BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment.
METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers.
RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients.
CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.

Pituitary tumors are generally intracranial neoplasms with high incidence and mortality rates. The investigation of novel factors involved in the tumorigenesis of pituitary tumors and the characterization of the underlying molecular mechanisms is urgently required for the diagnosis and treatment of pituitary tumors. Accumulating evidence has indicated that microRNAs (miRs) serve important roles in the initiation and progression of cancer. The present study found that miR‑1 was significantly downregulated in pituitary tumor tissues upon reverse transcription‑quantitative polymerase chain reaction analysis. Decreased expression of miR‑1 was associated with the progression and worse prognosis of patients with pituitary tumors. The MTT assay showed that overexpression of miR‑1 significantly suppressed proliferation. Highly expressed miR‑1 promoted the apoptosis of pituitary tumor cells upon fluorescence‑activated cell sorting analysis. Further molecular study revealed that glucose‑6‑phosphate dehydrogenase (G6PD), the first and rate‑limiting enzyme of the pentose phosphate pathway (PPP), was one of the targets of miR‑1. Western blot assays showed that overexpression of miR‑1 significantly decreased the protein level of G6PD in pituitary tumor cells without changing the mRNA level of G6PD. Consequently, oxidative PPP flux analysis revealed that suppression of G6PD by miR‑1 decreased the production of nicotinamide adenine dinucleotide phosphate and the glycolysis of pituitary cancer cells. Restoration of the expression of G6PD significantly reversed the inhibitory effect of miR‑1 on the PPP and the growth of pituitary tumor cells. Collectively, the present results uncovered the critical involvement of miR‑1 in pituitary tumors, indicating that miR‑1 is a potential therapeutic target for the treatment of pituitary tumors.

Lactate, which is regulated by gene expression, is largely believed to favor tumor growth and survival. Elevated lactate dehydrogenase (LDH) is a negative prognostic biomarker because it is a key enzyme involved in cancer metabolism. Our previous study revealed that special AT‑rich‑binding protein 1 (SATB1), a genome‑organizing protein, was strongly associated with high metastasis rates in ovarian cancer. However, its underlying molecular mechanisms in ovarian cancer are unclear. In the present study, we investigated whether SATB1 modulated LDH expression and examined the relationship between SATB1 and LDH in ovarian cancer. We employed transient siRNA‑mediated knockdown of SATB1 in ovarian cancer and explored the effects of this knockdown on the expression levels of key glucose metabolism‑related enzyme genes (G6PD, LDH, MDH1, PFK1 and TGM1) and the glucose metabolism‑related protein monocarboxylate transporter 1 (MCT1). We comprehensively analyzed the cellular and molecular role of LDH in ovarian cancer to determine whether it could be a conventional clinicopathological parameter. SATB1 knockdown significantly downregulated both LDH and MCT1 levels and markedly upregulated BRCA1 and BRCA2 levels in ovarian cancer cells (P<0.05). Serum LDH levels in ovarian cancer patients were significantly higher than those in patients with benign ovarian tumors (P<0.05). LDH levels at different stages and grades differed significantly in ovarian cancer. Survival curves revealed that higher LDH expression was correlated with shorter survival (P<0.05). SATB1 may reprogram energy metabolism in ovarian cancer by regulating LDH and MCT1 levels to promote metastasis. Serum LDH levels presented diagnostic accuracy with high specificity and may have potential as a conventional clinicopathological parameter for ovarian cancer.

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. Multiple studies have previously revealed that elevated G6PD levels promote cancer progression in numerous tumor types; however, the underlying mechanism remains unclear. In the present study, it was demonstrated that high G6PD expression is a poor prognostic factor in bladder cancer, and the levels of G6PD expression increase with increasing tumor stage. Patients with bladder cancer with high G6PD expression had worse survival rates compared with those with lower G6PD expression in resected tumors. In vitro experiments revealed that knockdown of G6PD suppressed cell viability and growth in Cell Counting Kit-8 and colony formation assays, and increased apoptosis in bladder cancer cell lines compared with normal cells. Further experiments indicated that the weakening of the survival ability in G6PD-knockdown bladder cancer cells may be explained by intracellular reactive oxygen species accumulation and protein kinase B pathway suppression. Furthermore, it was additionally revealed that 6-aminonicotinamide (6-AN), a competitive G6PD inhibitor, may be a potential therapy for bladder cancer, particularly in cases with high G6PD expression, and that the combination of cisplatin and 6-AN may optimize the clinical dose or minimize the side effects of cisplatin.

