Elucidation of Mixed Lineage Kinase 3 (MLK3) Signaling Pathway in Hepatocellular Carcinoma

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. In part I, TCGA databases suggest elevated MAP3K11 (MLK3 gene) expression, and TMA studies show MLK3 activation in human HCCs. We demonstrate that MLK3 activity is upregulated in carcinogen-induced HCC, and MLK3 deficiency (MLK3-/-) alleviates HCC progression. MLK3-/- reduces proliferation in vivo and MLK3 inhibition reduces proliferation and colony formation in vitro. In part II, RNA-sequencing analysis shows that MLK3-/- modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promote EMT via autocrine TGFβ signaling. Moreover, MLK3-/- attenuates activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes pSmad2 induction and HSC activation via paracrine TGFβ signaling. In part III, we report that MLK3 overexpression in HCC promotes interleukin-11 (IL-11) expression, and secretion dependent on JNK1, which promotes the abundance of AP-1 proteins, c-Jun, and JunB on the IL11 promoters. Furthermore, MLK3 and IL-11 induce MMP10 expression and might cooperate with each other in promoting migration/invasion. Interestingly, IL11 is also expressed in the DEN/PB induced HCC tumors. Finally, MLK3 promotes M2 macrophage infiltration in HCC in vivo and IL11 promotes M2 macrophage polarization in vitro, suggesting a potential collaboration between the two. These findings reveal that MLK3 regulates multiple steps of HCC, implying MLK3 to be an ideal drug target for HCC clinical management.