Role of Immune and Inflammation Mediators in Progression of Pancreatic Cancer

我们早些时候已经证明，肿瘤中MIF的高表达与PDAC患者较差的生存有关。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是治疗PDAC的潜在靶点。为了验证这一假设，我们使用了一种遗传策略，通过在LSL-KrasG12D/+；LSL-Trp53R172H/+；PDX-1-CRE(KPC)小鼠中删除MIF基因，这是一种PDAC的基因工程小鼠模型，它反映了人类疾病的发展、进展以及分子和组织病理学特征。Kaplan-Meier分析表明，MIF缺陷的KPC(MKPC)小鼠的存活时间明显长于野生型MIF的KPC小鼠。此外，MKPC小鼠的转移负担显著降低。KPC和MKPC小鼠的肝转移均以肝转移为主。此外，与KPC小鼠原代肿瘤细胞相比，MKPC小鼠原代肿瘤细胞E-钙粘蛋白表达增强，波形蛋白、ZEB1和N-钙粘蛋白表达降低，提示MKPC肿瘤的EMT减弱。这些发现提供了体内概念证明，MIF是PDAC的潜在治疗靶点。我们目前正在使用药物MIF抑制剂和抗MIF抗体来研究MIF抑制对胰腺癌生长、进展和生存的影响，使用几种临床前模型，包括KPC小鼠、PDTX和人胰腺癌有机物。此外，我们还评估了NOS2/NO信号在PDAC患者中的临床意义，并用qRT-PCR方法检测了107例早期手术切除肿瘤中NOS2的表达。然后根据NOS2表达的中位数将患者分为NOS2高表达组和NOS2低表达组。NOS2表达高于中位数的患者为NOS2高表达组，NOS2表达低于中位数的患者为NOS2低表达组。Kaplan-Meier分析显示，在切除的肿瘤中，与NOS2表达较低的患者相比，NOS2表达较高的患者的生存率明显较差。此外，通过单变量和多变量COX回归分析，肿瘤中NOS2越高，预后越差。此外，免疫组织化学染色显示，与非肿瘤胰管相比，肿瘤组织中NOS2蛋白的表达显著增加。这些结果表明，NOS2是手术切除早期PDAC患者的候选预后标志物，NOS2/NO信号转导通路可能在胰腺癌的进展中起作用。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，而在NKPC小鼠的肿瘤中检测不到NOS2蛋白。Kaplan-Meier分析表明，NKPC小鼠的存活时间明显长于KPC小鼠。与KPC小鼠的肿瘤相比，NKPC肿瘤的巨噬细胞浸润显著减少，这是通过对小鼠巨噬细胞标志物F4/80的免疫组织化学分析以及趋化因子配体2(CCL2)，也称为单核细胞化学吸引蛋白-1(MCP1)的表达显著降低而确定的。此外，qRT/PCR分析显示，与KPC小鼠肿瘤相比，NKPC肿瘤中mir-21的表达显著降低。这些发现利用遗传策略，在体内提供了靶向NOS2可能具有潜在治疗益处的概念证明。此外，NOS2/NO信号通路可能促进PDAC的炎症反应和miR-21的表达。我们目前正在多个PDAC的临床前模型中使用一种小分子的NOS2特异性抑制剂来研究NOS2的药理抑制作用，以评估NOS2抑制对PDAC的治疗效果。此外，基于我们观察到NOS2基因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性中的交互作用来进一步研究NOS2信号的机制作用。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的基础上。正如我们最近所描述的，我们最近发现，MIF和NOS2的高表达(高于中位数)与胰腺癌的低生存率有关。此外，在PDAC基因工程小鼠模型中，MIF或NOS2的基因消融延长了生存时间。我们假设MIF和NOS2是PDAC的候选治疗靶点。基于遗传学概念验证，如上所述，MIF和NOS2可能是PDAC的潜在靶点，我们已经启动了几项临床前研究，使用的是KPC小鼠、患者来源的肿瘤移植瘤和患者器官。这些临床前研究包括对MIF和NOS2的药理抑制，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临床中心启动临床试验提供关键支持。此外，我们正在扩大我们对MIF和NO介导的信号通路在胰腺癌进展中的调控的研究，包括NO和miR-21在胰腺癌进展中的交互作用，以及MIF活性在PDAC患者疾病侵袭性中的遗传调控。

We have earlier shown 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. Additionally, MKPC mice showed a significant reduction in metastatic burden. 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. Furthermore, a higher NOS2 in tumors predicted poor prognosis by both univariable and multivariable 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 build upon our current findings on the role of MIF and NO in pancreatic cancer. As described, we have recently shown that a higher ( above the median) 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 have initiated several preclinical studies using KPC mice, patient-derived tumor xenografts and patient organoids. These preclinical studies involve pharmacological inhibition of MIF and NOS2 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 are extending 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.