Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma*

Background: Genetic mutations in cancer-driver genes induce specific metabolic alterations in cancer cells. Results: EGF receptor signaling has an important role for glycolysis, pentose phosphate pathway, and pyrimidine biosynthesis in EGFR-mutated lung cancer. Conclusion: Our work reveals the relationship between the EGFR signaling axis and key metabolic changes. Significance: These data implicate a possible link between therapeutic response and regulation of metabolism in EGFR-mutated LAD.


Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated.
Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFR-TKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.
The discovery of oncogenic driver mutations allows us to identify druggable targets and develop new therapies using small molecule tyrosine kinase inhibitors (TKIs) 2 aimed at the relevant patient populations (1)(2)(3). More than 50% of lung adenocarcinomas (LAD) from East Asian non-smokers harbor EGFR mutations, and these tumors have been termed oncogene addicted to reflect their dependence on EGFR-mediated prosurvival signaling and their high susceptibility to apoptosis induced by EGFR-TKIs (e.g. gefitinib and erlotinib) (4 -7). The tyrosine kinase activity of EGFR is dysregulated by gene mutations that lead to aberrant EGFR signaling through pathways including the RAS/MAPK and PI3K/AKT pathways (8,9). The most frequently occurring mutations in the EGFR gene (inframe deletion in exon 19 at codons 746 -750 or a single-base substitution L858R in exon 21) predict an improved clinical response to first-line oral EGFR-TKIs compared with standard platinum-based chemotherapy in patients with advanced nonsmall-cell lung carcinoma (NSCLC) (4,8).
There is accumulating evidence that genetic mutations in cancer-driver genes, tumor suppressors, and amplified oncogenes are linked to specific alterations in metabolic activity in cancer cells, involving proteins such as isocitrate dehydrogenase (IDH), fumarate hydratase (FH), MYC, K-RAS, and BRAF (10 -13). The Warburg effect, the phenomenon in which cancer cells exhibit rapid glucose consumption with secretion of lactate despite abundant oxygen availability, has been recognized since the 1930s (14 -16). Indeed, glucose metabolism in cancer cells is tightly regulated by many molecules at the transcriptional, translational, and post-translational levels (10,17,18). c-MYC is critically involved in the regulation of many growth-* This work was supported by the National Cancer Center Research and promoting signal transduction pathways and glucose metabolism genes, including GLUT1, hexokinase 2 (HK2), pyruvate kinase muscle (PKM2), and lactate dehydrogenase A (LDHA) (10,19). Through the up-regulation of these genes, c-MYC contributes directly to the Warburg effect (19). The enzymatic activities of glycolytic enzymes such as HK2, phosphofructokinase (PFK), PKM2, and LDHA are modulated by post-translational modification (18). For example, PKM2 is phosphorylated in its tyrosine residue (Y105) with low activity in human cancer cells, resulting in increased lactate production, which is onestep downstream from PKM2 in glycolysis, even under aerobic conditions (14,17). Furthermore, PKM2 promotes the Warburg effect through EGF-stimulated EGFR activation and the MAPK signaling pathway (20,21). In brain cancer, the activating EGFRvIII mutation induces enhanced glycolysis by promoting glycolytic gene expression through the Myc/Max pathway (22). However, the specific role of mutated EGFR for aerobic glycolysis in lung cancer has not yet been clearly described.
In this work, we demonstrate that EGFR signaling is required for lactate production under aerobic growth conditions in LAD cells. EGFR signaling maintains key metabolites in glycolysis and PPP by regulating glucose transport through GLUT3 expression. In addition to glucose metabolism, we show that EGFR signaling up-regulates de novo pyrimidine biosynthesis. Moreover, we describe the altered metabolic profiles in TKIsensitive LAD cells in response to erlotinib. Our results imply that EGFR signaling plays a central role in modulating global metabolic pathways in EGFR-mutated LAD.
Cell Survival Assay and Proliferation Assay-EGFR mutant LAD cells were seeded in RPMI 1640 containing various concentrations of EGFR inhibitors in 96-well cell culture plates. After 72 h of incubation, cell viability was analyzed using a WST-8 assay using the Cell Counting Kit-8 (Dojindo, Japan). To count the number of viable cells, Trypan Blue-negative cells were counted using a Countess Automated Cell Counter (Invitrogen).
Lactate and Glucose Assay-Lactate and glucose in culture medium were measured with the respective lactate assay kit II and glucose assay kit II according to the manufacturer's instructions (BioVision, Mountain View, CA). Briefly, after centrifugation (3,500 rpm, 15 min, 4°C), cell culture medium supernatants were frozen at Ϫ20°C. Samples were later thawed, diluted in assay buffer, and mixed with lactate or glucose reaction mixture for 30 min. The optical density of the mixture in each well was read at 450 nm on a microplate reader (Molecular Devices). The lactate concentration was calculated from a standard curve and normalized to cell numbers and culture time. Glucose consumption was calculated from a standard curve, subtracting background from cell-free medium, and normalizing to cell numbers and time.
Measurement of ECAR and OCR-ECAR and OCR were measured with a XF glycolysis stress test kit according to the manufacturer's instructions (Seahorse Bioscience). In brief, 4.5 ϫ 10 4 cells were plated onto XF24 plates in RPMI 1640 (10% FBS, 2 mM glutamine) and incubated at 37°C, 5% CO 2 overnight. Cells were washed with assay medium (minus glucose and unbuffered RPMI 1640 (SIGMA R1383)), replaced with assay medium, and then placed at 37°C in a CO 2 -free incubator for 30 min. ECAR and OCR were monitored using a Seahorse Bioscience XF24 Extracellular Flux Analyzer over time and each cycle consisted of 3 min mixing, 3 min waiting and 3 min measuring. Glucose, oligomycin, and 2DG were diluted into XF24 media and loaded into the accompanying cartridge to achieve final concentrations of 10 mM, 5 M, and 100 mM, respectively. Injections of the drugs into the medium occurred at the time points specified.
Western Blotting-Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) on ice for 10 min, sonicated, and centrifuged at 15,000 ϫ g for 10 min. The protein content of supernatants was measured by BCA assay (Pierce). Identical amounts of protein samples were separated via 4 -20% SDS/PAGE, transferred to PVDF membranes, and incubated overnight with primary antibodies (1:1000 dilution). The primary antibodies used in this study are listed in the materials. ECL anti-rabbit IgG HRP-linked whole antibody (1:10,000; GE Healthcare) and ECL anti-mouse IgG HRP-linked whole antibody (1:10,000; GE Healthcare) were used as secondary antibodies. Signals were detected using ECL Western blotting detection reagent (GE Healthcare) and x-ray films (GE Healthcare).
Quantitative RT-PCR-Cells were washed with PBS and total RNA from the LAD cell lines was isolated with TRIzol Reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using the SuperScript VILO cDNA synthesis kit (Invitrogen). Synthesized primers were purchased from TaKaRa Bio (Japan). Real-time RT-PCR was carried out with specific primers and a 7500 detection system (Applied Biosystems). ␤-Actin was used for normalization as control and the relative quantitation value compared with the calibrator for that target is expressed as 2 Ϫ(CtϪCc) .
Metabolite Measurements-Metabolic extracts were prepared from 2-5 ϫ 10 6 cells with methanol containing Internal Standard Solution (Human Metabolome Technologies; HMT, Inc., Tsuruoka, Japan) and analyzed using a capillary electrophoresis (CE)-connected ESI-TOFMS and CE-MS/MS system (HMT, CARCINOSCOPE). 2-5 ϫ 10 6 cells were used for the extraction of intracellular metabolites. Culture medium was removed from the dish, and cells were washed twice in 5% mannitol solution (10 ml first and then 2 ml). Cells were then treated with 800 l of methanol and 550 l of Milli-Q water containing internal standards (H3304 -1002, HMT, Inc., Tsuruoka, Japan).
The metabolite extract was transferred into a microfuge tube and centrifuged at 2,300 ϫ g and 4°C for 5 min. Next, the upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 ϫ g and 4°C for 120 min to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 l of Milli-Q water for CE-MS analysis. Cationic compounds were analyzed in the positive mode of CE-TOFMS and anionic compounds were analyzed in the positive and negative modes of CE-MS/MS according to the methods developed by Soga et al. (23)(24)(25). To obtain peak information including m/z, migration time (MT), and peak area, detected peaks by CE-TOFMS and CE-MS/MS were extracted using automatic integration software (MasterHands, Keio University, Tsuruoka, Japan and MassHunter Quantitative Analysis B.04.00, Agilent Technologies, Santa Clara, CA, respectively). The peaks were annotated with putative metabolites from the HMT metabolite database based on their MTs in CE and m/z values determined by TOFMS. The tolerance range for the peak annotation was configured at Ϯ0.5 min for MT and Ϯ10 ppm for m/z. In addition, concentrations of metabolites were calculated by normalizing the peak area of each metabolite with respect to the area of the internal standard and by using standard curves, which were obtained by three-point calibrations.

Expression of Glucose Transporter and Glucose Transport
Assay-To detect expression of membrane-bound GLUTs, cells were fixed with 80% ethanol and incubated with anti-GLUT3 antibody (Abcam) and stained with the appropriate FITC-conjugated anti-rabbit IgG antibody (Jackson Immuno Research). Quantification of FITC-fluorescent intensity was performed using a FACSCanto II (BD Biosciences). Procedures for 3-OMeG uptake assay were previously described (26). LAD cells were treated with indicated TKIs for 6 h before glucose transport assay. Uptake was performed from 0.5 min to 10 min and radioactivity in the cells was quantified with Tri-Carb 3110TR low activity liquid scintillation analyzer (PerkinElmer).
Statistical Analyses-Unless otherwise indicated, results were reported as the mean Ϯ S.D. Statistical analyses were done by two-tailed Student's t test. For metabolomic data analysis we used Welch t test and p values were indicated as *, Ͻ0.05; **, Ͻ0.01; and ***, Ͻ0.001.

RESULTS
Lactate Production Was Decreased in TKI-sensitive LAD Cells after EGFR-TKI Treatment-We initially characterized the EGFR-mutated lung adenocarcinoma cell lines used in this study by measuring cell viability in the absence or presence of EGFR-TKIs after 72 h. All three LAD cell lines have the EGFR mutation in either exon 19 or exon 21. Cell line HCC827 carried the delE746-A750 mutation, PC-9 exhibited delE746-A750 and NCI-H1975 (H1975) carried EGFR L858RϩT790M (27,28). The H1975 cells have the T790M mutation, which causes resistance to gefitinib and erlotinib (29). HCC827 and PC-9 cell lines were highly sensitive to the EGFR-TKI gefitinib and erlotinib in the nanomolar range as compared with the TKIresistant cell H1975 (Fig. 1, A and B). These data are consistent with previous findings (27,28,30).
In dose response assays with EGFR inhibitors, we observed differences in the color of the culture medium in the presence of TKIs against EGFR, especially in the growth cultures of TKI-sensitive cell lines (Fig. 1C). In culture media, phenol red exhibits a gradual color transition from red to yellow at lower pH values as a result of lactate production (31). Therefore, we explored the glycolytic capacity of EGFR-mutated LAD cells. Since a 72-h incubation with TKIs leads to a dramatic reduction in cell viability in sensitive cell lines, we set up experimental conditions where TKI treatment was given at a relatively higher concentration (1 M) and shorter time (6 h) to allow all cells to grow equally and thereby standardize the number of viable cells analyzed (Fig. 1D). Interestingly, we discovered that exposure of the cells to TKIs for up to 6 h significantly lowered the rate of lactate accumulation in the medium of TKI-sensitive LAD cell lines but not in resistant cells (Fig. 1E, *, p Ͻ 0.05; **, p Ͻ 0.01 t test).
Glycolytic Activities Were Down-regulated in TKI-sensitive LAD Cells after Inhibition of EGFR Signaling-Next, we quantified the glucose consumption rate and found that inhibition of EGFR signaling significantly lowered the rate of glucose consumption from the growth medium of TKI-sensitive HCC827 and PC9 cells but not in the TKI-resistant H1975 cells ( Fig. 2A *, p Ͻ 0.05; **, p Ͻ 0.01 t test).
To better define lactate production derived from glucose, we measured the glucose-induced extracellular acidification rate (ECAR), an indicator of lactate production, and the oxygen consumption rate (OCR), an indicator of oxidative phosphorylation (OXPHOS), using a flux analyzer. Basal levels of ECAR at the beginning of measurements, which indicated non-glyco-lytic acidification, were low in HCC827 cells (Fig. 2B). Equivalent ECAR was observed in HCC827 cells both pre-and posttreatment with an ATPase inhibitor oligomycin to induce maximum cellular glycolytic capacity (Fig. 2B). At the final step, the addition of 2-deoxy-D-glucose (2DG), an inhibitor for glycolysis, completely shut down extracellular acidification (Fig.  2B). ECAR was statistically higher in DMSO controls compared with TKI-treated HCC827 and PC-9 cells (Fig. 2C *, p Ͻ 0.01; **, p Ͻ 0.005; ***, p Ͻ 0.001 t test). In contrast to ECAR, OCR was changed in TKI-treated HCC827, but not in PC-9 and H1975 cells (Fig. 2D).
EGFR Signaling Up-regulates Glycolysis and the Pentose Phosphate Pathway-To confirm the reduction of glycolysis metabolites by TKIs, we extracted intracellular metabolites with methanol and analyzed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) (25). Metabolome analysis revealed that intermediate metabolites in glycolysis and the pentose phosphate pathway (PPP) were down-regulated by erlotinib treatment for 6 h in both HCC827 and PC-9  Table 1. Fig. 3 and supplemental Table S1). We observed that key glycolysis and PPP metabolites such as fructose 1,6-bisphosphate (FBP), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate (3PG), phosphoenolpyruvate (PEP), lactate (LA), and 6-phosphogluconate (6PG) were decreased in TKI-sensitive HCC827 and PC9 cells after 6 h of erlotinib treatment, but not in TKI-resistant H1975 cells (Fig. 3 and supplemental Table  S1). Glucose 6-phosphate (G6P), glyceraldehyde 3-phosphate (G3P), pyruvate (PA), ribulose 5-phosphate (Ribu5P), and ribose 5-phosphate (R5P) were significantly reduced in both HCC827 and PC9 cells. The amount of adenosine triphosphate (ATP), which is the molecular unit of currency of intracellular energy transfer, was not changed in any of the tested three cell lines after erlotinib treatment. The reduction of glucose utilization after TKI treatment was observed in both glycolysis and pentose phosphate pathways, suggesting that EGFR signaling might regulate a glucose transport or hexokinase activity in TKI-sensitive LAD cells.

MYC-regulated Gene Expression for Glycolytic Enzymes-To test whether EGFR-TKIs inhibited EGFR activity and related
signaling molecules under our experimental conditions, we determined levels of total EGFR, phospho-EGFR (p-EGFR), ERK1/2, p-ERK1/2, AKT, p-AKT, MYC, and ␤-actin in cells treated with DMSO or gefitinib (1 M) for 2 h by Western blot (WB) analyses. Despite equivalent amounts of total EGFR, p-EGFR was clearly repressed in HCC827 and PC9 cells, but not in H1975 cells (Fig. 4A). Downstream signaling molecules such as ERK1/2 and AKT were also inactivated by the addition of gefitinib to HCC827 and PC9 cells as compared with H1975 cells (Fig. 4A).
We hypothesized that MYC regulates lactate production through transcriptional regulation, since MYC is an important regulator for cell cycle and glycolysis in cancer cells (19,32,33). We found that the levels of MYC were quickly down-regulated at both the mRNA and protein levels in response to EGFR-TKIs (Fig. 4, A and B). The MYC-regulated genes GLUT1 (glucose transporter 1) and HK2 (Hexokinase 2) were down-regulated in EGFR-TKI sensitive LAD cells, but not in the TKI-resistant H1975 cells (Fig. 4, C and D). Although previous studies reported that MYC up-regulated PKM2 (pyruvate kinase muscle isozyme 2) (34), we found that the loss of MYC did not affect mRNA expression of PKM2 in HCC827 or PC9 cells (Fig. 4E). These data suggest that the reduction of glycolysis after the inhibition of EGFR signaling is caused by down-regulation of the MYC pathway.
Protein Expression and Phosphorylation of Metabolic Enzymes-PKM2 is thought to be a key molecule for aerobic glycolysis in cancer cells (20,21). We hypothesized that PKM2 might play a critical role in the glycolysis pathway in the response to EGFR-TKI treatment, since PKM2 activity was down-regulated by EGF stimulation resulting in up-regulated lactate production (21). Although the phosphorylation of PKM2 at Tyr-105 was decreased at 24 h after EGFR-TKI addition in TKI-sensitive HCC827 and PC9 cells, this was not seen at the earlier time points (2 or 6 h) post-treatment (Fig. 5A). The phosphorylation of PKM2 was not changed in TKI-resistant H1975 cells over time (Fig. 5A). Therefore, there may be a more rapid, PKM2-independent molecular mechanism by which EGFR signaling regulates the glycolysis pathway.
To determine whether the changes in gene expression driven by MYC were associated with modifications of cellular metabolism, we analyzed phosphorylation and expression of EGFR signaling molecules and glycolytic enzymes by Western blot. We confirmed the effect of TKIs on molecular markers of the EGFR signaling cascade (EGFR, AKT, ERK, and c-Myc) in LAD cells incubated in the presence of gefitinib or erlotinib. We observed that phosphorylation of EGFR, AKT, and ERK was inhibited at 6 h after TKI treatment in HCC827 and PC9 cells (Fig. 5B). The expression level of enzymes for glucose metabolism such as GLUT1 and HK2 generally showed regulation in mRNA levels but not at the corresponding protein levels (Fig.  5B). In contrast, GLUT3 was decreased in HCC827 and PC-9 cells after 6 h treatment with TKIs (Figs. 4C and 5B). When we examined the expression level of MYC-regulated glutaminase (GLS) and glycogen synthase (GYS), we saw that GLS and p-GYS were modestly down-regulated in HCC827 and PC9  Table S2. Error bars indicate S. D. (n ϭ 6). *, p Ͻ 0.05; **, p Ͻ 0.01 versus control by two-tailed Student's t test. (Fig. 5B). Lactate dehydrogenase A (LDHA), p-LDHA, pyruvate dehydrogenase ␣1 (PDHA1), and p-PDHA1 were not altered in HCC827 and PC-9 cells after 6 h treatment with TKIs (Fig. 5B).
EGFR Signaling Pathway Down-regulates the Glucose Transporter GLUT3-GLUT1 (glucose transporter 1, SLC2A1) and GLUT3 (glucose transporter 3, SLC2A3) were mainly expressed in LAD cells (supplemental Table S2). Western blot analysis showed that GLUT3, but not GLUT1, was decreased in HCC827 and PC-9 cells after 6 h of treatment with TKIs. To further confirm membrane-bound GLUT3 expression levels, we investigated the effects of EGFR-TKIs on membrane-bound glucose transporters in HCC827, PC-9, and H1975 cells by flow cytometry. We observed reduction of membrane-bound GLUT3 in the EGFR TKI-sensitive cell lines HCC827 (Fig. 6, A  and B) and PC-9 (Fig. 6, C and D), although the expression of GLUT3 was unchanged in TKI-resistant H1975 cells (Fig. 6, E and F) after 6 h of TKI treatment.
