Transketolase counteracts oxidative stress to drive cancer development

Abstract

Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH-Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.

Keywords: HCC; PPP; ROS; TKT; metabolism.

Fig. 1.

TKT was overexpressed in HCC samples, and overexpression correlated with aggressive clinicopathological features. (A) Transcriptome sequencing data showing the expression of TKT, TKTL1, and TKTL2 in 16 pairs of HCC and NT liver samples. Gene expression is represented as FPKM. (B) qRT-PCR analysis of mRNA levels of TKT in 103 pairs of HCC tumor and NT samples. HPRT was used as the internal control. (C) Waterfall plot shows that TKT was up-regulated in 54.4% (56/103) of human HCC samples by at least twofold. –ΔΔCT = –[(CT_TKT – CT_HPRT) of HCC – (CT_TKT – CT_HPRT) of NT]. (D) Protein expression of TKT in representative cases of HCC and their corresponding NT liver tissues was determined by Western blotting. Intensities of bands were analyzed by Image J and normalized to the corresponding NT samples. (E) TKT expression in HCC correlated with aggressive clinicopathological features of HCC, including venous invasion, tumor microsatellite formation, tumor size, and absence of tumor encapsulation. *P < 0.05, **P < 0.01, ***P < 0.001. A, paired t test; B, Wilcoxon signed rank test; E, Mann–Whitney test.

Fig. S1.

TKT was significantly overexpressed in human HCC and other cancers. (A) Transcriptome sequencing from TCGA dataset showing the mRNA expression of genes in the PPP (TKT, G6PD, TKTL2, RPE, PGLS, RPIA, PGD, TALDO1, TKTL1) in 50 cases of paired HCC and NT liver tissues. **P < 0.01, ***P < 0.001, paired t test. (B) Box and whisker plots of Oncomine data on TKT mRNA levels (expressed as the log2 median-centered ratio) in various normal and cancerous tissues. P values, Student’s t test.

Fig. 2.

The NRF2/KEAP1/BACH1 pathway regulated TKT expression in HCC cells. (A) Positions and sequences of two NRF2 and BACH1 binding motifs in TKT. Positions refer to transcription start site (TSS). Exons are indicated as black blocks. (B and C) Recruitment of BACH1 (B) and NRF2 (C) to the two identified binding motifs in TKT. (D) Recruitment of NRF2 to the binding motifs in TKT in MHCC97L-NTC or -shBACH1 cells. ChIP assay was performed with antibodies against IgG, NRF2, or BACH1. Fold of enrichment was normalized to the according IgG controls. (E) Protein expression of TKT in MHCC97L-NTC, -shNRF2, -shKEAP1, and -shBACH1 subclones. Intensities of bands were analyzed by Image J and normalized to corresponding NTC. *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding IgG, Student’s t test. Data are presented as means ± SD.

Fig. S2.

The NRF2/KEAP1/BACH1 pathway regulated TKT expression in HCC cells. (A) Five additional NRF2 and BACH1 binding motifs were found in TKT. Positions refer to TSS. (B) ChIP assay showing the binding of BACH1 to the five identified binding motifs. Fold of enrichment is normalized to the corresponding IgG. (C) ChIP assay showing the binding of NRF2 to the five identified binding motifs. Fold of enrichment is normalized to the corresponding IgG. **P < 0.01, ***P < 0.001 versus IgG, Student’s t test. Data are presented as means ± SD.

Fig. S3.

Knockdown of the NRF2/KEAP1/BACH1 pathway members altered TKT mRNA expression. (A) mRNA expression of TKT in MHCC97L and SMMC-NTC and -shBACH1 subclones. (B) mRNA expression of TKT in MHCC97L and SMMC-NTC and -shNRF2 subclones. (C) mRNA expression of TKT in SMMC-NTC and -shKEAP1 subclones. **P < 0.01, ***P < 0.001 versus BACH1, NRF2, or KEAP1 mRNA expression in NTC, ^##P < 0.01, ^###P < 0.001 versus TKT mRNA expression in NTC, Student’s t test. Data are presented as means ± SD.

Fig. 3.

