GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080596
ISSN1525-2191
AutoresThomas Amann, Ulrike Maegdefrau, Arndt Hartmann, Abbas Agaimy, Jörg Marienhagen, Thomas S. Weiß, Oliver Stoeltzing, Christina Warnecke, Jürgen Schölmerich, Peter J. Oefner, Marina Kreutz, Anja‐Katrin Bosserhoff, Claus Hellerbrand,
Tópico(s)RNA modifications and cancer
ResumoAccelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1α. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor. Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1α. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor. Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver and one of the most common tumors worldwide.1El-Serag HB Hepatocellular carcinoma: recent trends in the United States.Gastroenterology. 2004; 127: S27-S34Abstract Full Text Full Text PDF PubMed Scopus (899) Google Scholar Liver cirrhosis is the main predisposing condition, but the molecular pathogenesis of HCC is still not well understood. Morbidity and mortality correlate directly with surgical resectability of the primary tumor. However, outcome is mostly poor, because the majority of patients are diagnosed at an advanced stage, and only 10 to 20% of HCCs can be resected completely.2Farazi PA DePinho RA Hepatocellular carcinoma pathogenesis: from genes to environment.Nat Rev Cancer. 2006; 6: 674-687Crossref PubMed Scopus (1706) Google Scholar, 3Bruix J Boix L Sala M Llovet JM Focus on hepatocellular carcinoma.Cancer Cell. 2004; 5: 215-219Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar In the 1920s Otto Warburg made the observation that tumor cells use glycolysis instead of mitochondrial oxidative phosphorylation for energy production even under oxygen-rich conditions. Recently, the Warburg effect has experienced a revival because it has been shown that aerobic glycolysis governs tumor cell biology.4Kim JW Dang CV Cancer's molecular sweet tooth and the Warburg effect.Cancer Res. 2006; 66: 8927-8930Crossref PubMed Scopus (1017) Google Scholar, 5Brahimi-Horn MC Chiche J Pouyssegur J Hypoxia signalling controls metabolic demand.Curr Opin Cell Biol. 2007; 19: 223-229Crossref PubMed Scopus (257) Google Scholar Previous studies found differences in glycolytic capacity between HCC cells and hepatocytes,6Hugo-Wissemann D Anundi I Lauchart W Viebahn R de Groot H Differences in glycolytic capacity and hypoxia tolerance between hepatoma cells and hepatocytes.Hepatology. 1991; 13: 297-303Crossref PubMed Scopus (46) Google Scholar and positron emission tomography (PET) revealed the fluorine-18-fluorodeoxyglucose uptake value as an independent prognostic factor for HCC.7Kong YH Han CJ Lee SD Sohn WS Kim MJ Ki SS Kim J Jeong SH Kim YC Lee JO Cheon GJ Choi CW Lim SM [Positron emission tomography with fluorine-18-fluorodeoxyglucose is useful for predicting the prognosis of patients with hepatocellular carcinoma].Korean J Hepatol. 2004; 10: 279-287PubMed Google Scholar Further, higher PET activity was shown to correlate with advanced tumor stages.8Khan MA Combs CS Brunt EM Lowe VJ Wolverson MK Solomon H Collins BT Di Bisceglie AM Positron emission tomography scanning in the evaluation of hepatocellular carcinoma.J Hepatol. 2000; 32: 792-797Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar The glucose transporter isoform 1 (GLUT1, also known as SLC2A1; MIM no. 138140) is a key rate-limiting factor in the transport and metabolism of glucose in cancer cells. GLUT1 expression is primarily undetectable in normal epithelial tissues and benign epithelial tumors. However, GLUT1 is overexpressed in a significant proportion of human carcinomas.9Airley RE Mobasheri A Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics.Chemotherapy. 2007; 53: 233-256Crossref PubMed Scopus (294) Google Scholar, 10Medina RA Owen GI Glucose transporters: expression, regulation and cancer.Biol Res. 2002; 35: 9-26Crossref PubMed Scopus (386) Google Scholar The apparent expression of a certain type of glucose transporter suggests an important role for this transporter in tumor biology. Therefore, it has been hypothesized that elevated GLUT1 expression by human carcinomas indicates an increased metabolic state, enhanced utilization of energy, and an associated increase in aggressive, metastatic behavior. Actually, Glut1 protein expression confers poor prognosis in a wide range of solid tumors.11Cooper R Sarioglu S Sokmen S Fuzun M Kupelioglu A Valentine H Gorken IB Airley R West C Glucose transporter-1 (GLUT-1): a potential marker of prognosis in rectal carcinoma?.Br J Cancer. 2003; 89: 870-876Crossref PubMed Scopus (109) Google Scholar, 12Oliver RJ Woodwards RT Sloan P Thakker NS Stratford IJ Airley RE Prognostic value of facilitative glucose transporter Glut-1 in oral squamous cell carcinomas treated by surgical resection; results of EORTC Translational Research Fund studies.Eur J Cancer. 2004; 40: 503-507Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Studies regarding GLUT1 expression in HCC have revealed inconclusive results, and the biological significance of GLUT1 expression in HCC remains unknown.13Yamamoto T Seino Y Fukumoto H Koh G Yano H Inagaki N Yamada Y Inoue K Manabe T Imura H Over-expression of facilitative glucose transporter genes in human cancer.Biochem Biophys Res Commun. 1990; 170: 223-230Crossref PubMed Scopus (453) Google Scholar, 14Grobholz R Hacker HJ Thorens B Bannasch P Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.Cancer Res. 1993; 53: 4204-4211PubMed Google Scholar, 15Su TS Tsai TF Chi CW Han SH Chou CK Elevation of facilitated glucose-transporter messenger RNA in human hepatocellular carcinoma.Hepatology. 1990; 11: 118-122Crossref PubMed Scopus (50) Google Scholar, 16Younes M Lechago LV Somoano JR Mosharaf M Lechago J Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers.Cancer Res. 1996; 56: 1164-1167PubMed Google Scholar, 17Zimmerman RL Fogt F Burke M Murakata LA Assessment of Glut-1 expression in cholangiocarcinoma, benign biliary lesions and hepatocellular carcinoma.Oncol Rep. 2002; 9: 689-692PubMed Google Scholar, 18Zimmerman RL Burke M Young NA Solomides CC Bibbo M Diagnostic utility of Glut-1 and CA 15-3 in discriminating adenocarcinoma from hepatocellular carcinoma in liver tumors biopsied by fine-needle aspiration.Cancer. 2002; 96: 53-57Crossref PubMed Scopus (34) Google Scholar, 19Roh MS Jeong JS Kim YH Kim MC Hong SH Diagnostic utility of GLUT1 in the differential diagnosis of liver carcinomas.Hepatogastroenterology. 2004; 51: 1315-1318PubMed Google Scholar Here, we show that GLUT1 expression is increased in a significant number of HCC cell lines and tissues, and high GLUT1 expression correlates with HCC proliferation and invasiveness. Furthermore, we found that siRNA-mediated abrogation of GLUT1 in HCC cell lines inhibits their proliferative and migratory potential. This suggests that increased GLUT1 expression in HCC does not only indicate an increased utilization of energy, which may correlate with an aggressive behavior, but directly causes tumorigenesis. Consequently, GLUT1 may serve as both a prognostic marker and a therapeutic target in HCC. The HCC cell lines HepG2 [American Type Culture Collection (Rockville, MD) HB-8065], PLC (American Type Culture Collection CRL-8024), and Hep3B (American Type Culture Collection HB-8064) were cultured as described.20Hellerbrand C Amann T Schlegel J Wild P Bataille F Spruss T Hartmann A Bosserhoff AK The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.Gut. 2008; 57: 243-251Crossref PubMed Scopus (65) Google Scholar Primary human hepatocytes (PHHs) were isolated and cultured as previously described.21Weiss TS Jahn B Cetto M Jauch KW Thasler WE Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes.Cell Prolif. 2002; 35: 257-267Crossref PubMed Scopus (54) Google Scholar Human liver tissue for cell isolation was obtained according to the guidelines of the charitable state-controlled foundation Human Tissue and Cell Research with the patient's informed consent. Hypoxia was induced by incubation with 2,2′-dipyridyl (DP) (100 μmol/L; Sigma Aldrich, Deisenhofen, Germany) or exposure to 1% O2 for the indicated periods of time. For pharmaceutical inhibition of hypoxia-inducible factor (HIF)-1 activity cells were incubated with 100 μmol/L of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1; Calbiochem, Darmstadt, Germany)22Chun YS Yeo EJ Choi E Teng CM Bae JM Kim MS Park JW Inhibitory effect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells.Biochem Pharmacol. 2001; 61: 947-954Crossref PubMed Scopus (154) Google Scholar, 23Yeo EJ Chun YS Cho YS Kim J Lee JC Kim MS Park JW YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.J Natl Cancer Inst. 2003; 95: 516-525Crossref PubMed Scopus (465) Google Scholar or 10 nmol/L of echinomycin (Alexis Biochemicals, Lörrach, Germany).24Kong D Park EJ Stephen AG Calvani M Cardellina JH Monks A Fisher RJ Shoemaker RH Melillo G Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity.Cancer Res. 2005; 65: 9047-9055Crossref PubMed Scopus (432) Google Scholar HCC tissues and corresponding nonneoplastic liver tissues were obtained from HCC patients (child A/B cirrhosis) undergoing surgical resection at the university hospitals Regensburg (n = 85) and Erlangen (n = 67). TMAs were constructed as described.20Hellerbrand C Amann T Schlegel J Wild P Bataille F Spruss T Hartmann A Bosserhoff AK The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.Gut. 2008; 57: 243-251Crossref PubMed Scopus (65) Google Scholar Further, tissue samples of 31 patients were immediately snap-frozen, stored at −80°C, and subsequently used for RNA isolation and analysis of mRNA expression. Clinicopathological patient characteristics are summarized in Table 1.Table 1Glut1 Immunoreactivity (IR) in HCC Tissue of 152 Patients in Relation to Clinicopathological Characteristics and Proliferation RateVariableCategorizationn%Glut1 IR-negativeGlut1 IR-positiveP*Fisher's exact test (two-sided); bold face represents P values <0.05.Clinicopathological characteristics Age at diagnosis<60 years5737.55160.621≥60 years9562.58114 SexFemale2214.51931.000Male13085.511317 Tumor stagepT15234.24570.023pT24429.0404pT34831.6426pT442.613nd42.640 Histological gradeG15737.5552 5 cm5334.9485nd2214.5184 CirrhosisNo3623.73420.348Yes9562.58312nd2113.8156Proliferation rate (MIB1 index)≤5%6442.16130.006>5%8153.3641774.670nd, no data available; IR, immunoreactivity.* Fisher's exact test (two-sided); bold face represents P values <0.05. Open table in a new tab nd, no data available; IR, immunoreactivity. Isolation of total cellular RNA from cultured cells and tissues and reverse transcription were performed as described previously.25Hellerbrand C Muhlbauer M Wallner S Schuierer M Behrmann I Bataille F Weiss T Scholmerich J Bosserhoff AK Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma.Carcinogenesis. 2006; 27: 64-72Crossref PubMed Scopus (69) Google Scholar Quantitative real-time polymerase chain reaction (PCR) was performed with primers specific for GLUT1 (forward: 5′-AACTCTTCAGCCAGGGTCCAC; reverse: 5′-CACAGTGAAGATGATGAAGAC) using LightCycler technology (Roche, Mannheim, Germany).26Hellerbrand C Bataille F Schlegel J Hartmann A Muhlbauer M Scholmerich J Buttner R Hofstadter F Bosserhoff AK In situ expression patterns of melanoma inhibitory activity 2 in healthy and diseased livers.Liver Int. 2005; 25: 357-366Crossref PubMed Scopus (19) Google Scholar Expression of MAZ was analyzed applying the QuantiTect primer assay according to the manufacturer's instructions (Qiagen, Hilden, Germany). Protein extraction and Western blotting were performed as described elsewhere,27Schuierer MM Bataille F Weiss TS Hellerbrand C Bosserhoff AK Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma.Oncol Rep. 2006; 16: 451-456PubMed Google Scholar applying the following primary antibodies: anti-Glut1 (1:600; Lab Vision, Wedel, Germany), anti-HIF-1 α (1:500; Novus Biologicals, Littleton, CO), and anti-β-actin (1:20,000, Sigma). Immunohistochemical staining of 5-μm sections of the TMA blocks was performed using polyclonal anti-Glut1 antibody (1:50) and an indirect immunoperoxidase protocol according to the LSAB2-kit (DAKO, Hamburg, Germany).20Hellerbrand C Amann T Schlegel J Wild P Bataille F Spruss T Hartmann A Bosserhoff AK The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.Gut. 2008; 57: 243-251Crossref PubMed Scopus (65) Google Scholar A surgical pathologist (A.H.) performed a blinded evaluation of the stained slides. For negative control, the primary antibody was omitted and IgG isotype control antibodies did not reveal any detectable staining. For analysis of the TMA, positivity for Glut1 was defined as any detectable membranous staining. Glut1 expression in carcinomas, when present, was variable as reported before in other studies,16Younes M Lechago LV Somoano JR Mosharaf M Lechago J Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers.Cancer Res. 1996; 56: 1164-1167PubMed Google Scholar ranging from at least 25 to almost 100% of the cells. In contrast, cases designated as Glut1-negative did not reveal any immunohistochemical staining for Glut1. MIB1 was analyzed applying anti-Ki-67 antibody (rabbit monoclonal, clone MIB1, 1:10, final concentration of 5 μg/ml; DAKO). Antibody binding was visualized using AEC-solution (LSAB2-Kit, DAKO). Finally, the tissues were counterstained by hemalaun. Applying the Lipofectamine plus method (Invitrogen, Carlsbad, CA) small interfering RNA (siRNA; GLUT1 Hs-SLC2A1-5 and GLUT1 Hs-SLC2A1-6; MAZ Hs_MAZ_6 and MAZ Hs_MAZ_8; all from Qiagen) was transiently transfected into HCC cells to deplete GLUT1 or MAZ expression. Transfection efficiency was determined by fluorescence-activated cell sorting analysis applying Alexa Fluor 488-labeled control siRNA (AllStars negative control siRNA; Qiagen). For luciferase reporter assays, cells were transfected with 0.5 μg of a reporter construct harboring six copies of the hypoxia-responsive element of the human phosphoglycerate kinase (PGK) gene (6×HRE) upstream of a HSV thymidine kinase promoter. To normalize transfection efficiency, 0.2 μg of a pRL-TK plasmid (Promega, Mannheim, Germany) was co-transfected and renilla luciferase activity measured by a luminometric assay (Promega). All transfections were performed in triplicate. Cell proliferation was measured using the XTT assay (Roche).25Hellerbrand C Muhlbauer M Wallner S Schuierer M Behrmann I Bataille F Weiss T Scholmerich J Bosserhoff AK Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma.Carcinogenesis. 2006; 27: 64-72Crossref PubMed Scopus (69) Google Scholar Further, cell number was determined by microscopic counting after trypsination of cells seeded in six-well plates (six per condition) at different time points. Migration assays were performed as previously described.20Hellerbrand C Amann T Schlegel J Wild P Bataille F Spruss T Hartmann A Bosserhoff AK The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.Gut. 2008; 57: 243-251Crossref PubMed Scopus (65) Google Scholar PET using 2-[18F]fluorodeoxyglucose (FDG-PET/CT) was performed by means of a Biograph16 PET/CT scanner (Siemens, Erlangen, Germany). After fasting for 4 to 6 hours to achieve blood glucose values <120 mg/dl, the patients received an intravenous dose of FDG (5 to 10 mCi, 185 to 