Fig 1.
PGM1 is down-regulated in HCC and inversely correlates with HCC malignance.
(A–B) IHC staining with anti-PGM1 antibody was performed on 69 pairs of HCC tumor tissues and corresponding peritumoral tissues. Representative images of tumors from two HCC patients are shown (panel A). Semiquantitative scoring was performed (panel B; paired t test, two-tailed, P < 0.0001). (C) IHC staining with anti-PGM1 antibody was performed on 272 tumor tissues from HCC patients with microvascular invasion (n = 169) or without microvascular invasion (n = 103). Semiquantitative scoring was performed (Mann-Whitney test, P = 0.0087). (D) IHC staining with anti-PGM1 antibody was performed on 272 HCC tumors with moderate to poor differentiation (n = 248) or with good differentiation (n = 24). Semiquantitative scoring was performed (Mann-Whitney test, P = 0.0018). (E–F) Kaplan-Meier curves of overall survival (panel E) and cumulative recurrence rates (panel F) were performed on 272 HCC patients after surgery. The optimal cutoff value of PGM1 staining is 9.6 × 107, as determined by X-title software. Significance was determined by the log-rank test for both overall survival and cumulative recurrence rates. (G) PGM1 expression in 17 HCC PDX tumors was measured. Representative images of PGM1 expression from two PDX tumors (left panel) and tumor growth curves (right panel) are shown. The data of tumor growth were adapted from one of our previous studies. Underlying data can be found in S1 Data. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; IOD, integrated optical density; PDX, patient-derived xenograft; PGM1, phosphoglucomutase 1.
Fig 2.
PGM1 inhibits tumor cell proliferation and tumor growth.
Immunoblotting analyses were performed with the indicated antibodies. (A–C) SK-Hep1 cells were infected with the lentivirus expressing EV or Flag-PGM1. Immunoblotting analyses were performed in these cells (panel A). Proliferation (left panel) and colony formation (right panel) were examined in these cells (panel B). These cells were subcutaneously injected into randomized athymic nude mice (5 mice per group). Nine weeks after the injection, tumors were dissected for HE staining or weight measurement. Representative images of HE staining of dissected tumors are shown in left panel. Quantitative analyses of dissected tumor weights were performed (right panel). Data represent the means ± SD of 5 mice (panel C). (D–F) SK-Hep1 cells were infected with the lentivirus expressing shNT or shPGM1. IB analyses were performed in these cells (panel D). Proliferation (left panel) and colony formation (right panel) were examined in these cells (panel E). These cells were subcutaneously injected into randomized athymic nude mice (5 mice per group). At 30 days after the injection, tumors were dissected for HE staining or weight measurement. Representative images of HE staining of dissected tumors are shown in left panel. Quantitative analyses of dissected tumor weights were performed (right panel). Data represent the means ± SD of 5 mice (panel F). (G–I) SK-Hep1 cells were depleted of endogenous PGM1 and rescued with Flag-rPGM1 WT or G121R. Proliferation of these cells was determined by SRB assay (panel G). Colony formation was examined in these cells (panel H). These cells were subcutaneously injected into randomized athymic nude mice (5 mice per group). At 21 days after the injection, tumors were dissected for weight measurement (panel I). Representative images of dissected tumors are shown in upper panel. Quantitative analyses of dissected tumor weights are shown in lower panel. Data represent the means ± SD of 5 mice. (J) SK-Hep1 cells stably expressing shNT or shPGM1 were subcutaneously injected into randomized athymic nude mice (5 mice per group), and D-galactose was administered via oral gavage at 0.1 g/kg body weight per day for 3 weeks. Then, tumors were dissected for weight measurement. Representative images of dissected tumors are shown in left panel. Quantitative analyses of dissected tumor weights were performed (right panel). Data represent the means ± SD of 5 mice. (K–L) SK-Hep1 cells stably expressing EV or Flag-PGM1 were treated with or without 0.1 ug/ml Tunicamycin, and cell proliferation was measured (panel K). These cells were subcutaneously injected into randomized athymic nude mice (5 mice per group), and 0.5 mg/kg body weight Tunicamycin was administered by intraperitoneal injection twice a week. At 30 days after the inoculation, tumors were dissected for weight measurement. Representative images of dissected tumors are shown in left panel. Quantitative analyses of dissected tumor weights were performed (right panel). Data represent the means ± SD of 5 mice. Underlying data can be found in S1 Data. EV, empty vector; HE, hematoxylin–eosin; IB, immunoblotting; PGM1, phosphoglucomutase 1; shNT, nontargeting shRNA; shPGM1, shRNA against PGM1; shRNA, short hairpin RNA; SRB, sulforhodamine B; WT, wild-type.
Fig 3.
PGM1 enhances glycogen synthesis but inhibits aerobic glycolysis.
(A–D) SK-Hep1 cells were infected with the lentivirus expressing EV, Flag-PGM1, shNT, or shPGM1. The culture media of these cells were collected for analysis of glucose consumption (panel A) and lactate production (panel B). Glycogen content (panel C) and G-1-P/G-6-P ratio (panel D) of these cells were measured. Data represent the means ± SD of 3 independent experiments. (E–F) SK-Hep1 cells stably expressing EV, Flag-PGM1, shNT, or shPGM1 were subcutaneously injected into randomized athymic nude mice (5 mice per group). Thirty days later, dissected tumors were homogenized and deproteinized for the measurement of lactate content using L-Lactate Assay kit (panel E). Glycogen content was measured in dissected tumors (panel F). Data represent the means ± SD of 5 mice. (G) SK-Hep1 cells stably expressing shNT or shPGM1 were treated with or without 0.5 mM 2-DG, and cell proliferation was measured. Data represent the means ± SD of 3 independent experiments. (H–J) The culture media of endogenous PGM1-depleted SK-Hep1 cells rescued with Flag-rPGM1 WT or G121R were collected for analysis of glucose consumption (panel H) and lactate production (panel I). Glycogen content (panel J) of these cells was measured. Data represent the means ± SD of 3 independent experiments. Underlying data can be found in S1 Data. 2-DG, 2-Deoxyglucose; EV, empty vector; G-1-P, glucose 1-phosphate; G-6-P, glucose 6-phosphate; PGM1, phosphoglucomutase 1; shNT, nontargeting shRNA; shPGM1, shRNA against PGM1; shRNA, short hairpin RNA; WT, wild-type.
Fig 4.
FOXJ2 enhances pgm1 promoter activity to increase PGM1 expression.
Immunoblotting analyses were performed with the indicated antibodies. (A) SK-Hep1 cells were stably transfected with EV or Flag-FOXJ2. Real-time PCR (upper panel) and immunoblotting analyses (lower panel) were performed to examine mRNA and protein levels of PGM1. Data represent the means ± SD of 3 independent experiments. (B) The luciferase reporter construct containing either FBM1 or FBM2 fragment was cotransfected with EV or Flag-FOXJ2 into SK-Hep1 cells. The relative levels of luciferase activity of Firefly were normalized to the levels of luciferase activity of control Renilla. Data represent the mean ± SD of 3 independent experiments (left panel). Flag-FOXJ2 protein was immunoprecipitated from SK-Hep1 cells stably expressing Flag-FOXJ2 using anti-Flag antibody and eluted with Flag peptides. EMSA was performed by mixing eluted Flag-FOXJ2 and biotin-labeled FBM1 or FBM2 (Biotin-FBM1 or Biotin-FBM2) oligonucleotides (middle and right panel). (C) The luciferase reporter construct containing either FBM1 WT or FBM1 MT fragment was cotransfected with EV or Flag-FOXJ2 into SK-Hep1 cells. The relative levels of luciferase activity of Firefly were normalized to the levels of luciferase activity of control Renilla. Data represent the mean ± SD of 3 independent experiments (left panel). Flag-FOXJ2 protein was immunoprecipitated from SK-Hep1 cells stably expressing Flag-FOXJ2 using anti-Flag antibody and eluted with Flag peptides. EMSA was performed by mixing eluted Flag-FOXJ2 and Biotin-FBM1 WT or Biotin-FBM1 MT oligonucleotides (right panel). (D) ChIP analyses were performed with an anti-FOXJ2 antibody in SK-Hep1 cells. Real-time PCR analysis was carried out to amplify PGM1 promoter. Data represent the means ± SD of 3 independent experiments. (E–H) SK-Hep1 cells were infected with the lentivirus expressing shNT or shFOXJ2 (panel E). The culture media of these cells were collected for analysis of glucose consumption (panel F) and lactate production (panel G). Glycogen content (panel H) of these cells was measured. Data represent the means ± SD of 3 independent experiments. (I) IHC staining with anti-FOXJ2 antibody was performed in 69 pairs of tumor tissues and corresponding peritumoral tissues from HCC patients. Representative images of tumor tissues and paired peritumoral tissues are shown in left panel. Semiquantitative scoring was performed (right panel, paired t test, two-tailed, P < 0.0001). (J) Real-time PCR was performed to examine relative genomic copy number of FOXJ2 in paired tumor tissues and corresponding peritumoral tissues from 9 HCC patients. Data represent the means ± SD of 9 patients. Underlying data can be found in S1 Data. ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; EV, empty vector; FBM1, FOXJ2-binding motif 1; FOXJ2, forkhead box protein J2; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; MT, mutant; PGM1, phosphoglucomutase 1; shNT, nontargeting shRNA; shPGM1, shRNA against PGM1; shRNA, short hairpin RNA; WT, wild-type.
Fig 5.
FOXJ2 disrupts the binding of CNBP to PGM1 promoter.
