A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma

Abstract

Abnormal expression of the E3 ubiquitin ligase A20 has been found in some malignant cancers, including hepatocellular carcinoma (HCC). Here, we discovered that A20 is an E3 ubiquitin ligase for phosphofructokinase, liver type (PFKL) in HCC A20 interacts with PFKL and promotes its degradation, therefore inhibiting glycolysis in HCC cell lines. Downregulation of A20 in HCC cells promotes proliferation, migration, and glycolysis, all of which can be inhibited by targeting PFKL with RNA interference. Importantly, A20 is downregulated in advanced HCC tissues and inversely correlated with PFKL expression. Thus, our findings establish A20 as a critical regulator of glycolysis and reveal a novel mechanism for A20 in tumor suppression and PFKL regulation. Given that an increased level of glycolysis is linked with HCC, this study also identifies potential therapeutic targets for HCC treatment.

Fig. 1. A20 is linked to glucose metabolism in HCC cells.

a A20 suppresses proliferation in Huh7 and LM3 cells. Cell proliferation was determined by cell counting. The two-tailed Student’s t-test was used. b A20 decreases HCC tumor growth in vivo. Huh7 cells stably expressing A20 and empty vector counterpart cells were subcutaneously injected into the bilateral flanks of nude mice. At 4 weeks after injection, tumors from 10 mice were extracted and photographed (left panel). Tumor diameters were measured at the indicated time points, and tumor volumes were calculated (right panel). Significant differences were evaluated by t-test. c A20 suppresses the migration of Huh7 and LM3 cells. Huh7 and LM3 cells were transfected with pcDNA3-A20 or pMKO-shA20 plasmids, and cell migration was analyzed by Transwell experiments. d Quantitative analysis of cell migration was performed by ImageJ. The numbers of migrated cells (mean ± S.D.) from three independent experiments. e, f A20 suppresses cell glucose uptake and lactate production. pcDNA3-A20 or pMKO-shA20 plasmids was transfected in Huh7 and LM3 cells, respectively, and cellular glucose uptake and lactate production were detected via glucose uptake assay and lactate colorimetric assay, respectively. Error bars represent ± S.D. for triplicate experiments. g A20 inhibits cell glycolysis. The real-time assessment of the extracellular acidification rate (ECAR) in cultured cells was examined by Seahorse XFe96 analyzer. h Relative glycolytic capacity was normalized to the cell number (means ± S.D., n = 3). (The two-tailed Student’s t-test was used. The symbol * shows statistically significant differences with *p < 0.05, **p < 0.01, and ***p < 0.001).

Fig. 2. The metabolic enzyme PFKL is an A20-interacting protein.

a LC-MS/MS identified an interaction between PFKL and A20. b, c PFKL binds with A20 directly. The interaction between endogenous A20 and PFKL was determined by co-IP and western blotting (b). Exogenous VC155-A20 and VN173-PFKL plasmids were transfected into Huh7 cells. Representative confocal pictures of BiFC signals (green) in Huh7 cells are shown (c). d GST-PFKL can readily pull-down A20. BL21 E. Coli were transformed with pGEX-6P-1-GST-PFKL plasmid and induced by isopropyl-b-D-thiogalactoside. Protein was purified by GST antibody-conjugated columns and incubated with Huh7 cell lysates and then repurified through immunoprecipitation and subjected to western blotting. e, f LPS enhances the interaction between A20 and PFKL. Huh7 cells were cultured with or without LPS for 4 h as indicated and then processed for double immunofluorescence with antibodies against PFKL (green) and A20 (red). Merged images of both channels are shown on the right. Bar: 10 mm (e). LPS promotes endogenous PFKL binding with A20 in Huh7 cells. Huh7 cells were pretreated with MG132 for 6 h, then with or without LPS for 4 h as indicated, followed by proximity ligation (Duolink®) assay. Confocal images of the PLA reaction between A20 and PFKL in Huh7 cells. The PLA signal is in red, and DAPI is in blue. Representative data from 3 independent biological experiments (f).

Fig. 3. A20 downregulates PFKL protein levels by post-transcriptional modification.

