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.

微囊藻毒素-LR(Microcystin-LR，MCLR)是由淡水蓝藻产生的效力最强、含量最高的氰化毒素。 接触MCLR与非酒精性脂肪性肝病(NAFLD)和肝细胞癌相关 (肝细胞癌)。非酒精性脂肪肝在许多国家迅速上升为最常见的慢性肝病，而肝细胞癌 是最常见的一种原发性肝癌。我的K99/R00数据表明MCLR推动了 非酒精性脂肪性肝炎(NASH)，NAFLD的高级形式，到更严重的倦怠表型 (纤维化增加，脂肪变性减少)。烧毁阶段是致病的重要一步 不可逆转的肝硬变和肝癌。SpragueDawley大鼠的初步数据表明，MCLR诱导的倦怠 NASH在MCLR暴露4周后持续存在，而对照组饮食组恢复到基线 表型。此外，MCLR持续失调的母体基因(与纤维化有关)和 NASH中的致癌基因(去分化/干细胞标志物)。基于这些数据，我们假设 MCLR介导的肝纤维化和致癌重编程在NASH中得到加强，并促进 肝硬变和肝细胞癌的发展。这一假设将在两个目标上得到检验。目标1将决定 蛋白磷酸酶2A(PP2A)相关通路在MCLR介导的星状细胞活化和凋亡中的作用 肝细胞去分化。考虑到肝脏是MCLR的主要靶点，令人惊讶的是缺乏 对这些途径进行了研究，但没有一项研究检查PP2A对细胞类型的特定影响。学习 1.1将定义负责MCLR诱导的肝细胞的特定PP2A相关通路的贡献 使用MCLR和PP2调节剂在人肝细胞和人Lx2星状细胞中的星状效应 在静态培养或微流控条件下，细胞共培养为3D球体。研究1.2将检验这一假设 肝细胞是主要的MCLR靶点，星状细胞通过使用HepaRG的肝细胞损伤而被激活 和Lx2细胞单独或组合培养在Transwell板上。研究1.3将定义PP2A的影响- MCLR-和NASH饮食上的途径调节剂-在喂养对照组或NASH饮食的大鼠中引发肝脏病理。 目的2将确定MCLR诱导的纤维化和致癌的机制和长期影响 健康与非酒精性脂肪肝患者的肝硬变和肝细胞癌发展的再编程。研究2.1将模仿重要的 原始MCLR每日可耐受摄入量研究的各个方面，包括剂量(40微克/公斤)、间隔(每日)和 持续时间(3个月)在肿瘤启动剂(二乙基亚硝胺)和NASH饮食的情况下。这些机械式的 研究将与磷酸蛋白质组学和单核转录组学分析相补充，可能 提供了可下药的靶点。这项研究将产生持续的影响，因为它将是第一个 全面评估既往肝病中MCLR诱发的肝硬变和肝细胞癌。通过模仿TDI 这项研究可能表明，患有既往肝病的人需要较低的TDI。