Immunoblotting analyses and coimmunoprecipitation were performed with the indicated antibodies. (A) SK-Hep1 cells were transiently transfected with EV or Flag-FOXJ2. (B) Real-time PCR (left panel) and immunoblotting analyses (right panel) were performed to examine mRNA and protein levels of PGM1 in SK-Hep1 cells stably expressing shNT or shCNBP. Data represent the means ± SD of 3 independent experiments. (C) EMSA was carried out by mixing bacteria-purified recombinant human CNBP with biotin-G20 oligonucleotides. (D) ChIP analyses with an anti-CNBP antibody were performed in SK-Hep1 cells. Data represent the means ± SD of 3 independent experiments. (E) SK-Hep1 cells stably expressing EV or HA-FOXJ2 were infected with a lentivirus expressing EV or Flag-CNBP. Real-time PCR (left panel) and immunoblotting analyses (right panel) were performed to examine mRNA and protein levels of PGM1. Data represent the means ± SD of 3 independent experiments. (F) Flag-FOXJ2 was immunoprecipitated from SK-Hep1 cells stably expressing Flag-FOXJ2 and eluted with Flag peptides. EMSA was performed by mixing bacteria-purified recombinant CNBP and biotin-G20 oligonucleotides in the presence or absence of eluted Flag-FOXJ2 protein. (G) SK-Hep1 cells were infected with a lentivirus expressing EV or HA-FOXJ2. ChIP analyses with an anti-CNBP antibody were performed in these cells. Data represent the means ± SD of 3 independent experiments. (H–K) SK-Hep1 cells were infected with the lentivirus expressing EV or Flag-CNBP. The culture media of these cells were collected for analysis of glucose consumption (panel H) and lactate production (panel I). Glycogen content (panel J) and colony formation (panel K) of these cells were measured. Data represent the means ± SD of 3 independent experiments. Underlying data can be found in S1 Data. ChIP, chromatin immunoprecipitation; CNBP, cellular nucleic acid-binding protein; EMSA, electrophoretic mobility shift assay; EV, empty vector; FOXJ2, forkhead box protein 2; HA-FOXJ2, HA-tagged FOXJ2; IP, immunoprecipitation; PGM1, phosphoglucomutase 1; shNT, non-targeting shRNA; shPGM1, shRNA against PGM1; shRNA, short hairpin RNA.
Fig 6.
FOXJ2-up-regulated PGM1 expression inhibits glycolysis, cell proliferation, and HCC development.
(A–D) SK-Hep1 or Huh7 cells stably expressing shNT or shPGM1 were infected with a lentivirus expressing EV or Flag-FOXJ2. The culture media of these cells were collected for analysis of glucose consumption (panel A) and lactate production (panel B). Glycogen content (panel C) and colony formation (panel D) of these cells were measured. Data represent the means ± SD of 3 independent experiments. (E–F) SK-Hep1 cells stably expressing shNT or shPGM1 were infected with a lentivirus expressing EV or Flag-FOXJ2. These cells were subcutaneously injected into randomized athymic nude mice (6 mice per group). At 30 days after the injection, tumors were dissected for weight (panel E) and glycogen content (panel F) measurement. Representative images of Periodic Acid-Schiff staining of the tumors were presented (panel F, left panel). Glycogen content of the tumors was measured. Data represent the means ± SD of 6 mice (panel F, right panel). Underlying data can be found in S1 Data. EV, empty vector; FOXJ2, forkhead box protein J2; HCC, hepatocellular carcinoma; PGM1, phosphoglucomutase 1; shNT, nontargeting shRNA; shPGM1, shRNA against PGM1; shRNA, short hairpin RNA.
Fig 7.
PGM1 and FOXJ2 expression correlates with the prognosis of HCC patients.
IHC staining using anti-PGM1 or anti-FOXJ2 antibodies were performed in 272 HCC tissues, respectively. (A) Representative images of IHC staining of tumor tissues from three HCC patients were shown. (B) Pearson correlation analysis was performed to determine correlation between PGM1 and FOXJ2 expression in HCC tissues (Pearson correlation coefficient = 0.601, P < 0.0001). (C–D) Kaplan-Meier curves of overall survival (panel C) and cumulative recurrence rates (panel D) were performed on HCC patients. The optimal cutoff values of PGM1 staining (9.6 × 107) and FOXJ2 staining (27.3 × 107) were determined by X-title software. Significance was determined by the log-rank test for both overall survival and cumulative recurrence rates. (E) Schematic model of mechanism that FOXJ2-dependent PGM1 up-regulation inhibits HCC growth. Upon sufficient extracellular glucose, FOXJ2 binds to PGM1 promoter and interacts with CNBP, which subsequently prevents CNBP-mediated G4-DNA formation in PGM1 promoter, thereby increasing PGM1 expression. This FOXJ2-dependent PGM1 up-regulation enhances glycogen synthesis in HCC cells, which consequently reduces glucose flux into glycolysis for biosynthesis processes and therefore inhibits tumor cell proliferation. Underlying data can be found in S1 Data. CNBP, cellular nucleic acid-binding protein; FOXJ2, forkhead box protein J2; HCC, hepatocellular carcinoma; PGM1, phosphoglucomutase 1.