TDO2 Overexpression Is Associated with Cancer Stem Cells and Poor Prognosis in Esophageal Squamous Cell Carcinoma

概要

TDO2 Overexpression Is Associated
With Cancer Stem Cells And Poor
Prognosis In Esophageal Squamous
Cell Carcinoma
ৱಕἹฑ৏ൿ‫ؠ‬Ͷ͕͜Ζ TDO2 ͹գ৔൅‫ݳ‬ͺ‫ࡋײؠ‬๖͕Γ;෈ྒྷ͵༩‫ޛ‬
ͳ‫ؖ‬࿊ͤΖ

PHAM QUOC THANG

Abstract
Objective: Esophageal cancer is one of the deadliest cancers in the world, and the main
subtype is esophageal squamous cell carcinoma (ESCC), which comprises 90% of cases.
Expression of tryptophan 2,3-dioxygenase (TDO2), an enzyme involved in tryptophan
catabolism, has been linked with tumor survival and poor prognosis of brain and breast
cancer. However, no studies have invested the potential role of TDO2 in esophageal cancer.
Here we explored the expression and biological significance of TDO2 in ESCC.
Methods: TDO2 protein expression was evaluated in 90 ESCC tissue samples by
immunohistochemistry. TDO2 function in ESCC cell lines and spheroid colony formation
was evaluated by RNA interference (RNAi).
Results: TDO2 overexpression was associated with tumor stage, recurrence status and the
CD44 cancer stem cell marker in ESCC. TDO2 overexpression was correlated with poor
outcome of ESCC patients. Inhibition of TDO2 expression by RNAi in TE-10 and TE-11 cell
lines reduced both the number and the size of spheroid colonies as well as cell proliferation.
Knockdown of TDO2 expression also induced inactivation of the EGFR signaling pathway.
Conclusion: Our results imply that TDO2 could play an important role in the progression of
ESCC. Furthermore, TDO2 may be a potential therapeutic target in ESCC.

Introduction
Esophageal cancer is the eighth most common cancer and the sixth leading cause of
cancer death worldwide [1]. Esophageal cancer is classified into two main subtypes:
esophageal squamous cell carcinoma (ESCC), which accounts for approximately 90% of
esophageal cancer cases worldwide, and esophageal adenocarcinoma (EAC), which shows
increasing rates of incidence and mortality in several regions in North America and Europe [2].
Studies have illustrated the distinction in the molecular characteristics of EAC and ESCC [3].
Patients with esophageal cancer have poor prognosis, and the 5-year overall survival rate of
ESCC patients ranges from 10% to 30% [4,5]. Targeted therapies for esophageal cancer
treatment currently remain limited [6]. Although several clinical trials for targeted treatments
of esophageal cancer have launched, only one study has enrolled patients with ESCC [7].
Therefore, it is urgent to identify new biomarkers and develop a novel therapeutic target for
esophageal cancer.
Cancer stem cells (CSCs) have been reported in many solid tumors [8]. Spheroid
colony formation assays, an in vivo technique of plating a limited number cells in culture
dishes specifically coated for non-attachment in serum-free media, have been used to
investigate CSC characteristics [9]. We previously analyzed the gene expression profile of
spheroid colonies and parental cells derived from gastric cancer (GC) cell lines by microarray
analysis [10]. We identified several genes upregulated in spheroid colonies, such as KIF11,
KIFC1 and IQGAP3, that are required for spheroid colony formation in GC cell lines and are
associated with poor survival of GC patients [10-12]. These genes are also required for
spheroid colony formation in esophageal and colorectal cancer cells [13,14]. Among the
genes upregulated in spheroid colonies, tryptophan 2,3-dioxygenase (TDO2) is dramatically
upregulated in MKN-1 cells (derived from adenosquamous cell carcinoma) compared with
other cell lines.

TDO2, indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 are three enzymes that
catalyze the amino acid tryptophan into kynurenine in the kynurenine pathway, which
accounts for 95% of tryptophan catabolism [15,16]. TDO2 is mainly expressed in the liver
and exists in the brain, while IDO1 and IDO2 have wider tissue expression, including in
peripheral blood and immune cells [16]. TDO2 plays an important role in neurological
diseases such as Alzheimer’s disease, Parkinson’s disease and autism [15,16]. TDO2 is
expressed in several established human cancer cell lines, including glioblastoma, colorectal
carcinoma, head-and-neck carcinoma, and gallbladder carcinoma cells [17]. Overexpression
of TDO2 promoted tumor cell survival and was correlated with tumor grade and poor
prognosis in triple negative breast cancer [18] and in brain tumors [19]. Furthermore,
overexpression of TDO2 has been reported in human colorectal cancer and leiomyosarcoma
[20, 21]. However, the expression and biological significance of TDO2 in esophageal cancer
have not been investigated.
In this study, we analyzed the expression and distribution of TDO2 in ESCC by
immunohistochemistry and examined the relationship between TDO2 expression and
clinicopathologic characteristics of ESCC. We also evaluated the effect of inhibiting TDO2
expression by RNA interference (RNAi) on spheroid colony formation and cell proliferation.

