Abstract 4147: Role of MLK3-mediated phosphorylation of β-catenin in regulating cellular senescence and osteopontin expression in hepatocellular carcinoma

Abstract The canonical Wnt/β-catenin signaling pathway is important for normal liver growth and development, abnormal activation of which can lead to various liver pathologies, including hepatocellular carcinoma (HCC). Usually, β-catenin signaling is regulated by a degradation complex that includes adenomatous polyposis coli, Axin, Glycogen Synthase Kinase β, and Casein kinase 1α. Mutations of β-catenin itself and other genes regulating this pathway can cause aberrant activation of this axis. We have reported earlier that Mixed Lineage Kinase 3 (MLK3), a member of the MAPK kinase Kinase (MAP3K) family, can phosphorylate β-catenin and regulate its downstream pathway. The current studies were aimed to elucidate the biological consequences of MLK3-induced β-catenin phosphorylation in HCC. The experimental procedures included creating stable cell lines, RT2 Profiler PCR Arrays, qPCR analysis, Immunofluorescence (IF), ELISA, IHC, MTT and β-Galactosidase (β-Gal) assays. Using Mass Spectrometry, we identified that MLK3 can phosphorylate the N-terminal region of β-catenin at Serine 129 amino acid residue. To understand the significance of β-catenin S129 phosphorylation, stable HCC cell lines overexpressing β-catenin-WT or its phospho-mutants (S129A, S129D) were generated. While MTT assays showed no significant differences between β-catenin-WT or mutant overexpression, β-Gal assays revealed that overexpression of β-catenin WT and S129D can promote a senescence phenotype, which was inhibited in the S129A mutant. These cells also showed increased expression of a set of unique senescent genes including COL1A1, COL3A1, and IGFBP7 which were dependent on S129 phosphorylation. Analysis by RT2 Profiler PCR Array for cytokine and chemokine genes showed an increased expression of SPP1 (Osteopontin gene) in the WT compared to S129A cells. Further validation by qPCR, IF, and ELISA confirmed the induction and secretion of Osteopontin by β-catenin WT and S129D but not by S129A, suggesting a senescence-induced secretory phenotype (SASP) mediated by β-catenin-S129 phosphorylation. TMA analysis showed significant increase in Osteopontin expression in HCC patient samples compared to normal. Database analysis suggested that the SPP1 gene is induced in HCC, and high expression of SPP1 is associated with poor prognosis. In addition, there was a strong correlation between CTNNB1 (β-catenin gene) and SPP1 expressions in HCCs. Considering a well-established role of Osteopontin in HCC and other liver pathologies, these results suggest a potentially novel paracrine signaling network operating in liver cells mediated via a crosstalk of MLK3-β-catenin-Osteopontin axis. These also suggest MLK3 to be a viable drug target for antagonizing this axis and in clinical management of HCC. Citation Format: Tanushree Kashyap, Rong Ke, Deepti Srivastava, Sunil K. Singh, Subhasis Das, Piush Srivastava, Sandeep Kumar, Ajay Rana, Basabi Rana. Role of MLK3-mediated phosphorylation of β-catenin in regulating cellular senescence and osteopontin expression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4147.