Unraveling the Role of NADPH Oxidase in Inflammation-associated Pancreatic Diseases

摘要 胰腺炎症是胃肠相关临床发病率和死亡率的主要原因， 针对胰腺炎性疾病的有效治疗策略仍然是未满足的医疗需求。 炎症是胰腺炎及相关腺泡-导管化生发病机制的共同特征 (ADM)胰腺上皮内瘤变（PanIN）病变和胰腺导管腺癌（PDAC）。 鉴于炎症在胰腺病理学谱中的突出作用，了解胰腺- 特定的炎症机制对于相关疾病的预防和治疗至关重要。 已知炎症诱导活性氧（ROS）。在已知的致病因素中，ROS既起作用， 信号分子和炎症介质。野生型KRAS在半胱氨酸上经历ROS修饰 氧化还原敏感的NKC 118 D基序导致短暂的超活化。在约30%的患者中观察到KRAS突变体 慢性胰腺炎患者中，提示KRAS突变在胰腺炎症中的关键作用。 在过去的三十年里，突变KRAS被认为是被锁定在一个组成性的活跃状态。然而，在这方面， 最近的研究发现，突变的KRAS在内源性水平上不是组成型活性的，但可以是 被炎性损伤过度激活，导致持续的炎症、不可逆的ADM和PanIN损伤。 然而，将炎症与突变型KRAS过度活化和 相关的病理学仍然难以捉摸。研究表明，NADPH氧化酶（NOX）是 胰腺炎诱导的炎症和突变KRAS激活的主要酶产生ROS。 然而，突变型KRAS是否与野生型KRAS经历相同的ROS修饰，从而导致KRAS突变。 在炎症情况下观察到的活动过度是未知的。本建议的目的是确定 分子介质，并阐明炎症如何促进突变KRAS的潜在机制 过度活化和相关的胰腺病理学。值得注意的是，通过切除NOX对接亚基p22 phox 在胰腺腺泡细胞中表达内源性水平KRASG 12 D/+的小鼠中，我们证明NOX 抑制显著抑制KRASG 12 D/+的过度活化，表明NOX不仅是下游效应物 而且是KRASG 12 D的潜在上游调节剂。因此，NOX和KRASG 12 D潜在地形成共活化 前馈回路是诱导慢性炎症、不可逆ADM和PanIN损伤所必需的。我们 假设在炎症条件下，NOX产生ROS以修饰KRASG 12 D的氧化还原敏感基序， 导致KRASG 12 D过度活化和相关的病理。我们的假设将通过三个具体的 目的是在小鼠模型中的炎症损伤的背景下。目的：1.探讨一氧化氮（NOX）在胰腺癌中的作用。 炎症目的2是确定NOX是否是KRASG 12 D过度活化、持续炎症 不可逆ADM和PanIN病变。目的3是确定氧化还原敏感的NKC 118 D基序是否是 KRASG 12 D超活化和相关病理学。在我们的研究中获得的信息将填补突出的 关于炎症性损伤如何促进突变KRAS过度活化和相关病理学的知识缺口 并指导针对人类这些疾病的新型预防和治疗策略的设计。

Abstract Pancreatic inflammation is the major cause of gastrointestinal-related clinical morbidity and mortality, and effective therapeutic strategies against pancreatic inflammatory diseases remain an unmet medical need. Inflammation is a common feature in the pathogenesis of pancreatitis and associated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) lesions, and pancreatic ductal adenocarcinoma (PDAC). Given the prominent role of inflammation in the spectrum of pancreatic pathologies, understanding pancreas- specific inflammatory mechanisms is critical for the prevention and treatment of the associated diseases. Inflammation is known to induce reactive oxygen species (ROS). Among known causal factors, ROS act as both a signaling molecule and a mediator of inflammation. Wild-type KRAS is subject to ROS modification on cysteine of the redox-sensitive NKC118D motif leading to a transient hyperactivation. KRAS mutants are observed in ~30% of patients with chronic pancreatitis, suggesting the critical role of KRAS mutations in pancreatic inflammation. In the past thirty years, mutant KRAS has been viewed as being locked in a constitutively active state. However, recent studies have found that mutant KRAS, at an endogenous level, is not constitutively active but can be hyper-activated by inflammatory insults leading to sustained inflammation, irreversible ADM, and PanIN lesions. However, the molecular mediator and mechanism linking inflammation to mutant KRAS hyperactivation and associated pathologies remain elusive. Studies have shown that NADPH oxidases (NOXs) are a major mediator of pancreatitis-induced inflammation and major enzymes activated by mutant KRAS for the generation of ROS. However, whether mutant KRAS is subject to the same ROS modification as wild-type KRAS leading to the observed hyperactivity in the context of inflammation is unknown. The objective of this proposal is to identify the molecular mediator and unravel the underlying mechanism on how inflammation promotes mutant KRAS hyperactivation and associated pancreatic pathologies. Notably, by ablating the NOX-docking subunit p22phox in mice expressing an endogenous level of KRASG12D/+ in pancreatic acinar cells, we demonstrate that NOX inhibition considerably curbs KRASG12D/+ hyperactivation, suggesting that NOX is not only a downstream effector but also a potential upstream regulator of KRASG12D. Thus, NOX and KRASG12D potentially form a co-activation feed-forward loop necessary to induce chronic inflammation, irreversible ADM, and PanIN lesions. We hypothesize that under inflammation, NOX generates ROS to modify the redox-sensitive motif of KRASG12D, leading to KRASG12D hyperactivation and associated pathologies. Our hypothesis will be tested with three specific aims in mouse models in the context of inflammatory insults. Aim 1 is to determine the role of NOX in pancreatic inflammation. Aim 2 is to determine if NOX is necessary for KRASG12D hyperactivation, sustained inflammation, irreversible ADM, and PanIN lesions. Aim 3 is to determine if the redox-sensitive NKC118D motif is required for KRASG12D hyperactivation and associated pathologies. Information gained in our studies will fill the outstanding knowledge gap on how inflammatory insults promote mutant KRAS hyperactivation and associated pathologies and guide the design of novel preventive and therapeutic strategies against these diseases in humans.