Analysis of MLK3-dependent gene expression in carcinogen-induced murine hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Although Mixed Lineage Kinase 3 (MLK3), a MAPKKK, has emerged as a key regulator of acute liver injury, inflammation, and non-alcoholic steatohepatitis (NASH), its role in HCC remains unclear. TCGA databases suggest an elevated expression of MAP3K11 (gene of MLK3) and TMA studies show higher MLK3 activation in human HCCs compared to the normal livers. We demonstrate here that MLK3 kinase activity is upregulated in Diethylnitrosamine/phenobarbital (DEN/PB)-induced HCC, and MLK3 deficiency (MLK3-/-) alleviates HCC progression. Histological and biochemical analyses reveal that MLK3-/- reduces DEN/PB induced cell proliferation in vivo and MLK3 inhibition by si/shRNA or pharmacological inhibitors reduces cell proliferation and colony formation in vitro. Interestingly, MLK3-/- does not impair DEN-induced liver injury phenotypes at the early stage. RNA-sequencing analysis shows that MLK3-/- modulates the gene signature of various biological pathways, including epithelial-mesenchymal transition (EMT), and reduces TGFß1&2 expressions. HCC cells overexpressing MLK3 promote EMT, and induction of TGFß, suggesting MLK3 to be an upstream regulator of TGFß signaling. In addition, MLK3-/- significantly reduces the activated hepatic stellate cell (HSC) signature, which is increased in DEN/PB-Wild-Type livers. Further analysis suggested that MLK3 might promote a paracrine increase in TGFß expression leading to pSmad2 induction and HSC activation. Our work thus shows that MLK3 promotes HCC proliferation, TGFß expression, and HSC activation, implying that pharmacological inhibition of MLK3 may have a significant clinical impact on HCC progression. Overall design: A total of 10 samples were analyzed. There were 4 experimental groups: water treated Wild-type (WT) and MLK3-Knockout (MLK3-KO) and DEN/PB-treated WT and MLK3-KO. 2 biological replicates were included for each water treated sample, and 3 biological replicates were included for each DEN/PB-treated sample.