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. 2020 May 22;5(1):54.
doi: 10.1038/s41392-020-0152-8.

Hypoxia-induced GBE1 expression promotes tumor progression through metabolic reprogramming in lung adenocarcinoma

Lifeng Li #  1   2   3 , Li Yang #  1   2 , Zhirui Fan #  4 , Wenhua Xue  5 , Zhibo Shen  1   2   5 , Yongliang Yuan  5 , Xiangdong Sun  6 , Dan Wang  1   2 , Jingyao Lian  1   2 , Liping Wang  2 , Jie Zhao  7   8 , Yi Zhang  9   10
Affiliations

Hypoxia-induced GBE1 expression promotes tumor progression through metabolic reprogramming in lung adenocarcinoma

Lifeng Li et al. Signal Transduct Target Ther. .

Abstract

Hypoxia mediates a metabolic switch from oxidative phosphorylation to glycolysis and increases glycogen synthesis. We previously found that glycogen branching enzyme (GBE1) is downstream of the hypoxia-inducible factor-1 (HIF1) signaling pathway in lung adenocarcinoma (LUAD) cells; however, the molecular mechanism underlying HIF1 regulation of GBE1 expression remains unknown. Herein, the effect of GBE1 on tumor progression via changes in metabolic signaling under hypoxia in vitro and in vivo was evaluated, and GBE1-related genes from human specimens and data sets were analyzed. Hypoxia induced GBE1 upregulation in LUAD cells. GBE1-knockdown A549 cells showed impaired cell proliferation, clone formation, cell migration and invasion, angiogenesis, tumor growth, and metastasis. GBE1 mediated the metabolic reprogramming of LUAD cells. The expression of gluconeogenesis pathway molecules, especially fructose-1,6-bisphosphatase (FBP1), was markedly higher in shGBE1 A549 cells than it was in the control cells. FBP1 inhibited the tumor progression of LUAD. GBE1-mediated FBP1 suppression via promoter methylation enhanced HIF1α levels through NF-κB signaling. GBE1 may be a negative prognostic biomarker for LUAD patients. Altogether, hypoxia-induced HIF1α mediated GBE1 upregulation, suppressing FBP1 expression by promoter methylation via NF-κB signaling in LUAD cells. FBP1 blockade upregulated HIF1α, triggered the switch to anaerobic glycolysis, and enhanced glucose uptake. Therefore, targeting HIF1α/GBE1/NF-κB/FBP1 signaling may be a potential therapeutic strategy for LUAD.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Hypoxia elevates GBE1 levels and glycogen production in LUAD cells. a Scatter plots showing the correlation between HIF1α and GBE1 expression. The red line represents the linear interpolation curve between both genes in the samples from LUAD patients. The correlation coefficient R value between two genes was computed using Pearson’s coefficient correlation. b Gene set enrichment analysis of The Cancer Genome Atlas (TCGA) data set revealed that GBE1 expression was significantly correlated with hallmark hypoxia and the nucleotide sugar biosynthetic process pathway. c Immunohistochemistry (IHC) staining of primary LUAD samples with high or low HIF1α and GBE1 expression scores and PAS staining for glycogen. d Immunofluorescence images of LUAD tissues stained for DNA (DAPI), HIF1α (green), and GBE1 (red) were merged. The scale bar represents 20 μm. e Protein expression of GBE1 and HIF1α in the LUAD and adjacent tissues was analyzed by western blotting. f mRNA expression of GBE1 and HIF1α in normal lung (16HBE) and cancer (H460 and A549) cell lines was analyzed by qPCR. g mRNA expression of GBE1 and HIF1α in A549 cells under hypoxia or normoxia was analyzed by qPCR. h Protein expression of GBE1 and HIF1α in the A549 cells under hypoxia or normoxia was analyzed by western blotting. i Immunofluorescence images of the A549 cells under hypoxia and normoxia stained for DNA (DAPI), HIF1α (green), and GBE1 (red) were merged. Scale bar represents 20 μm. j Transmission electron microscopy and PAS staining of glycogen under hypoxia and normoxia. Data are represented as the means ± SD. **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
GBE1 is a direct target gene of HIF1α. a The PROMO and JASPAR websites predicted the possible binding sequences of HIF1α to the GBE1 promoter region. b A549 cells were exposed to hypoxia and normoxia for 24 h, and ChIP assays were performed using IgG or antibodies against HIF1α. Primers flanking the entire sequence are shown and used for qPCR; the results were normalized to those for IgG at normoxic O2. c HIF1α primers were used to detect the relative expression levels of GBE1 by qPCR. d Schematic representation of the GBE1 promoter region. HIF1α in the yellow box indicates the location of the primer on the GBE1 promoter. e A549 cells were cotransfected with pSV-Renilla and firefly luciferase reporter pGL2-HRE1 (containing an oligonucleotide encompassing HIF-binding sites) and exposed to hypoxic and normoxic O2 for 24 h. Data are represented as the means ± SD. **P < 0.01
Fig. 3
Fig. 3
The effect of GBE1 on the biological behavior of LUAD in vitro and in vivo. a qPCR and western blot analysis confirming the effects of knocking down GBE1 in the siGBE1 A549 cells compared with those of the negative control cells. a–g siGBE1 A549 cell proliferation was analyzed by CCK-8 assay (b), apoptosis by flow cytometry (c), cell cycle by flow cytometry (d), migration and invasion by Transwell assays (e), migration by wound-healing assays (f), and angiogenesis by tube formation assays (g). h qPCR and western blot analysis confirming the knockdown of GBE1 in the shGBE1 A549 cells compared with the levels in the negative control cells. i Estimating the cell proliferation rate in shGBE1 cells was performed by IncuCyte ZOOM™ assay. j, k shGBE1 A549 cell colony formation (j), migration (k), and sphere formation ability (l). m Representative macroscopic tumor images upon necropsy of mice with postimplant shGBE1 and shNC A549 cells. Tumor volumes and body weights were measured at the indicated time points in the tumor-implanted mice after cell implantation. Tumor volumes and weights of xenografts at the final time point after cell implantation were also measured. n Representative IHC imaging of GBE1, caspase-3, and Ki67 expression and HE staining of tissues from xenografts in formalin-fixed paraffin-embedded sections. Data are represented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
GBE1 induces metabolic reprogramming in LUAD cells. a Seahorse metabolic analysis of ECARs and OCRs in the shGBE1 and shNC A549 cells. b Intracellular 2-NBDG accumulation evaluation in the shGBE1 and shNC A549 cells. c, d The effect of GBE1 knockdown in the A549 cells on lactate production (c) and ATP secretion (d). e The correlation between GBE1 expression levels in the LUAD tissues with Glut1 and LDHA expression levels in the samples in the TCGA data set. f ROS expression in the shGBE1 and shNC A549 cells was analyzed by fluorescence imaging and flow cytometry. g The ratio of NADP/NADPH in the shGBE1 and shNC A549 cells. h RNA-seq and i pan-metabolomic analysis of GBE1 knockdown in the A549 cells. j Heat map showing the fold changes of differentially expressed genes based on the glucose metabolism PCR array. k Schematic illustration of changes in metabolic signaling pathways induced by GBE1 knockdown. Data are represented as the means ± SD. **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
FBP1 prevents LUAD tumor progression. a Kaplan−Meier OS and RFS curves based on FBP1 expression as determined using the TCGA data set. b Box plots of FBP1 expression in the LUAD tissues at different tumor stages, according to the TCGA data set. c Scatter plot of FBP1 methylation levels in the tumor and adjacent tissues from samples in the TCGA data set. d The correlation between FBP1 mRNA levels in the LUAD tumor samples and FBP1 methylation levels in the TCGA data set. e Scatter plot of FBP1 expression was based on FBP1 copy number alterations in the TCGA data set. f Western blotting and qPCR analysis confirming the overexpression of FBP1 in the A549 cells compared with the level in the control cells. g–l The effect of FBP1 overexpression on cell apoptosis (g); cell proliferation (h); colony formation (i); cell migration, as determined by wound-healing assays (j); cell migration and invasion, as determined by Transwell assays (k); and angiogenesis (l). Data are represented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
GBE1-mediated FBP1 suppression via promoter methylation enhances HIF1α levels through NF-κB signaling. a qPCR and western blotting were performed to examine the mRNA and protein levels of FBP1 in the shGBE1 A549 cells. b FBP1 methylation levels in the shGBE1 and shNC A549 cells were analyzed by MethylRAD technology. c MSP analysis of the FBP1 promoter in the A549 cells treated with or without decitabine (5 μmol L−1, treated for 6 d). M methylated-specific primers, U unmethylated specific primers. d FBP1 expression was detected by qPCR and western blotting in the A549 cells after treatment with decitabine. e Protein levels of phospho-p65 (p-p65) and total p65 (p65) in the shGBE1 and shNC A549 cells were analyzed by western blotting. f Protein levels of phospho-p65 and total P65 in the A549 cells treated with QNZ were analyzed by western blotting. g Protein levels of FBP1 in the A549 cells treated with QNZ were analyzed by western blotting. h MSP analysis of the FBP1 promoter in the A549 cells treated with QNZ. i Correlation between FBP1 expression levels in the LUAD samples and HIF1α expression in the TCGA data set. j IHC of the LUAD tissues with high or low HIF1α and FBP1 expression. k HIF1α expression in the A549 cells with or without FBP1 overexpression under normoxia and hypoxia (1% O2) following 24 h incubation was analyzed by western blotting. l mRNA levels of GLUT1, HK2, LDHA, PDK1, and VEGFA in the A549 cells with or without FBP1 overexpression were analyzed by qPCR. m Heat map showing the expression of HIF1α, GBE1, and FBP1 in the LUAD tissues in the TCGA data set. n The signaling pathway of hypoxia-induced tumor progression. Hypoxia drives the Warburg effect to promote tumor progression via GBE1 induction, and GBE1-mediated FBP1 suppression by FBP1 promoter methylation enhances HIF1α levels via NF-κB signaling in LUAD. Data are represented as the means ± SD. *P < 0.05, **P < 0.01
Fig. 7
Fig. 7
GBE1 is a negative prognostic biomarker for LUAD patients. a IHC staining of GBE1 expression from a representative human LUAD tissue microarray. C cancer tissue, N adjacent normal lung. b Quantification of the IHC staining showing GBE1 expression in 75 LUAD and paired normal lung tissues. c Kaplan−Meier OS curve based on high or low GBE1 expression. d–f Scatter plot of GBE1 expression based on IHC score in the LUAD tissues with primary tumors (d) in regional lymph nodes (e) and by tumor stage (f). g, h Correlation between survivin and VEGF expression with GBE1 in the LUAD tissues. i, j IHC score of GBE1 in the LUAD tissues according to EGFR or ALK mutation status. k ROC curve based on GBE1 expression in the LUAD tissues. l Graphical summary of the metabolic pathway of GBE1 under hypoxia in the LUAD cells. Data are represented as the means ± SD. *P < 0.05, ***P < 0.001
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