Abstract 108: Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma
Authors: Ke Rong , Viswakarma Navin , Vishnoi Kanchan , Kumar Sandeep , Rana Ajay , Rana Basabi 
Published: September 2020

Abstract
Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Evidence suggest that Mixed Lineage Kinase 3 (MLK3), a member of MAPK pathway, is a key regulator of tumorigenesis and metastasis in different cancer-types. Previous studies from our laboratory indicated that, knocking out MLK3 in mice alleviates hepatocarcinogenesis when treated with Diethylnitrosamine (DEN) in combination with phenobarbital (PB). While these suggested an involvement of MLK3 in HCC progression, the detailed mechanism by which this is accomplished is still unclear. To address the mechanism of MLK3-induced HCC, liver cancer cells stably overexpressing MLK3 wildtype (WT) or kinase dead (KA) in a Doxycycline (Dox)-inducible manner, were established. Our RT2 Profiler PCR array analysis with a cytokine and chemokine array (PAHS-150Z from QIAGEN) showed that MLK3-WT overexpression can significantly upregulate mRNA levels of inflammatory cytokines, such as IL11, BMP6, TNFα and IL6, which were absent with MLK3-KA overexpression. Q-PCR and ELISA analysis confirmed the increased IL11 mRNA levels and IL11 release following MLK3 overexpression. Interestingly, STAT3, the downstream signal transducer of Janus kinases (JAKs), which are activated by cytokine signals was also activated by MLK3 overexpression, suggesting that MLK3 may have a novel crosstalk with JAK-STAT3 signaling pathway. To explore this possibility, cells were treated with inhibitors of JAKs (AZD1480 and Ruxolitinib) in the presence of Dox (to induce MLK3), which significantly decreased STAT3 activation as well as IL11 release, suggesting the involvement of JAKs in MLK3-induced IL11 signaling. Furthermore, we also demonstrated a significant reduction of IL11 release following treatment with the JNK inhibitor, SP600125. To elucidate the detailed mecahnism and identify potential transcription factors that mediate the effect of MLK3 overexpression on IL11 promoter activity, deletion mutants of pGL4.16 IL11-luciferase reporter (-877/+169), (-249/+169), (-153/+169) and (-78/+169) were generated. Luciferase assays confirmed that MLK3-WT overexpression indeed increased IL11 promoter reporter activity, which required the fragment from -153 to -78. More studies are currently undergoing with site-directed mutagenesis to explore the detailed mechanism. In addition, our studies demonstrated an induction of epithelial to mesenchymal transition (EMT) during later stages of Dox treatment, suggesting that MLK3-WT overexpression may regulate EMT, cell migration and invasion. These results indicated that both JAKs, and MLK3/JNK signaling converge to mediate IL11 release in HCC, but the mechanism of crosstalk between MLK3 and JAK-STAT3 signaling pathway is still unclear. Taken together, our current studies indicate that overexpression of MLK3 may not only activate JNK/c-Jun pathway, but also regulate JAK-STAT3 signaling pathway to induce IL11 release and downstream signaling in hepatocellular carcinoma.
Citation Format: Rong Ke, Navin Viswakarma, Kanchan Vishnoi, Sandeep Kumar, Ajay Rana, Basabi Rana. Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 108.