379 MBq). Whole-body image acquisition from the skull base to the proximal thigh started ∼60 minutes later (axial field 90 cm; seven bed positions for 3 minutes each, one head/neck position). The total time required was ∼20 minutes (40 seconds CT scanning and subsequent CT attenuation-corrected PET scanning, low-dose CT). Statistical analyses were performed using SPSS version 10.0 (SPSS, Chicago, IL) and GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA). Results are expressed as mean ± SE (range) or percent. P values <0.05 were considered statistically significant. Comparisons between groups were made using the Mann-Whitney test. Contingency table analysis and the two-sided Fisher's exact test were used to study the statistical association between clinicopathological and immunohistochemical variables. Initially, we analyzed GLUT1 mRNA expression in three different HCC cell lines (HepG2, PLC, and Hep3B) and PHHs by quantitative real-time PCR. In all three HCC cell lines a significantly increased GLUT1 mRNA expression was observed compared with PHHs (Figure 1A). This result was confirmed by Western blotting (Figure 1B). Next, we analyzed a panel of 22 paired specimens obtained from patients with HCC. From each HCC patient, RNA was isolated from cancerous tissue and adjacent nontumorous liver tissue, and GLUT1 mRNA expression was measured by quantitative real-time PCR (Figure 1C). In 15 HCC specimens, GLUT1 mRNA expression was increased compared with matched nontumorous tissue. In six patients (nos. 2, 9, 13, and 18 to 20) GLUT1 mRNA expression was not significantly different from control tissue. Only two HCC specimens (nos. 4 and 10) revealed decreased GLUT1 mRNA expression levels compared with nontumorous tissue. To assess GLUT1 expression in HCC in situ, we performed immunohistochemical staining for Glut1 protein. A representative immunohistochemical staining of a Glut1-positive tumor is presented in Figure 1D. Immunohistochemistry revealed a strong membranous signal in HCC cells (Figure 1DII). In contrast, no Glut1 staining was detectable in nontumorous hepatic tissue. Next, we analyzed Glut1 expression in a series of 152 HCCs and corresponding nontumorous tissue of the same patients (n = 146) using TMA technology. Investigation of Glut1 protein expression was informative in all HCC and nontumorous tissue samples. In 13.2% (20 of 152) of the HCC, a Glut1 immunosignal was detectable. In contrast, Glut1 expression was found in none of the noncancerous tissue samples (P < 0.0001). Matched data of mRNA expression and semiquantitative protein expression analyzed on the TMA were available from 31 HCC patients. GLUT1 mRNA expression was significantly higher in HCC cases with positive Glut1 immunosignal (n = 7) compared with cases in which no GLUT1 was detectable (n = 24; 3.4 ± 1.1-fold; P = 0.0015). This finding indicates that highly increased Glut1 expression is accurately detected by immunohistochemistry (IHC). However, lower expression may be below the detection limit of IHC. Herewith, differences between HCC and nontumorous liver may be missed, and probably, Glut1 protein is increased in even more cases than now shown by IHC. For descriptive data analysis, clinicopathological characteristics were compared with GLUT1 mRNA and protein expression. GLUT1 mRNA expression correlated significantly with tumor stage (r = 0.37, P = 0.039), grading (r = 0.48, P = 0.007), and proliferation rate (MiB-1 index; r = 0.62, P = 0.0002). Immunohistochemistry confirmed these data on the protein level (Table 1). Glut1 expression was significantly associated with higher tumor stage (P = 0.023) and tumor grading (P < 0.0001). Furthermore, Glut1-positive HCCs had a significantly higher proliferation rate (MiB-1 index) compared with Glut1-negative HCCs (P = 0.006, Figure 1E). No