TGF‐β1 targets Smad, p38 MAPK, and PI3K/Akt signaling pathways to induce PFKFB3 gene expression and glycolysis in glioblastoma cells

In human cancers, transforming growth factor-β1 (TGF-β1) plays a dual role by acting as both a tumor suppressor and a promoter of tumor metastasis. Although TGF-β1 contributes to the metabolic reprogramming of cancer cells and tumor-associated stromal cells, little is known of the molecular mechanisms connecting this cytokine with enhanced glycolysis. PFKFB3 is a homodymeric bifunctional enzyme, belonging to the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, that controls the conversion of fructose-6-phosphate (Fru-6-P) to fructose-2,6-bisphosphate (Fru-2,6-P₂ ). This metabolite is important for the dynamic regulation of glycolytic flux by allosterically activating phosphofructokinase-1, a rate-limiting enzyme in glycolysis. The PFKFB3 gene is involved in cell proliferation via its role in carbohydrate metabolism. Here, we studied the mechanisms connecting TGF-β1, glucose metabolism, and PFKFB3 in glioblastoma cell lines. We demonstrate that TGF-β1 upregulates PFKFB3 mRNA and protein expression resulting in an increase in fructose 2,6-bisphosphate concentration, glucose uptake, glycolytic flux and lactate production. Moreover, these increases in PFKFB3 mRNA and protein expression and Fru-2,6-P₂ concentration were reduced when the Smad3, p38 mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways were inhibited. We demonstrate that inhibition of PFKFB3 activity with 3PO or siRNA-mediated knockdown of PFKFB3 significantly eliminated the capacity of the T98G cells to form colonies by TGF-β1, one of the hallmarks of transformation. Taken together, these results show that TGF-β1 induces PFKFB3 expression through activation of the p38 MAPK and PI3K/Akt signaling pathways that complement and converge with early activation of Smad signaling. This suggests that PFKFB3 induction by TGF-β1 can be one of the main mechanisms mediating the reprogramming of glioma cells.