Materials and Methods
Tissue samples
In a retrospective study design, 100 primary tumors were collected from patients
diagnosed with ESCC who underwent surgery between 2000 and 2014 at Hiroshima
University Hospital (Hiroshima, Japan). All patients underwent curative resection. All
patients underwent right transthoracic esophagectomy with extensive lymph node dissection.
Reconstruction was performed with a gastric tube positioned in the posterior mediastinum.
Adjuvant chemotherapy was performed on all patients [22]. Only patients without
preoperative radiotherapy or chemotherapy were enrolled in the study. Operative mortality
was defined as death within 30 days of patients leaving the hospital. Postoperative follow-up
was scheduled every 1, 2 or 3 months during the first 2 years after surgery and every 6
months thereafter unless more frequent follow-up was deemed necessary. Chest X-ray, chestabdominal computed tomographic scan and serum chemistry analysis were performed at
every follow-up visit. Patients were followed by their physician until death or the date of the
last documented contact. Tumor staging was determined according to the TNM classification
system [23]. This study was approved (No. IRINHI66) by the Ethical Committee for Human
Genome Research of Hiroshima University (Hiroshima, Japan).
For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we
used 10 ESCC samples. Tumor tissues and the corresponding non-neoplastic tissue were
surgically removed, frozen immediately in liquid nitrogen, and stored at −80°C until use. A
total of 12 types of normal tissue samples were purchased from Clontech Laboratories, Inc.
(Mountainview, CA, USA), including: heart (catalog no. 636532), lung (catalog no. 636524),
stomach (catalog no. 636578), small intestine (catalog no. 636539), colon (catalog no.
636553), liver (catalog no. 636531), pancreas (catalog no. 636577), kidney (catalog no.
636529), bone marrow (catalog no. 636591), leukocytes (catalog no. 636592), skeletal

muscle (catalog no. 636547) and brain (catalog no. 636530).
For immunohistochemical analysis, we used archival formalin-fixed, paraffinembedded tissues from the 90 patients who had undergone surgical excision for ESCC. One
or two representative tumor blocks, including the tumor center, invading front, and tumorassociated non-neoplastic squamous epithelial, were examined in each patient using
immunohistochemistry. In cases of large, late-stage tumors, two different sections were
examined to include representative areas of the tumor center as well as in the lateral and deep
tumor invasive fronts.

Analysis of TCGA datasets
To explore the expression of TDO2 in cancer and normal samples, we used an online
analytical tool, the Broad Institute TCGA Genome Data Analysis Center,
http://firebrowse.org/ [24]. TDO2 was set up as the target gene and the cohort dataset
included liver hepatocellular carcinoma; pancreatic adenocarcinoma; head and neck
squamous cell carcinoma; bladder urothelial carcinoma; esophageal carcinoma; colon
adenocarcinoma; stomach adenocarcinoma; rectum adenocarcinoma; breast invasive
carcinoma; kidney renal clear cell carcinoma; and prostate adenocarcinoma.

qRT-PCR analysis
Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA),
and 1 μg of total RNA was converted to cDNA using the First Strand cDNA Synthesis Kit
(Amersham Biosciences, Piscataway, NJ, USA). Quantitation of TDO2 mRNA level was
performed by real-time fluorescence detection as previously described [25]. PCR was
conducted using the SYBR Green PCR Core Reagents Kit (Applied Biosystems; Thermo
Fisher Scientific, Inc., Waltham, MA, USA). Real-time detection of the emission intensity of

SYBR green bound to double-stranded DNA was performed with the ABI PRISM 7700
Sequence Detection System (Applied Biosystems). Actin-beta (ACTB)-specific PCR
products were amplified from the same RNA samples and served as an internal control.
Primer sequences for TDO2 were forward, 5′-CGG TGG TTC CTC AGG CTA TC-3′ and
reverse, 5′-CTT CGG TAT CCA GTG TCG GG-3′. Primer sequences for CD44 were
forward, 5′-TAC AGC ATC TCT CGG ACG GA-3′ and reverse, 5′-CAC CCC TGT GTT
GTT TGC TG-3′. Primer sequences for ACTB were forward, 5′-CTG TCT GGC GGC ACC
ACC AT-3′ and reverse, 5′-GCA ACT AAG TCA TAG TCC GC-3′.

