Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism

Abstract

BackgroundUnlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors.ResultsWe found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors.ConclusionsWe conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.

Metabolic interplay between glycolysis and mitochondrial oxidation: The reverse Warburg effect and its therapeutic implication

“…An enhanced antioxidant capacity allows cancer cells to not a universal finding, and mitochondrial respiration impairment is not a fixed feature of cancer cells [41] . Although the glycolytic inhibitors targeting the Warburg effect have been investigated in various cancer types, the glycolytic inhibitors with the exception of 3-BP (a lactate analog) [18,19, , 3-BrOP (a 3-bromopyruvate derivative) [71][72][73][74] , and dichloroacetate (DCA) have demonstrated low efficacy in arresting tumor growth when used alone [99] ; these inhibitors include 2-deoxy-D-glucose (a glucose analog) [70,[100][101][102][103][104][105][106][107][108][109][110][111][112] , lonidamine (a derivative of indazole-3-carboxylic acid) [113][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132] , methyl jasmonate on HK , 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one on PFK [162]…”

Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway

“…To further characterize this kinase in 3T3-L1 adipocytes, we employed 3-PO, a competitive inhibitor of PFKFB3 that binds to the substrate binding site in PFKFB3 (53). 3-PO is specific for PFKFB3 as verified in cells with reduced PFKFB3 (53,54), and it has been widely used to inhibit PFKFB3 in vitro and in vivo (53)(54)(55)(56)(57)(58)(59). In 3T3-L1 adipocytes, insulin significantly increased the levels of Fru-2,6-BP, the product of PFKFB3 activity, by 1.7-fold and incubation of cells with 3-PO blocked this increase, thus confirming that 3-PO inhibited PFKFB3 (Fig.…”

Glutamine at focus: versatile roles in cancer

“…In this context, some studies have addressed the key role of some metabolic enzymes that participated directly or not, on the gluconeogenesis; for example, phosphoenol pyruvate carboxylase kinase (PEPCK) activation or PFKFB3 inhibition. These enzymes are important steps required to control metabolism of tumor cells during nutrient deprivation [113,114]. Overall, these data suggest that tumor cells have much more complex metabolic repertoire than previous appreciated, and that some tumor cells are reliant on glutamine toward anabolic metabolism.…”