAC patients undergoing surgical resection and NOS2/NO signaling may play a role in pancreatic cancer progression. To examine the role of NOS2/NO signaling in pancreatic cancer progression, we used a genetic strategy by deleting the NOS2 gene in KPC mouse model of PDAC and generating NOS2-deficient NKPC mice. Pancreatic tumors in KPC mice expressed a high level of NOS2 protein, which as expected was undetectable in tumors from NKPC mice. Kaplan-Meier analysis showed that NKPC mice survived significantly longer than KPC mice. Compared to tumors in KPC mice, NKPC tumors showed significantly reduced macrophages infiltration, as determined by immunohistochemical analysis of F4/80, a murine macrophage marker and a marked decrease in the expression of chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP1). Furthermore, qRT/PCR analysis revealed a significant decrease in the expression of mir-21 in NKPC tumors as compared to tumors from KPC mice. These findings, using a genetic strategy, provided in vivo proof-of-concept that targeting NOS2 may have potential therapeutic benefits. Furthermore, NOS2/NO signaling may enhance inflammation and miR-21 expression in PDAC. We are currently pursuing pharmacological inhibition of NOS2 using a small molecule NOS2-specific inhibitor in multiple preclinical models of PDAC to assess the therapeutic effect of NOS2 inhibition on PDAC. Furthermore, based on our observation of a lower expression of miR-21 in pancreatic tumors from NOS2-deficient mice, we are extending our investigation on the mechanistic role of NOS2 signaling by investigating the interactive role of NO and miR-21 in pancreatic cancer progression and disease aggressiveness. Our ongoing research builds upon our current findings on the role of MIF and NO in pancreatic cancer. As described, we have recently shown that a higher expression of MIF and NOS2 are associated with poor survival in pancreatic cancer. Furthermore, genetic ablation of either MIF or NOS2 in a genetically engineered mouse model of PDAC prolonged survival. We hypothesize that MIF and NOS2 are candidate therapeutic targets in PDAC. Based on the genetic proof-of-concept, as described above that MIF and NOS2 may be potential targets for PDAC, we are pursuing several preclinical studies using KPC mice, patient-derived tumor xenografts and patient organoids. These preclinical studies involve pharmacological inhibition of MIF and NOS2 as single agents or in combination of the standard of care drug gemcitabine to evaluate their effects on the pancreatic cancer growth, progression, and survival. Our major goal is to establish strong preclinical evidence providing key support to initiate a clinical trial at CCR/NCI clinical center. Furthermore, we have extended our investigation on the regulation of MIF- and NO-mediated signaling pathways in pancreatic cancer progression, which includes the interactive role of NO and miR-21 in pancreatic cancer progression, and genetic regulation of MIF activity in disease aggressiveness in PDAC patients. These investigations are being pursued by the generation of NOS2 and mir-21 double knockout mice in the background of pancreas specific p53 and Kras mutation. To examine the genetic regulation of MIF, we have focused on the presence of a higher number of CATT microsatellite repeat in MIF promoter. A higher than 5 CATT repeat enhances the MIF production and severity of several inflammatory diseases. We are testing the hypothesis that a higher than 5 CATT repeats in MIF promoter is associated with poorer survival in pancreatic cancer patients. To test this hypothesis DNA of patients from PLCO cohorts have been examined in collaboration with the DCEG, NCI.