Immunohistochemistry
Immunohistochemical analysis was performed with the EnVision+ Mouse
Peroxidase Detection System (Dako Cytomation, Carpinteria, CA, USA) as previously
described [23]. A mouse polyclonal anti-TDO2 antibody was used as primary antibody
(1:250; catalogue no. H00006999-B01P, Abnova, Jhouzih St., Taipei, Taiwan). The
percentage and the intensity of staining of tumor cells was scored from 0–100% and from 0
(no immunoreactivity) to 3+ (intense staining), respectively. The expression score was
calculated by the formula: Ax1+Bx2+Cx3, in which A represents the percentage of weakly
stained (score of 1) cells, B represents the percentage of moderately stained (score of 2) cells,
and C represents the percentage of strongly stained (score of 3) cells. The expression score
ranged from 0 to 300. Two surgical pathologists, without knowledge of the clinical and
pathologic parameters or the patients’ outcomes, independently reviewed immunoreactivity
in each specimen. Interobserver differences were resolved by consensus review at a doubleheaded microscope after independent review. Immunostaining of CD44 was performed as
previously described [26].

Cell lines
Four ESCC cell lines (TE-1, TE-5, TE-10 and TE-11) were purchased from the
Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). All cell lines were
maintained in RPMI-1640 (Nissui Pharmaceutical Co, Ltd, Tokyo, Japan) containing 10%
fetal bovine serum (BioWhittaker, Walkersville, MD, USA) in a humidified atmosphere of
5% CO2 and 95% air at 37°C.

Western blot analysis
Cells were lysed as previously described [27]. The lysates (40 μg) were solubilized
in Laemmli sample buffer by boiling and then subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis followed by electrotransfer onto a nitrocellulose filter.
Western blot procedures were performed as previously described [27]. Anti-TDO2 antibody
was purchased from Abnova. Anti-epidermal growth factor receptor (EGFR), anti-phosphoEGFR (pEGFR), anti-Erk, anti-phospho-Erk1/2 (pErk1/2), anti-Akt, and anti-phospho-Akt
(pAkt) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG was used in the secondary reaction.
Immunocomplexes were visualized with an ECL Western Blot Detection System (Amersham
Biosciences). β-Actin (Sigma) was used as a loading control.

RNAi
Short interfering RNA (siRNA) oligonucleotides targeting TDO2 and a negative
control were purchased from Invitrogen (Carlsbad, CA, USA). We used two independent
TDO2 siRNA oligonucleotide sequences (catalog no. 10620318 and 10620319). Transfection
was performed using Lipofectamine RNAiMAX (Invitrogen) as previously described [25].
Briefly, 60 pmol of siRNA and 10 μl of Lipofectamine RNAiMAX were mixed in 1 ml of

RMPI medium (10 nmol/l final siRNA concentration). After 20 min of incubation, the
mixture was added to cells and then cells were plated in culture dishes. Forty-eight hours
after transfection, cells were analyzed.

Cell growth assays
We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assays as previously described [28]. The cells were seeded at a density of 2,000 cells per well
in 96-well plates. Cell growth was examined after 1, 2, and 4 days. Three independent
experiments were performed. The mean and standard deviation (SD) were calculated for each
experiment.

Spheroid colony formation assay
For the generation of spheres, 2,000 cells were plated in each well of 24-well ultralow attachment plates (Corning) containing mTeSR medium (STEMCELL Technologies
Inc., Cambridge, MA, USA). The plates were incubated at 37°C in a 5% CO2 incubator for
15 days. Sphere number and size were determined and counted under a microscope.

Statistical methods
Associations between clinicopathological parameters and TDO2 expression were
analyzed by Chi-Squared test. Kaplan–Meier survival curves were constructed for TDO2
high expression and TDO2 low expression patients. Survival rates were compared between
TDO2 high expression and TDO2 low expression groups. Differences between survival
curves were tested for statistical significance by a log-rank test. Differences between the two
groups (TDO2 siRNA-transfected cells and negative control siRNA-transfected cells) were
tested by Student t-test.

Results
Expression of TDO2 in ESCC
We first examined the expression of TDO2 in various types of cancers using the
TCGA dataset. TDO2 expression was upregulated in most cancers except for liver cancer and
pancreas cancer (Supplementary Fig. 1). The fold change expression of TDO2 between
normal and cancer tissue was the highest in esophageal cancer (Supplementary Fig. 1).
We next evaluated the expression of TDO2 in 12 types of normal tissue samples and 10
ESCC tissue samples that contained both tumor tissue (T) and corresponding non-neoplastic
tissue (N) using qRT-PCR. Among the 12 normal tissue samples, TDO2 mRNA levels were
expressed the highest in liver (Fig. 1a). We also observed high TDO2 mRNA levels in ESCC
tissue samples compared with normal tissues. We also calculated the T/N ratios for the 10
ESCC cases and defined upregulation as a change of more than two-fold. We found that
expression of TDO2 was upregulated in 8 out of the 10 ESCC cases (80%) (Fig. 1b).
Next, we performed immunohistochemistry on 90 ESCC tissue samples, using
normal liver tissue as a positive control (Fig. 1c). Normal squamous epithelial cells showed
weak staining for TDO2 (Fig. 1d). In contrast, TDO2 expression was increased in ESCC
tissues, and TDO2 staining was observed in the cytoplasm of tumor cells (Fig. 1e). Most
ESCC cases presented heterogeneity of TDO2 staining, both in the intensity and the
percentage of TDO2-stained tumor cells. The percentage and the intensity of staining of
tumor cells was calculated as described in the Methods section and representative images are
shown in Supplementary Fig. 2a. There was no difference in the TDO2 expression score
between invasive fronts and the central/superficial areas.
We also used receiver operating characteristic (ROC) curve analysis to define the
cut-off point for the TDO2 expression score correlating with clinicopathological

