Mechanisms of microcystin-induced hepatocellular carcinoma in nonalcoholic steatohepatitis

非酒精性脂肪性肝炎微囊藻毒素诱发肝细胞癌的机制

• 批准号: 10116789
• 负责人: John Daniel Clarke
• 金额: $ 45.58万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-19 至 2025-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10116789
• 关键词: 3-Dimensional; Address; Age; Alcohols; Algae; Animals; Attenuated; Automobile Driving; Award; Big Data; Career Mobility; Cell Nucleus; Cells; Cholesterol; Chronic Disease; Cirrhosis; Coculture Techniques; Complement; Country; Cultured Cells; Cyanobacterium; Cyanotoxin; DNA Methylation; Data; Development; Diet; Diethylnitrosamine; Differentiation Antigens; Dose; Environmental Health; Exposure to; Fatty acid glycerol esters; Fibrosis; Fresh Water; Genes; Hepatocyte; Histopathology; Human; In Vitro; Individual; Injections; Intake; Liver; Liver diseases; Long-Term Effects; Malignant neoplasm of liver; Measures; Mediating; Methods; Microfluidics; Modeling; National Institute of Environmental Health Sciences; Pathogenicity; Pathologist; Pathology; Pathway interactions; Phenotype; Predisposition; Primary Malignant Neoplasm of Liver; Primary carcinoma of the liver cells; Protein Inhibition; Protein phosphatase; Proteomics; Proto-Oncogene Proteins c-akt; Rattus; Research; Role; Science; Scientist; Sprague-Dawley Rats; Strategic Planning; Testing; Toxin; Unhealthy Diet; base; biological research; carcinogenesis; carcinogenicity; cell determination; cell type; chronic liver disease; cyanoginosin LR; defined contribution; dietary constituent; dietary control; druggable target; in vivo; inhibitor/antagonist; innovation; microcystin; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; phosphoproteomics; stellate cell; stem cell biomarkers; stem cell differentiation; transcriptome sequencing; transcriptomics; treatment group; tumor initiators; uptake

Microcystin-LR (MCLR) is the most potent and abundant cyanotoxin produced by freshwater blue-green algae. MCLR exposure is associated with nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). NAFLD has quickly risen to become the most common chronic liver disease in many countries, and HCC is the most common type of primary liver cancer. My K99/R00 data indicate MCLR drives progression of nonalcoholic steatohepatitis (NASH), an advanced form of NAFLD, to a more severe burnt out phenotype (increased fibrosis and reduced steatosis). The burnt out stage is an important pathogenic step towards irreversible cirrhosis and HCC. Preliminary data in Sprague Dawley rats indicate that MCLR-elicited burnt out NASH persists 4 weeks after MCLR exposure, in contrast to the control diet group that returned to a baseline phenotype. In addition, MCLR persistently dysregulated matrisome genes (related to fibrosis) and carcinogenesis genes (de-differentiation/stem cell markers) in NASH. Based on these data we hypothesize MCLR-mediated fibrotic and carcinogenic reprogramming of the liver is enhanced in NASH and promotes the development of cirrhosis and HCC. This hypothesis will be tested in two aims. Aim 1 will determine the role of the protein phosphatase 2A (PP2A)-associated pathways in MCLR-mediated stellate cell activation and hepatocyte de-differentiation. Considering the liver is the primary target for MCLR, there is surprising paucity of research investigating these pathways and none have examined the cell type specific effects of PP2A. STUDY 1.1 will define the contribution of specific PP2A-associated pathways responsible for MCLR-elicited hepatocyte and stellate effects using MCLR and PP2 modulators in human HepaRG hepatocytes and human LX2 stellate cells co-cultured as 3D spheroids in static culture or microfluidics. STUDY 1.2 will test the hypothesis that hepatocytes are the primary MCLR target and stellate cells are activated by hepatocyte damage using HepaRG and LX2 cells cultured individually or in combination in transwell plates. STUDY 1.3 will define the effects of PP2A- pathway modulators on MCLR- and NASH diet-elicited liver pathology in rats fed either a control or a NASH diet. Aim 2 will determine the mechanisms and long-term effects of MCLR-elicited fibrotic and carcinogenic reprogramming on cirrhosis and HCC development in healthy versus NASH. STUDY 2.1 will mimic important aspects of the original MCLR tolerable daily intake study including the dose (40 µg/kg), interval (daily), and duration (3 months) in the context of a tumor initiator (diethylnitrosamine) and a NASH diet. These mechanistic studies will be complemented with phosphoproteomics and single nuclei transcriptomics analyses, potentially providing druggable targets. This research will have sustained impact because it will be the first to comprehensively assess MCLR-elicited cirrhosis and HCC in pre-existing liver disease. By mimicking the TDI study, this research may indicate a lower TDI is required for people with pre-existing liver disease.