PURPOSE: The present study investigated the dynamics and prognostic role of messenger RNA (mRNA) expression responsible for ¹⁸F-fluorodeoxyglucose (FDG) uptake in FDG positron emission tomography (PET) and radioactive iodine (¹³¹I) uptake in whole-body radioactive iodine scans (WBS) in papillary thyroid cancer (PTC) patients.
MATERIALS AND METHODS: The primary and processed data were downloaded from the Genomic Data Commons Data Portal. Expression data for sodium/iodide symporter (solute carrier family 5 member 5, SLC5A5), hexokinase (HK1-3), glucose-6-phosphate dehydrogenase (G6PD), and glucose transporter (solute carrier family 2, SLC2A1-4) mRNA were collected.
RESULTS: Expression of SLC5A5 mRNA were negatively correlated with SLC2A1 mRNA and positively correlated with SLC2A4 mRNA. In PTC with BRAF mutations, expressions of SLC2A1, SLC2A3, HK2, and HK3 mRNA were higher than those in PTC without BRAF mutations. Expression of SLC5A5, SLC2A4, HK1, and G6PD mRNA was lower in PTC without BRAF mutation. PTCs with higher expression of SLC5A5 mRNA had more favorable disease-free survival, but no association with overall survival.
CONCLUSION: Expression of SLC5A5 mRNA was negatively correlated with SLC2A1 mRNA. This finding provides a molecular basis for the management of PTC with negative WBS using ¹⁸F-FDG PET scans. In addition, higher expression of SLC5A5 mRNA was associated with less PTC recurrence, but not with deaths.

BACKGROUND: Circulating tumor cells (CTCs), an advantageous target of liquid biopsy, is an important biomarker for the prognosis and monitoring of cancer. Currently, detection techniques for CTCs are mainly based on the physical and/or epithelial characteristics of tumor cells. However, biofunctional activity markers that can indicate the high metastatic capacity of CTCs are lacking.
METHODS: Functional microarray, quantitative real-time polymerase chain reaction, and Western blot were used on five prostate cancer cell lines with different metastatic capacities to identify the metastasis-related metabolic genes. The identified genes were detected in the CTCs of 64 clinical samples using the RNA in situ hybridization. A multi-criteria weighted model was used to determine the optimal metabolic markers for the CTCs test. Based on five fluorescent signals targeting DAPI, CD45, metabolic, epithelial (EpCAM/CKs), and mesenchymal (Vimentin/Twist) markers, the filtration-enriched CTCs were classified as GM
RESULTS: Eight metastasis-related metabolic genes were identified, including HK2, PDP2, G6PD, PGK1, PHKA1, PYGL, PDK1, and PKM2. Among them, PGK1 and G6PD were determined as optimal glucose metabolic (GM) markers for CTCs. GM
CONCLUSIONS: The metabolic marker (PGK1/G6PD) is determined as the indicator for the biofunctional activity analysis of CTCs, compared with the existing morphological (EMT) classification on CTCs. The metabolic characterization of CTCs demonstrates that hypermetabolic GM

PURPOSE: Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme preventing cells from oxidative damage and has been reported to have tumor-promoting roles. This study aims to comprehensively evaluate the predictive values of G6PD on brain tumor risk, prognosis and chemo-resistance.
METHODS: A retrospective 13-year cohort study analyzing cancer risk using the Taiwan National Health Insurance Research Database (4066 G6PD deficiency patients and 16,264 controls) was conducted. Furthermore, RNAseq and clinical data of grade II-III glioma (LGG, n = 515) and glioblastoma (GBM, n = 155) were downloaded from The Cancer Genome Atlas (TCGA) and analyzed. Bioinformatics methods were applied to build a glioma prognostication model and to predict response to chemotherapy based on tumor G6PD-related gene expressions. The predicted results were validated in another glioma cohort GSE 16011 and in KALS1 cell line.
RESULTS: G6PD-dificient patients were found to have an increased risk for cancers, especially for brain tumor (adjusted hazard ratio (HR) 10.5, 95% CI 1.03-7.60). Furthermore, higher tumor G6PD expression was associated with poor patient survival in LGG, but not in GBM. A prognostication model using expression levels of G6PD and 9 related genes (PSMA2, PSMB8, SHFM1, GSS, GSTK1, MGST2, POLD3, MSH2, MSH6) could independently predict LGG patient survival. Boosted decision tree analysis on 213 cancer cell line database revealed predictive values of G6PD expression on response to gemcitabine and bortezomib. Knockdown of G6PD in KALS1 cell line enhanced its sensitivity to both chemotherapeutic agents.
CONCLUSIONS: Our study suggests that G6PD could be a marker predicting glioma risk, prognosis and chemo-sensitivity.

Breast cancer is one of the most lethal tumors in the world, among which 15% are triple-negative breast cancers (TNBCs) with higher metastasis and lower survival rate. Anoikis resistance is a key process during tumor metastasis, which is usually accompanied with metabolism reprogram. In this study, we established an anchorage independent growth model for MDA-MB-231 cells and investigated the changes in metabolism and redox homeostasis. Results showed that during detached-growth, MDA-MB-231 cells tend to generate ATP through fatty acid oxidation (FAO), instead of glycolysis. Amount of glucose was used for pentose phosphate pathway (PPP) to keep redox balance. Moreover, we discovered that a synthesized flavonoid derivative GL-V9, exhibited a potent inhibitory effect on the anchorage independent growth of TNBCs in vitro and anti-metastasis effect in vivo. In terms of the mechanism, GL-V9 could promote the expression and activity of AMPK, leading to the decrease of G6PD and the increase of p-ACC. Thus, the level of PPP was suppressed, whereas FAO was highly enhanced. The reprogram of glycolipid metabolism destroyed the redox balance ultimately and induced cell death. This paper indicated a novel regulating mechanism of redox homeostasis involving with glycolipid metabolism, and provided a potential candidate for the anti-metastatic therapy of TNBCs.

Multiple myeloma (MM), the second most common hematologic malignancy, is an incurable disease characterized by the accumulation of malignant plasma cells within the bone marrow. Though great progresses have been made in understanding the mechanisms of MM, metabolic plasticity and drug resistance remain largely unknown. In this study, we found lncRNA Protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P) is highly expressed in MM and is associated with the survival rate of MM patients. PDIA3P regulates MM growth and drug resistance through Glucose 6-phosphate dehydrogenase (G6PD) and the pentose phosphate pathway (PPP). Mechanistically, we revealed that PDIA3P interacts with c-Myc to enhance its transactivation activity and binding to G6PD promoter, stimulating G6PD expression and PPP flux. Our study identified PDIA3P as a novel c-Myc interacting lncRNA and elucidated crucial roles for PDIA3P in metabolic regulation of MM, providing a potential therapeutic target for MM patients.

Altered metabolism is one of the hallmarks of cancer cells. Pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is elevated in many cancers and contributes to tumor growth by producing ribose-5-phosphate and NADPH through PPP. However, the role of G6PD in hepatocellular carcinoma (HCC) metastasis and the clinical significance of G6PD in HCC progression and prognosis have not been well determined. In this study, by investigating tissue samples from HCC patients and HCC cell lines, we found that elevated G6PD expression is significantly associated with HCC metastasis and poor prognosis of HCCs, and that knockdown of G6PD inhibits in vitro proliferation, migration and invasion of HCC cell lines. Further studies reveal that G6PD contributes to HCC migration and invasion of hepatocellular carcinoma cells by inducing epithelial-mesenchymal transition through activation of signal transducer and activator of transcription 3 (STAT3) pathway. Our findings suggest that targeting G6PD could open up possibilities for metastasis intervention and improve the patients' outcomes for HCC.

Although metabolic reprogramming and redox imbalance are widely reported to be involved in chemo-resistance in cancer treatment, much more attention was paid to anti-cancer drug induced effect. Our previous studies showed that cancer cells can develop P-gp overexpression-mediated intrinsic drug resistance in the formation of 3D MCF-7 multi-cellular layers (MCLs) without any drug induction. However, whether metabolic reprogramming and redox imbalance functioned during this progress remained unrevealed. In our present study, LC-Q/TOF-MS and GC-MS were used in combination for analysing intracellular metabolites. The contribution of pentose phosphate pathway (PPP) and its related redox status were checked by chemical interfering and silencing/over-expression of glucose-6-phosphate dehydrogenase (G6PD). The downstream products of G6PD were assayed by quantitative real-time PCR, western blot and flow cytometry. Results showed that not only G6PD expression but also G6PD activity was significantly lowered along with 3D MCF-7 cells culture time. Impaired PPP disturbed redox-cycling, generated reactive oxygen species (ROS), which triggered cell cycle arrest and caused the switch to Chk2/p53/NF-κB pathway-mediated P-gp induction. Our results provided a new attempt to associate intrinsic small molecule metabolites (impaired PPP) communicating with cell signalling pathways through disturbed intracellular redox status to elucidate multi-cellular resistance (MCR) in 3D MCF-7 cells, which improved the understanding of the mechanisms of P-gp up-regulation in MCR with metabolomic and related redox status support.

BACKGROUND: Drug resistance is one of the major concerns in the treatment of hepatocellular carcinoma (HCC). The aim of the present study was to determine whether aberrant high expression of the inhibitor of differentiation 1(ID1) confers oxaliplatin-resistance to HCC by activating the pentose phosphate pathway (PPP).
METHODS: Aberrant high expression of ID1 was detected in two oxaliplatin-resistant cell lines MHCC97H-OXA(97H-OXA) and Hep3B-OXA(3B-OXA). The lentiviral shRNA or control shRNA was introduced into the two oxaliplatin-resistant cell lines. The effects of ID1 on cell proliferation, apoptosis and chemoresistance were evaluated in vitro and vivo. The molecular signaling mechanism underlying the induction of HCC proliferation and oxaliplatin resistance by ID1 was explored. The prognostic value of ID1/G6PD signaling in HCC patients was assessed using the Cancer Genome Atlas (TCGA) database.
RESULTS: ID1 was upregulated in oxaliplaitin-resistant HCC cells and promoted HCC cell proliferation and oxaliplatin resistance. Silencing ID1 expression in oxaliplaitin-resistant HCC cell lines inhibited cell proliferation and sensitized oxaliplaitin-resistant cells to death. ID1 knockdown significantly decreased the expression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the PPP. Silencing ID1 expression blocked the activation of G6PD, decreased the production of PPP NADPH, and augmented reactive oxygen and species (ROS), thus inducing cell apoptosis. Study of the molecular mechanism showed that ID1 induced G6PD promoter transcription and activated PPP through Wnt/β-catenin/c-MYC signaling. In addition, ID1/G6PD signaling predicted unfavorable prognosis of HCC patients on the basis of TCGA.
CONCLUSIONS: Our study provided the first evidence that ID1 conferred oxaliplatin resistance in HCC by activating the PPP. This newly defined pathway may have important implications in the research and development of new more effective anti-cancer drugs.

Glioblastomas are characterized by fast uncontrolled growth leading to hypoxic areas and necrosis. Signalling from EGFR via mammalian target of rapamycin complex 1 (mTORC1) is a major driver of cell growth and proliferation and one of the most commonly altered signalling pathways in glioblastomas. Therefore, epidermal growth factor receptor and mTORC1 signalling are plausible therapeutic targets and clinical trials with inhibitors are in progress. However, we have previously shown that epidermal growth factor receptor and mTORC1 inhibition triggers metabolic changes leading to adverse effects under the conditions of the tumour microenvironment by protecting from hypoxia-induced cell death. We hypothesized that conversely mTORC1 activation sensitizes glioma cells to hypoxia-induced cell death. As a model for mTORC1 activation we used gene suppression of its physiological inhibitor TSC2 (TSC2sh). TSC2sh glioma cells showed increased sensitivity to hypoxia-induced cell death that was accompanied by an earlier ATP depletion and an increase in reactive oxygen species. There was no difference in extracellular glucose consumption but an altered intracellular metabolic profile with an increase of intermediates of the pentose phosphate pathway. Mechanistically, mTORC1 upregulated the first and rate limiting enzyme of the pentose phosphate pathway, G6PD. Furthermore, an increase in oxygen consumption in TSC2sh cells was detected. This appeared to be due to higher transcription rates of genes involved in mitochondrial respiratory function including PPARGC1A and PPARGC1B (also known as PGC-1α and -β). The finding that mTORC1 activation causes an increase in oxygen consumption and renders malignant glioma cells susceptible to hypoxia and nutrient deprivation could help identify glioblastoma patient cohorts more likely to benefit from hypoxia-inducing therapies such as the VEGFA-targeting antibody bevacizumab in future clinical evaluations.

Malignant tumors, such as colorectal cancer (CRC), are heterogeneous diseases characterized by distinct metabolic phenotypes. These include Warburg- and reverse Warburg phenotypes depending on differential distribution of the lactate carrier proteins monocarboxylate transporter-4 and -1 (MCT4 and MCT1). Here, we elucidated the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) as the key regulator of cellular adaptation to inflammatory/environmental stress in shaping the metabolism toward a reverse Warburg phenotype in malignant and premalignant colonic epithelial cells. Immunohistochemistry of human CRC tissues revealed reciprocal expression of MCT1 and MCT4 in carcinoma and stroma cells, respectively, accompanied by strong epithelial Nrf2 activation. In colorectal tissue from inflammatory bowel disease patients, MCT1 and Nrf2 were coexpressed as well, relating to CD68+inflammatory infiltrates. Indirect coculture of human NCM460 colonocytes with M1- but not M2 macrophages induces MCT1 as well as G6PD, LDHB and TALDO expression, whereas MCT4 expression was decreased. Nrf2 knockdown or reactive oxygen species (ROS) scavenging blocked these coculture effects in NCM460 cells. Likewise, Nrf2 knockdown inhibited similar effects of tBHQ-mediated Nrf2 activation on NCM460 and HCT15 CRC cells. M1 coculture or Nrf2 activation/overexpression greatly altered the lactate uptake but not glucose uptake and mitochondrial activities in these cells, reflecting the reverse Warburg phenotype. Depending on MCT1-mediated lactate uptake, Nrf2 conferred protection from TRAIL-induced apoptosis in NCM460 and HCT15 cells. Moreover, metabolism-dependent clonal growth of HCT15 cells was induced by Nrf2-dependent activation of MCT1-driven lactate exchange. These findings indicate that Nrf2 has an impact on the metabolism already in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations. Favoring the reverse Warburg effect, these Nrf2-dependent alterations add to malignant transformation of the colonic epithelium.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that generates NADPH to maintain reduced glutathione (GSH), which scavenges reactive oxygen species (ROS) to protect cancer cell from oxidative damage. In this study, we mainly investigate the potential roles of G6PD in colorectal cancer (CRC) development and chemoresistance. We discover that G6PD is overexpressed in CRC cells and patient specimens. High expression of G6PD predicts poor prognosis and correlated with poor outcome of oxaliplatin-based first-line chemotherapy in patients with CRC. Suppressing G6PD decreases NADPH production, lowers GSH levels, impairs the ability to scavenge ROS levels, and enhances oxaliplatin-induced apoptosis in CRC via ROS-mediated damage in vitro. In vivo experiments further shows that silencing G6PD with lentivirus or non-viral gene delivery vector enhances oxaliplatin anti-tumor effects in cell based xenografts and PDX models. In summary, our finding indicated that disrupting G6PD-mediated NADPH homeostasis enhances oxaliplatin-induced apoptosis in CRC through redox modulation. Thus, this study indicates that G6PD is a potential prognostic biomarker and a promising target for CRC therapy.

Purpose/Aim: Cancer cells are addicted to glycolytic anaerobic pathways, in presence or in absence of a functional Krebs' cycle (phenomenon Warburg). This metabolic predilection relies on both extracellular (impaired vascularization and oxygenation) and intracellular (oncogenic activation of genes) causes.
MATERIALS AND METHODS: We investigated the expression and prognostic relevance of enzymes involved in the glucose absorption and metabolism, monocarboxylate transporter (MCT) expression, MCT1 and MCT2, pentose pathway (Glucose-6-phospahte dehydrogenase G6PD), glycogene synthesis (glycogene synthase GYS1), glycolysis (Hexokinase HXKII, phosphofructokinase PFK1, fructose biphosphate aldolase), fate of pyruvate (pyruvate dehydrogenase PDH, phosphorylated pPDH, PDH kinase PDK1, lactate dehydrogenase LDH5 and LDH1) and key Kreb's cycle enzymes (citrate synthase CSynth and isocitrate dehydrogenase IDH).
RESULTS: A strong overexpression of the above enzymes/proteins was noted in a varying percentage of cases examined. An interesting significant correlation between the enzymes involved in glycolysis and with the LDH5 was noted. Adenocarcinomas expressed higher levels of GLUT1 and MCT2 compared to other subtypes. Stage (p = 0.0001), aldolase (p = 0.004), LDH5 (p = 0.008), GLUT2 (p = 0.008), MCT2 (p = 0.009), GSYS1 (p = 0.04), and GLUT1 (p = 0.05) were significantly related with poor disease specific overall survival. In multivariate analysis stage (p = 0.001), LDH5 (p = 0.04), pPDH (p = 0.04), and aldolase (p = 0.04) were independent prognostic variables.
CONCLUSION: It is concluded that an orchestrated activation of glucose absorption and metabolism towards anaerobic pathways characterize the majority of NSCLC, and this phenotype is strongly linked with an aggressive clinical behavior. This glycolytic addiction of lung cancer cell is revealed as a key therapeutic target.