characteristics (Supplementary Fig. 2b–e). Youden’s criterion, which maximizes the sum of
sensitivity and specificity, was applied for the optimization cut-off point [29]. By using the
Youden’s index, the cut-off score for T classification, N classification, stage and recurrence
status was 55. TDO2 immunostaining was considered high expression if the expression score
was over 55 and TDO2 immunostaining was considered as low expression if the expression
score was below or equal to 55. We then examined the relationship of TDO2 expression,
classified using the cut-off score, with clinicopathological characteristics of the 90 ESCC
patients (Table 1). TDO2 high expression was associated with advanced T classification,
tumor stage and recurrence status. These results suggested that TDO2 may play an important
role in the progression of ESCC.
Kaplan-Meier analysis further demonstrated that TDO2 high expression ESCC cases
showed poorer survival than TDO2 low expression ESCC cases (p=0.015; Fig. 1f). We next
performed univariate and multivariate Cox proportional hazards analyses to evaluate the
potential role of TDO2 expression as an independent predictor in ESCC (Table 2). In
univariate analysis, T classification, N classification, stage, histological classification, and
TDO2 expression were associated with poor survival. However, in the multivariate model,
only N classification was found to be an independent predictor of survival in ESCC patients
(Table 2).

Relation between TDO2 expression and the CSC marker CD44
A previous report showed that TDO2 facilitates anoikis resistance in triple-negative
breast cancer [18] and another study indicated that resistance to anoikis was related with
CSC-like cells [30]. Thus, we hypothesized that TDO2 expression may be related with CSC
marker expression in ESCC. Numerous studies have identified CSC markers in esophageal
cancer, such as CD44, ALDH1, and CD133, and the pathways involved in CSCs, such as

Hippo, Wnt/β-catenin, and Notch [31]. Among these CSC markers, CD44 is a well-known
CSC marker in esophageal cancer [31]. We thus performed immunohistochemical analysis of
CD44 in 90 ESCC cases. In normal squamous epithelium, CD44 displayed uniform
membranous staining in basal cell layers. Interestingly, we found that TDO2-expressing
cancer cells also expressed CD44 (Fig. 2). Furthermore, ESCCs with high TDO2 expression
showed significantly enriched numbers of CD44-positive cells (Table 3).

Effect of TDO2 inhibition on spheroid formation
The immunohistochemical results indicated that CD44-positive ESCC cases showed
increased expression of TDO2. However, the significance of TDO2 expression in ESCC
CSCs was unknown. Therefore, we next investigated the effect of inhibiting TDO2
expression on spheroid colony formation. We first established spheroid body-forming cells
from ESCC cell lines. qRT-PCR revealed that spheroid body-forming cells showed enriched
CD44 expression in TE-5, TE-10 and TE-11 cell lines (Supplementary Fig. 3a). We then
measured the expression of TDO2 mRNA in spheroid body-forming cells from ESCC cell
lines and the parental cells. TDO2 mRNA levels were dramatically upregulated in spheroid
body-forming cells compared with the parental cells in all four ESCC cell lines (Fig. 3a).
We next examined the effect of TDO2 inhibition by siRNA transfection on sphere
number and size. We selected TE-10 and TE-11 cells, which exhibited high levels of TDO2
mRNA expression in spheroid body-forming and parental cells, for analysis. Two different
siRNAs targeting TDO2 were transfected into TE-10 and TE-11 cells, and TDO2 mRNA
expression was successfully suppressed by siRNA1 and siRNA2 transfection (Fig. 3b,
Supplementary Fig. 3b). We next evaluated the impact of TDO2 knockdown by evaluating
the number and size of spheres at day 15 after transfection. TE-11 and TE-10 cells transfected
with TDO2 siRNA showed reduced number and size of spheres compared with negative

control transfected cells (Fig. 3c, Supplementary Fig. 3c). These results suggest that TDO2
is required for spheroid colony formation in ESCC.

TDO2 inhibition reduced cell growth and mediated EGFR pathway signaling
We next examined the effect of TDO2 inhibition on cell growth using MTT assays.
As shown in Fig. 4a and Supplementary Fig. 4a, TDO2 siRNA1- and siRNA2-transfected
TE-11 and TE-10 cells showed significantly reduced cell growth compared with negative
control siRNA-transfected TE-11 and TE-10 cells (p < 0.05). We also examined the effect of
TDO2 inhibition on drug resistance. Since 5-fluorouracil is an important treat- ment option for
patients with ESCC, resistance to 5-fluorouracil was examined in the present study. MTT
assays were performed to measure the cell viability of TDO2 siRNA- and negative control
siRNA-transfected cells under various concentrations of 5-FU for 48 h. However, the 50%
inhibitory concentration value of TDO2 siRNA1- and siRNA2-transfected TE-11 and TE-10
cells was not significantly different (Supplementary Fig. 5a-b).

