Abstract 3450: Elucidating the role of mixed lineage kinase 3 (MLK3)-β-catenin axis in hepatocellular carcinoma

Abstract

Abstract Hepatocellular carcinoma (HCC) is one of the leading cause of cancer-related deaths worldwide. Of the estimated 40,710 new cases of liver cancers diagnosed in 2017, about three-fourths were HCCs. Surgery, liver transplantation, tumor ablation and chemotherapy are available treatment options if detected at an early stage. However, the only therapeutic option currently available for advanced HCCs is Sorafenib which is only effective for a few months. There is thus an urgent need for the development of safe and more efficacious treatment options for HCC. MLK3 is a member of the mitogen-activated protein kinase (MAPK) family that can activate JNK pathway to induce cell death in neuronal and non-neuronal cells. Several studies have shown that MLK3 plays a significant role in cancer invasion, migration and metastasis. Earlier studies from our laboratory have demonstrated that MLK3 can induce the expression of β-catenin and regulate β-catenin downstream signaling. The current studies were designed to obtain further mechanistic insight on MLK3-β-catenin crosstalk and elucidate the detailed mechanism. To address this, our first goal was to map the domain(s) of β-catenin that mediate MLK3-induced stabilization and interaction by creating β-catenin deletion constructs with N terminal or C-terminal deletions. Overexpression studies designed with these revealed that AA 51-130 of β-catenin was important to mediate MLK3-induced stabilization. In addition, initial Immunoprecipitation and Immunoblotting combination studies also suggest this domain of β-catenin might be mediating the interaction with MLK3. Since our earlier studies suggested that MLK3-induced stabilization of β-catenin was dependent on its kinase activity, studies are currently underway to identify potential phosphorylation sites that might be mediating this stabilization of β-catenin. To obtain a broader picture on the potential effects of MLK3 in HCC disease progression, studies were performed with HCC cell lines following treatment with a pan MLK inhibitor CEP-1347 (CEP). These studies revealed a decrease in PCNA and c-myc expression along with increased PARP cleavage upon CEP treatment, in a time-dependent manner, indicative of a role in antagonizing cell proliferation and induction of cell death respectively. Cell cycle analysis revealed that CEP arrests HCC cells at G2/M phase of cell cycle. Flow cytometric studies also revealed a time-dependent increase in cell apoptosis and increased mitochondrial depolarization following treatment with CEP, associated with increased cleavage of Caspases 3, 8 and PARP. Further validation following siRNA-mediated knockdown of MLK3 showed a potentiation of the apoptotic response following CEP treatment. Taken together these findings indicate that MLK3 plays a major role in HCC progression and CEP-induced targeting of this axis might be efficacious in the management of HCC. Citation Format: Karan S. Saini, Taher Gheewala, Subhasis Das, Gautam Sondarva, Ajay Rana, Basabi Rana. Elucidating the role of mixed lineage kinase 3 (MLK3)-β-catenin axis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3450.