High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue
Otsuka et al. Cell Communication and Signaling
High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue
Yutaro Otsuka 2
Hiroki Sato 0
Tsukasa Oikawa 2
Yasuhito Onodera 2
Jin-Min Nam 1
Ari Hashimoto 2
Kiyoshi Fukunaga 2
Kanako C. Hatanaka 3
Yutaka Hatanaka 3
Yoshihiro Matsuno 3
Satoshi Fukuda 0
Hisataka Sabe 2
0 Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Hokkaido University , North 15, West 7, Kita-ku, Sapporo, Hokkaido 060-8638 , Japan
1 Global Station for Quantum Medical Science and Engineering, Global Institution for Collaborative Research and Education Hokkaido University , Sapporo, Hokkaido 060-8648 , Japan
2 Department of Molecular Biology, Graduate School of Medicine, Hokkaido University , North 15, West 7, Kita-ku, Sapporo, Hokkaido 060-8638 , Japan
3 Department of Surgical Pathology, Hokkaido University Hospital , North 15, West 7, Kita-ku, Sapporo, Hokkaido 060-8638 , Japan
Background: Squamous cell carcinoma of the tongue (tongue SCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer under current therapeutics. ARF6 and its effector AMAP1 are often overexpressed in different types of cancers, such as breast cancer and renal cancer, and in these cancers, AMAP1 binds to EPB41L5 to promote invasion, metastasis, and drug resistance. EPB41L5 is a mesenchymal-specific protein, normally induced during epithelial-mesenchymal transition (EMT) to promote focal adhesion dynamics. Similarly to breast cancer and renal cancer, the acquisition of mesenchymal phenotypes is the key process that drives the malignancy of HNSCC. We previously showed that the overexpression of AMAP1 in tongue SCC is statistically correlated with the poor outcome of patients. In this study, we examined whether tongue SCC also expresses EPB41L5 at high levels. Results: Immunohistochemical staining of clinical specimens of tongue SCC demonstrated that high expression levels of EPB41L5 statistically correlate with poor disease-free survival and poor overall survival rates of patients. The tongue SCC cell line SCC-9, which overexpress Arf6 and AMAP1, also expressed EPB41L5 at high levels to promote invasiveness, whereas the weakly invasive SCC-25 cells did not express EPB41L5 at notable levels. Among the different EMT-associated transcriptional factors, ZEB1 was previously found to be most crucial in inducing EPB41L5 in breast cancer and renal cancer. In contrast, expression levels of ZEB1 did not correlate with the expression levels of EPB41L5 in tongue SCC, whereas KLF8 and FOXO3 levels showed positive correlations with EPB41L5 levels. Moreover, silencing of EPB41L5 only marginally improved the drug resistance of SCC-9 cells, even when coupled with ionizing radiation. (Continued on next page) © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Conclusion: Our results indicate that activation of the cancer mesenchymal program in tongue SCC, which leads to
EPB41L5 expression, closely correlates with the poor prognosis of patients. However, ZEB1 was not the major
inducer of EPB41L5 in tongue SCC, unlike in breast cancer and renal cancer. Thus, processes that trigger the
mesenchymal program of tongue SCC, which drives their malignancies, seem to be substantially different from
those of other cancers.
Head and neck squamous cell carcinoma (HNSCC) is
one of the most common cancers in the world . Over
600,000 new cases of HNSCC are reported annually .
Despite the recent advancements of cancer therapeutics,
over 350,000 HNSCC patients die each year .
Squamous cell carcinoma of the tongue (tongue SCC) is a
major subtype of HNSCC, accounting for approximately
25% of all HNSCC cases . In general, clinical
characteristics and treatment strategy of tongue SCC are
similar to those of other HNSCCs. Surgical resection is the
primary choice for the treatment of HNSCC. However,
many cases of HNSCC are non-operative, due to a late
diagnosis with locally advanced malignancy, or surgery
should be avoided to maintain the patients’ quality of
life. The classical drug cisplatin is still the major drug
used for the treatment of HNSCC, often coupled with
radiation [2, 3]. Whereas these treatments generally
exhibit cancer-reducing effects, there are many cases that
show relapse, primarily due to the acquisition of
resistance to such treatments.
A number of studies have indicated that the
acquisition of mesenchymal properties by cancer cells is closely
associated with the generation of cancer stem cell-like
cells, as well as the acquisition of drug resistance .
Although many such studies primarily dealt with breast
cancer, the acquisition of mesenchymal properties has
also been highly implicated in the malignancy of
HNSCC . We previously showed that the small
GTPase ARF6 and its effector AMAP1 are often
overexpressed in different types of cancers, including breast
cancer and renal cancer, and play pivotal roles in
promoting invasion, metastasis, and drug resistance [6–18].
We have moreover shown that AMAP1 binds to
EPB41L5 , which is normally induced during
epithelial-mesenchymal transition (EMT) . Thus, the
ARF6-AMAP1-EPB41L5 pathway appears to be a
cancer-specific pathway that is not expressed in cancer
cells unless they undergo EMT-like changes. The ARF6
pathway promotes β1 integrin recycling and the
downregulation of E-cadherin, thus enhancing cell adhesion
dynamics and motile phenotypes [10, 13, 18].
We previously reported that the AMAP1 protein is
often expressed at high levels in clinical specimens of
tongue SCC, and is statistically correlated with the poor
prognosis of patients . We here examined whether
tongue SCC also express the mesenchymal component
of the ARF6-based pathway, namely EPB41L5, and
whether its expression correlates with malignancy.
Tongue SCC patients, having been subjected to the
curative resection of primary sites (Table 1), were
classified into two groups based on the presence (positive) or
the absence (negative) of lymph node metastasis; and
EPB41L5 protein expression at the primary lesions were
evaluated by the H-score (Fig. 1a).
We found that the average H-score of the “positive”
group was 0.45, whereas that of the “negative” group
Table 1 Clinicopathological characteristics of patients
Location of tumor
Oral cavity (tongue)
Score 0 Score 1 Score 2
Negative Lymph node metastasis
Fig. 1 (See legend on next page.)
(See figure on previous page.)
Fig. 1 Immunohistochemical staining of EPB41L5 in primary tongue SCCs. a Specimens were immunohistochemically stained with polyclonal
anti-EPB41L5 antibodies. Each tissue section was scored for staining intensity on a scale of 0–2, as described in Methods (H-score). A
representative image for each staining is shown. Bars, 50 μm. b Patients were classified into two groups by the existence of lymph node metastasis. The
Hscore of each patient was plotted, and the t-test was performed. Black circles indicate patients without any adjuvant therapy. Black lines indicate
the means. ***P < 0.001. c and d Patients with H-scores in the top 33% were classified into the “high expression” group, and the others into the
“low expression” group. Kaplan-Meier curves were drawn regarding disease-free survival (c) and overall survival (d) of the patients. To evaluate the
P-values of these analyses, the logrank test was performed
was 0.15 (P < 0.001, Fig. 1b). These results suggested
a statistical correlation between the enhanced
expression of EPB41L5 and the metastatic properties of
We next investigated whether EPB41L5 expression
levels are correlated with disease-free survival rates and
overall survival rates of tongue SCC patients. The
abovementioned cohort of patients were then classified into
two groups based on their expression levels of EPB41L5:
patients with H-scores in the top 33% were classified
into the “high expression” group, and the others into the
“low expression” group (Fig. 1c, also see Methods). At
60 months after diagnosis, none of the patients of the
“high expression” group were found to be disease-free,
whereas 54% of the “low expression” group remained
disease-free (P = 0.0182, Fig. 1c). Consistently, 69% of
the “low expression” group survived until 60 months
after diagnosis, whereas only 14% of the “high
expression” group survived until that time (P = 0.0312, Fig. 1d).
These results indicated a tight statistical correlation
between high expression levels of EPB41L5 and the poor
prognosis of tongue SCC patients. Thus, we propose
that immunohistochemical staining of EPB41L5 provides
a biomarker predictive for the prognosis of tongue SCC.
We then examined cultured cell lines, and found that
SCC-9 cells clearly express EPB41L5 whereas SCC-25
cells do not (Fig. 2a). Both of these cell lines are negative
for HPV16/18. SCC-9 cells originated from a tongue
carcinoma of a patient with cancer stage T2N1 (‘M’ was not
described), and similarly, SCC-25 cells also originated
from a tongue carcinoma of a patient with cancer stage
T1N1M0 . SCC-9 cells, but not SCC-25 cells,
exhibited significant Matrigel invasion activity in vitro
(Fig. 2b). siRNA-mediated silencing of EPB41L5
significantly, but only partially, inhibited the Matrigel invasion
of SCC-9 cells (Fig. 2c). Silencing of EPB41L5 did not
notably affect cell morphology (data not shown). These
results suggested that the EPB41L5 pathway and the
ARF6-based pathway might not be the only pathways
driving the invasiveness of SCC-9 cells.
We have previously shown that silencing of EPB41L5
in other types of cancers, such as breast cancer and renal
cancer, drastically improves their drug-resistance [16,
18]. We hence examined whether the same is the case
with SCC-9 cells. Contrary to our expectation, however,
silencing of EPB41L5 did not at all improve sensitivity of
SCC-9 cells towards cisplatin (50 nM for 72 h) (Fig. 2d).
This silencing of SCC-9 cells neither improved
sensitivity for ionizing radiation (4 Gy: 2 Gy x 2 times) (Fig. 2d).
Anti-cancer drugs are often used in combination with
radiation to be effective treatments for HNSCCs [2, 3].
Death rates only by about 20% could be observed with
SCC-9 cells upon treatment by cisplatin and radiation in
combination, even if their EPB41L5 was silenced
(Fig. 2d). Thus, similar to the above results, these results
indicate that although the EPB41L5-based mesenchymal
property might in some way participate in the
therapeutic resistance of tongue SCC cells, its contribution is
very limited, unlike in the case of breast cancer and
Among the different transcriptional factors driving
cancer EMT (i.e., EMT-TFs), ZEB1 has been found to be
crucial for the induction of cancer stem cell-like
properties and also for the therapeutic resistance of different
cancers . Consistently, we have shown that ZEB1 is
central in inducing EPB41L5 in breast cancer cells and
renal cancer cells [16, 18]. However, analysis of The
Cancer Genome Atlas (TCGA) RNA-Seq datasets on
human primary tongue SCCs showed that ZEB1 mRNA
levels do not statistically correlate with EPB41L5 mRNA
levels (Fig. 3a), whereas a tight correlation between
ZEB1 mRNA levels and EPB41L5 mRNA levels was
observed in our TCGA RNA-Seq analysis on human
primary breast cancers . In addition to the ZEB1
binding sites, the promoter region of the EPB41L5 gene
contains putative binding sites for the other EMT-TFs,
including SNAIL and TWIST (http://jaspar.genereg.net/;
see also Fig. 3b). However, the levels of these
wellknown EMT-TFs also did not exhibit any statistical
correlation with EPB41L5 levels (Fig. 3c). On the other
hand, we found that KLF8 and FOXO3 show positive
correlations with EPB41L5 with statistical significance
(Fig. 3c). However, we were unable to demonstrate that
the siRNA-mediated silencing of KLF8 or FOXO3, as
well as of ZEB1, notably reduces EPB41L5 expression in
SCC-9 cells (data not shown).
In this study, we showed that tongue SCC frequently
express EPB41L5, and that its expression levels
statistically correlate with the node-positivity and poor
outcome of patients. We previously showed that EPB41L5
Cisplatin: 0 nM
Radiation: 0 Gy
Cisplatin: 50 nM
Radiation: 0 Gy
Cisplatin: 0 nM
Radiation: 2 Gy x 2 times Cisplatin: 50 nM
Radiation: 2 Gy x 2 times
Fig. 2 EPB41L5 promotes cell invasiveness of a tongue SCC cell line. a Expression of EPB41L5 in the tongue SCC cell lines SCC-9 and SCC-25 was
examined by western blotting. b The invasive ability of each tongue SCC cell line was assessed by the Matrigel invasion assay. After 12 h of
incubation, invaded cells were fixed and stained with crystal violet. The numbers of cells in six distinct regions of a single chamber were counted.
Data are shown as means ± SEM. ***P < 0.001. c SCC-9 cells were transfected with two different sequences of siRNAs against EPB41L5, and then
the Matrigel invasion assay was performed. Data are means ± SEM. *P < 0.05. d Cell viabilities of SCC-9 cells with or without the silencing of
EPB41L5 was assessed following the exposure to cisplatin and/or radiation. Cisplatin concentrations and radiation doses are as indicated. Data are
shown as means ± SEM. ***P < 0.001
Fig. 3 (See legend on next page.)
(See figure on previous page.)
Fig. 3 In silico search for EMT-TFs that upregulate EPB41L5. a Scatter plot of ZEB1-EPB41L5 expression levels. Data were obtained from TCGA
RNA-Seq datasets. b Putative binding sites of the indicated transcription factors at the EPB41L5 locus. JASPAR databases were used. c Scatter plots
of mRNA expression levels of the indicated EMT-TFs and EPB41L5. All expression data were converted to log2 values before analysis. To calculate
P-values, the Spearman rank correlation test was performed
is the key molecule that drives mesenchymal
malignancies in significant populations of breast cancer patients
and renal cancer patients [16, 18]. However, unlike these
types of cancers, we were unable to obtain evidence
supporting the idea that EPB41L5 is the key molecule that
drives the invasion and chemoresistance of tongue SCC.
Consistently, database analysis suggested that the
EMT-TFs that induce EPB41L5 in tongue SCC appear
to be different from those of breast cancer and renal
cancer. Given the fact that the high expression of
EPB41L5 in primary tongue SCC s tightly correlates
with poor outcome of the patient, it is likely that the
cancer mesenchymal programs, that lead to the
EPB41L5 expression as a result of inducing EMT, is
more crucial than the EPB41L5 expression itself in
promoting malignancies of tongue SCC. Tongue SCC
has a high incidence among the different HNSCCs,
and similar protocols are generally used for the
treatment of the various HNSCCs. Nevertheless, similar
studies on other types of HNSCC await to be
performed in order to understanding further the
mesenchymal programs driving HNSCC malignancies.
The locally advanced invasiveness of HNSCC appears
to be the major cause of treatment resistance. Moreover,
although chemotherapies and radiotherapies result in
notable cancer-reducing effects in most primary lesions
of HNSCC, a significant number of HNSCC relapse
within a few years, as mentioned earlier. Such treatment
resistance may not be merely due to the robustness of
HNSCC cells acquired by their mesenchymal and motile
phenotypes, but maybe simply due to the fact that the
head and neck are vital for the feeding, breathing, and
articulation of individuals, and hence areas that can be
physically resected or irradiated are tightly restricted. On
the other hand, regions within the head and neck,
particularly the tongue, are consistently exposed to physical
tension as well as many different chemicals from the
intake of food and air. Thus, it is possible that epithelial
cells of these organs, as well as HNSCC cells in general,
may have an innate mechanism to be highly tolerant to
such stresses. We found that SCC-9 cells did not die
under intensive radiation or a platinum-based drug
treatment, which is an unusual property and is unlike
the other malignant cancer cells we have examined so
far. Thus, the molecular mechanisms as to how HNSCC,
such as SCC-9 cells, can tolerate such harsh conditions
of genotoxic stress, even in the absence of EPB41L5,
deserve further experimental scrutiny, towards achieving a
more precise understanding of the molecular profiles of
the treatment-resistance of HNSCC.
SCC-9 and SCC-25 cells were purchased from the
American Type Culture Collection and cultured under
5% CO2 at 37 °C in Dulbecco’s modified Eagle’s
medium/F12 medium (1:1), supplemented with 10%
fetal calf serum and 400 ng/mL of hydrocortisone.
Antibiotics were not used in our cell cultures.
siRNA-mediated gene silencing was performed as
described previously [6, 10]. In brief, 2 × 105 cells were
transfected with 48 nM siRNAs using RNAi MAX kit
(Invitrogen), and incubated for 48 h before being
subjected to assays. siRNAs targeting EPB41L5 were
chemically synthesized and purified by Japan BioService. An
irrelevant siRNA was purchased from GE Healthcare.
The specificity and efficacy of the siRNAs were
confirmed previously [17, 18].
Antibodies and immunoblotting
The mouse monoclonal antibody against β-actin was
purchased from a commercial source (EMD Millipore).
Rabbit polyclonal antibodies against EPB41L5 were
established as described previously .
Peroxidaseconjugated donkey antibodies against mouse or rabbit
IgGs were purchased from Jackson ImmunoResearch
Laboratories, Inc. Immunoblotting analysis was performed
as described previously  using ECL Western detection
reagents (GE Healthcare).
Invasion assay was performed using 24-well BD BioCoat
Matrigel Invasion Chambers with a pore size of 8 μm
(BD Bioscience), as described previously . Briefly, 105
cells were seeded in the upper chamber in 500 μL of
medium without serum. The lower chamber was filled
with 750 μL of complete medium. After 12 h of
incubation, cells were fixed with 4% paraformaldehyde for
30 min, and then stained with crystal violet. The
numbers of invaded cells were counted in six different areas
for every chamber. More than three independent
experiments were performed. The Welch t-test was performed
for group comparisons. Cell viabilities were analyzed
using CellTiter 96 (Promega) in accordance with the
Immunohistochemistry and scoring
Immunohistochemical staining was performed as
described previously [8, 10, 15]. Briefly, specimens were
fixed with formalin and embedded in paraffin, and then
sliced sequentially at a thickness of 3 μm. Samples were
deparaffinized with xylene and rehydrated with graded
alcohol. After rinsing with Tris-buffered saline, samples
were processed for antigen retrieval with Dako EnVision
FLEX Target Retrieval Solution (pH 9.0), using Dako PT
Link at 97 °C for 20 min according to the manufacturer’s
instructions. Samples were incubated with antibodies
against EPB41L5 (1:1,000) for 30 min, and the Dako
Envision FLEX system was used for visualization. The
coloring reaction was performed with 3,3′-Diaminobenzidine
(Dojin) for 5 min. Hematoxylin was used as a
counterstain. The scoring of immunohistochemical samples was
performed as described previously  by two different
investigators, including one pathologist (K.C.H). The
Hscore value 33% from the top was chosen as the cutoff
point for the high-expression group.
Chemoradiation resistance assay
Cells (1.5 × 104 each) were seeded onto collagen-coated
48-well dishes and then treated with siRNAs for
EPB41L5. After a 24 h incubation, cells were treated
with 50 nM of cisplatin (Sigma), followed by an X-ray
irradiation (MBR-1520R-3 HITACHI, 150 kV with a
0.5 mm aluminum filter. 2Gy x 2 times: 48 and 72 h post
cell seeding). After a further 24 h incubation, cell
viabilities were assessed by using a calcein solution (Dojin).
Each assay was performed at least three times, each in
duplicate. The Welch t-test was performed for group
Patients and specimens
All clinical specimens used in this study were the same
as those described in a previous study .
Characteristics of the patients were described in a previous report
 and in Table 1.
Disease-free and overall survival rates were analyzed
using the Kaplan-Meier method, and P-values were
calculated by the logrank test. The starting points of the
survival rates were the date of surgical resection. The
endpoint of disease-free survival was the date of the first
diagnosis of disease progression, relapse, or death due to
any cause. The endpoint of overall survival was the date
of death or the most recent follow-up.
RNA-Seq data sets on tongue SCC were obtained from
The Cancer Genome Atlas (URL:
http://cancergenome.nih.gov/, n = 129). The Spearman rank correlation test
was used to calculate P-values.
All statistical analyses were performed by R software
EMT: Epithelial-mesenchymal transition; EMT-TF: EMT-transcription factor;
HNSCC: Head and neck squamous cell carcinoma; TCGA: The Cancer
Genome Atlas; Tongue SCC: Squamous cell carcinoma of the tongue
This study was supported in part by Grants-in-aid from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (MEXT) to H. Sabe.
YO, HS, TO, Y. Onodera, JMN, AH, YM, SF, and H. Sabe contributed to the
design of the study. YO, HS, KF, KCH, YH, and H. Sabe performed the
experiments and analysed the data. YO, HS, TO, and H. Sabe compiled the
data and prepared a first manuscript. YO, HS, TO, Y. Onodera, JMN, AH, and
H. Sabe contributed to the writing of the manuscript. All authors read and
approved the final version of this manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All study participants provided informed consent, and the study design was
approved by the Institutional Review Board of Hokkaido University Hospital,
Japan (study number 012–0166).
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