Knockdown of TKT reduced HCC cell proliferation and glucose uptake in vitro. (A) mRNA expression of TKT in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. HPRT was used as the internal control. (B) Protein expression of TKT in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. β-actin was used as the loading control. (C) Cell proliferation assay in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. (D) Glucose uptake assay was performed in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. (Left) Representative flow cytometry analysis showing intensities of 2-NBDG in the indicated HCC subclones. (Right) Histograms summarize the 2-NBDG intensities (glucose uptake) in different HCC subclones. Relative values were calculated based on the according NTC subclones. *P < 0.05, ***P < 0.001 versus NTC. A, C, and D, Student’s t test. Data are presented as means ± SD.

Fig. 4.

Knockdown of TKT increased the intracellular ROS level and induced oxidative stress-associated G1 phase arrest. (A and B) Flow cytometry was performed to analyze the ROS levels in MHCC97L and SMMC-NTC, –shTKT-1, and –shTKT-2 subclones. (Left) Representative flow cytometry analysis showing intensities of ROS (CM-H₂DCFDA) in the indicated subclones. (Right) Histograms summarize the ROS levels in different HCC subclones. Relative values were calculated based on the according NTC subclones. (C) Relative NADPH/NADP⁺ ratio in MHCC97L and SMMC-NTC and –shTKT-2 cells. The NADPH/NADP⁺ ratio was normalized to the corresponding NTC subclones. (D, Left) Representative flow cytometry analysis showing intensities of ROS (CM-H₂DCFDA) in MHCC97L-NTC and –shTKT-2 cells with vehicle or 10 μM GSH. (Right) Histogram summarizes the ROS levels. Relative values were calculated based on the ROS level in the NTC. (E) Representative flow cytometry analysis showing intensities of PI in MHCC97L-NTC and –shTKT-2 cells treated with vehicle, 10 μM GSH, or 2.5 mM NAC. Cells were synchronized with nocodazole and released from nocodazole for 9 h for cell-cycle analysis. *P < 0.05, **P < 0.01, ***P < 0.001 versus NTC or as indicated by brackets, Student’s t test. Data are presented as means ± SD.

Fig. S4.

Synchronization of HCC cells in the G2/M phase. Representative flow cytometry analysis shows the intensities of PI in MHCC97L-NTC and –shTKT-2 cells right after nocodazole treatment. All cells were synchronized at the G2/M phase.

Fig. S5.

Knockdown of TKT in normal liver cell lines. (A, Left) mRNA expression of TKT in MIHA-NTC, –shTKT-1, and –shTKT-2 subclones. 18S was used as the internal normalizing control, and values were normalized to NTC. (Right) Protein expression of TKT in MIHA-NTC, –shTKT-1, and –shTKT-2 subclones. (B) Cell proliferation assay in MIHA-NTC, –shTKT-1, and –shTKT-2 subclones. (C) Flow cytometry was performed to analyze the ROS levels in MIHA-NTC, –shTKT-1, and –shTKT-2 subclones. (Left) Representative flow cytometry analysis showing intensities of CM-H₂DCFDA (ROS) in the indicated subclones. (Right) Histograms summarize the ROS levels in different MIHA subclones. Values were normalized to NTC. (D) Relative NADPH/NADP⁺ ratio in MIHA-NTC, –shTKT-1, and –shTKT-2 cells. Values were normalized to NTC. ***P < 0.001 versus NTC, Student’s t test. Data are presented as means ± SD.

Fig. 5.

Knockdown of TKT altered glucose metabolism and glutathione metabolism. (A) Metabolic intermediates and reactions in the glycolysis and PPP. (B and C) Quantification of metabolic intermediates in glycolysis (B) and the PPP (C). (D, Left) 1,2-¹³C₂-glucose is converted to M+1 Ru5P/R5P through the oxidative arm and M+2 Ru5P/R5p through the nonoxidative arm. (Right) Quantification of mass isotopomer distribution of Ru5P/R5P in MHCC97L-NTC and -shTKT cells cultured in 1,2-¹³C₂-glucose for 12 h. M+0 indicates unlabeled Ru5P/R5P. M+1 indicates one carbon-labeled Ru5P/R5P. M+2 indicates two carbon-labeled Ru5P/R5P. 3PG, 3-phosphoglyceric acid; E4P, erythrose 4-phosphate; F1,6P, fructose 1,6-bisphosphate; F6P, frutose-6-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose-6-phosphate; PRPP, phosphoribosyl pyrophosphate; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; X5P, xylulose-5-phosphate. *P < 0.05, **P < 0.01, ***P < 0.001 versus NTC, Student’s t test. Data are presented as means ± SD.