EGFR activates the RAS-MEK-ERK and AKT-PI3K pathways, leading to cancer
cell proliferation and survival [32]. Additionally, kynurenine is the product of downstream
metabolites of tryptophan by TDO2 and acts as both a regulatory molecule and endogenous
ligand for the aryl hydrocarbon receptor (AhR) [19]. A previous report demonstrated that the
crosstalk between AhR and EGFR is involved in regulating colon cancer cell proliferation
[33]. Therefore, we next analyzed the effect of TDO2 inhibition on the EGFR signaling
pathway. Western blot analysis confirmed successful TDO2 knockdown in TE-11 and TE-10
cells transfected with TDO2 siRNA (Fig. 4b, Supplementary Fig. 4b). We also found that
the levels of phosphorylated EGFR, Erk and Akt were lower in TDO2 siRNA1- and siRNA2transfected TE-11 and TE-10 cells compared with control cells (Fig. 4b, Supplementary
Fig. 4b).

Discussion
CSCs play an essential role in tumor progression, metastasis, and cancer recurrence,
as well as resistance to chemotherapy and radiation therapy [8]. We previously identified the
expression of CD44 and two other CSC markers, ALDH1 and CD133, in GC and found that
CD44 is an independent prognosis marker for GC patients [26]. In ESCC, CD44 expression
is a predictive marker in patients treated with neoadjuvant chemotherapy after radical
esophagectomy [34]. In the current study, we found that the overexpression of TDO2 was

associated with advanced disease and poor outcome of ESCC patients. Additionally, the
expression of TDO2 was correlated with CD44 expression in ESCC tumor tissues. Inhibition
of TDO2 expression decreased the number and size of spheroid colonies in ESCC cell lines.
Together our results reveal that TDO2 expression may play an indispensable role in ESCC
stem cells.
Immune-targeted therapy is now becoming a new strategy for cancer treatment, with
various drugs approved by the Food and Drug Administration [35]. A previous report showed
that tryptophan metabolism by IDO1 mediated tumor immune tolerance, and an inhibitor
targeting IDO1 is currently under investigation in a clinical trial [36]. Another study using an
immunized mouse model showed that tumors acquired immune tolerance by inducing TDO2
expression [17]. An inhibitor of TDO2 was previously developed and has been used in in
vitro studies [37]. Here we show that TDO2 may play a crucial role in cancer cell survival, as
inhibition of TDO2 by siRNA suppressed ESCC cell line proliferation. Research in brain
tumors corroborates our data, and TDO2 expression not only suppresses anti-tumor immune
responses and but also promotes tumor cell survival [19]. Furthermore, IDO1 was reported to
play a role in the progression of ESCC and serve as a predictor for poor prognosis in ESCC
[38]. Together these data indicate that the relationship between TDO2 expression and IDO1
expression and the tumor immune response in ESCC should be analyzed in future studies.

TDO2 catalyzes tryptophan into kynurenine in the kynurenine pathway [15, 16]. It is
well known that tryp- tophan derivatives such as 6-formylindolo[3, 2-b]car- bazole,
kynurenine, and 2-(1′H-indole-3′-carbonyl)- thiazole-4-carboxylic acid methyl ester (ITE) are
natural ligands of the AhR [39]. The synthetic agonist of the AhR can downregulate the
master pluripotency factor Oct4 in stem-like breast cancer cell lines and inhibit their
proliferation and metastasis [39], indicating that synthetic ITE can induce the differentiation
of stem-like cancer cells and reduces their tumorigenic potential. In fact, it has been reported
that reduction of endogenous ITE levels in cancer cells by tryptophan deprivation leads to
elevation of Oct4, which is the master pluripotency factor [39]. Therefore, overexpression of
TDO2 could reduce the tryptophan level in cancer cells and induce Oct4 expression.
EGFR overexpression is frequently observed in 50% of ESCCs [40]. A clinical trial
phase II in advanced ESCCs showed that the combination of cetuximab, an EGFR-blocking
monoclonal antibody, with cisplatin/5-fluorouracil increased the efficacy of standard
chemotherapy [41]. Our results showed that the EGFR signaling pathway was dramatically
inactivated by the inhibition of TDO2 expression in ESCC. These findings indicate that
TDO2 could participate in the activation of EGFR and imply that EGFR signaling may be
important for ESCC cell growth in TDO2 high expression ESCCs.
Interestingly, a previous study reported that inhibition of EGFR could prevent the
induction of cancer stem-like cells in ESCC through suppressing epithelial-mesenchymal
transition (EMT) [42]. Recent research has revealed the correlation between cancer stemness
and EMT [42,439]. In this study we showed that transient knockdown of TDO2 by RNAi
reduced activation of the EGFR signaling pathway and reduced spheroid formation in ESCC
cell lines, although the EMT profiles were not examined. Therefore, further research is
required for the specificity of TDO2.
In summary, in this study, we found that TDO2 overexpression was related with a

poor prognosis and associated with cancer cell proliferation and CSCs in ESCC. Suppression
of TDO2 inhibited activation of the EGFR signaling pathway and spheroid formation. Our
data indicates that TDO2 inhibition may be an essential target for clinical trial research in
ESCC.

