Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

Metabolic reprogramming is a pivotal characteristic of cancer, yet the intricate interplay between glycolysis and the pentose phosphate pathway (PPP) remains elusive. This study unveils the pivotal role of 6-phosphofructokinase liver type (PFKL) in glycolysis and ribose 5-phosphate isomerase A (RPIA) in PPP, orchestrating liver tumorigenesis. PFKL, the rate-limiting enzyme in glycolysis, stabilizes RPIA by impeding ubiquitination/proteasome activity. The pro-inflammatory and tumor cytokine interleukin 6 activates pSTAT3 which binds to the promoter region and activates AMPK and PFKL transcription. Furthermore, pAMPK stabilizes PFKL protein by preventing proteasome degradation in hepatoma cells. Inhibiting PFKL, AMPK, and STAT3 genetically or pharmacologically can reduce glycolysis, ATP production, resulting in reduction of hepatoma cell proliferation and migration. Intriguingly, the PFKL, AMPK, RPIA, and PKM2 are co-localized in the Glycolytic body (G-body) which starts forming at chronic hepatitis, dramatically increases during active hepatitis, and the size of G-bodies becomes bigger from cirrhosis to hepatocellular carcinoma. Furthermore, using Bimolecular fluorescence complementation (BiFC) assay, we demonstrated that PFKL and RPIA direct interacts. Targeting AMPK or STAT3 significantly reduced tumor formation and lipid accumulation in zebrafish models, suggesting the STAT3/AMPK/PFKL axis as a potential therapeutic avenue for liver cancer treatment. ### Competing Interest Statement The authors have declared no competing interest. * PFKL : 6-phosphofructokinase liver type RPIA : Ribose 5-Phosphate Isomerase A PPP : pentose phosphate pathway IL6 : interleukin 6 G body : Glycolytic body BiFC : Bimolecular fluorescence complementation assay HCC : Hepatocellular carcinoma MALFD : Metabolic dysfunction-associated fatty liver disease NAFLD : Nonalcoholic fatty liver disease NASH : Nonalcoholic steatohepatitis PFK1 : Phosphofructokinase-1 STAT3 : Signal transducer and activator of transcription 3 PFKFB3 : 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 ERK : Extracellular signal-regulated kinase AMPK : AMP-activated protein kinase mTOR : target of rapamycin NHRI : National Health Research Institutes IRS : immunoreactivity scores FACS : fluorescence-activated cell sorting pERK : phosphorylated extracellular signal-regulated kinase PK : pyruvate kinase PGI : phosphoglucose isomerase TPI : triose-phosphate isomerase G6PD : glucose-6-phosphate dehydrogenase TALDO : transaldolase TKL : transketolase CSCs : cancer stem cells FBP : fructose 1,6-bisphosphate DHAP : dihydroxyacetone phosphate 3PG : 3-phosphoglycerate cDNA : Complementary DNA qPCR : Quantitative polymerase chain reaction siRNA : small interfering RNA shRNA : short-hairpin RNA DMEM : Dulbecco’s modified Eagle’s medium FBS : fetal bovine serum MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide WST-1 : Water-Soluble Tetrazolium 1 OCR : oxygen consumption rate ChIP : Chromatin immunoprecipitation IHC : Immunohistochemistry IF : immunofluorescence TZCF : Taiwan Zebrafish Core Facility IACUC : Institutional Animal Care and Use Committee AAALAC : Association for Assessment and Accreditation of Laboratory Animal Care International hpf : hours post-fertilization HFD : high-fat diet dpf : days post fertilization DMSO : Dimethyl sulfoxide PBS : phosphate-buffered saline H&E : Hematoxylin & eosin staining dpi : day post-injection