GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer
© Masin et al.; licensee BioMed Central Ltd. 2014
Received: 27 March 2014
Accepted: 11 July 2014
Published: 29 July 2014
Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT.
Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival.
Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.
KeywordsEpithelial-mesenchymal transition Glucose transporter GLUT3 Non-small cell lung cancer SLC2A3 ZEB1
The reprogramming of energy metabolism constitutes one of the hallmarks of cancer . In order to build the necessary biomass required for proliferation, tumor cells increase their glucose consumption . Glucose transporters of the GLUT (SLC2A) family are at the first step of cellular glucose utilization, mediating glucose entry by facilitative diffusion. The GLUT family is composed of 14 members that transport glucose or other substrates in different tissues, with different efficiencies . In various tumor types, an increased expression of GLUT1 has been reported . Furthermore, GLUT1 levels are higher in colorectal cancer cell lines harboring KRAS or BRAF mutations compared with isogenic clones and confer cell survival properties in low-glucose conditions . Interestingly, in an oncogenic Kras(G12D)-dependent mouse model of pancreatic ductal adenocarcinoma (PDAC), oncogene withdrawal led to reduced Glut1 expression and glucose uptake . In contrast to GLUT1, little is known about the regulation and function of another glucose transporter, GLUT3, in cancer. GLUT3 was originally referred to as the neuronal GLUT ; with a high affinity for glucose (KM approximately 1.5 mM) and the highest calculated turnover number of all glucose transporters, it ensures efficient glucose uptake by neurons of the central nervous system. With the exception of neurons and a few hematopoietic cell types, GLUT3 is lowly or not expressed in most organs of healthy adults. However, pathological GLUT3 expression has been reported in gastric, testicular, ovarian, and non-small cell lung cancer (NSCLC) [8, 9]. Recently, GLUT3 was shown to be highly expressed in glioblastoma and to promote the growth of brain tumor initiating cells . GLUT3 was also reported to be a transcriptional target of NF-κB and HMGA1, in mouse embryonic fibroblasts and human colorectal tumor cells, respectively [11, 12]. However, the regulation and contribution of GLUT3 to lung tumor progression remain unknown.
The epithelial-mesenchymal transition (EMT) is a process that occurs early in embryonic development, notably during gastrulation, in which epithelial cells undergo cytoskeletal changes and lose cell-cell contacts to gain mesenchymal traits and become more motile . During carcinoma progression, pathological EMT occurs to promote tumor cell invasion and metastasis. One of the essential steps in the EMT process is the loss of E-cadherin, an adherens junction protein that maintains cell-cell adhesion and epithelial tissue integrity. Mechanistically, the EMT process can be triggered by different transcription factors that include TWIST, ZEB1, ZEB2, SNAIL, and SLUG . In our study, we discovered a strong association between the EMT program and the induction of the glucose transporter GLUT3 in NSCLC and extended this observation to tumor cells from another cancer type, hepatocellular carcinoma (HCC). We demonstrate that GLUT3 is a direct transcriptional target of ZEB1. We further show that GLUT3 expression contributes to proliferation of lung tumor cells and is an independent prognostic factor of poor overall survival in NSCLC.