Statement of Ethics
This study was approved (No. IRINHI66) by the Ethical Committee for Human
Genome Research of Hiroshima University (Hiroshima, Japan).

Disclosure Statement
The authors report no potential conflicts of interest.

Funding Sources
The present study was supported by Grants-in-Aid for Scientific Research (B15H04713) and Challenging Exploratory Research (grant nos. 26670175 and 16K15247)
(both from the Japan Society for the Promotion of Science) and by the Takeda Science
Foundation.

References
1.

Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM,

Forman D, Bray F: Cancer incidence and mortality worldwide: sources, methods and major
patterns in GLOBOCAN 2012. Int J Cancer 2015;136(5):E359-86.
2.

Rustgi AK, El-Serag HB: Esophageal carcinoma. N Engl J Med 2014;371(26):2499-

509.
3.

Montgomery E, Field JK, Boffetta P, Daigo Y, Shimizu M, Shimoda T: Squamous

cell carcinoma of the oesophagus; In: Bosman FT, Carneiro F, Hruban RH, Theise ND (Eds):
WHO Classification of Tumors of the Digestive System, IARC, Lyon, 2010, pp 18-24.
4.

Morita M, Yoshida R, Ikeda K, Egashira A, Oki E, Sadanaga N, Kakeji Y,

Yamanaka T, Maehara Y: Advances in esophageal cancer surgery in Japan: an analysis of
1000 consecutive patients treated at a single institute. Surgery 2008;143(4):499-508.
5.

The Cancer Genome Atlas Research Network: Integrated genomic characterization

of oesophageal carcinoma. Nature 2017;541(7636):169-175.
6.

National Comprehensive Cancer Network: Clinical Practice Guidelines in Oncology.

Esophageal and esophagogastric junction cancers, version 1.2017.
https://www.nccn.org/professionals/physician_gls/pdf/esophageal.pdf (accessed July 30th,
2017).
7.

Smyth EC, Lagergren J, Fitzgerald RC, Lordick F, Shah MA, Lagergren P,

Cunningham D: Oesophageal cancer. Nat Rev Dis Primers 2017;3:17048.
8.

Batlle E, Clevers H: Cancer stem cells revisited. Nat Med 2017;23(10):1124-1134.

9.

Takaishi S, Okumura T and Wang TC: Gastric cancer stem cells. J Clin Oncol

2008;26(17):2876-82.
10.

Oue N, Mukai S, Imai T, Pham TT, Oshima T, Sentani K, Sakamoto N, Yoshida K,

Yasui W: Induction of KIFC1 expression in gastric cancer spheroids. Oncol Rep

2016;36(1):349-55.
11.

Oue N, Yamamoto Y, Oshima T, Asai R, Ishikawa A, Uraoka N, Sakamoto N,

Sentani K, Yasui W: Overexpression of the Transmembrane Protein IQGAP3 Is Associated
with Poor Survival of Patients with Gastric Cancer. Pathobiology, Epub ahead of print.
12.

Imai T, Oue N, Nishioka M, Mukai S, Oshima T, Sakamoto N, Sentani K, Matsusaki

K, Yoshida K, Yasui W: Overexpression of KIF11 in Gastric Cancer with Intestinal Mucin
Phenotype. Pathobiology 2017;84(1):16-24.
13.

Imai T, Oue N, Yamamoto Y, Asai R, Uraoka N, Sentani K, Yoshida K, Yasui W:

Overexpression of KIFC1 and its association with spheroid formation in esophageal
squamous cell carcinoma. Pathol Res Pract 2017;213(11):1388-1393.
14.

Imai T, Oue N, Sentani K, Sakamoto N, Uraoka N, Egi H, Hinoi T, Ohdan H,

Yoshida K, Yasui W: KIF11 Is Required for Spheroid Formation by Oesophageal and
Colorectal Cancer Cells. Anticancer Res 2017;37(1):47-55.
15.

Platten M, Wick W, Van den Eynde BJ: Tryptophan catabolism in cancer: beyond

IDO and tryptophan depletion. Cancer Res 2012;72(21):5435-40.
16.

Badawy AA: Kynurenine Pathway of Tryptophan Metabolism: Regulatory and

Functional Aspects. Int J Tryptophan Res DOI: 10.1177/1178646917691938.
17.

Pilotte L, Larrieu P, Stroobant V, Colau D, Dolusic E, Frédérick R, De Plaen E,

Uyttenhove C, Wouters J, Masereel B, Van den Eynde BJ: Reversal of tumoral immune
resistance by inhibition of tryptophan 2,3-dioxygenase. Proc Natl Acad Sci U S A
2012;109(7):2497-502.
18.

