Role of Immune and Inflammation Mediators in Progression of Pancreatic Cancer

我们先前已经表明，肿瘤中MIF的高表达与PDAC患者的生存率较差相关。此外，我们在另外3个队列中验证了这些发现，包括公开可用的数据集。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-KrasG 12 D/+; LSL-Trp 53 R172 H/+; Pdx-1-Cre（KPC）小鼠中的MIF基因，这是一种PDAC的遗传工程小鼠模型，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分析显示，MIF缺陷型KPC（MKPC）小鼠的存活时间显著长于野生型MIF的KPC小鼠。此外，MKPC小鼠显示转移负荷显著降低。KPC和MKPC小鼠的转移主要发生在肝脏。此外，从MKPC小鼠中分离的原发性肿瘤细胞在蛋白质和mRNA水平上显示出E-cadherin的表达增强和波形蛋白、zeb 1和N-cadherin的表达降低，与来自KPC小鼠的原发性肿瘤细胞相比，这表明MKPC肿瘤中的EMT减弱。这些发现提供了体内概念验证，即MIF是PDAC的潜在治疗靶点。我们目前正在使用药理学MIF抑制剂和抗MIF抗体，使用几种临床前模型（包括KPC小鼠、PDTX和人胰腺癌类器官）来检查MIF抑制对胰腺癌生长、进展和存活的影响。此外，我们评估了NOS 2/NO信号在PDAC患者中的临床相关性，并通过qRT-PCR检测了107例早期切除病例肿瘤中NOS 2的表达。然后根据NOS 2表达的中位数将患者分为NOS 2-高和NOS 2-低组。将肿瘤组织中NOS 2表达高于中位数的患者定义为NOS 2高表达组，将肿瘤组织中NOS 2表达低于中位数的患者定义为NOS 2低表达组。Kaplan-Meier分析显示，在切除的肿瘤中，具有较高NOS 2表达的患者的生存率显著低于具有较低NOS 2表达的患者。此外，通过单变量和多变量Cox回归分析，肿瘤中较高的NOS 2预测预后不良。此外，免疫组化染色显示，与非肿瘤胰管相比，肿瘤中的NOS 2蛋白表达显著更高。这些发现表明，NOS 2是接受手术切除的早期PDAC患者的候选预后标志物，并且NOS 2/NO信号传导可能在胰腺癌进展中起作用。为了研究NOS 2/NO信号传导在胰腺癌进展中的作用，我们使用了一种遗传策略，通过删除PDAC的KPC小鼠模型中的NOS 2基因并产生NOS 2缺陷的NKPC小鼠。KPC小鼠的胰腺肿瘤表达高水平的NOS 2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小鼠的存活时间显著长于KPC小鼠。与KPC小鼠中的肿瘤相比，NKPC肿瘤显示巨噬细胞浸润显着减少，这是通过F4/80（小鼠巨噬细胞标志物）的免疫组织化学分析确定的，并且趋化因子配体2（CCL 2）的表达显着减少，也称为单核细胞趋化蛋白-1（MCP 1）。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。这些发现，使用遗传策略，提供了体内概念验证，靶向NOS 2可能具有潜在的治疗益处。此外，NOS 2/NO信号可能增强PDAC中的炎症和miR-21表达。我们目前正在寻求使用小分子NOS 2特异性抑制剂在PDAC的多个临床前模型中药理学抑制NOS 2，以评估NOS 2抑制对PDAC的治疗效果。此外，基于我们在NOS 2缺陷小鼠胰腺肿瘤中观察到miR-21表达较低，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相互作用来扩展我们对NOS 2信号传导机制作用的研究。我们正在进行的研究建立在我们目前对MIF和NO在胰腺癌中的作用的研究结果的基础上。如前所述，我们最近发现MIF和NOS 2的高表达（高于中位数）与胰腺癌的生存率相关。此外，在PDAC的基因工程小鼠模型中，MIF或NOS 2的基因切除延长了生存期。我们推测MIF和NOS 2是PDAC的候选治疗靶点。基于遗传概念验证，如上所述，MIF和NOS 2可能是PDAC的潜在靶标，我们已经使用KPC小鼠、患者来源的肿瘤异种移植物和患者类器官启动了几项临床前研究。这些临床前研究涉及MIF和NOS 2的药理学抑制，以评估其对胰腺癌生长、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，我们正在扩大我们的调查对调节MIF和NO介导的信号通路在胰腺癌的进展，其中包括NO和miR-21在胰腺癌进展中的相互作用，和遗传调节的MIF活性在疾病的侵袭性在PDAC患者。

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.