Microcystin-LR activates MAPKs and alters the phosphoproteome in human HepaRG cells

Abstract ID 129698 Poster Board 018 Hepatocellular carcinoma (HCC) accounts for about 90% of all liver cancer cases and has poor prognosis for survival in advanced stages. Microcystin-LR (MCLR) is a potent hepatotoxin that has been linked to the development of HCC. Many of the carcinogenic mechanisms for MCLR have been attributed to the induction of cell survival and proliferation through altered protein phosphorylation pathways. Despite the fact that MCLR is a covalent inhibitor of protein phosphatases-1 (PP1) and PP2A, and indirectly activates serine/threonine kinases, minimal studies have explored phosphoproteomics after MCLR exposure. The current study determined MCLR effects on the phosphoproteome in human HepaRG cells. Differentiated HepaRG cells were treated with either vehicle or MCLR followed by phosphoproteomic analysis and western blotting of MAPK-activated proteins. MCLR had no effect on cell viability at 2 h up to 10 μM, whereas it decreased cell viability at 24 h at doses as low as 0.03 μM. MCLR exposure resulted in a dose-dependent increase in phosphorylation of extracellular signal-regulated kinase 1/2, (ERK1/2), p38, c-Jun-N-terminal kinase (JNK), and AKT. The number of decreased phosphosites by 0.1 μM MCLR was similar between the 2 and 24 h timepoints (212 and 154, respectively). In contrast, a greater number of phosphosites were increased at the 24 h timepoint (i.e., 567) versus the 2 h timepoint (i.e., 136), indicating the hyperphosphorylation state caused by MCLR-mediated inhibition of PPs is time-dependent. A kinase perturbation analysis predicted that MCLR exposure at both 2 and 24 h increased the function of multiple kinases associated with cell cycle and proliferation, including aurora kinase B (AURKB), checkpoint kinase 1 (CHEK1), serum and glucocorticoid-regulated kinase 1 (SGK1), and cyclin-dependent kinase 1 (CDK1). String database analysis of the phosphosites altered by MCLR exposure revealed pathways associated with cell proliferation and survival, including RSK, apoptosis-related proteins, Ahr signaling, and VEGFR2-mediated vascular permeability. In addition, several cancer-related KEGG pathways were enriched at both 2 and 24 h timepoints, and multiple cancer-related disease-gene associations were identified at the 24 h timepoint. Many of the kinases and pathways described above play crucial roles in the development of HCC by affecting processes such as invasion, and metastasis. Overall, our data indicate that MCLR-mediated changes in protein phosphorylation involve biological pathways related to carcinogenesis that may contribute to the development of HCC (Funding: R01ES032558).