D'Amato NC, Rogers TJ, Gordon MA, Greene LI, Cochrane DR, Spoelstra NS,

Nemkov TG, D'Alessandro A, Hansen KC, Richer JK: A TDO2-AhR signaling axis
facilitates anoikis resistance and metastasis in triple-negative breast cancer. Cancer Res
2015;75(21):4651-64.

19.

Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, Trump S, Schumacher T,

Jestaedt L, Schrenk D, Weller M, Jugold M, Guillemin GJ, Miller CL, Lutz C, Radlwimmer
B, Lehmann I, von Deimling A, Wick W, Platten M: An endogenous tumour-promoting
ligand of the human aryl hydrocarbon receptor. Nature 2011;478(7368):197-203.
20.

Puccetti P, Fallarino F, Italiano A, Soubeyran I, MacGrogan G, Debled M, Velasco

V, Bo- det D, Eimer S, Veldhoen M, Prendergast GC, Platten M, Bessede A, Guillemin GJ:
Accumulation of an endogenous tryptophan derived metabolite in colorectal and breast
cancers. PLoS One 2015;10:e0122046.
21.

Cuppens T, Moisse M, Depreeuw J, Anniba- li D, Colas E, Gil-Moreno A, Huvila J,

Car- pén O, Zikán M, Matias-Guiu X, Moerman P, Croce S, Lambrechts D, Amant F:
Integrated genome analysis of uterine leiomyosarcoma to identify novel driver genes and
targetable pathways. Int J Cancer 2018;142: 1230–1243.
22.

Shimizu K, Hihara J, Yoshida K, Toge T: Clinical evaluation of low-dose cisplatin

and 5-fluorouracil as adjuvant chemoradiothera- py for advanced squamous cell carcinoma of
the esophagus. Hiroshima J Med Sci 2005;54: 67–71.
23.

Sobin LH, Gospodarowicz MK, Wittekind CH: TMN classification of malignant

tumors, ed 7th, Wiley-Liss, New York, 2009, pp 66-72.
24.

Broad Institute TCGA Genome Data Analysis Center,

http://firebrowse.org/viewGene.html?gene=tdo2 (accessed July 30th, 2017).
25.

Sakamoto N, Oue N, Sentani K, Anami K, Uraoka N, Naito Y, Oo HZ, Hinoi T,

Ohdan H, Yanagihara K, Aoyagi K, Sasaki H, Yasui W: Liver-intestine cadherin induction
by epidermal growth factor receptor is associated with intestinal differentiation of gastric
cancer. Cancer Sci 2012;103(9):1744-50.
26.

Wakamatsu Y, Sakamoto N, Oo HZ, Naito Y, Uraoka N, Anami K, Sentani K, Oue

N, Yasui W: Expression of cancer stem cell markers ALDH1, CD44 and CD133 in primary

tumor and lymph node metastasis of gastric cancer. Pathol Int 2012;62(2):112-9.
27.

Yasui W, Ayhan A, Kitadai Y, Nishimura K, Yokozaki H, Ito H, Tahara E:

Increased expression of p34cdc2 and its kinase activity in human gastric and colonic
carcinomas. Int J Cancer 1993:53(1):36-41.
28.

Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott

BJ, Mayo JG, Shoemaker RH, Boyd MR: Feasibility of drug screening with panels of human
tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988;48(3):589-601.
29.

Perkins NJ, Schisterman EF: The Inconsistency of “Optimal” Cut-points Using Two

ROC Based Criteria. Am J Epidemiol 2006;163(7):670-5.
30.

Kim SY, Hong SH, Basse PH, Wu C, Bartlett DL, Kwon YT, Lee YJ: Cancer Stem

Cells Protect Non-Stem Cells From Anoikis: Bystander Effects. J Cell Biochem
2016;117(10):2289-301.
31.

Wang D, Plukker JTM, Coppes RP: Cancer stem cells with increased metastatic

potential as a therapeutic target for esophageal cancer. Semin Cancer Biol 2017;44:60-66.
32.

Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P, Ferguson KM: Structural basis

for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell
2005;7(4):301-11.
33.

Xie G, Peng Z, Raufman JP: Src-mediated aryl hydrocarbon and epidermal growth

factor receptor cross talk stimulates colon cancer cell proliferation. Am J Physiol Gastrointest
Liver Physiol 2012;302(9):G1006-15.
34.

Okamoto K, Ninomiya I, Ohbatake Y, Hirose A, Tsukada T, Nakanuma S, Sakai S,

Kinoshita J, Makino I, Nakamura K, Hayashi H, Oyama K, Inokuchi M, Nakagawara H,
Miyashita T, Hidehiro T, Takamura H, Fushida S, Ohta T: Expression status of CD44 and
CD133 as a prognostic marker in esophageal squamous cell carcinoma treated with
neoadjuvant chemotherapy followed by radical esophagectomy. Oncol Rep 2016;36(6):3333-

3342.
35.