Human GLUT3, mouse Snail, and mouse Zeb1 cDNAs, purchased from Thermo Scientific (Waltham, MA, USA; MHS1010-7429646, MMM1013-7510291, and MMM1013-99828709, respectively), were amplified by polymerase chain reaction (PCR) using forward 5′-CTCATCGATGCCACCATGGGGACACAGAAGGT-3′ and reverse 5′-CTCCCCGGGTTAGACATTGGTGGTGG-3′ (GLUT3), forward 5′-CTCATCGATGCCACCATGCCGCGCTCCTTC-3′ and reverse 5′-CTCGAATTCTCAGCGAGGGCCTCC-3′ (Snail), and forward 5′-CTCATCGATGCCACCATGGCGGATGGCCCC-3′ and reverse 5′-CTCGAATTCCTAAGCTTCATTTGT-3′ (Zeb1) oligos. The PCR products were digested with Cla I and Xma I (GLUT3) or Cla I and Eco RI (Snail and Zeb1) and cloned into identically digested pRDI292-CMV lentiviral vector (gift of D. Trono, EPFL, Lausanne). Genomic DNA was used to amplify by PCR (a) an approximately 1,000-bp region ending close to the transcription start site of human SLC2A3 (gene encoding GLUT3), with the oligos forward 5′-CTCGAGCTCGAGACTAGCAGAAAGTG-3′ and reverse 5′-CTCCTCGAGCGACAAGCCCCCAGCCCCACCCCACCCCACCCCACCCCCCTGAAGCAA-3′, or (b) a region containing the SLC2A3 intron 2 sequence, with the oligos forward 5′-CTCGAGCTCACTGGGGTCATCAATGCTCC-3′ and reverse 5′-CTCCTCGAGGGTTGGTGGAAGAACAGAC-3′. After Sac I and Xho I digestion, the fragments were cloned into an identically digested luciferase reporter plasmid containing a minimal promoter (kindly provided by J. Huelsken, EPFL, Lausanne) to generate prom.-LUC or int.2-LUC constructs, respectively. Deletion of the E-box-like motif CACCTC from the intron 2 sequence was achieved by site-directed mutagenesis, using oligos forward 5′-CCACTCTTTATAGTGATGCACATCCTG-3′ and reverse 5′-CATCACTATAAAGAGTGGGAGGAAGAAC-3′, combined with the oligos indicated above in (b). shRNAs specific to GLUT3 were either from Thermo Scientific (TRCN0000042880) or designed using the pSICOLIGOMAKER 1.5 program (created by A. Ventura, Memorial Sloan-Kettering Cancer Center, New York). In the latter case, forward 5′-TGCAAGGATGTCACAAGAAATTCAAGAGATTTCTTGTGACATCCTTGCTTTTTTC-3′ and reverse 5′-TCGAGAAAAAAGCAAGGATGTCACAAGAAATCTCTTGAATTTCTTGTGACATCCTTGCA-3′ oligos were annealed and ligated into a pSicoR lentiviral vector. The fidelity of all the PCR amplifications and oligo syntheses was confirmed by sequencing. Control pLKO.1 was from Thermo Scientific.
Cells (8 × 107 per immunoprecipitation) were lysed in NP-40 buffer (0.2% NP-40, 150 mM NaCl, 20 mM Tris pH 8.0, 10 mM EDTA) containing a protease inhibitor cocktail (complete, Roche, Basel, Switzerland) and 1 mM Na3VO4 for 15 min on ice, followed by three quick steps of freezing in liquid N2 and thawing at 37°C. Pre-clearing was achieved using sepharose-6B (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 4°C on a rotating wheel. Immunoprecipitation was performed using a 1:1 mixture of sepharose-6B and protein-G sepharose (Sigma-Aldrich), together with 2 μg control or ZEB1 antibody, overnight at 4°C on a rotating wheel. After four steps of washing in lysis buffer, sample buffer was added, and the samples were boiled and loaded on a polyacrylamide gel for electrophoresis followed by Western blot.
Except when used for immunoprecipitation, cells were lysed in RIPA buffer (20 mM Tris pH 8, 50 mM NaCl, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM Na3VO4, protease inhibitor cocktail (complete, Roche)) for 5 min on ice. Proteins were loaded on 8% or 10% polyacrylamide gels for electrophoresis (150 V, 1 h). Transfer was performed on PVDF membranes (100 V, 1 h).
Cell culture conditions
The human embryonic kidney (HEK) 293 T cells and the human hepatoma cell lines, which include HLF, HLE, Huh-1 (all three kindly provided by K. Morikawa, Showa University, Tokyo, Japan), Huh-7, and Hep3B, were grown in DMEM. The human non-small cell lung cancer cell lines include A549, SW1573, NCI-H23, NCI-H2122, NCI-H441, NCI-H460, NCI-H727, NCI-H2009, NCI-H1944, and Calu-6; they were all obtained from ATCC (Manassas, VA, USA) and all grown in RPMI. All cell media were supplemented with 10% FBS. For the experiments with high or low glucose, cells were cultured for 4 days in normal RPMI (containing 11 mM glucose), supplemented by 10% FBS, or with RPMI without glucose, supplemented by 10% FBS, resulting in a final concentration of 11.5 mM and 0.5 mM glucose, respectively. After 2 days of culture, the medium was replaced with fresh medium to maintain the initial concentrations of glucose.
To measure cell number, a trypan blue exclusion assay was performed. Cells were mixed with trypan blue, and the number of live, dead, and total cells was counted on an automated cell counter (Countess, Life Technologies, Carlsbad, CA, USA).