correlation was found between Glut1 expression and age, gender, tumor size, and the existence of liver cirrhosis. The etiology of the underlying liver disease was known in only approximately half of the patients (73 of 152). In most cases, HCC had developed in alcohol-related cirrhosis (52 of 73, 71%), and this percentage was similar in the groups of Glut1-negative (45 of 64, 70%) and -positive (7 of 9, 78%) HCCs. The transcription factor HIF-1α is an important mediator of hypoxic adaptation of tumor cells and controls several genes that have been implicated in tumor growth including GLUT1. Therefore, we analyzed HIF-1α protein expression in three different HCC cell lines (HepG2, PLC, and Hep3B) grown under aerobic conditions in vitro. Interestingly, no HIF-1α expression could be detected in HCC cells by Western blotting (Figure 2A). In contrast, after pharmacological HIF induction by DP, strong HIF-1α protein expression was detected in all three HCC cell lines by Western blotting (Figure 2A). In line with this finding, transfection of Hep3B cells with a luciferase reporter plasmid containing six copies of a functional hypoxia-responsive element (HRE) revealed only baseline activity under aerobic conditions (Figure 2B). In contrast, strong HRE reporter gene activity was observed in Hep3B cells under DP-induced hypoxia, and this activity was completely abrogated by pharmaceutical inhibition of HIF-1α activity with YC-122Chun YS Yeo EJ Choi E Teng CM Bae JM Kim MS Park JW Inhibitory effect of YC-1 on the hypoxic induction of erythropoietin and vascular endothelial growth factor in Hep3B cells.Biochem Pharmacol. 2001; 61: 947-954Crossref PubMed Scopus (154) Google Scholar, 23Yeo EJ Chun YS Cho YS Kim J Lee JC Kim MS Park JW YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.J Natl Cancer Inst. 2003; 95: 516-525Crossref PubMed Scopus (465) Google Scholar or echinomycin.24Kong D Park EJ Stephen AG Calvani M Cardellina JH Monks A Fisher RJ Shoemaker RH Melillo G Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity.Cancer Res. 2005; 65: 9047-9055Crossref PubMed Scopus (432) Google Scholar Similar results were obtained with HepG2 and PLC cells (data not shown). Interestingly, GLUT1 expression was further increased in HCC cells under DP-induced hypoxia, and this induction was strongly repressed by inhibition of HIF-1α activity with YC-1 or echinomycin (Figure 2C). Similarly as DP-induced hypoxia, also culture of HCC cells under hypoxic conditions led to a significant increase of GLUT1 mRNA expression in all three HCC cell lines (Figure 2D). Previous studies in glia cells have shown that hypoxia alters GLUT1 expression post–transcriptionally by enhancing GLUT1 mRNA stability.28Bruckner BA Ammini CV Otal MP Raizada MK Stacpoole PW Regulation of brain glucose transporters by glucose and oxygen deprivation.Metabolism. 1999; 48: 422-431Abstract Full Text PDF PubMed Scopus (79) Google Scholar Therefore, we analyzed GLUT1 mRNA expression in HCC cells at different time point after exposure to DP-induced hypoxia with or without pretreatment with actinomycin D (ActD), an inhibitor of transcription. ActD treatment resulted in a decline of GLUT1 mRNA levels with time indicating a GLUT1 mRNA half-live larger than 12 hours, similarly as previously reported in neurons (Figure 2E).28Bruckner BA Ammini CV Otal MP Raizada MK Stacpoole PW Regulation of brain glucose transporters by glucose and oxygen deprivation.Metabolism. 