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: 10515346
• 负责人: John Daniel Clarke
• 金额: $ 42.08万
• 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/10515346
• 关键词: 3-Dimensional; Acceleration; Address; Age; Alcohols; Algae; Animals; Attenuated; Automobile Driving; Award; Big Data; Career Mobility; Cell Nucleus; Cells; Certification; Cholesterol; Chronic Disease; Cirrhosis; Coculture Techniques; Complement; Country; Cyanobacterium; Cyanotoxin; DNA Methylation; Data; Development; Diet; Diethylnitrosamine; Differentiation Antigens; Dissection; Dose; Environmental Health; Euthanasia; 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; Microfluidics; Modeling; National Institute of Environmental Health Sciences; Pathogenicity; Pathologist; Pathology; Pathway interactions; Persons; 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; biological research; carcinogenesis; carcinogenicity; cell determination; cell type; chronic liver disease; cyanoginosin LR; defined contribution; dietary constituent; dietary control; druggable target; in vivo; inhibitor; innovation; microcystin; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; phosphoproteomics; single nucleus RNA-sequencing; stellate cell; stem cell biomarkers; 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.

微囊藻蛋白LR（MCLR）是淡水蓝绿色藻类产生的最潜力和丰富的氰毒素。 MCLR暴露与非酒精性脂肪肝病（NAFLD）和肝细胞癌有关 （HCC）。 NAFLD迅速成为许多国家中最常见的慢性肝病，HCC 是原发性肝癌的最常见类型。我的K99/R00数据表明MCLR驱动了进展 非酒精性脂肪性肝炎（NASH），一种先进的NAFLD形式，更严重地烧毁了表型 （纤维化增加和减少脂肪变性）。烧毁的阶段是迈向的重要致病步骤 不可逆的肝硬化和HCC。 Sprague Dawley大鼠中的初步数据表明MCLR引用的烧伤 MCLR暴露后4周持续存在，与返回基线的控制饮食组相比 表型。此外，MCLR持续失调的基质组基因（与纤维化有关）和 NASH中的致癌基因（脱不同/干细胞标记）。基于这些数据，我们假设 MCLR介导的肝脏的纤维化和致癌重编程在NASH中得到增强，并促进 肝硬化和HCC的发展。该假设将以两个目的进行检验。 AIM 1将决定 MCLR介导的星状细胞激活中与蛋白质磷酸酶2a（PP2A）相关的途径 肝细胞脱不同。考虑到肝脏是MCLR的主要目标，令人惊讶的是 研究这些途径的研究，没有一个研究了PP2A的细胞类型特异性效应。学习 1.1将定义负责MCLR引起的肝细胞的特定PP2A相关途径的贡献 使用MCLR和PP2调节剂在人Heparg肝细胞和人LX2星级中使用星状效应 细胞在静态培养物或微流体中共培养为3D球体。研究1.2将检验以下假设 肝细胞是主要的MCLR靶标，而肝细胞损伤则使用肝细胞激活。 和LX2细胞单独或在Transwell板中培养。研究1.3将定义pp2a-的影响 大鼠喂养对照或纳什饮食的大鼠MCLR和NASH饮食引起的肝脏病理学的途径调节剂。 AIM 2将确定MCLR引起的纤维化和致癌的机制和长期影响 对健康与纳什的肝硬化和HCC发育进行重新编程。研究2.1将模仿重要的 原始MCLR可耐受的每日摄入研究的各个方面，包括剂量（40 µg/kg），间隔（每日）和 在肿瘤引发剂（二乙基硝基胺）和纳什饮食的情况下，持续时间（3个月）。这些机械 研究将通过磷酸蛋白质组学和单核转录组学分析完成，可能有可能 提供可吸毒的目标。这项研究将持续影响，因为它将是第一个 在先前存在的肝病中，全面评估了MCLR引起的肝硬化和HCC。通过模仿TDI 研究，这项研究可能表明患有肝病的患者需要较低的TDI。