Cells (1 × 106 per well, seeded the day before the experiment) were cultured on six-well plates. 2-Deoxyglucose uptake assays were performed as described previously . Radioactivity was determined by scintillation counting. For sample normalization, protein concentration was measured by BCA protein assay.
siRNAs were from Life Technologies. siRNA transfection was performed with RNAiMAX transfection reagent (Life Technologies), according to the manufacturer’s instructions. For each siRNA, 10 nM was used. cDNA transfection of 293 T cells was performed with Lipofectamine 2000 (Life Technologies), according to the manufacturer’s instructions. For each well of a 12-well plate, a mix of 125 ng Renilla-Luciferase (phRL-TK, Promega, Madison, WI, USA) and 1.6 μg GLUT3-Luciferase (promoter, intron 2-WT or intron 2-Δ-CACCTC) was used. Twenty-four hours after transfection, cells were lysed and a luciferase reporter assay was performed (Dual-Luciferase Reporter Assay System, Promega). The reported luciferase activity was the ratio between Firefly (GLUT3 construct)-Luciferase and Renilla-Luciferase, which was then normalized to 1 to get fold activities. For double transfection, 293 T cells were transfected first with siRNAs and re-transfected 72 h later with plasmids, 24 h prior to lysis.
Anchorage-independent growth assays
Cells (9 × 104) were plated in triplicates in six-well plates in 0.4% agar in RPMI on top of a layer of 0.8% agar with RPMI. Cells were allowed to grow at 37°C for 3 weeks. Colonies were counted using a microscope. A colony was defined as an aggregate of 50 or more cells.
RNA purification, reverse transcription, and real-time PCR amplification
RNA was purified using Trizol (Life Technologies), according to the manufacturer’s instructions. RNA (1 μg) was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). cDNA (5 ng) was used for real-time PCR amplification, using commercially available Taqman probes for human 18S, GAPDH, SLC2A3, SLC2A1, SLC2A4, SLC2A12, VIM, ZEB1, SNAI1, and CDH1 (Life Technologies). Data were normalized to GAPDH or 18S levels. For measurement of mature miR-200b, reverse transcription was performed with the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies), according to the manufacturer’s instructions, separately for miR-200b and the normalization control, RNU24. cDNA (0.9 ng) was used for real-time PCR amplification.
Cells (4 × 107) were fixed in 4% paraformaldehyde, embedded in paraffin, and mounted on slides. Following antigen retrieval with 10 mM Na-citrate and blocking, cells were stained with anti-GLUT3 or anti-E-cadherin antibodies overnight at 4°C, followed by washing and staining with biotin-conjugated secondary antibodies for 1 h at room temperature. After washing, avidin-biotin horseradish peroxidase complexes were added for 30 min (ABC kit, Vectastain), and the complexes were revealed with a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). Counterstain was performed using Harris hematoxylin.
Recombinant human TGF-β2 was from PeproTech (Rocky Hill, NJ, USA). Antibodies used were anti-GLUT3 (#400062, Calbiochem, San Diego, CA, USA), anti-E-cadherin (#3195, Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (#5741, Cell Signaling), anti-SNAIL (#3879, Cell Signaling), anti-ZEB1 (#sc-25388, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-β-tubulin (#sc-9104, Santa Cruz Biotechnology), anti-CtBP (#sc-17759, Santa Cruz Biotechnology), anti-p300 (#sc-585, Santa Cruz Biotechnology), and normal rabbit IgG (#12-370, Millipore, Billerica, MA, USA).
Virus production and infection
293 T cells were transfected using the calcium-phosphate precipitation method, co-transfecting the lentiviral plasmid of interest in conjunction with pMD2G (VSV-G protein) and pCMVR8.74 (lentivirus packaging vector, kind gift of D. Trono, EPFL, Lausanne). Viral supernatants were harvested 24 and 36 h post-transfection, filtered, and used directly for infection of cell lines, as described. Puromycin selection was performed to select cells with stable pRDI292-CMV (delivering control, human GLUT3, mouse Snail, or mouse Zeb1 cDNA) or pLKO.1 (with control or GLUT3 shRNA) genomic integration. Fluorescence-activated cell sorting was used to select cells with stable pSicoR constructs, based on GFP expression.
Unless specified differently, P values were determined by Student’s t tests.
Publicly available replicate ChIP-seq data for ZEB1 (GSM803411), RNA polymerase II (GSM803485), H3K27ac (GSM733771), as well as naked DNA (Input) (GSM733742)  were re-analyzed using Bowtie 2  for mapping to the human GRCh37 genome with the parameters ‘--very-sensitive -M 10 -p 8’ and CCAT 3.0  for peak-calling using the parameters ‘fragmentSize 200 slidingWinSize 100 movingStep 50 isStrandSensitiveMode 1 minCount 13 outputNum 100000 randomSeed 123456 minScore 5 bootstrapPass 80’. Data were visualized using IGV Browser .