BACKGROUND: 18F-fluoro-2-deoxy-glucose (18F-FDG) positron emission tomography (PET) is a functional imaging modality based on glucose metabolism. The correlation between EGFR or KRAS mutation status and the standardized uptake value (SUV) of 18F-FDG PET scanning has not been fully elucidated.
METHODS: Correlations between EGFR or KRAS mutation status and clinicopathological factors including SUVmax were statistically analyzed in 734 surgically resected lung adenocarcinoma patients. Molecular causal relationships between EGFR or KRAS mutation status and glucose metabolism were then elucidated in 62 lung adenocarcinomas using cap analysis of gene expression (CAGE), a method to determine and quantify the transcription initiation activities of mRNA across the genome.
RESULTS: EGFR and KRAS mutations were detected in 334 (46%) and 83 (11%) of the 734 lung adenocarcinomas, respectively. The remaining 317 (43%) patients had wild-type tumors for both genes. EGFR mutations were more frequent in tumors with lower SUVmax. In contrast, no relationship was noted between KRAS mutation status and SUVmax. CAGE revealed that 4 genes associated with glucose metabolism (GPI, G6PD, PKM2, and GAPDH) and 5 associated with the cell cycle (ANLN, PTTG1, CIT, KPNA2, and CDC25A) were positively correlated with SUVmax, although expression levels were lower in EGFR-mutated than in wild-type tumors. No similar relationships were noted with KRAS mutations.
CONCLUSIONS: EGFR-mutated adenocarcinomas are biologically indolent with potentially lower levels of glucose metabolism than wild-type tumors. Several genes associated with glucose metabolism and the cell cycle were specifically down-regulated in EGFR-mutated adenocarcinomas.

Chemoresistance in breast cancer is a major obstacle, especially in p53 mutation types. The aim of this study was to evaluate if a combination therapy of l-arginine with 5-fluorouracil (5-FU) can alter the effect of this chemotherapy drug on breast cancer cells. The study was performed on BT-20 and MCF-7 cell lines. The effects of l-arginine alone and in combination with 5-FU were investigated on cell viability, apoptosis and nitric oxide (NO) production. Drugs effects on the cellular energetic metabolism were investigated through the lactate production and glucose-6-phosphate dehydrogenase (G6PD) activity assay. Migration and invasion of treated cells were assessed. Real- time PCR was used for analyzing the changes in the expression level of CXCL12 and CXCR4 as two important genes involved in migration and metastasis of breast cancer cells. l-arginine increased 5-FU effect on BT-20 and MCF-7 cell lines by reducing cell viability and increasing apoptosis and NO production. Lactate production and G6PD activity assays showed that cellular energetic metabolism of both cells was altered in favor of cell death. Moreover, l-arginine decreased the metastatic activity of both cells which was confirmed through migration, invasion and gene expression results performed for both cell lines. However, drugs effect on MCF-7 (p53 wild-type) was greater than that of BT-20 (p53 mutation) in all sets of experiments. Our findings indicated that l-arginine increased the anticancer effect of 5-FU in BT-20 and MCF-7 cell lines. So, combination therapy with l-arginine and 5-FU could be considered as an effective strategy in breast cancer therapy.

