Inflammasomes in pathogenesis of mesothelioma

描述(由申请人提供)：石棉纤维损伤肺间皮细胞，触发代偿性增殖反应、炎症和恶性间皮瘤(MM)。在这项提案中，我们将重点关注炎症小体成分，Nod样受体蛋白3(NLRP3)，含有卡片的凋亡相关斑点样蛋白(ASC)和Caspase-1，我们发现它们在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。在这里，我们假设，MM细胞和肿瘤组织中NLRP3和caspase-1活性的结构性减弱会增强对该肿瘤的耐药性。我们还假设，长期接触石棉会导致多发性骨髓瘤的发生，导致NLRP3的表达和活性下调。在特定的目标1：a)我们将评估NLRP3和ASC在各种人MM细胞和原发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，NLRP3在MM肿瘤组织阵列中的蛋白表达水平也将通过免疫荧光进行鉴定。B)我们还将研究石棉暴露对永生化细胞(LP9)和人间皮细胞原代分离株(HMC)炎症体表达和激活的影响。与caspase-1活性一起，IL-1、IL-18和IL-33水平将被评估为炎性小体激活的指标。C)为了证明长期暴露在石棉中的炎性小体激活产生的IL-1会导致间皮细胞的代偿性增殖和转化，我们将IL-1作用于LP9细胞不同的时间点，并评估这些细胞在软琼脂中的增殖、间皮细胞向成纤维细胞转化(MFT)的标志物和集落形成能力。此外，IL-1受体阻滞剂Anakinra也将用于确认IL-1在这些过程中的作用。在特定的目标2：我们将研究长期接触石棉对NLRP3调控的作用，了解在MMS中观察到低水平NLRP3的原因，为此，我们将A)通过使用小分子抑制物、小干扰/短发夹状si/sh RNA以及过表达方法来确定细胞外信号调节激酶(ERK1/2)在间皮和MM细胞的炎症表达、活性和功能调节中的作用，以及B)评估NLRP3启动子在MM细胞和肿瘤中是否甲基化，并研究长期石棉暴露是否导致LP9细胞中NLRP3启动子的甲基化。最后，在特定的目标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)将稳定高表达NLRP3的人MM细胞或空载体注射到SCID小鼠体内。细胞注射两周后，小鼠将接受生理盐水或化疗药物(多柔比星、顺铂)或药物加Anakinra治疗4-6周。将通过收集腹膜灌洗液(PLF)、测定细胞分类计数和细胞因子图谱来评估炎症对有无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，获得SCID-NLRP3-/-小鼠。利用这些小鼠，我们将能够研究宿主NLRP3蛋白对人类多发性骨髓瘤发生的影响。这些研究的目标将是证明炎性小体在多发性骨髓瘤发展中的作用，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.