To gain additional insight into the functional role of glucose transport, we measured 3-O-( 3 H-methyl)-D-glucose (3-OMeG) uptake in the absence or presence of EGFR-TKIs. Following 6-hr treatment with gefitinib or erlotinib, the 3-OMeG transport rate in HCC827 and PC-9 cells significantly decreased (Fig.  6, G and H).
Alterations in Additional Metabolic Pathways other than Glycolysis and PPP in the Response to Erlotinib Treatment-To further characterize whether EGFR signaling regulates additional metabolic pathways other than glycolysis and PPP, we quantified metabolites in tricarboxylic acid (TCA), amino acids, and redox. Despite equivalent amount of acetyl-CoA and citrate, fumarate (FA) and malate (MA) were decreased in HCC827 and PC-9 cells but not in H1975 cells after 6 h treat-ment with erlotinib ( Fig. 7A and supplemental Table S1). This result suggests that glutaminolysis was down-regulated after inhibition of EGFR signaling, consistent with the lower expression levels of GLS (Fig. 5B). In contrast, amino acids such as proline (Pro) and aspartate (Asp) were increased in TKI-sensitive HCC827 and PC9 cells as compared with TKI-resistant H1975 cells (Fig. 7A and supplemental Table S1). Additionally, reductive nicotinamide adenine dinucleotide (NADH) and reductive glutathione (GSH) were significantly reduced in HCC827 and PC9 cells, while conversely oxidative glutathione (GSSG) was increased in H1975 cells (Fig. 7A and supplemental  Table S1).
EGFR Signaling Is Required for de Novo Pyrimidine Biosynthesis-N-Carbamoyl-aspartate (NC-Asp) levels were decreased after EGFR-TKI treatment as determined by metabolome analysis (Fig. 7A). Consistent with this observation, the phosphorylation of ribosomal protein S6 kinase 1 (S6K) and carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase (CAD) were obviously down-regulated in HCC827 and PC9 cells but not in H1975 cells (Fig. 7B). The phosphorylation of the Ser1859 residue in CAD protein is required for the first step in the de novo synthesis of pyrimidines (35). These data imply that the inhibition of EGFR signaling alters de novo pyrimidine biosynthesis in EGFR-mutated LAD cells.

DISCUSSION
In this report, we demonstrated that EGFR signaling up-regulated aerobic glycolysis in EGFR-mutated LAD cells. EGFR signaling regulates functional GLUT3 to control the glycolysis and pentose phosphate pathways. Moreover, EGFR signaling activated de novo pyrimidine synthesis, which is regulated by

Regulation of Cancer Metabolism in EGFR-mutated Lung Cancer
CAD activity. We conclude that EGFR signaling regulates global metabolic pathways in EGFR-mutated LAD cells. Our data provide evidence that may link the EGFR-TKI response to the regulation of metabolism in EGFR-mutated LAD. Inhibition of EGFR signaling abrogated the Warburg effect by inhibiting multiple steps including MYC-driven transcription and phosphorylation of PKM2 to regulate glycolysis in LAD. We comprehensively quantified the key metabolites in glycolysis and PPP and identified glucose transport as the most important regulatory step for controlling glucose metabolism in LAD cells during EGFR signaling. This observation is consistent with a previous study demonstrating that gefitinib treatment decreased glucose transport efficiency and hexokinase activity in TKI-sensitive LAD cells (26).
The molecular mechanism by which EGFR signaling regulates glucose transport is still unclear. Weihua et al. found that EGFR physically associated with and stabilized the sodium/glucose transporter (SGLT1) to promote glucose uptake into cancer  cells (36). However, this function did not require EGFR kinase activity. In this report, we found that TKIs to EGFR, gefitinib, and erlotinib, repressed aerobic glycolysis and PPP in EGFR-mutated LAD cells. Although SGLT1 directly interacts with EGFR, EGFR signaling may regulate GLUT translocation in an indirect manner. Mutated EGFRs found in LAD have constitutive tyrosine kinase activity, resulting in activation of downstream RAS/MAPK and PI3K/AKT pathways (Figs. 4A and 5B). In adipocytes and skeletal muscle, insulin and the PI3K/AKT pathway mediate GLUT4 translocation (37,38). To promote glucose uptake into muscle and fat cells, insulin stimulates the translocation of GLUT4 from intracellular membranes to the cell surface. Insulin signals go through AS160 (Akt substrate of 160 kDa) and Tbc1Ds to modulate Rab GTPase, and through Rho GTPase TC10a to act on other targets (37,38). The EGFR-PI3K/AKT axis might control GLUT translocation to the plasma membrane in EGFR-mutated LAD cells. To prove this, we would need to characterize in more detail the molecular mechanisms that control GLUT expression, activity, and translocation.
