Inflammasomes in pathogenesis of mesothelioma

描述（由申请方提供）：天冬氨酸纤维损伤肺间皮瘤细胞，引发代偿性增殖反应、炎症和恶性间皮瘤（MM）。在这项提案中，我们将重点关注炎性小体组分，NOD样受体蛋白3（NLRP 3），包含CARD（ASC）和Caspase-1的骨化相关斑点样蛋白，我们显示它们在MM细胞和肿瘤中减弱，并在间皮瘤细胞中被石棉激活。NLRP 3和caspase-1水平的降低可能是这些肿瘤中耐药性增强的原因。在这里，我们假设，组成性衰减水平的NLRP 3和半胱天冬酶-1活性在MM细胞和肿瘤组织赋予增强的耐药性，这种肿瘤。我们还假设，慢性暴露于石棉导致MM肿瘤的结果在下调NLRP 3的表达和活性。具体目标1：A）我们将评估与间皮瘤细胞和正常匹配组织相比，NLRP 3、ASC在各种人MM细胞和原发性肿瘤中的表达和活性。此外，与良性组织相比，还将通过免疫荧光在MM肿瘤组织阵列中鉴定NLRP 3的蛋白表达水平。B）我们还将研究石棉暴露对永生化（LP 9）和人间皮细胞（HMC）原代分离物中炎性体表达和激活的影响。沿着半胱天冬酶-1活性，IL-1、IL-18和IL-33的水平将被评估为炎性小体活化的量度。C）为了证明由于长期暴露于石棉而激活炎性小体而产生的IL-1 <$导致间皮细胞的补偿性增殖和转化，我们将用IL-1 <$处理LP 9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。具体目标2：我们将研究长期石棉暴露对NLRP 3调节的作用，以了解观察到的低NLRP 3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以及过表达方法来确定细胞外信号调节激酶（ERK 1/2）在间皮瘤和MM细胞中炎性小体表达、活性和功能调节中的作用，和B）评估NLRP 3启动子在MM细胞和肿瘤中是否被甲基化，并研究长期石棉暴露是否导致LP 9细胞中NLRP 3启动子的甲基化。最后，在具体目标3中，我们将使用小鼠模型评估NLRP 3在MM肿瘤发展中的作用。A）将稳定过表达NLRP 3或空载体的人MM细胞腹膜内（IP）注射到SCID小鼠中。细胞注射后两周，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4-6周。将通过收集腹膜灌洗液（PLF）并确定差异细胞计数和细胞因子谱来评估炎症对有和无药物的肿瘤发展过程的影响。B）我们还将NLRP 3-/-小鼠与SCID小鼠杂交以获得SCID-NLRP 3-/-小鼠。使用这些小鼠，我们将能够研究宿主NLRP 3蛋白对人MM肿瘤发生的影响。这些研究的目的是证明炎性小体在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.