AMPK-HIF-1α signaling enhances glucose-derived de novo serine biosynthesis to promote glioblastoma growth.

BACKGROUND: Cancer cells undergo cellular adaptation through metabolic reprogramming to sustain survival and rapid growth under various stress conditions. However, how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment remain unknown. METHODS: We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify the regulatory mechanisms of S/G deprivation-induced metabolic flexibility, we employed high-throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. RESULTS: The high-throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK-dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes the expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, the HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and the proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that the levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and the poor prognosis of GBM patients. CONCLUSION: Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells, and highlight the potential to target SSP for treating human GBM.

Mutual regulation between phosphofructokinase 1 platelet isoform and VEGF promotes glioblastoma tumor growth.

Glioblastoma (GBM) is a highly vascular malignant brain tumor that overexpresses vascular endothelial growth factor (VEGF) and phosphofructokinase 1 platelet isoform (PFKP), which catalyzes a rate-limiting reaction in glycolysis. However, whether PFKP and VEGF are reciprocally regulated during GBM tumor growth remains unknown. Here, we show that PFKP can promote EGFR activation-induced VEGF expression in HIF-1α-dependent and -independent manners in GBM cells. Importantly, we demonstrate that EGFR-phosphorylated PFKP Y64 has critical roles in both AKT/SP1-mediated transcriptional expression of HIF-1α and in the AKT-mediated ß-catenin S552 phosphorylation, to fully enhance VEGF transcription, subsequently promoting blood vessel formation and brain tumor growth. Levels of PFKP Y64 phosphorylation in human GBM specimens are positively correlated with HIF-1α expression, ß-catenin S552 phosphorylation, and VEGF expression. Conversely, VEGF upregulates PFKP expression in a PFKP S386 phosphorylation-dependent manner, leading to increased PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells. These findings highlight a novel mechanism underlying the mutual regulation that occurs between PFKP and VEGF for promoting GBM tumor growth and also suggest that targeting the PFKP/VEGF regulatory loop might show therapeutic potential for treating GBM patients.

Blockade of PD-L1/PD-1 signaling promotes osteo-/odontogenic differentiation through Ras activation.

The programmed cell death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) deliver inhibitory signals to regulate immunological tolerance during immune-mediated diseases. However, the role of PD-1 signaling and its blockade effect on human dental pulp stem cells (hDPSCs) differentiation into the osteo-/odontogenic lineage remain unknown. We show here that PD-L1 expression, but not PD-1, is downregulated during osteo-/odontogenic differentiation of hDPSCs. Importantly, PD-L1/PD-1 signaling has been shown to negatively regulate the osteo-/odontogenic differentiation of hDPSCs. Mechanistically, depletion of either PD-L1 or PD-1 expression increased ERK and AKT phosphorylation levels through the upregulation of Ras enzyme activity, which plays a pivotal role during hDPSCs osteo-/odontogenic differentiation. Treatment with nivolumab (a human anti-PD-1 monoclonal antibody), which targets PD-1 to prevent PD-L1 binding, successfully enhanced osteo-/odontogenic differentiation of hDPSCs through enhanced Ras activity-mediated phosphorylation of ERK and AKT. Our findings underscore that downregulation of PD-L1 expression accompanies during osteo-/odontogenic differentiation, and hDPSCs-intrinsic PD-1 signaling inhibits osteo-/odontogenic differentiation. These findings provide a significant basis that PD-1 blockade could be effective immunotherapeutic strategies in hDPSCs-mediated dental pulp regeneration.

RIOX1-demethylated cGAS regulates ionizing radiation-elicited DNA repair.

Exposure to radiation causes DNA damage; hence, continuous surveillance and timely DNA repair are important for genome stability. Epigenetic modifications alter the chromatin architecture, thereby affecting the efficiency of DNA repair. However, how epigenetic modifiers coordinate with the DNA repair machinery to modulate cellular radiosensitivity is relatively unknown. Here, we report that loss of the demethylase ribosomal oxygenase 1 (RIOX1) restores cell proliferation and reduces cell death after exposure to ionizing radiation. Furthermore, RIOX1 depletion enhances homologous recombination (HR) repair but not nonhomologous end-joining (NHEJ) repair in irradiated bone marrow cells and oral mucosal epithelial cells. Mechanistic study demonstrates that RIOX1 removes monomethylation at K491 of cyclic GMP-AMP synthase (cGAS) to release cGAS from its interaction with the methyl-lysine reader protein SAGA complex-associated factor 29 (SGF29), which subsequently enables cGAS to interact with poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 (PARP1) at DNA break sites, thereby blocking PARP1-mediated recruitment of Timeless. High expression of RIOX1 maintains cGAS K491me at a low level, which impedes HR repair and reduces cellular tolerance to ionizing radiation. This study highlights a novel RIOX1-dependent mechanism involved in the non-immune function of cGAS that is essential for the regulation of ionizing radiation-elicited HR repair.

Wnt signaling promotes tumor development in part through phosphofructokinase 1 platelet isoform upregulation.

The activation of Wnt signaling has been detected in various types of human cancer and has been shown to be associated with cancer development. In the present study, it was revealed that Wnt signaling induced the expression of phosphofructokinase 1 platelet isoform (PFKP), which has been reported to catalyze a rate­limiting reaction in glycolysis and is important for the Warburg effect, proliferation, colony formation and cancer cell migration. Moreover, it was demonstrated that Wnt3A induced PFKP expression in a ß­catenin­independent manner, resulting in increased PFK enzyme activity. Wnt3A­induced epidermal growth factor receptor transactivation activated PI3K/AKT, which stabilized PFKP through PFKP S386 phosphorylation and subsequent PFKP upregulation. Wnt3A­induced PFKP S386 phosphorylation increased PFKP expression and promoted the Warburg effect, cell proliferation, colony formation and the migratory ability of cancer cells. On the whole, the findings of the present study underscore the potential role of PFKP in Wnt signaling­induced tumor development.

PFK activation is essential for the odontogenic differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair.