Head and neck squamous cell carcinoma (HNSCC) ranks among the top ten cancers by occurrence worldwide [
]. For local HNSCC, recurrence and metastasis (R/M) have been regarded as the clinical factors associated with the poorest outcomes. Once a patient is diagnosed with R/M HNSCC, the prognosis is very poor, and the overall survival is often less than 1 year [
]. The underlying reasons for why relatively localized HNSCC becomes increasingly invasive and metastatic remain unclear and urgently need to be addressed. Previous reports have suggested that hypoxia could induce HNSCC cell migration and invasion [
] and cause a switch to anaerobic glycolysis for energy and survival (known as the “Warburg effect”) [
]. This switch increases tumor cell proliferation rates by generating not only sufficient amounts of ATP but also high amounts of macromolecules [
]. In recent studies, such metabolic reprogramming has also been shown to contribute to cancer progression and metastasis [
]. However, how tumor cells establish this metabolic reprogramming and its influence on aggressive phenotypes are as yet unknown.
Glucose transporters (GLUTs) are membrane proteins that can facilitate glucose uptake and are found in most mammalian cells. There are 12 subtypes of GLUTs that have been identified in the human genome. Recently, the expression of GLUTs has been found in different cancers to modulate glucose metabolism and correlate with epithelial-mesenchymal transition (EMT) [
], chemotherapy resistance [
], and cell proliferation [
]. In this study, we first identified the expression of GLUT4 in oral squamous cell carcinoma and its prognostic impact on HNSCC patients. The overexpression of GLUT4 in the HNSCC cell lines Ca9-22 and HSC-3-M3 elevated the proliferation rate and migration ability. In vivo animal models validated that GLUT4-overexpressing HNSCC cells exhibited enhanced lymph node and lung metastasis. Finally, an in silico analysis found that the novel GLUT4–TRIM24 signaling pathway may contribute to these aggressive cancer phenotypes possibly through DDX58 downregulation.
Cell culture and stable clone establishment
The human head and neck squamous cancer cell lines FaDu, Detroit-562, HSC-2, HSC-3, HSC-M3, HSC-4, RPMI-650, and Ca-922 were grown in MEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). All cells were incubated in a humidified atmosphere of 5% CO
at 37 °C. All cell lines were purchased from the JCRB cell bank. The pGIPZ lentiviral shRNAmir system (Thermo, Waltham, MA, USA), virus-backboned short hairpin RNA (shRNA) clones, and the GLUT4 sequence were used to establish stable cell lines (Additional file
: Table S5). Lentiviruses were used to infect the cells for 2 days. Stable clones were selected by treating the cells with 1 μg/ml puromycin (Sigma, St. Louis, MO, USA) for 2 weeks.
Western blot analysis
HNSCC cell pellets were lysed in RIPA buffer with protease/phosphatase inhibitors on ice. The protein content was quantified using a BCA assay kit (Thermo, Waltham, MA, USA), and equal protein amounts (30 μg) of each sample were used for western blot analysis. PVDF membranes (Millipore, Bedford, MA, USA) were blocked with 5% fat-free milk and then incubated with primary antibodies directed against GLUT4 (Epitomics, Cambridge, MA, USA), GLUT1 (GeneTex, Hsinchu, Taiwan), DDX58 (GeneTex, Hsinchu, Taiwan) or OASL (GeneTex, Hsinchu, Taiwan), and α-tubulin (Sigma, St. Louis, MO, USA). Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) system (Amersham ECL Plus™, GE Healthcare Life Sciences, Chalfont St. Giles, UK).
Total RNA was extracted and purified using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) and qualified with a model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). All RNAs were labeled using a GeneChip 3′IVT Expression Kit & Hybridization Wash and Stain Kit (Affymetrix, Santa Clara, CA, USA) and analyzed using Affymetrix GeneChip Human Genome U133 plus 2.0 arrays (Affymetrix, Santa Clara, CA, USA). The gene expression levels were normalized as log2 values using GeneSpring software (Agilent Technologies, Palo Alto, CA, USA). Genes that were up- or downregulated with greater than 1.5-fold changes in response to GLUT4 overexpression were further subjected to computational simulation by Ingenuity Pathway Analysis (IPA; QIAGEN, Valencia, CA, USA) online tools to predict potential upstream regulators and canonical pathways. The microarray data were uploaded to the National Center for Biotechnology Information Gene Expression Omnibus (GEO, NCBI) (GSE89631).
Glucose uptake and lactate production analyses and compounds
Glucose consumption and lactate production were measured using colorimetric glucose and lactate assay kits (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocols. Briefly, cells from the designated experiments were incubated with assay buffer containing enzyme and glucose/lactate probes. Then, the optical densities were measured at 570/450 nm wavelengths. The glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Sigma, St. Louis, MO, USA) was also used to analyze glucose uptake. In addition, cells were treated with the GLUT4 transport inhibitors indinavir or ritonavir (Sigma, St. Louis, MO, USA) at 100 and 50 μM, respectively, for 60 min, and the uptake of 2-NBDG was measured using Vector 3 (Bruker, MA, USA) to detect relative fluorescence counts.