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF. MUC1-ARF, like its MUC1-TM 'parent' protein, contains a tandem repeat (VNTR) domain. However, the amino acid sequence of the MUC1-ARF tandem repeat as well as N- and C- sequences flanking it differ entirely from those of MUC1-TM. In vitro protein synthesis assays and extensive immunohistochemical as well as western blot analyses with MUC1-ARF specific monoclonal antibodies confirmed MUC1-ARF expression. Rather than being expressed at the cell membrane like MUC1-TM, immunostaining showed that MUC1-ARF protein localizes mainly in the nucleus: Immunohistochemical analyses of MUC1-expressing tissues demonstrated MUC1-ARF expression in the nuclei of secretory luminal epithelial cells. MUC1-ARF expression varies in different malignancies. While the malignant epithelial cells of pancreatic cancer show limited expression, in breast cancer tissue MUC1-ARF demonstrates strong nuclear expression. Proinflammatory cytokines upregulate expression of MUC1-ARF protein and co-immunoprecipitation analyses demonstrate association of MUC1-ARF with SH3 domain-containing proteins. Mass spectrometry performed on proteins coprecipitating with MUC1-ARF demonstrated Glucose-6-phosphate 1-dehydrogenase (G6PD) and Dynamin 2 (DNM2). These studies not only reveal that the MUC1 gene generates a previously unidentified MUC1-ARF protein, they also show that just like its 'parent' MUC1-TM protein, MUC1-ARF is apparently linked to signaling and malignancy, yet a definitive link to these processes and the roles it plays awaits a precise identification of its molecular functions. Comprising at least 524 amino acids, MUC1-ARF is, furthermore, the longest ARF protein heretofore described.

Like most other types of cancer cells, leukaemia cells undergo metabolic reprogramming to support rapid proliferation through enhancing biosynthetic processes. Pentose phosphate pathway (PPP) plays a pivotal role in meeting the anabolic demands for cancer cells. However, the molecular mechanism by which PPP contributes to leukaemia remains elusive. Here, we report that leukaemia cell proliferation is dependent on the oxidative branch of PPP, in particular the first and rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD). Knockdown of G6PD reduces NADPH level in acute myeloid leukaemia (AML) cell lines. Exogenous lipid supplements partially restore the proliferation of G6PD-depleted cells. Deacetylase SIRT2 promotes NADPH production through deacetylating G6PD at lysine 403 (K403). Activation of G6PD by SIRT2 supports the proliferation and clonogenic activity of leukaemia cells. Chemical inhibitors against SIRT2 suppress G6PD activity, leading to reduced cell proliferation of leukaemia cells, but not normal hematopoietic stem and progenitor cells. Importantly, SIRT2 is overexpressed in clinical AML samples, while K403 acetylation is downregulated and G6PD catalytic activity is increased comparing to that of normal control. Together, our study reveals that acetylation regulation of G6PD is involved in the metabolic reprogramming of AML, and SIRT2 serves as a promising target for further therapeutic investigations.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer.

REC8 meiotic recombination protein (REC8) was found to be preferentially methylated in gastric cancer (GC) using promoter methylation array. We aimed to elucidate the epigenetic alteration and biological function of REC8 in GC. REC8 was downregulated in 100% (3/3) of Epstein-Barr virus (EBV)-positive and 80% (8/10) of EBV-negative GC cell lines by promoter methylation, but the expression could be restored through demethylation treatment. Protein expression of REC8 was significantly lower in human primary gastric tumors than in adjacent non-tumor tissues. A negative correlation between methylation and mRNA expression of REC8 was observed in 223 gastric samples of The Cancer Genome Atlas study (r=-0.7018, P<0.001). The methylation level (%) of the REC8 promoter was significantly higher in EBV-positive gastric tumors than in EBV-negative gastric tumors, as shown by bisulfite genomic sequencing (77.6 (69.3-80.5) vs 51.4 (39.5-62.3), median (interquartile range); P<0.001); methylation levels in both subtypes of tumors were significantly higher than in normal stomach tissues (14.8 (4.2-24.0)) (both P<0.001). Multivariate analysis revealed that REC8 methylation was an independent factor for poor survival in GC patients (hazard ratio=1.68, P<0.05). REC8 expression significantly suppressed cell viability, clonogenicity and cell cycle progression; it induced apoptosis and inhibited migration of AGS-EBV (EBV-positive) and BGC823 (EBV-negative) GC cells, and it suppressed tumorigenicity in nude mice. In contrast, knockdown of REC8 in gastric epithelial immortalized GES-1 cells significantly increased cell viability, clonogenicity and migration ability. The tumor-suppressive effect of REC8 is mediated at least in part by the downregulation of genes involved in cell growth (G6PD, SLC2A1, NOL3, MCM2, SNAI1 and SNAI2), and the upregulation of apoptosis/migration inhibitors (GADD45G and LDHA) and tumor suppressors (PinX1, IGFBP3 and ETS2). In conclusion, REC8 is a novel tumor suppressor that is commonly downregulated by promoter methylation in GC, especially in the EBV-associated subtype. Promoter methylation of REC8 is an independent risk factor for the shortened survival of GC patients.

Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance.

Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer.

Given the involvement of telomerase activation and dysregulated metabolism in glioma progression, the connection between these two critical players was investigated. Pharmacological inhibition of human Telomerase reverse transcriptase (hTERT) by Costunolide induced glioma cell apoptosis in a reactive oxygen species (ROS)-dependent manner. Costunolide induced an ROS-dependent increase in p53 abrogated telomerase activity. Costunolide decreased Nrf2 level; and ectopic Nrf2 expression decreased Costunolide-induced ROS generation. While TERT knock-down abrogated Nrf2 levels, overexpression of Nrf2 increased TERT expression. Inhibition of hTERT either by Costunolide, or by siRNA or dominant-negative hTERT (DN-hTERT) abrogated (i) expression of Glucose-6-phosphate dehydrogenase (G6PD) and Transketolase (TKT) - two major nodes in the pentose phosphate (PPP) pathway; and (ii) phosphorylation of glycogen synthase (GS). hTERT knock-down decreased TKT activity and increased glycogen accumulation. Interestingly, siRNA-mediated knock-down of TKT elevated glycogen accumulation. Coherent with the in vitro findings, Costunolide reduced tumor burden in heterotypic xenograft glioma mouse model. Costunolide-treated tumors exhibited diminished TKT activity, heightened glycogen accumulation, and increased senescence. Importantly, glioblastoma multiforme (GBM) patient tumors bearing TERT promoter mutations (C228T and C250T) known to be associated with increased telomerase activity; exhibited elevated Nrf2 and TKT expression and decreased glycogen accumulation. Taken together, our findings highlight the previously unknown (i) role of telomerase in the regulation of PPP and glycogen accumulation and (ii) the involvement of Nrf2-TERT loop in maintaining oxidative defense responses in glioma cells.

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions.

Quinoxaline 1,4-dioxides (QdNOs) are widely used as a kind of antibacterial growth promoter in animal husbandry. The adrenal cortex was found to be one of the main toxic targets of QdNOs, accompanied by a decreased aldosterone level. However, the way in which QdNOs decrease production of the hormone aldosterone is far from clear. To illustrate the mechanism by which QdNOs damage the adrenal cortex and decrease aldosterone hormone levels, the QdNOs were screened to choose the drug with most toxic effects on aldosterone production, and then to reveal the mechanism between the gene and protein profiles in human adrenocortical cells (NCI-H295R cells). The results found that quinocetone (QCT) showed the highest adrenal toxic effect among QdNOs. After exposing H295R cells to 10 and 20μM QCT for 24h, compared with blank cells, the gene and protein expression profiles obtained were analyzed by microarray and MALDI TOF/TOF mass spectrometry, respectively. The results of microarray analysis suggested that ABCG1 and SREBF1, which were involved in the cholesterol biosynthetic and metabolic processes, and CYP17A1, NR4A2 and G6PD, which were related to aldosterone biosynthesis, were important molecular targets. It has been speculated that PKC and ERK pathways might be involved in the reduction of aldosterone production caused by QCT, through enhanced mRNA expression of CYP17A1. Additionally, JNK and p38MAPK signal transduction pathways might participate in apoptosis induced by QCT. Twenty-nine and 32 protein spots were successfully identified when cells were treated with 10 and 20μM QCT, respectively. These identified proteins mainly included material synthesis and energy metabolism-related proteins, transcription/translation processing-related proteins, signal transduction proteins, cytoskeletal proteins, molecular chaperones, proteins related to response to stress, and transport proteins. Further investigations suggested that oxidative stress caused by QCT was exacerbated through disruption of the Keap1/Nrf2/ARE anti-oxidative stress pathway. Taken together, the data demonstrated for the first time that the Keap1/Nrf2/ARE pathway plays a crucial role in adrenal toxicity, and that CYP17A1 was the key switch to reduce the aldosterone production induced by QCT. Furthermore, large numbers of genes and proteins and entry points for research in the inhibition of aldosterone synthesis induced by QCT were offered, which will provide new insight into the adrenal toxicity of QdNOs and help to provide a theoretical foundation for the formulation of safety controls for products obtained from animals and to design new QdNOs with less harmful effects.