Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis
Chang et al. Journal of Hematology & Oncology
Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through the TRIM24-DDX58 axis
Yu-Chan Chang 0 1
Li-Hsing Chi 0
Wei-Ming Chang 0
Chia-Yi Su 0
Peter Mu-Hsin Chang
Alex T. H. Wu
Michael Hsiao 0 1
0 Genomics Research Center , Academia Sinica, Taipei , Taiwan
1 Graduate Institute of Life Sciences, National Defense Medical Center , Taipei , Taiwan
Background: Head and neck squamous cell carcinoma (HNSCC) represents a unique and major health concern worldwide. Significant increases in glucose uptake and aerobic glycolysis have been observed in HNSCC cells. Glucose transporters (GLUTs) represent a major hub in the glycolysis pathway, with GLUT4 having the highest glucose affinity. However, GLUT4's role in HNSCC has not been fully appreciated. Methods: An in silico analysis was performed in HNSCC cohorts to identify the most significant glucose transporter associated with HNSCC patient prognosis. An immunohistochemical analysis of a tissue microarray with samples from 90 HNSCC patients was used to determine the association of GLUT4 with prognosis. Complementary functional expression and knockdown studies of GLUT4 were performed to investigate whether GLUT4 plays a role in HNSCC cell migration and invasion in vitro and in vivo. The detailed molecular mechanism of the function of GLUT4 in inducing HNSCC cell metastasis was determined. Results: Our clinicopathologic analysis showed that increased GLUT4 expression in oral squamous cell carcinoma patients was significantly associated with a poor overall survival (OS, P = 0.035) and recurrence-free survival (RFS, P = 0. 001). Furthermore, the ectopic overexpression of GLUT4 in cell lines with low endogenous GLUT4 expression resulted in a significant increase in migratory ability both in vitro and in vivo, whereas the reverse phenotype was observed in GLUT4-silenced cells. Utilizing a GLUT4 overexpression model, we performed gene expression microarray and Ingenuity Pathway Analysis (IPA) to determine that the transcription factor tripartite motif-containing 24 (TRIM24) was the main downstream regulator of GLUT4. In addition, DDX58 was confirmed to be the downstream target of TRIM24, whose downregulation is essential for the migratory phenotype induced by GLUT4-TRIM24 activation in HNSCC cells. Conclusions: Here, we identified altered glucose metabolism in the progression of HNSCC and showed that it could be partially attributed to the novel link between GLUT4 and TRIM24. This novel signaling axis may be used for the prognosis and therapeutic treatment of HNSCC in the future.
GLUT4; HNSCC; TRIM24; DDX58; Metastasis
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
[3, 4] 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
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% CO2 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 1: 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)
Glucose uptake and lactate production analyses and
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-diazol4-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-Prkdcscid Il2rgtm1Wjl/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 × 106 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 × 106
cells were resuspended in 100 μL of PBS and then
subcutaneously injected into the flanks of the mice, and 5 ×
106 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
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
logrank 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.
Increased expression of GLUT4 is significantly correlated
with metastasis and poor prognosis in HNSCC patients
To determine the clinical association between glucose
transporters (GLUTs) in HNSCC patients, we utilized a
previously developed HNSCC microarray database to
examine and compare the expression of 10 major GLUTs
using the Oncomine website. GLUT4 was found to be the
only GLUT family member to have a significant
correlation with metastatic status compared with other GLUT
family members in the clinical cohort (Fig. 1a, 3.59-fold
change, P = 5.20E-5). We then compared the correlations
of all the GLUT family members with the prognosis of
patients in the Petel HNSCC cohort (E-MTAB-1328, n = 89)
Fig. 1 Overexpression of GLUT4 correlates with poor survival in HNSCC patients. a The heatmap indicates the correlation between the mRNA
expression level of glucose transporters and HNSCC metastasis. Note that GLUT4 is the only gene that is significantly correlated with metastasis
events in the Rickman Head–Neck cohort (n = 36) in the analysis by the Oncomine online tool. b The box plot shows that higher GLUT4 expression
was correlated with a poor survival rate in patients in the Petel HNSCC cohort (E-MTAB-1328, n = 89) from the SurvExpress database (HR = 3.37,
P = 0.043). c The expression level of the GLUT4 protein in tumor tissue compared to the corresponding normal adjacent tissue. d Scores (0~3)
indicating GLUT4 levels in representative head and neck squamous tumor tissues. e Kaplan–Meier curves of overall and disease-free survival of
90 patients with HNSCC, stratified by a high or low GLUT4 protein expression level (P = 0.017 and P = 0.001, respectively)
in the SurvExpress database. The number of cases was
divided approximately in half based on the expression
(low or high) of the GLUT family member of interest,
and a Kaplan–Meier survival analysis was performed
on both groups using the SurvExpress website. The
results showed that GLUT4 is the only GLUT family
member whose RNA expression is significantly
correlated with HNSCC overall survival (Fig. 1b, HR = 3.37,
P value =0.043, other GLUT family data in Additional
file 1: Figure S1). Forest plots of GLUT family members
and their corresponding hazard ratios and Cox-P values
were generated for another HNSCC microarray cohort
(GSE2837, n = 40), and these results also showed GLUT4
to be the strongest prognosis marker with the highest
hazard ratio. The Cox-P value for GLUT4 was
calculated to be 0.07 by the Pronoscan website (Additional
file 1: Figure S2). Together, these data show that of the
GLUT family members, GLUT4 is the most
significantly correlated with the clinical outcomes of HNSCC.
We next validated these findings by examining GLUT4
protein expression using our own clinical HNSCC tissue
cohort. The immunohistochemical staining results showed
stronger staining of the GLUT4 protein in tumor tissues
than in the adjacent normal tissues (Fig. 1c). After scoring,
we determined the correlation between patient survival
and either low-level GLUT4 staining (Fig. 1d, IHC scores 0
and 1) or high-level GLUT4 staining (Fig. 1d, IHC scores 2
and 3). The results indicated that high-level GLUT4
staining was significantly correlated with the poor overall and
disease-free survival probabilities (Fig. 1e, P = 0.017, P =
0.001, respectively). A clinicopathological analysis showed
that high GLUT expression is significantly correlated with
recurrence (Table 1, P = 0.001). The patient demographic
Table 1 Correlation of clinicopathological features of HNSCC
patients with GLUT4 expression
GLUT4 expression, n (%)
Low (n = 23)
High (n = 67)
features are shown in Additional file 1: Table S1. Univariate
and multivariate analyses of the disease-free survival
probability showed that high-level GLUT4 expression served as
the strongest independent prognostic marker in both the
univariate analysis (Table 2, HR = 3.35, P = 0.001) and the
multivariate analysis (Table 2, HR = 3.76, P < 0.001). These
data indicate that the upregulation of GLUT4 is
significantly associated with the distant metastasis and
diseaserelated progression in HNSCC patients.
GLUT4 ectopic overexpression promotes the migration
and invasion abilities of HNSCC cells
To determine the functional attributes of GLUT4 in
promoting HNSCC cellular migration and invasion, we
first examined the GLUT4 protein expression levels in
HNSCC cell lines. Our results showed varied
expression levels of the GLUT4 protein in the eight HNSCC
cell lines examined (Fig. 2a). We then determined the
migration and invasion potentials of these HNSCC cell
lines (Fig. 2b, c). The migration and invasion potentials
of these cell lines were compared with their respective
GLUT4 protein expression levels. Our results showed
that GLUT4 expression appeared to be causally
associated with metastatic potentials in HNSCC cells (Fig. 2d,
Spearman rho = 0.81, P = 0.015). The GLUT4 gene was
ectopically overexpressed in the low GLUT4-expressing
cell lines HSC-3 and FaDu to determine whether GLUT4
overexpression induces HNSCC cell migration and
invasion. The results in the left panel of Fig. 2e show the
overexpression of the GLUT4 protein in the HSC-3 and
FaDu cells. GLUT4 overexpression indeed significantly
promoted the migration and invasion capabilities of the
low-metastatic FaDu and HSC-3 cells (Fig. 2e, right panel,
P < 0.01). In a complementary model, GLUT4 gene
silencing significantly reduced the GLUT4 protein levels in
HSC-3-M3 and HSC-2 cells, which expressed a high level
of endogenous GLUT4 (Fig. 2f, left panel). GLUT4
knockdown in these two cell lines significantly inhibited
the migratory/invasive capabilities of the highly
metastatic HSC-3-M3 and HSC-2 cells (Fig. 2f, right panel).
Increased GLUT4 expression promotes in vivo lung
metastasis and in situ neck lymph node invasion
Next, we examined the role of GLUT4 in the promotion
of metastasis in vivo using xenograft mouse models by
intravenously injecting GLUT4-overexpressing and
vectorcontrol FaDu cells into mice. Six weeks after injection, the
lungs were removed and examined for metastatic foci. Mice
injected with GLUT4-overexpressing FaDu cells exhibited
significantly higher numbers of metastatic foci compared to
the vector control group by gross and histopathological
examinations (Fig. 3a). There was a 4-fold increase in
foci number in the GLUT4 overexpression group
compared to the vector control group (Fig. 3b, P < 0.001).
*P value <0.05 was considered statistically significant (Student’s t test for
continuous variables and Pearson’s chi-square test for variables). SD represents the
standard deviation. The tumor stage, tumor, lymph node, and distal metastasis
status were classified according to the international system for staging HNSCC
Table 2 Univariate and multivariate analysis of GLUT4 expression and HNSCC patients
*P value <0.05 was considered significant
To mimic clinical HNSCC metastasis, we established an
orthotopic xenograft HNSCC mouse model by injecting
mice intrabuccally with luciferase-expressing FaDu cells
that expressed either the GLUT gene or a vector control.
Our results showed that stronger bioluminescence could
be observed in 4 out of 5 mice injected with the
GLUT4overexpressing cells compared to only 1 mouse exhibiting
weak bioluminescence in the vector control group (Fig. 3c).
The average bioluminescence counts were obtained from
the neck lymph nodes of all 10 mice, and the results
showed that the GLUT4-overexpressing group had
significantly higher counts compared to the vector control
group (Fig. 3d, P < 0.05). In addition, we also established a
xenograft model by subcutaneous injection of
GLUT4overexpressing FaDu cells. We observed that GLUT4 did
not significantly increase the tumorigenicity of FaDu cells
in vivo, and this result is consistent with the cell
proliferation rate in vitro (Additional file 1: Figure S3). These data
suggest that GLUT4 overexpression promotes HNSCC
metastasis in vivo and in situ.
HNSCC cell migration and invasion induced by GLUT4
overexpression is independent of glucose transporter
To determine whether the GLUT4-mediated promotion
of HNSCC cell migration and invasion requires glucose
transporter activity, we screened glucose uptake and
lactate production in a panel of HNSCC cells. The data
showed that metabolic events may be correlated with
metastasis ability in several cell lines (Additional file 1:
Figure S4), but no significant P values were obtained.
Therefore, we added the glucose transport inhibitors
ritonavir and indinavir to block glucose transport
efficiency in a GLUT4-overexpressing cell model. We first
used 2-NBDG treatment to demonstrate that ritonavir
did indeed block transporter function. We observed the
uptake of the glucose analog 2-NBDG by its
autofluorescence. The GLUT4-overexpressing FaDu and HSC-3
cells treated with ritonavir had lower fluorescence
counts (Fig. 4a, P < 0.01, left panel and P = 0.016, right
panel) than did the vector-control FaDu and HSC-3
cells (Fig. 4a, P = 0.021, left panel and P < 0.01, right panel).
We further confirmed the decrease in glucose uptake after
inhibitor treatment by analyzing the culture medium
(Fig. 4b). However, the results showed that ritonavir/
indinavir did not significantly reduce the GLUT4-induced
migration and invasion abilities of FaDu and HSC-3 cells
compared to control cells (Fig. 4c). These results
suggested that GLUT4 promotes HNSCC cell migration and
invasion only partially through the transportation of
glucose to the cancer cells.
TRIM24-DDX58 axis is involved in GLUT4-mediated
HNSCC cell migration
To determine whether another novel pathway or network
by plays a transporter function-independent role in the
GLUT4-mediated promotion of HNSCC cell migration
and invasion, we next performed a microarray analysis
using the low-metastatic HNSCC FaDu cells with or
without GLUT4 overexpression. The normalized data from
the microarray database analysis were subjected to
Ingenuity Pathway Analysis (IPA) to identify molecules that
are activated upon GLUT4 overexpression in FaDu cells.
The results showed that the transcription factor TRIM24
is the top predicted candidate to be activated in response
to GLUT4 overexpression, as verified by the
transcriptional activity of its target genes with a Z-score of 2.868
and P value =1.55E-05 (Fig. 5a and Additional file 1:
Table S2). The top 11 activated transcription factors with
Z-scores higher than 2 and their respective downstream
genes are shown in Additional file 1: Table S2. Similarly,
the top 7 inhibited transcription factors and their
respective downstream genes are shown in Additional file 1:
Because TRIM24 was the most activated transcription
factor upon GLUT4 overexpression in FaDu cells, we
compared our GLUT4 microarray datasets with the
TRIM24related signature obtained by IPA analysis. Our results
Fig. 2 GLUT4 expression is positively correlated with metastasis ability in HNSCC cells and complementary models showed that GLUT overexpression
promotes HNSCC migration and invasion. a Western blot analysis of GLUT4 and tubulin protein expression in various HNSCC cells. Tubulin was used as
an internal control for protein loading. b The correlation between the GLUT4 protein expression level and the migration and invasion abilities of various
HNSCC cell lines. c The significance of the correlation was analyzed using the nonparametric Spearman method. d Giemsa staining for evaluating the
migration and invasion abilities of a panel of various HNSCC cell lines. e Left panel: western blot analysis of GLUT4 and tubulin protein expression after
GLUT4 overexpression in FaDu cells and HSC-3 cells. Right panel: the migration and invasion abilities of FaDu cells and HSC-3 cells after the overexpression
of the exogenous GLUT4 gene. f Western blot analysis of GLUT4 knockdown in HSC-2 cells and HSC-3-M3 cells. Tubulin was used as an internal control for
protein loading. Right panel: the migration and invasion abilities of HSC-2 and HSC-3-M3 after GLUT4 knockdown. NS represents the nonsilenced control
showed that DDX58 and OASL were the most
significantly downregulated transcriptional targets of TRIM24
(Fig. 5b; −2.42- and −2.99-fold for DDX58 and
−2.96and −2.34-fold for OASL in Additional file 1: Table S4).
We further validated the expression levels of DDX58
and OASL in cell models of GLUT4 overexpression and
knockdown. Our western blot results showed that DDX58
and OASL were downregulated in GLUT4-overexpressing
FaDu and HSC-3 cells (Fig. 5c, left panel) and that the
knockdown of GLUT4 expression in HSC-2 cells resulted
Fig. 3 GLUT4 promotes in vivo metastasis and in situ invasion phenotypes. a Metastatic lung foci appearance as indicated by arrows (left panel)
and foci morphologies (middle panel, ×12.5 magnification, and right panel, ×100 magnification) in mice (n = 5) implanted with control (vector only) or
GLUT4-overexpressing FaDu cells through tail vein injection. b The quantified plot of metastatic lung foci numbers from Fig. 3a. c Bioluminescence
images of the vector and GLUT4-overexpressed groups of the orthotopic FaDu xenograft mouse model. FaDu-GL-VC and –GLUT4 cells were
intrabuccally injected into NSG mice. Luminescence was measured using a noninvasive bioluminescence imaging system (IVIS spectrum) at
6 weeks after injection. Lymph node metastasis is expressed as the bioluminescence intensity (BLI) change (five mice per group). d Quantitation of
photon counts of each group from Fig. 3c. (P = 0.04). The significance of the difference was analyzed using the nonparametric Mann–Whitney U test
in the higher expression of the DDX58 and OASL
proteins (Fig. 5c, right panel). We further knocked down
the gene expression of DDX58 and OASL by their
respective shRNAs in the GLUT4-knockdown highly
metastatic HSC-2 cells. A western blot analysis showed
that the DDX58 and OASL protein expression was
significantly reduced (Fig. 5d). The subsequent
knockdown of DDX58 could significantly restore the
migration potential of GLUT4-knockdown HSC-2 cells
by 1.5-fold (Fig. 5e, P < 0.001); however, OASL
knockdown did not restore the migration capability (Additional
file 1: Figure S5). These data suggested that DDX58 may
be the primary negatively regulated downstream target of
TRIM24 mediating the GLUT4-induced HNSCC cell
migration. This in vitro inverse relationship of GLUT4
and DDX58 expression was then validated in in silico
clinical HNSCC cohorts. The results showed that high
GLUT4 RNA expression in combination with low
DDX58 RNA expression levels was significantly
correlated with the worst HNSCC patient survival (Fig. 5f
and Additional file 1: Figure S6, P = 0.029, P < 0.001,
GLUT4, encoded by the SLC2A4 gene, is a high-capacity
transporter that is normally restricted to nondividing cells,
Fig. 4 GLUT4 promotes HNSCC metastasis. a Relative fluorescence units after GLUT4 overexpression in FaDu (left panel) and HSC-3 cells (right panel)
with or without ritonavir treatment. b The migration and invasion abilities of FaDu cells and HSC-3 cells were demonstrated after the overexpression of
the exogenous GLUT4 gene, with and without the addition of ritonavir or indinavir. The data were the average of three independent experiments and
are presented as the mean ± SEM. The significance of the difference was analyzed using the nonparametric Mann–Whitney U test. The blue and green
columns represent cellular migration and invasion abilities, respectively. c The glucose uptake abilities of FaDu cells and HSC-3 cells were demonstrated
after the overexpression of the exogenous GLUT4 gene, with and without the addition of ritonavir or indinavir. The data were the average of three
independent experiments and are presented as the mean ± SEM. The significance of the difference was analyzed using the nonparametric
Mann–Whitney U test. The black and red columns represent ritonavir and indinavir treatment, respectively
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.
Fig. 5 GLUT4 triggers TRIM24 activation to promote HNSCC metastasis. a The bar chart indicates the potential upstream regulators predicted by
Ingenuity Pathway Analysis (IPA) software based on microarray from GLUT4-overexpressing FaDu cells with a 1.5-fold change cutoff compared to
vector control cells. b The TRIM24 network was predicted based on the common signature from the Ingenuity (IPA) database overlaid with
microarray data from GLUT4-overexpressing FaDu cells with a 1.5-fold change cutoff compared with vector control cells. The intensity of the
node color indicates the degree of activating (orange) and inhibiting (blue) regulation following GLUT4 interactomics. c Western blot analysis
of DDX58, OASL, and tubulin protein expression after GLUT4 overexpression in FaDu and HSC-3 cells (left panel) or GLUT4 knockdown in HSC-2
cells (right panel). Tubulin was used as an internal control for protein loading. d Western blot analysis of DDX58 or OASL knockdown combined with
GLUT4 knockdown in HSC-2 cells. Tubulin was used as an internal control for protein loading. e The migration capabilities of HSC-2 cells with DDX58
or OASL knockdown combined with GLUT4 knockdown. f Kaplan–Meier survival curve analysis of HNSCC patients with high GLUT4 and low DDX58 or
OASL levels as determined by IHC staining at the endpoint of overall survival (P = 0.029 and P = 0.362, respectively)
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) [20–22]. 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 TGFB-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. 5a and Additional file
1: 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 [24, 25]. TRIM24 was also reported to
be a regulator of interferon signal transducers to activate
the STAT pathway through retinoic acid receptor
inhibition [26, 27]. TRIM24 serves as a co-factor for binding
the STAT1 promoter region to enhance cell
proliferation through the induction of the IFN/STAT1 pathway
[26, 28]. 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.
In this study, we showed that GLUT4 overexpression
promotes tumor metastasis and is significantly associated with
poor prognosis in HNSCC patients through a
glucoseindirect pathway in cancer cells that leads to the activation
of the TRIM24 pathway. Furthermore, we validated the
downstream target DDX58 as the suppressor of GLUT4–
TRIM24-induced migration and invasion. The inverse
correlation of GLUT4 and DDX58 may be used as a
significant predictor of poor prognosis in HNSCC patients.
The GLUT4–TRIM24 axis may serve as a new target for
drug development to treat HNSCC patients with metastasis.
Additional file 1: Table S1. Demographic features of HNCC patient
cohort. Table S2. GLUT overexpression activated transcription factors and
their downstream targets ranked by Z-Score. Table S3. GLUT overexpression
inhibited transcription factors and their downstream targets ranked by
Z-Score. Table S4. List of TRIM24 downstream genes and their fold
changes. Table S5. List of primers and knockdown clones’ information.
Table S6. List of candidate probes >2.0-fold change cutoff by GLUT4
vs. control in FaDu cells. Figure S1. Box plot showing the expression of
the GLUT family members correlated with the survival rate of the patients in
the Petel HNSCC cohort (E-MTAB-1328, n = 89) in the SurvExpress database
(HR = 3.37, P = 0.043). Figure S2. Forest plot of GLUT family members and
their corresponding hazard ratios, probes and Cox-P values. Figure S3. GLUT4
overexpression model in vitro and in vivo. (A) Cell proliferation rate and (B)
tumorigenicity ability in animal model GLUT4-overexpressing FaDu cells.
Figure S4. Glucose uptake and lactate production in a panel of HNSCC cell
lines. Figure S5. The migration abilities of with or without GLUT4 knockdown
combined DDX58 or OASL knockdown in HSC-2 cells. Figure S6. Correlation
plot of GLUT4 expression with the (A) OASL or (B) DDX58 RNA level in a
clinical cohort (Pearson r = −0.7146, P < 0.001 and Pearson r = −0.6246,
P < 0.001, respectively). (DOCX 2697 kb)
DDX58: DEXD-H-box helicase 58; GLUT: Glucose transporter; IPA: Ingenuity
Pathway Analysis; OASL: 2′-5′-Oligoadenylate synthetase Like;
TRIM24: Tripartite motif-containing 24
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.
Availability of data and materials
Additional data are available in Additional files 1.
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.
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.
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