Integrin alpha x stimulates cancer angiogenesis through PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression in blood vessel endothelial cells

Integrin alpha x (ITGAX), a member of the integrin family, usually serves as a receptor of the extracellular matrix. Recently, accumulating evidence suggests that ITGAX may be involved in angiogenesis in dendritic cells. Herein, we report a direct role of ITGAX in angiogenesis during tumor development. Overexpression of ITGAX in human umbilical vein endothelial cells (HUVECs) enhanced their proliferation, migration, and tube formation and promoted xenograft ovarian tumor angiogenesis and growth. Further study showed that overexpression of ITGAX activated the PI3k/Akt pathway, leading to the enhanced expression of c-Myc, vascular endothelial growth factor-A (VEGF-A), and VEGF receptor 2 (VEGFR2), whereas, the treatment of cells with PI3K inhibitor diminished these effects. Besides, c-Myc was observed to bind to the VEGF-A promoter. By Co-Immunoprecipitation (Co-IP) assay, we manifested the interaction between ITGAX and VEGFR2 or the phosphorylated VEGFR2. Immunostaining of human ovarian cancer specimens suggested that endothelial cells of micro–blood vessels displayed strong expression of VEGF-A, c-Myc, VEGFR2, and the PI3K signaling molecules. Also, overexpression of ITGAX in HUVECs could stimulate the spheroid formation of ovarian cancer cells. Our study uncovered that ITGAX stimulates angiogenesis through the PI3K/Akt signaling–mediated VEGFR2/VEGF-A overexpression during cancer development.     

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

Neuromedin B induces angiogenesis via activation of ERK and Akt in endothelial cells

Neuromedin B (NMB) is one of the bombesin-like peptides in mammals. Recently, bombesin-like peptides have been characterized as growth factors in highly vascularized tumors. In this study, we report that NMB potently stimulates in vivo neovascularization in a mouse Matrigel plug and the sprouting of endothelial cells ex vivo in rat aortic rings. In addition, NMB increases the migration and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with NMB activates the extracellular signal-regulated kinase 1/2 (ERK_1/2), Akt, and endothelial nitric oxide synthase (eNOS) and increases the level of NO production in a dose- and time-dependent manner. Furthermore, ERK activation and angiogenic sprouting in response to NMB are significantly blocked by the MEK inhibitor. Inhibition of phosphatidylinositol 3-kinase (PI3K) suppresses the NMB-stimulated tubular formation of HUVECs, along with reduction in the phosphorylation of Akt and eNOS. Taken together, these results indicate that NMB is a novel angiogenic peptide, and its angiogenic activity is mediated by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent pathways. This study suggests that NMB may play important roles in mediating a variety of pathophysiological angiogenesis.     

Pro-angiogenic activity and vasculogenic mimicry in the tumor microenvironment by leptin in cancer

The acquired ability to induce the formation of a functional vasculature is a hallmark of cancer. Blood vessels in tumors are formed through various mechanisms, among the most important in cancer biology, angiogenesis, and vasculogenic mimicry have been described. Leptin is one of the main adipokines secreted by adipocytes in normal breast tissue and the tumor microenvironment. Here, we provide information on the relationship between leptin and the development of angiogenesis and vasculogenic mimicry in different types of cancer. Here, we report that leptin activates different pathways such as JAK-STAT3, MAPK/ERK, PKC, JNK, p38, and PI3K-Akt to induce the expression of various angiogenic factors and vasculogenic mimicry. In vivo models, leptin induces blood vessel formation through the PI3K-Akt-mTOR pathway. Interestingly, the relationship between leptin and vasculogenic mimicry was more significant in breast cancer. The information obtained suggests that leptin could be playing an essential role in tumor survival and metastasis through the induction of vascular mechanisms such as angiogenesis and vasculogenic mimicry; thus, leptin-induced pathways could be suggested as a promising therapeutic target.     

Liraglutide restores angiogenesis in palmitate-impaired human endothelial cells through PI3K/Akt-Foxo1-GTPCH1 pathway

Glucagon-like peptide-1 (GLP-1) and its analogues have a beneficial role in cardiovascular system. Here, we aimed to investigate whether liraglutide, a GLP-1 analogue, modulated angiogenesis impaired by palmitic acid (PA) in cultured human umbilical vein endothelial cells (HUVECs). Cells were incubated with liraglutide (3–100 nmol/L) in the presence of PA (0.5 mmol/L), and endothelial tube formation was observed and quantified. The protein levels of signaling molecules were analyzed and the specific inhibitors were used to identify the signaling pathways through which liraglutide affected angiogenesis. Results showed that liraglutide ameliorated endothelial tube formation impaired by PA in HUVECs in a dose-dependent manner. Meanwhile, liraglutide increased the phosphorylation of Akt and forkhead box O1 (Foxo1), and upregulated the levels of guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) and endothelial nitric oxide synthase (eNOS) in PA-impaired HUVECs. Notably, addition of the PI3K inhibitor LY294002, Foxo1 nuclear export inhibitor trifluoperazine dihydrochloride (TFP), GTPCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) or NOS inhibitor N-nitro-l-arginine-methyl ester (L-NAME) eliminated the angiogenic effect of liraglutide. Moreover, either LY294002 or TFP abolished the liraglutide-induced upregulation of GTPCH1 and eNOS protein levels. In conclusion, liraglutide restores angiogenesis in PA-impaired HUVECs. The effect is mediated via upregulation of GTPCH1 and eNOS levels in a PI3K/Akt-Foxo1-dependent mechanism.     

