GSK3β-Mediated Phosphorylation of SOX4 and Its Interaction with STAT6 to Regulate MTHFD2 Expression, NADPH Production, Cancer Stemness, and Prognosis in Hepatocellular Carcinoma

SOX4 is associated with poor prognosis in various cancers, including hepatocellular carcinoma (HCC). SOX4 plays a crucial role in angiogenesis, cellular differentiation, and cell growth through interactions with transcription factors in response to stimuli. Despite its overexpression and link to poor prognosis, SOX4 remains an undruggable target. Therefore, targeting its interacting or regulatory proteins might disclose a therapeutic strategy in HCC. Our aim was to elucidate SOX4 regulation and interactions in HCC cells to provide further insights into its role in tumorigenesis. GSK3β phosphorylated SOX4 at serine residues 379, 383, 387, 391, and 395, as revealed by in vitro kinase assays, which stabilized SOX4 protein. Interactions between SOX4, STAT6, and GSK3β in HCC cells were confirmed by co-immunoprecipitation, immunofluorescence, and proximal-ligation assay. Knockout SOX4 or inhibition of STAT6 repressed cell growth, sphere formation, and NADPH production through modulating MTHFD2 expression, which in turn repressed expression of PD-L1 in HCC cells. The significant elevated NADPH was found in SOX4high HCC tumor specimen as compared with the SOX4low tumors. Positive correlations of GSK3β & SOX4, STAT6 & SOX4, and SOX4 & MTHFD2 proteins were observed in liver cancer specimens through western blot analysis. Furthermore, tumor lesion with elevated of SOX4, MTHFD2, or both SOX4 and MTHFD2 transcripts tend to have poor prognosis in patients with HCC using TCGA-LIHC database. Treatment with inhibitors targeting STAT6 or MTHFD2 reduced cell proliferation in vitro and attenuated tumor growth in an HCC patient-derived xenograft (PDX) mice model in vivo. The GSK3β/SOX4/STAT6/MTHFD2 regulatory axis played a critical role in modulating NADPH production and maintaining cancer stemness, associated with poor prognosis in HCC that might be a potential therapeutic target.