Compound 3k(Saitama Ika Daigaku)

Previously, we showed that lactate promoted the proliferation and mobility of hepatocellular carcinoma (HCC) cells by increasing the expression of ornithine decarboxylase 1 (ODC1). In this study, we determined the relationship between ODC1 and pyruvate kinase M2 (PKM2, a key lactate metabolism enzyme), and determined the combined effects of difluoromethylornithine (DFMO; an ODC1 inhibitor) and compound 3k (a PKM2 inhibitor) on HCC cells.

First, the relationship between PKM2 and ODC1 was analyzed using Western blotting, Cell Counting Kit (CCK)-8 assays, transwell assays, bioinformatics, quantitative real-time fluorescent PCR (qRT-PCR), and immunohistochemical staining. Thereafter, the ODC1 inhibitor DFMO and the PKM2 inhibitor compound 3k were employed. Their combined effects on HCC cell proliferation and mobility were evaluated via CCK-8 assay, flow cytometry, a subcutaneous xenograft tumor model in mice, wound healing assays, and transwell assays. Additionally, the effects of DFMO and compound 3k on the epithelial-mesenchymal transition phenotype and the AKT/GSK-3β/β-catenin pathway were explored using Western blotting and immunofluorescence.

PKM2 knockdown significantly decreased the ODC1 expression, and the proliferation and invasion of HCC cells, while ODC1 overexpression reversed the inhibitory effects of PKM2 knockdown. Similarly, inhibition of ODC1 also decreased the expression of PKM2 via reducing the c-myc-induced transcription. PKM2 was co-expressed with ODC1 in HCC samples, while simultaneously upregulated PKM2 and ODC1 led to the poorest survival outcome. DFMO and compound 3k synergistically inhibited HCC cell proliferation, induced apoptosis, and suppressed cell mobility, as well as the EMT phenotype and the AKT/GSK-3β/β-catenin pathway. The AKT activator SC79 reversed the inhibitory effects.

PKM2/ODC1 are involved in a positive feedback loop. The simultaneous inhibition of ODC1 and PKM2 using DFMO and compound 3k exerts synergistic effects against HCC cells via the AKT/GSK-3β/β-catenin pathway. Thus, DFMO combined with compound 3k may be a novel effective strategy for treating HCC.

Deficiency in GLI-similar 3 (GLIS3) in humans and mice causes polycystic kidney disease (PKD). We previously reported that Glis3- knockout (KO) kidneys undergo metabolic reprogramming and remain more reliant on aerobic glycolysis. Here, to understand the mechanism by which GLIS3 controls this process, we analyzed its role in regulating glycolytic gene expression using transcriptomics and cistromics. Transcriptomic analysis revealed increased expression of key glycolytic genes and reduced expression of several gluconeogenic genes in Glis3 -KO compared with wild-type kidneys. Chromatin immunoprecipitation followed by sequencing analysis showed that many of these genes were directly regulated by GLIS3 in coordination with the transcription factor, HNF-1B. Pkm emerged as a key upregulated glycolytic GLIS3 target gene, which via alternative splicing generates two protein isoforms, PKM1 and PKM2. Expression of Pkm and the dimeric form of PKM2, which promotes aerobic glycolysis, was elevated in Glis3 -KO kidneys, whereas exogenous GLIS3 expression in primary Glis3 -KO renal epithelial cells suppressed Pkm expression. Phosphorylation of PKM2 at Y105 and S37, which stimulate dimer formation, nuclear localization and aerobic glycolysis, is increased in Glis3 -KO kidneys, consistent with a role for PKM2 in metabolic reprogramming and cyst formation. Consistent with this, siRNA-mediated knockdown of PKM2 in Glis3 -KO renal epithelial cells suppressed spheroid growth and glycolytic activity. In addition, pharmacological inhibition of PKM2 with compound 3K in GLIS3-deficient spheroid cultures and Glis3 -KO kidneys significantly reduced spheroid size, cyst number and cystic area. Our study identifies GLIS3 as a novel regulator of glycolysis and PKM2 function, whose dysregulation contributes to metabolic reprogramming and cystogenesis in Glis3 -KO kidneys.