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.     

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background

The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.

Methodology/Principal Findings

We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P₃ using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.

Conclusion/Significance

These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.     

Over-expression of PDGFR-β promotes PDGF-induced proliferation, migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway

The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes.     

Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis

Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.     

Thematic review series: sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.     

Huaier shows anti-cancer activities by inhibition of cell growth,migration and energy metabolism in lung cancer through PI3K/AKT/HIF-1α pathway

Huaier has been verified to have anti-cancer effects on many tumours. However, little information is available about the effects of Huaier on non-small cell lung cancer (NSCLC). We sought to probe the anti-cancer effects and related mechanisms of Huaier on lung cancer. A549 cells were pre-treated with 2, 4 and 8 mg/mL Huaier at different time points. Thereafter, cell viability was analysed by CCK-8 and the migration and invasion were detected by Scratch test and Transwell chamber migration assay. Moreover, ELISA, Western blot, shRNA transfection and RT-PCR were conducted to discover the related gene and protein expressions of energy metabolism and phosphatidylinositol 3-kinase (PI3K)/AKT/hypoxia-inducible factor 1α (HIF-1α) pathway. Furthermore, tumour xenografts were accomplished to inspect the anti-cancer effects of Huaier. Our consequences suggested that Huaier considerably repressed cell viability and migration in a dose-dependent way. In addition, Huaier statistically suppressed glycolysis, glucose transport and lactic acid (LA) accumulation. Besides, we detected that Huaier could inactivate the PI3K/AKT/HIF-1α pathway. The in vivo data confirmed that Huaier obviously decreased tumour volume and tumour growth, reduced the glycolysis, glucose transport and HIF-1α expression in the tumour-bearing tissues. Our results suggested Huaier revealed anti-tumour effects in both in vivo and in vitro possibly through PI3K/AKT/HIF-1α pathway.     

Pravastatin-induced proangiogenic effects depend upon extracellular FGF-2

The HMG-CoA reductase inhibitors (statins) have been shown to exert several protective effects on the vasculature that are unrelated to changes in the cholesterol profile, and to induce angiogenesis. The proangiogenic effect exerted by statins has been attributed to the activation of the PI3K/Akt pathway in endothelial cells; however, it is unclear how statins activate this pathway. Pravastatin-mediated activation of Akt and MAPK occurs rapidly (within 10 min.) and at low doses (10 nM). Here, we hypothesized that FGF-2 contributes to the proangiogenic effect of statins. We found that pravastatin, a hydrophilic statin, induced phosphorylation of the FGF receptor (FGFR) in human umbilical vein endothelial cells. SU5402, an inhibitor of FGFR, abolished pravastatin-induced PI3K/Akt and MAPK activity. Likewise, anti-FGF-2 function-blocking antibodies inhibited Akt and MAPK activity. Moreover, depletion of extracellular FGF-2 by heparin prevented pravastatin-induced phosphorylation of Akt and MAPK. Treatment with FGF-2 antibody inhibited pravastatin-enhanced endothelial cell proliferation, migration and tube formation. These observations indicate that pravastatin exerts proangiogenic effects in endothelial cells depending upon the extracellular FGF-2.     

Targeting integrins and PI3K/Akt-mediated signal transduction pathways enhances radiation-induced anti-angiogenesis

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.     

Insulin regulates monocyte trans-endothelial migration through surface expression of macrophage-1 antigen

During the pathogenesis of atherosclerosis, adhesion of monocytes to vascular endothelium and subsequent migration across the endothelium has been recognized as a key process in the chronic inflammatory response in atherosclerosis. As type 2 diabetes is closely associated with the pathogenesis of atherosclerosis, we investigated whether monocyte adhesion and migration were affected by insulin. We found that insulin activated Akt and induced subsequent migration in THP-1. However, glucose and insulin-like growth factor-1, which is a growth factor that is structurally similar to insulin, were not effective. Insulin-dependent migration of THP-1 was blocked by inhibition of PI3K or Akt and by silencing of Akt1. Insulin-dependent migration of bone marrow-derived monocytic cells (BDMCs) was attenuated by inhibition of PI3K and Akt. In addition, BDMCs from Akt1^−/− mice showed defects in insulin-dependent migration. Stimulation of THP-1 with insulin caused adhesion with human vein endothelial cells (HUVECs) that was blocked by silencing of Akt1. However, stimulation of HUVECs did not cause adhesion with THP-1. Moreover, BDMCs from Akt1^−/− mice showed defects in insulin-dependent adhesion with HUVECs. Insulin induced surface expression of Mac-1, and neutralization of Mac-1 blocked insulin-induced adhesion of THP-1 as well as BDMCs. Surface expression of Mac-1 was blocked in THP-1 with silenced Akt1, and in BDMCs isolated from mice lacking Akt1. Finally, trans-endothelial migration of THP-1 and BDMCs was blocked by Mac-1-neutralizing antibody, in THP-1 with silenced Akt1 and in BDMCs from Akt1^−/− mice. These results suggest that insulin stimulates monocyte trans-endothelial migration through Akt-dependent surface expression of Mac-1, which may be part of the atherogenesis in type 2 diabetes.     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.