Fig. S6.

Profiles of metabolites in TKT knockdown cells. (A) Hierarchical clustering analysis of 108 metabolites in MHCC97L-NTC and -shTKT cells in triplicate is presented as a heat map. Each row represents one intracellular metabolite. Red–green color scale depicts the amount of metabolites relative to internal control in the CE-TOFMS analysis. Increasing levels of metabolites compared with internal control are represented as increasing intensity of red. Decreasing levels of metabolites compared with internal control are represented as increasing intensity of green. (B) G6PD enzymatic activity in MHCC97L and SMMC cells. Cell lysates of MHCC97L cells were incubated with 100 μM R5P and 100 μM Ru5P before measurement. Cell lysates of SMMC cells were incubated with 500 μM R5P and 500 μM Ru5P before measurement. (C) G6PD enzymatic activity in MHCC97L and SMMC-NTC and -shTKT cells. (D) Quantification of metabolic intermediates in the purine metabolism. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. Data are presented as means ± SD.

Fig. 6.

Knockdown of TKT suppressed tumor growth and lung metastasis in vivo. (A and B) SMMC-NTC and –shTKT-1 cells (A) or SMMC-NTC and –shTKT-2 cells (B) were s.c. injected into flanks of nude mice. Tumor volumes were monitored for 28 d. (Left) Representative pictures of tumors formed in nude mice. (Scale bar, 1 cm.) (Middle) Growth curves of s.c. xenografts. (Right) Quantification of tumor mass on day 28. (C) Luciferase-labeled MHCC97L-NTC, –shTKT-1, or –shTKT-2 cells were orthotopically injected into left hepatic lobes of nude mice and allowed to grow for 42 d. (Left) Representative pictures of orthotopic xenografts. (Scale bar, 1 cm.) Right: quantification of tumor volume. (D, Left) Bioluminescent images of lung tissues. (Right) Quantification of bioluminescent intensities of lung tissues. (E) Representative pictures of H&E staining of tumor xenografts. Arrows indicate irregular growth front in the NTC group. (Scale bars, 200 μm.) (F) Representative pictures of H&E staining of lung tissues. Arrows indicate tumor cells found in the lung tissue. (Scale bars, 200 μm.) *P < 0.05, **P < 0.01 versus NTC, Student’s t test (N ≥ 5). Data are presented as means ± SD.

Fig. 7.

Genetic knockdown and pharmacological inhibition of TKT sensitized HCC cells to Sorafenib treatment via enhancing oxidative stress. (A) Proliferation curves of SMMC-NTC and –shTKT-2 cells in the presence of vehicle DMSO (Ctrl) or 6 μM Sorafenib (Sora). (B) Representative flow cytometry histogram (Left) and quantification (Right) of ROS staining in SMMC-NTC and –shTKT-2 cells treated with vehicle (Ctrl) or 6 μM Sora for 24 h. (C and D) Growth curves of s.c. xenografts derived from SMMC-NTC or –shTKT-1 cells in nude mice that were administered the control vehicle (Ctrl) or 30 mg/kg/d Sora for 14 d. (D, Left) Representative picture of tumor xenografts. (Right) Quantification of tumor mass (n = 6). (Scale bar, 1 cm.) (E) Proliferation curves of SMMC cells treated with vehicles, 6 μM Sora, 5 mM OT, or 6 μM Sora and 5 mM OT (Sora + OT). (F) Representative flow cytometry histograms of ROS staining in MHCC97L (Left) and SMMC (Right) cells treated with vehicles, 6 μM Sora, 5 mM OT, or 6 μM Sora and 5 mM OT (Sora + OT) for 24 h. (G) Growth curves of s.c. xenografts derived from parental SMMC cells in mice that were administered the vehicles (Ctrl), 30 mg/kg/d Sora, 80 mg/kg/d OT, or 30 mg/kg/d Sora and 80 mg/kg/d OT (Sora + OT) for 18 d. (H, Left) Representative pictures of tumor xenografts. (Right) Quantification of tumor mass (n = 12). (Scale bar, 1 cm.) *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl. Student’s t test. Data are presented as means ± SD.