微囊藻毒素-LR (MCLR) 是淡水蓝绿藻产生的最有效、最丰富的蓝藻毒素。 MCLR 暴露与非酒精性脂肪性肝病 (NAFLD) 和肝细胞癌相关 （肝癌）。 NAFLD 已迅速成为许多国家最常见的慢性肝病，而 HCC 是最常见的原发性肝癌类型。我的 K99/R00 数据表明 MCLR 推动了 非酒精性脂肪性肝炎 (NASH) 是 NAFLD 的一种高级形式，可导致更严重的倦怠表型 （增加纤维化和减少脂肪变性）。倦怠阶段是重要的致病步骤 不可逆的肝硬化和肝癌。 Sprague Dawley 大鼠的初步数据表明 MCLR 引起的倦怠 NASH 在 MCLR 暴露后持续 4 周，而对照组饮食组则恢复到基线 表型。此外，MCLR 持续失调基质体基因（与纤维化相关）和 NASH 中的致癌基因（去分化/干细胞标记）。根据这些数据我们假设 MCLR 介导的肝脏纤维化和致癌性重编程在 NASH 中得到增强，并促进 肝硬化和肝癌的发展。该假设将在两个目标上进行检验。目标 1 将确定以下角色： MCLR 介导的星状细胞激活中蛋白磷酸酶 2A (PP2A) 相关通路 肝细胞去分化。考虑到肝脏是 MCLR 的主要目标，令人惊讶的是， 研究调查了这些途径，但没有一个研究检查 PP2A 对细胞类型的特异性影响。学习 1.1 将定义负责 MCLR 诱导肝细胞的特定 PP2A 相关途径的贡献 在人 HepaRG 肝细胞和人 LX2 星状细胞中使用 MCLR 和 PP2 调节剂的星状效应 在静态培养或微流体中将细胞共培养为 3D 球体。研究 1.2 将检验以下假设： 肝细胞是主要 MCLR 目标，使用 HepaRG 因肝细胞损伤而激活星状细胞 LX2细胞在transwell板中单独或组合培养。研究 1.3 将定义 PP2A- 的影响 MCLR 和 NASH 饮食的通路调节剂在喂食对照或 NASH 饮食的大鼠中引发肝脏病理学。 目标 2 将确定 MCLR 引起的纤维化和致癌的机制和长期影响 健康与 NASH 中肝硬化和 HCC 发展的重编程。研究 2.1 将模仿重要的 原始 MCLR 每日耐受摄入量研究的各个方面，包括剂量（40 µg/kg）、间隔（每日）和 肿瘤引发剂（二乙基亚硝胺）和 NASH 饮食的持续时间（3 个月）。这些机械性的 研究将得到磷酸蛋白质组学和单核转录组学分析的补充，可能 提供可药物靶标。这项研究将产生持续的影响，因为它将是第一个 全面评估 MCLR 引起的肝硬化和 HCC 先前存在的肝病。通过模仿TDI 研究表明，患有肝病的人需要较低的 TDI。