1999; 48: 422-431Abstract Full Text PDF PubMed Scopus (79) Google Scholar No significant difference was found between HCC cells grown under normoxic and hypoxic conditions, indicating that hypoxia does not affect mRNA stability in HCC cells. Together, these data indicate that basal GLUT1 expression in HCC is further increased under hypoxic conditions, and this induction is regulated on the transcriptional level by HIF-1α activation. In search for the molecular mechanisms that cause the increased expression of GLUT1 in HCC cells under normoxic conditions we performed in silico promotor studies using the Genomatix software. Alignment of the promotor sequence of the murine, rat, and human GLUT1 gene revealed that a binding site for the transcription factor MAZ (Myc-associated zinc finger protein, located 310 bp upstream of the transcriptional start site of the human GLUT1 gene) was highly conserved in all three species. Similarly as previously described29Dudas J Mansuroglu T Moriconi F Haller F Wilting J Lorf T Fuzesi L Ramadori G Altered regulation of Prox1-gene-expression in liver tumors.BMC Cancer. 2008; 8: 92Crossref PubMed Scopus (50) Google Scholar we found increased MAZ expression in HCC cells compared with PHHs (data not shown), and noteworthy, transient transfection with two different MAZ siRNAs significantly inhibited GLUT1 mRNA expression in Hep3B cells (Figure 2F) as well as in HepG2 and PLC cells (data not shown). In summary, these data indicate that constitutively high GLUT1 mRNA expression in HCC cells under normoxic conditions is at least in part dependent on the transcription factor MAZ, and that GLUT1 mRNA expression is further increased under hypoxic conditions by HIF-1α activation. To gain insight into the functional role of increased GLUT1 in HCC, we inhibited GLUT1 expression in HCC cells by transient transfection with two different GLUT1 siRNAs. Quantitative real-time PCR analysis revealed a strong down-regulation of GLUT1 mRNA in Hep3B cells transfected with GLUT1 siRNA (siRNA1 and siRNA2) as compared with Hep3B cells transfected with control siRNA and nontransfected cells, respectively (Figure 3A). Down-regulation of GLUT1 expression in Hep3B cells transfected with GLUT1 siRNA was also confirmed at the protein level (Figure 3B). FACS analysis of Alexa Fluor 488-labeled control siRNA revealed a transfection efficiency of ∼90.6 ± 0.8% (data not shown). Furthermore, we studied the duration of the inhibitory effect of GLUT1 siRNA on GLUT1 mRNA expression in HCC cells and found that GLUT1 mRNA remained reduced for at least 4 days after transfection (data not shown). Comparable results regarding efficiency of transfection and GLUT1 expression with GLUT1 siRNA were obtained in PLC and HepG2 cells (data not shown). To further characterize the role of GLUT1 in HCC cells, we performed functional in vitro assays with HCC cells by suppressing GLUT1 expression (GLUT1 siRNA1 and GLUT1 siRNA2) in comparison with HCC cells transfected with control siRNA and nontransfected HCC cells. Inhibition of GLUT1 expression caused significantly impaired migration as analyzed in Boyden chamber assays (Figure 3C). Next, we analyzed whether GLUT1 expression affected the proliferation of HCC cells in vitro. HCC cells with suppressed GLUT1 expression grew significantly slower compared with controls cultured in monolayers (Figure 3D). In addition, we compared the growth of HCC cells with suppressed GLUT1 expression and control cells in a three-dimensional cell culture model. Transfection with GLUT1 siRNA resulted in the formation of significantly smaller spheroids compared with spheroids of control cells (Figure 3E). HCC cells secreted significantly more lactate into the supernatant than PHHs (Figure 4A). In Hep3B cells transfected with GLUT1 siRNA lactate secretion was significantly reduced as compared with control cells (Figure 4B). Further, suppression of GLUT1 expression reduced glucose uptake into Hep3B cells significantly (Figure 4C). Similar results for glucose uptake and lactate secretion were obtained for HepG2 and PLC cells after GLUT1 suppression (data not shown). Together, these
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