Inflammasomes in pathogenesis of mesothelioma

描述（由申请人提供）：石棉纤维会损伤肺间皮细胞，从而引发代偿性增殖反应、炎症和恶性间皮瘤（MM）。在本提案中，我们将重点关注炎性体成分、NOD 样受体蛋白 3 (NLRP3)、含有 CARD (ASC) 的凋亡相关斑点样蛋白和 Caspase-1，我们发现这些成分在 MM 细胞和肿瘤中减弱，并在间皮细胞中被石棉激活。 NLRP3 和 caspase-1 水平降低可能是这些肿瘤耐药性增强的原因。在此，我们假设 MM 细胞和肿瘤组织中 NLRP3 和 caspase-1 活性水平的组成性减弱可增强对该肿瘤的耐药性。我们还假设长期接触石棉会导致 MM 肿瘤发生，导致 NLRP3 表达和活性下调。具体目标 1：A) 我们将评估 NLRP3、ASC 在各种人类 MM 细胞和原发性肿瘤中的表达和活性，并与间皮细胞和正常匹配组织进行比较。此外，与良性组织相比，通过免疫荧光也可以鉴定 MM 肿瘤组织阵列中 NLRP3 的蛋白表达水平。 B) 我们还将研究石棉暴露对永生化细胞 (LP9) 和原代分离的人间皮细胞 (HMC) 中炎症小体表达和激活的影响。除了 caspase-1 活性外，IL-1¿、IL-18 和 IL-33 的水平也将作为炎症小体激活的指标进行评估。 C) 为了证明长期接触石棉而激活炎症小体而产生的 IL-1¿ 会导致间皮细胞的代偿性增殖和转化，我们将在不同时间点用 IL-1¿ 处理 LP9 细胞，并评估软琼脂中这些细胞的增殖、间皮向成纤维细胞转变 (MFT) 标志物和集落形成能力。此外，IL-1¿ 受体阻断剂 Anakinra 也将用于确认 IL-1 在这些过程中的作用。具体目标 2：我们将研究长期石棉暴露对 NLRP3 调节的作用，以了解 MM 中观察到的低 NLRP3 水平的原因，为此，我们将 A) 通过使用小分子抑制剂、小干扰/短发夹 si/sh RNA 以及过表达方法，确定细胞外信号调节激酶 (ERK1/2) 在间皮和 MM 细胞炎症小体表达、活性和功能调节中的作用，以及B) 评估 MM 细胞和肿瘤中的 NLRP3 启动子是否甲基化，并研究长期石棉暴露是否会导致 LP9 细胞中的 NLRP3 启动子甲基化。最后，在具体目标 3 中，我们将使用小鼠模型评估 NLRP3 在 MM 肿瘤发展中的作用。 A) 将稳定过表达 NLRP3 或空载体的人 MM 细胞腹膜内 (IP) 注射到 SCID 小鼠中。细胞注射两周后，小鼠将接受盐水或化疗药物（阿霉素、顺铂）或药物加阿那白滞素，持续 4-6 周。通过收集腹腔灌洗液（PLF）并确定差异细胞计数和细胞因子分析来评估炎症对有或没有药物的肿瘤发展过程的影响。 B) 我们还将 NLRP3-/- 小鼠与 SCID 小鼠杂交以获得 SCID-NLRP3-/- 小鼠。使用这些小鼠，我们将能够研究宿主 NLRP3 蛋白对人类 MM 肿瘤发生的影响。这些研究的目标是证明炎症小体在 MM 发展中的作用，并可能有助于为患者设计未来的治疗策略。

DESCRIPTION (provided by applicant): Asbestos fibers damage lung mesothelial cells which triggers a compensatory proliferative response, inflammation, and malignant mesothelioma (MM). In this proposal we will focus on inflammasome components, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1, which we show are attenuated in MM cells and tumors and activated by asbestos in mesothelial cells. Decreased levels of NLRP3 and caspase-1 may be responsible for enhanced drug resistance in these tumors. Here we hypothesize that constitutively attenuated levels of NLRP3 and caspase-1 activity in MM cells and tumor tissues impart enhanced drug resistance to this tumor. We also hypothesize that chronic exposure to asbestos leading to MM tumorigenesis results in downregulation of NLRP3 expression and activity. In Specific Aim 1: A) We will assess expression and activity of NLRP3, ASC in various human MM cells and primary tumors as compared to mesothelial cells and normal matching tissues. In addition, protein expression levels of NLRP3 will also be identified in MM tumor tissue arrays by immunofluorescence as compared to benign tissues. B) We will also study the effect of asbestos exposure on inflammasome expression and activation in immortalized (LP9) and a primary isolate of human mesothelial cells (HMC). Along with caspase-1 activity, levels of IL-1¿, IL-18 and IL-33 will be assessed as measures of inflammasome activation. C) To show that IL-1¿ generated as a result of activation of inflammasomes by long term exposure to asbestos causes compensatory proliferation and transformation of mesothelial cells, we will treat LP9 cells with IL-1¿ for different time points and assess proliferation, markers of mesothelial to fibroblast transition (MFT) and colony formation ability of these cells in soft agar. Also, the IL-1¿ receptor blocker Anakinra, will also be used to confirm the role of IL-1 in these processes. In Specific Aim 2: We will study the role of long term asbestos exposure on NLRP3 regulation, to understand the cause of observed low NLRP3 levels in MMs, for this, we will A) determine the role of extracellular signal regulated kinase (ERK1/2) in inflammasome expression, activity and functional regulation in mesothelial and MM cells by using small molecule inhibitor, small interfering/short hairpin si/sh RNA as well as overexpression approaches, and B) evaluate if the NLRP3 promoter is methylated in MM cells and tumors and study if long term asbestos exposure causes methylation of the NLRP3 promoter in LP9 cells. Lastly in Specific Aim 3, we will evaluate the role of NLRP3 in MM tumor development using mouse models. A) Human MM cells stably overexpressing NLRP3 or empty vector will be injected intraperitoneally (IP) into SCID mice. Two weeks after cell injection mice will receive either saline or chemotherapeutic drugs (Doxorubicin, Cisplatin) or drugs plus Anakinra for 4-6 weeks. Effects of inflammation on the process of tumor development with and without drug will be assessed by collecting peritoneal lavage fluid (PLF) and determining differential cell counts and cytokine profiling. B) We will also cross NLRP3-/- mice with SCID mice to get SCID-NLRP3-/- mice. Using these mice, we will be able to study the effects of host NLRP3 protein on human MM tumorigenesis. The goal of these studies will be to demonstrate the role of inflammasomes in MM development and may help in designing future therapeutic strategies for patients.