Fig. S7.

Genetic knockdown and pharmacological inhibition of TKT sensitized MHCC97L cells to Sorafenib treatment via enhancing oxidative stress. (A) Proliferation curves of MHCC97L-NTC, –shTKT-1, or –shTKT-2 cells in the presence of control vehicle (Ctrl) or 2.5 μM Sorafenib (Sora). (B) Representative flow cytometry histogram (Left) and quantification (Right) of ROS staining in MHCC97L-NTC and –shTKT-2 cells in the presence of Ctrl or 10 μM Sora. (C) Growth curves of s.c. tumors derived from SMMC-NTC and –shTKT-2 cells in nude mice that were administered the vehicle control (Ctrl) or 30 mg/kg/d Sora for 14 d. (D, Left) Representative pictures of tumor xenografts from C. (Scale bar, 1 cm.) (Right) Quantification of tumor mass. (E) Proliferation curves of parental MHCC97L cells treated with DMSO (Ctrl), 2.5 μM Sora, 5 mM OT, or 2.5 μM Sora with 5 mM OT (Sora + OT). *P < 0.05, **P < 0.01, ***P < 0.001 versus Ctrl NTC or Ctrl, Student’s t test. Data are presented as means ± SD.

Fig. S8.

Effect of Sorafenib on cell death and cell proliferation in TKT knockdown cells. (A) Annexin V assay was performed to analyze the percentage of cell death in SMMC-NTC and –shTKT-2 cells after Sorafenib (Sora) treatment. Cells were incubated in serum-free DMEM-HG medium containing 10 μM Sorafenib for 24 h. (B) BrdU assay was performed to examine the cell proliferation of MHCC97L cells treated with vehicle (Ctrl) or 4 μM Sora with or without 10 μM GSH treatment. (C) BrdU assay was performed to examine cell proliferation of SMMC cells treated with vehicles (Ctrl) or 6 μM Sora with or without 10 μM GSH treatment. *P < 0.05, **P < 0.01, ***P < 0.001. Student’s t test.

Fig. S9.

Inhibition of TKT by OT in normal liver cell lines did not sensitize cells to Sorafenib treatment. (A) Proliferation curves of MIHA cells treated with vehicles (Ctrl), 2.5 μM Sorafenib (Sora), 5 mM OT, or 2.5 μM Sora and 5 mM OT (Sora + OT). (B) Proliferation curves of LO2 cells treated with vehicles (Ctrl), 2.5 μM Sorafenib (Sora), 5 mM OT, or 2.5 μM Sora and 5 mM OT (Sora + OT). (C) Flow cytometry was performed to analyze the ROS levels in LO2 cells treated with vehicles (Ctrl), 2.5 μM Sora, 5 mM OT, or 2.5 μM Sora and 5 mM OT (Sora + OT). *P < 0.05, **P < 0.01, ***P < 0.001. Student’s t test.

Fig. S10.

Effect of knockdown or inhibition of TKT on HCC cells upon tBHP treatment. (A) Representative flow cytometry histograms of ROS staining in MHCC97L-NTC or -shTKT cells treated with 1000 μM tert-Butyl hydroperoxide (tBHP). (B) Representative flow cytometry histograms of ROS staining in SMMC-NTC or -shTKT cells treated with 500 μM tBHP. (C) Representative flow cytometry histograms of ROS staining in MHCC97L cells treated with vehicle (Ctrl), 1,000 μM tBHP, 5 mM OT, and 1,000 μM tBHP with 5 mM OT (tBHP + OT). (D) Representative flow cytometry histograms of ROS staining in SMMC cells treated with vehicle (Ctrl), 500 μM tBHP, 5 mM OT, and 500 μM tBHP with 5 mM OT (tBHP + OT).