E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation

Abstract

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

Fig. 1.

Glycolysis and proliferation in human neuroblastoma SH-SY5Y cells. S phase (BrdU-staining analysis by flow cytometry) and glycolysis correlated positively in SH-SY5Y cells at different stages of proliferation.

Fig. 2.

Effect of overexpression of Cdh1 on glycolysis, proliferation, and amount of PFKFB3 in different cell types. (A) The increase in S phase and glycolysis in SH-SY5Y cells, following exit from quiescence by removal of RA, were both prevented by Cdh1 overexpression. Glycolysis, but not proliferation, was restored by expression of PFKFB3mut, which is not recognized by Cdh1, or by overexpression of 6-phosphofructo-1-kinase, human muscle isoform (PFK1). (B) S phase and glycolysis were both increased following exit from quiescence by addition of serum to HEK 293 cells. Both responses were prevented by Cdh1 overexpression. Glycolysis, but not proliferation, was restored by expression of PFKFB3mut or by overexpression of PFK1. (C) Serum addition to HEK 293 cells increased their PFKFB3 protein abundance, an effect that was abolished by Cdh1 overexpression. The detected protein was quantified by densitometry from three independent experiments. *P < 0.05 versus the corresponding 0-h points.

Fig. 3.

Cdh1 promotes PFKFB3 degradation in the nucleus. (A) Cdh1 overexpression promoted the degradation of coexpressed rat PFKFB3 cDNA (RB2K6 splice variant), but did not affect its KEN-box-mutant form (PFKFB3mut). (B) In Cdh1-overexpressing cells, coexpressed rat PFKFB3 (RB2K6 splice variant) was present only in the nucleus, whereas its KEN-box-mutant form (PFKFB3mut) accumulated in the nucleus and was spread throughout the cytosol. (C) Serum-induced exit from quiescence promoted PFKFB3 accumulation, both in the nucleus and in the cytosol, as well as a decrease in Cdh1 in the nucleus. The detected protein in A and C was quantified by densitometry from three independent experiments.

Fig. 4.

Cdh1 coordinates glycolysis with S-phase entry. (A) Treatment of HEK 293 cells with an shRNA against Cdh1 efficiently promoted a decrease in Cdh1 protein abundance and a concomitant increase in PFKFB3; the increase in PFKFB3 protein resulting from Cdh1 silencing was prevented by cotreatment with an shRNA against PFKFB3. The detected protein was quantified by densitometry from three independent experiments. (B) S phase and glycolysis in serum-treated HEK 293 cells were increased by Cdh1 silencing. This was counteracted by cosilencing PFKFB3 (shPFKFB3), but not when PFK1 was overexpressed. ^#P < 0.05 versus the corresponding control (no plasmids added).