Three representative 1-mm-diameter cores from each tumor, taken from formalin-fixed paraffin-embedded tissues, were selected for morphology typical of the diagnosis. Assessable cores were obtained in 90 cases. The histopathological diagnoses of all samples were reviewed and confirmed by a pathologist, Michael Hsiao. IHC staining was performed on serial 5-μm-thick tissue sections cut from the tissue microarray (TMA) using an automated immunostainer (Ventana, Tucson, AZ, USA). Briefly, the sections were first dewaxed in a 60 °C oven, deparaffinized in xylene, and rehydrated in graded alcohol. Antigens were retrieved by heat-induced antigen retrieval for 30 min in Tris-EDTA buffer. The slides were stained with a polyclonal rabbit anti-human GLUT4 antibody (1:750, Epitomics, Cambridge, MA, USA). The sections were subsequently counterstained with hematoxylin, dehydrated, and mounted. The IHC staining intensity was scored by two pathologists as follows: no cytoplasmic staining or cytoplasmic staining in <10% of tumor cells was defined as score 0; faint/barely perceptible partial cytoplasmic staining in >10% of tumor cells was defined as score 1+; moderate cytoplasmic staining in >10% of tumor cells was defined as score 2+; and strong cytoplasmic staining in >10% of tumor cells was defined as score 3+. Scores of 0 and 1+ were defined as low GLUT4 expression, while scores of 2+ and 3+ were defined as high GLUT4 expression.
In vivo model
Age-matched, nonobese diabetic-severe combined immunodeficient gamma (NOD.Cg-Prkdc
tm1Wjl/SzJ JAX®, NOD-SCID γ) male mice (6–8 weeks old, 20–25 g body weight) were used. To evaluate lung colony-forming ability, 1 × 10
6 cells were resuspended in 100 μL of PBS and injected into the lateral tail vein. Lung nodule formation was quantified after H&E staining using a dissecting microscope at the endpoint. To evaluate in vivo tumorigenicity ability and establish an orthotopic model, 5 × 10
6 cells were resuspended in 100 μL of PBS and then subcutaneously injected into the flanks of the mice, and 5 × 10
6 cells were resuspended in 10 μL of PBS and injected into the buccal submucosa. All animal experiments were conducted in accordance with a protocol approved by the Academia Sinica Institutional Animal Care and Utilization Committee (IACUC).
In total, 90 patients diagnosed with head and neck squamous cell carcinoma at the Taipei Medical University Hospital in Taiwan from 1991 to 2010 were included in this study. Patients who received preoperative chemotherapy or radiation therapy were excluded. Clinical information and pathology data were collected via a retrospective review of patient medical records. All cases were staged according to the 7th edition of the Cancer Staging Manual of the American Joint Committee on Cancer (AJCC), and the histological cancer type was classified according to the World Health Organization (WHO) 2004 classification guidelines. Follow-up data were available in all cases, and the longest clinical follow-up time was 190 months. Overall survival and disease-free survival were defined as the intervals from surgery to death caused by head and neck squamous cell carcinoma and recurrence or distant metastasis, respectively. The study was performed with the approval of the Institutional Review Board and with permission from the ethics committee of the institution involved (TMU-IRB 99049).
The nonparametric Mann–Whitney
U test was used to analyze the statistical significance of results from three independent experiments. Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) 17.0 software (SPSS, Chicago, IL, USA). A paired
t test was performed to compare the GLUT4 IHC expression levels in cancer tissues and in the corresponding normal adjacent tissues. The association between clinicopathological categorical variables and the GLUT4 IHC expression levels were analyzed by Pearson’s chi-square test. Estimates of the survival rates were calculated using the Kaplan–Meier method and compared using the log-rank test. The follow-up time was censored if the patient was lost during follow-up. Univariate and multivariate analyses were performed using Cox proportional hazards regression analysis with and without an adjustment for GLUT4 IHC expression level, tumor stage, lymph node stage, and recurrence status. For all analyses, a
P value of <0.05 was considered significant.
GLUT4, encoded by the SLC2A4 gene, is a high-capacity transporter that is normally restricted to nondividing cells, including adipose tissue, skeletal muscle, and myocardium [
]. GLUT4 is not detectable in normal oral epithelial cell lines [
], whereas GLUT1 is ubiquitously expressed and is constitutively located on the cell membrane [
]. Evidence from intensive research in the field of diabetes shows that GLUT4 traffics between the plasma membrane and intracellular vesicles (termed GLUT4-storage vesicles, GSVs) and that this activity is regulated by the PI3k/Akt pathway in an insulin-responsive manner [
] or by the AMPK pathway [
] in response to muscle contraction. Surprisingly, evidence has suggested that GLUT4 is present for basal glucose consumption and cell growth and survival in multiple myeloma [
] and breast cancer cells [
]. To date, little is known about the involvement of GLUT4 in cancer metabolism. This raises the question of whether the regulation of GLUT4 in cancer cells is due to a cancer-specific glucose transporter or a cancer-specific signaling mechanism. In this study, we first confirmed the role of GLUT4 in cancer metastasis and the possible signaling network involved.
TRIM24 controls gene expression through several mechanisms. First, TRIM24 promotes AKT phosphorylation to promote cell proliferation [
]. Second, TRIM24 interacts with nuclear receptor, such as RAR or ER, to regulate gene expression [
]. Third, TRIM24 contains a RING domain and E3 ligase activity that degrades p53, which controls gene expression [
]. Because our signaling analysis was generated using GLUT4-silenced HNSCC cells, we proposed that GLUT4 triggers TRIM24 to repress several downstream tumor suppressors. We hypothesized that the GLUT4–TRIM24 axis had a positive correlation and represented a powerful biomarker for clinical treatment and prognosis.
One of the most interesting observations we made in this study was that GLUT4-mediated HNSCC metastasis was independent of glucose concentration and the innate glucose transport function of GLUT4. It may be that the ectopic overexpression of GLUT4 leads to a lower threshold for activating its downstream molecules, rendering the ligand (glucose) concentration nonconsequential. This phenomenon was reported in the case of epidermal growth factor (EGF) and its receptor (EGFR) [
]. It is also plausible that alternative ligands (other than glucose) of GLUT4 may be present and responsible for this phenomenon. Thus, further investigation is required.
Based on its tissue and function specificity, GLUT4 has long been thought to be an insulin-dependent glucose transporter in muscle and fat cells. Our study thus uncovers a new role for GLUT4 as a metastatic promoter and prognostic biomarker for HNSCC patients. However, the reason why GLUT4 expression is elevated in HNSCC cells remains unclear. A plausible explanation could be the deranged metabolism in cancer cells. Because the upper aerodigestive tract is susceptible to environmental carcinogens, such as tobacco, alcohol, and betel nuts [
], cellular stress and damages generated by these agents may result in malignant transformation and metabolism. Our IPA analysis revealed that hypoxia-induced factors (EPAS1, HIF1A) and TGF
-associated genes (SKIL, TGIF1, SMAD4) as well as genes involved in stemness and tumorigenesis (MYB, FOXL2, FOXO1, NOTCH3) were all upregulated, which supported the hypothesis that GLUT4 may be an abnormal responder to environmental carcinogens and result in carcinogenesis, cancer progression, and metabolic shifts. (Fig.
and Additional file
: Table S2).
According to recently published reports, TRIM24 was found to be correlated with poor survival and was involved in cell proliferation and metastasis in colon cancer and breast cancer [
]. TRIM24 was also reported to be a regulator of interferon signal transducers to activate the STAT pathway through retinoic acid receptor inhibition [
]. TRIM24 serves as a co-factor for binding the
promoter region to enhance cell proliferation through the induction of the IFN/STAT1 pathway [
]. Interestingly, DDX58 was found to be significantly upregulated in TRIM24-deficient mice [
]. Here, in our study, we further provided direct evidence that GLUT4 overexpression significantly activates TRIM24 to downregulate DDX58 expression and consequently promotes HNSCC cell motility and invasion. The detailed mechanism regarding how GLUT4 modulates TRIM24 activity remains to be elucidated.
We like to thank Miss Tracy Tsai for her assistance in the immunohistochemistry works. We would also like to thank the Genomics Research Center Instrument Core Facilities for their support for the Affymetrix microarray, IVIS spectrum, and Aperio digital pathology analyses.
This study is supported by Academia Sinica and Ministry of Science and Technology grants MOST 104-0210-01-09-02 and MOST 105-0210-01-13-01 to Michael Hsiao.
Availability of data and materials
Additional data are available in Additional files
PM-HC, ATHW, and MH designed and supervised the study and experiments, analyzed the data, and co-wrote the manuscript. Y-CC developed the methodologies, performed the experiments, analyzed the data, and co-wrote the manuscript. L-HC, W-MC, M-HC, and Y-FL performed the experiments and analyzed the data. C-YS and MH performed the histopathological analysis. C-LC provided the clinical specimens. M-HC provided the compound. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Paraffin tissues used to generate tissue microarrays were collected from Taipei Medical University Hospital Wan Fang Hospital. The study protocol was approved by the institutional review board (IRB) at Taipei Medical University Wan Fang Hospital (approval number 99049). Written informed consent was obtained from all participants.