Mechanisms of microcystin-induced hepatocellular carcinoma in nonalcoholic steatohepatitis

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.