ChIP was performed as described previously , using 6 × 107 SW1573 and 5 μg of each antibody. To amplify regions within the promoter region of SLC2A3, the following primers were used: 5′-ACTGCCCTGATAGTTGGTCTGG-3′ with 5′-TTTGCCAGTGTTCCTTTCTTCG-3′ (−608 and −523 bp upstream of SLC2A3 TSS); 5′-ACTGCCCTGATAGTTGGTCTGG-3′ with 5′-GAGGGAAAGACAGCCTGAGAGA-3′ (−608 and −482 bp upstream of SLC2A3 TSS). To amplify regions from the intron 2, the following primers were used: 5′-CATCACAGTTGCTACAATCGGC-3′ with 5′-ACCATGCCTGGCCTTAAATTCT-3′ (2,396 and 2,559 bp downstream of SLC2A3 TSS); 5′-ACCATGCCTGGCCTTAAATTCT-3′ with 5′-AGCCTCAGGAGTAGCTGGGACT-3′ (2,452 and 2,594 bp downstream of SLC2A3 TSS); 5′-GTGAGTGCCAGGCCACAATAAT-3′ with 5′-TGTGTTGCTCAGGATGGTGTTT-3′ (2,466 and 2,647 bp downstream of SLC2A3 TSS); 5′-AGTCCCAGCTACTCCTGAGGCT-3′ with 5′-TTCCGGGAGTAAGTGAGCTTTG-3′ (2,573 and 2,791 bp downstream of SLC2A3 TSS); 5′-AAACACCATCCTGAGCAACACA-3′ with 5′-TGAGGTGAAAGAGTGGGAGGAA-3′ (2,626 and 2,831 bp downstream of SLC2A3 TSS); 5′-TTCCTCCCACTCTTTCACCTCA-3′ with 5′-GCACCGATGTTCACAGTCTACC-3′ (2,810 and 2,926 bp downstream of SLC2A3 TSS); 5′-AAGCTGGGTTCCCTTAGCAGAG-3′ with 5′-AAAGGGTTGGTGGAAGAACAGA-3′ (2,877 and 3,117 bp downstream of SLC2A3 TSS); 5′-CAGTCTGTTCTTCCACCAACCC-3′ with 5′-AGGACCAGAGAGACGTGAGCAG-3′ (3,093 and 3,212 bp downstream of SLC2A3 TSS). The intergenic region upstream of GAPDH was amplified, using primers 5′-ATGGGTGCCACTGGGGATCT-3′ and 5′-TGCCAAAGCCTAGGGGAAGA-3′, as described previously . As positive control, a DNA sequence of CDH1 known to bind ZEB1 was amplified using primers 5′-GGCCGGCAGGTGAACCCTCA-3′ and 5′-GGGCTGGAGTCTGAACTGA-3′, as described previously . Real-time PCR was performed using SyBR green. Fold enrichment was calculated as follows: 2^ΔΔct (ΔΔct = Δctspecific antibody − ΔctIgG). Data were normalized using the negative control, an untranscribed region upstream of GAPDH, and represented as fold enrichment/fold enrichment of the negative control locus.
We collected publicly available datasets from journal articles and Gene Expression Omnibus (GEO) repository, selecting those with medium to large sample size [23, 24]. The .CEL files were imported into R/Bioconductor (http://www.bioconductor.org/) and RMA normalized . A total of 462 early-stage untreated lung NSCLC samples were available, with associated patients’ overall survival. The probe IDs with higher variation for each gene were retained from a total of 13,960 genes. The normalized data together with the clinico-pathological variables were then used for further analysis. Pairwise Spearman correlations between GLUT3 and other available genes were calculated. Univariate and multivariate survival analyses were performed using proportional Cox regression (package ‘survival’, R). Kaplan-Meier figure was reported showing hazard ratio (HR), 95% confidence intervals, and Wald p_value from Cox regression model for a specific comparison of interest. The continuous GLUT3 expression was dichotomized in high and low risk levels by the median of the expression for visualization purposes. Multivariate analysis was performed with the following variables: GLUT3 continuous expression, stage, histology, gender, and age (dichotomized by the median of 62 years).