a A20 decreases the PFKL protein level. Huh7 and LM3 cells were transfected with pcDNA3-A20 or pMKO-shA20 plasmids. The protein levels of PFKL were determined by standard western blotting (left panel). The relative PFKL protein compared with the β-actin level was quantified (right panel). b A20 reduces PFKL protein level in a dose-dependent manner. Huh7 cells were transfected with the PFKL vector together with varying amounts of A20 or empty control (Con) vectors, and the protein levels of PFKL were determined by standard western blotting (left panel). The relative PFKL protein compared with the β-actin level and relative A20 protein compared with the β-actin level was quantified (right panel). c LPS treatment increases the endogenous A20 protein level and decreases the PFKL protein level. Huh7 cells were treated with 9 and 18 μg/mL LPS for 4 h. Untreated cells were used as controls. The expression level of A20 protein was determined by western blotting. d–f A20 decreases PFKL expression at the post-transcriptional level. PFKL mRNA was determined by qPCR and normalized against β-actin. Error bars represent ± S.D. of triplicate experiments. The two-tailed Student’s t-test was used. NS denotes no significance (d). Scatterplots revealed that A20 and PFKL were not correlated at the mRNA level. Clinical data of non-cancerous liver samples and hepatocellular carcinoma samples were based on GSE364 (e). Data from the GEPIA website showed that A20 has no correlation with PFKL at the mRNA level (r = 0.16) (f). (The two-tailed Student’s t-test was used. The symbol * shows statistically significant differences with *p < 0.05, **p < 0.01, and ***p < 0.001).

Fig. 4. A20 promotes ubiquitination and degradation of PFKL.

a MG132 treatment led to the accumulation of PFKL protein levels. Huh7 and LM3 cells were treated with MG132. Cell lysates were directly subjected to western blotting. b MG132 rescues PFKL protein reduction induced by A20 overexpression. Huh7 and LM3 cells were transfected with pcDNA3-A20 plasmid, followed by MG132 treatment. The protein expression level of PFKL and A20 was determined by western blotting. c A20 overexpression shortens the half-life of PFKL. Huh7 cells were transfected with pcDNA3-A20 or vector plasmids. A CHX chase experiment was performed and PFKL protein was determined by western blotting (left panel). The right panel showcases the relative protein amounts of different groups. Error bars represent ± S.D. of triplicate experiments. The two-tailed Student’s t-test was used. *p < 0.05, **p < 0.01, and ***p < 0.001. d A20 promotes PFKL degradation through ubiquitination. Huh7 cells were transfected with the indicated plasmids. After MG132 treatment, the ubiquitination level of purified flag-PFKL protein was determined. e A20 knockdown decreases the ubiquitination level of endogenous PFKL. Huh7 cells were transfected with the indicated plasmids. After MG132 treatment for 6 h, cells were lysed for co-IP assay. Endogenous PFKL was immunoprecipitated by anti- PFKL antibody and probed for total ubiquitin.

Fig. 5. A20 inhibits cell proliferation and migration by downregulating glucose metabolism.

a, b A20 inhibits HCC cell proliferation through PFKL. Huh7 and LM3 cells were transfected with the indicated plasmids respectively. Proliferative ability was determined by cell counting assay at the indicated times. Data represent the means ± S.D. of three independent experiments. c A20 inhibits HCC cell colony formation through PFKL. The indicated plasmids were co-transfected into Huh7 cells. Proliferative ability was determined by colony formation experiment at 10 days in culture. Error bars represent ± S.D. for triplicate experiments. d A20 inhibits HCC cell migration through PFKL. The indicated plasmids were co-transfected into Huh7 cells. Cell migration capacity was analyzed by Transwell experiments. The numbers of migrated cells (mean ± S.D.) from three independent experiments. e, f A20 inhibits HCC cell glucose consumption and lactate excretion by downregulating PFKL. The indicated plasmids were co-transfected into Huh7 cells. Cell glucose consumption and lactate excretion were detected by glucose uptake assay and lactate colorimetric assay, respectively. Error bars represent ± S.D. of triplicate experiments. g A20 inhibits HCC cell glycolysis through PFKL. ECAR was examined using a Seahorse XFe96 analyzer. (The two-tailed Student’s t-test was used. The symbol * shows statistically significant differences with *p < 0.05, **p < 0.01 and ***p < 0.001).

Fig. 6. A20 expression is inversely correlated with PFKL in HCC patients.

a A20 expression is inversely with PFKL. Human HCC samples were paired as tumor tissue (designated as T) and adjacent non-tumor tissue (designated as NT). Samples were lysed and directly subjected to western blotting. 10 pairs of samples showcasing an inverse correlation are shown. b Immunohistochemical staining of A20 and PFKL proteins in tumor and adjacent non-tumor tissues. A20 expression was negatively correlated with the PFKL level in human HCC specimens (magnification, left panel, ×100; right panel, ×400). c A20 and PFKL mRNA levels are inversely correlated in non-metastatic tissues and metastatic tissues. Scatterplot data are from clinical data sets from GSE364. d Kaplan–Meier overall survival curves of HCC patients in the Protein Atlas website showing that high expression of PFKL significantly correlates with poor overall survival.

Fig. 7. Working model.

A20 upregulation promotes PFKL degradation through UPS. Malignant transformation of HCC results in decreased expression of A20 and subsequently reduced PFKL degradation. Accumulation of PFKL facilitates rapid cell proliferation and migration, therefore promoting HCC growth.