A recent study showed that AMPK-dependent degradation of thioredoxin-interacting protein (TXNIP) upon stress led to enhanced glucose uptake via GLUT1 (39). Another research report showed that tumor-associated mutant p53 (mutp53) stimulated the Warburg effect in cancer cells as a new mutp53 gain of function (40). Mutp53 did not affect the expression of GLUT1, but promoted aerobic glycolysis by inducing GLUT1 translocation to the plasma membrane, which was mediated by activated RHOA and its downstream effector ROCK. In this study, the EGFR-TKI-sensitive LAD cell lines HCC827 and PC9 possess mutp53, but not the H1975 cell line. A possible molecular mechanism is that either EGFR signaling may regulate GLUT translocation by directly activating the RHOA/ROCK pathway or the mutp53 pathway that in turn activates RHOA/ ROCK function. Further experiments would be required to determine whether EGFR signaling controls glucose transport through the TXNIP or mutp53 pathway.
New therapeutic strategies are currently needed to overcome the EGFR T790M-mediated acquired resistance observed in the clinic (8). A recent Phase III study of afatinib monotherapy failed to show overall survival benefit in patients with acquired resistance to reversible EGFR-TKIs (41). Kim et al. showed that targeting of glycolysis was an effective therapeutic option to overcome the limited efficacy of afatinib in LAD cells with EGFR T790M (42). Treatment with 2DG completely shut down lactate production in EGFR-mutated LAD cells (Fig. 2B), since 2DG is a glucose analog that competes with glucose for cellular uptake. Therefore, combination therapies of EGFR-TKIs and drugs that block the glycolysis pathway such as GLUT-inhibitors would be expected to be much more effective for TKI-resistant cases.
Molecular targeting therapy using TKIs is currently one of the most successful forms of treatment in the clinic, and includes imatinib targeting BCR-ABL in chronic myeloid leukemia (CML) and gefitinib/erlotinib in EGFR-mutated LAD (3). Despite high therapeutic responses to EGFR-TKI treatment, it is clear that not all patients experience benefit; thus, there is still a need to identify potential non-responders and match patients with the most effective therapies (4). Monitoring of tumor glucose utilization by [ 18 F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) was implemented for the early prediction of treatment response to EGFR-TKIs in NSCLC (26,43). In this report, we demonstrate that TKIs to EGFR, gefitinib and erlotinib, repress aerobic glycolysis in EGFR-mutated LAD cells. Those correlations strongly suggest that intermediate metabolites in the pentose phosphate pathway, glycolysis, and pyrimidine biosynthesis such as FBP, DHAP, LA, 6PG, and NC-Asp could serve as well-defined biomarkers to predict response to EGFR-TKI therapy.
The application of metabolomics in oncology has focused its ability to identify biomarkers for cancer diagnosis, prognosis, and therapeutic efficacy (44). In our previous study, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung patients using CE-TOFMS to elucidate tumor-specific metabolism (45). Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis (45). Here we show that EGFR signaling regulates many metabolites in EGFR-mutated LAD cells under in vitro culture conditions; however, whether EGFR-TKIs have the same effects in vivo is still unknown. To build upon this work, further investigations will explore these concepts in relevant animal models and in LAD tissue biopsy samples using bronchoscope before and after EGFR-TKI therapy. In vivo validation of these concepts will have significant implications for future diagnostic and therapeutic possibilities for patients.