GLUT3 expression correlates with EMT
GLUT3 is induced during EMT
ZEB1 induces GLUT3 through direct binding and transcriptional activation
GLUT3 promotes glucose uptake and proliferation of mesenchymal lung tumor cells
GLUT3 expression in human NSCLC correlates with poor overall survival and EMT signatures
Univariate and multivariate analyses of overall survival in NSCLC
P value (Wald)
1.14E − 02
Stage (II vs. I)
2.19E − 02
Histology LCC vs. AD
7.89E − 01
Histology SCC vs. AD
5.91E − 03
Gender (F vs. M)
6.06E − 03
Age (>62 vs. ≤62)
7.47E − 05
3.16E − 02
Stage (II vs. I)
9.04E − 02
Histology LCC vs. AD
9.90E − 01
Histology SCC vs. AD
2.03E − 02
Gender (F vs. M)
6.33E − 02
Age (>62 vs. ≤62)
2.56E − 04
In this study, we have identified a unique role for glucose transporter GLUT3 in the proliferation of lung tumor cells with mesenchymal characteristics. We found that GLUT3 is strongly up-regulated during EMT and contributes to glucose uptake specifically in mesenchymal-like lung tumor cells. Furthermore, GLUT3 overexpression is sufficient to substantially enhance the proliferation of lung tumor cells with epithelial traits specifically in the context of low glucose. Interestingly, increased glucose consumption was shown to promote EMT via stabilization of SNAIL , suggesting that increased expression of GLUT family members promotes the induction and/or maintenance of EMT. In our experiments, we did not detect an EMT occurring in response to ectopic GLUT3 protein expression in epithelial cells (MM and EM, unpublished observations), but we found a decrease in vimentin expression upon GLUT3 knockdown in mesenchymal cells. Collectively, these data suggest the intriguing possibility that GLUT3 plays a role in the successful establishment or maintenance of the EMT, when tumor cells become more motile and invasive, or in tumor cell survival upon loss of cell-cell adhesion.
EMT in cancer is a complex phenomenon that is orchestrated by different transcription factors with partially overlapping gene targets. These transcription factors, like SNAIL or ZEB1, often act as transcriptional repressors. However, the mechanisms of target gene regulation are not fully elucidated. For example, ZEB1 inhibits the transcription of target genes when bound to CtBP [29, 31]. In contrast, ZEB1 binding to R-SMADs and the acetyltransferases p300 and P/CAF promotes target gene transcription [30, 32]. Likewise, in our study, we have demonstrated that ZEB1 can interact with each of CtBP and p300 in mesenchymal lung tumor cells and that it is a direct activator of SLC2A3 gene transcription. Of note, ZEB1 expression was higher in the mesenchymal, GLUT3-expressing cell lines compared to the epithelial cell lines, and ZEB1 was induced prior to GLUT3 in response to TGF-β (see Figures 2A and 4A, and Additional files 1 and 4). This is in agreement with a previous finding reporting that the SLC2A3 gene was one of the top 50 genes correlating positively with ZEB1 gene expression in a panel of human lung tumor cell lines . In the future, it will be important to decipher all of the genes that are directly targeted—either repressed or induced—by ZEB1 in lung cancer and to determine if this includes additional genes that regulate tumor cell metabolism.
The increased GLUT3 expression observed in tumor cells upon EMT may have important implications for cancer treatment. Indeed, not only is EMT well recognized as a crucial process that increases invasive capacities of tumor cells and promotes the acquisition of stem cell characteristics , it may occur earlier in tumor development than previously anticipated. For example, in a K-ras LSL-G12D/+ ; p53 Flox/Flox mouse model of PDAC, EMT and invasiveness precede the detection of tumors in situ. Interestingly, a molecular classification of human PDAC was performed recently, where three subtypes were defined: classical, exocrine-like, and quasi-mesenchymal (which has the worst prognosis of all three) ; SLC2A3 was one of a 20-gene signature of the quasi-mesenchymal subtype. This observation, together with our findings of a connection between GLUT3 and EMT in human liver cells from HCC, indicates that the regulation of GLUT3 by EMT extends beyond lung cancer.
By uncovering direct regulation of GLUT3 by the transcription factor ZEB1, our study provides evidence for a tight association between two central characteristics of carcinoma development: EMT and glucose metabolism. Because GLUT3 expression is mainly restricted to the brain of healthy individuals, the future development of small molecule compounds that selectively block GLUT3 and that do not cross the blood-brain barrier may become a viable strategy to treat patients with NSCLC and possibly other cancer types.
Non-small cell lung cancer
Pancreatic ductal adenocarcinoma
Solute carrier family 2 (facilitated glucose transporter), member 3
Zinc finger E-box-binding homeobox 1.
We thank Anja Irmisch and Ute Koch (ISREC, EPFL) for their help on the ChIP-PCR and 2-DOG uptake experiments, respectively, and the EPFL SV Histology Core Facility for histological sectioning. We thank Tyler Jacks (Massachusetts Institute of Technology) for insightful comments, and Mikael J. Pittet (Harvard Medical School) and Inder M. Verma (The Salk Institute) for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (PP00P3_133661) and by a ‘Molecular Life Sciences’ grant from the ISREC Foundation. The funding bodies played no role in the design, collection, analysis, interpretation of data, writing, or decision to submit the manuscript for publication.
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