Shekarian T, Valsesia-Wittmann S, Caux C, Marabelle A: Paradigm shift in

oncology: targeting the immune system rather than cancer cells. Mutagenesis
2015;30(2):205-11.
36.

Munn DH, Mellor AL: IDO in the Tumor Microenvironment: Inflammation,

Counter-Regulation, and Tolerance. Trends Immunol 2016;37(3):193-207.
37.

Pantouris G, Mowat CG: Antitumour agents as inhibitors of tryptophan 2,3-

dioxygenase. Biochem Biophys Res Commun 2014;443(1):28-31.
38.

Jia Y, Wang H, Wang Y, Wang T, Wang M, Ma M, Duan Y, Meng X, Liu L: Low

expression of Bin1, along with high expression of IDO in tumor tissue and draining lymph
nodes, are predictors of poor prognosis for esophageal squamous cell cancer patients. Int J
Cancer 2015;137(5):1095-106.
39.

Cheng J, Li W, Kang B, Zhou Y, Song J, Dan S, Yang Y, Zhang X, Li J, Yin S, Cao

H, Yao H, Zhu C, Yi W, Zhao Q, Xu X, Zheng M, Zheng S, Li L, Shen B, Wang YJ:
Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells. Nat
Commun 2015; 6:7209
40.

Hanawa M, Suzuki S, Dobashi Y, Yamane T, Kono K, Enomoto N, Ooi A: EGFR

protein overexpression and gene amplification in squamous cell carcinomas of the esophagus.
Int J Cancer 2006;118(5):1173-80.
41.

Lorenzen S, Schuster T, Porschen R, Al-Batran SE, Hofheinz R, Thuss-Patience P,

Moehler M, Grabowski P, Arnold D, Greten T, Müller L, Röthling N, Peschel C, Langer R,
Lordick F: Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in
first-line metastatic squamous cell carcinoma of the esophagus: a randomized phase II study
of the Arbeitsgemeinschaft Internistische Onkologie. Ann Oncol 2009;20(10):1667-73.

Figure legends
Fig. 1. Expression of TDO2 in ESCC. (a) Quantitative reverse transcription-polymerase chain
reaction (qRT-PCR) analysis of TDO2 mRNA in 10 ESCC samples (T1–T10) and 13 normal
tissue samples. The bars represent individual samples. (b) qRT-PCR of TDO2 in 10 ESCC
samples. The bars represent individual samples. Fold difference is the ratio of TDO2 mRNA
level in ESCC compared with that in corresponding non-neoplastic tissue. Upregulation was
defined as a difference in expression of more than two-fold (dotted line). (c)
Immunohistochemical analysis of TDO2 in normal liver. Original magnification, x400. (d)
Immunohistochemical analysis of TDO2 in ESCC. Weak cytoplasmic staining was observed
in normal squamous epithelial cells (arrowhead), whereas strong cytoplasmic TDO2 staining
was detected in ESCC cells (arrow). Original magnification, x20. (e) Immunohistochemical
analysis of TDO2 in ESCC. Strong cytoplasmic TDO2 staining was detected in ESCC cells.
Original magnification, x400. (f) Kaplan–Meier plot of survival in ESCC patients according
to tumor TDO2 expression.

Fig. 2. Immunohistochemical analysis of TDO2 and CD44 in ESCC. (a)
Immunohistochemical analysis of TDO2. Original magnification, x100. (b)
Immunohistochemical analysis of CD44. Original magnification, x100.

Fig. 3. Effect of TDO2 inhibition on spheroid formation. (a) qRT-PCR analysis of TDO2
mRNA levels in spheroid body-forming cells and parental cells in ESCC cell lines. (b) qRTPCR analysis of TDO2 mRNA in TE-11 cells transfected with two siRNAs targeting TDO2.
(c) The number and size of spheres from TE-11 cells transfected with TDO2 siRNA or
negative control siRNA. Bars and error bars indicate mean ± SD, respectively, of three
different experiments. Scale bar, 100 μm. *p < 0.05.

Fig. 4. Effect of TDO2 inhibition on cell growth in TE-11 cells. (a) Effect of TDO2
knockdown on cell growth of TE-11 cells. Cell growth was assessed by MTT assays at 1, 2,
and 4 days after seeding TDO2 siRNA-transfected TE-11 cells on 96-well plates. Bars and
error bars indicate the mean and standard error (SE). (b) Western blot analysis of TDO2,
EGFR, phospho-EGFR (pEGFR), Erk1/2, phospho-Erk1/2 (pErk1/2), Akt, and phospho-Akt
(pAkt) in cell lysates from TDO2 cells transfected with TDO2 siRNAs or negative control
siRNA. β-Actin was included as a loading control. *p < 0.05.

Table 1. Relationship between TDO2 expression and clinicopathological characteristics in
esophageal squamous cell carcinoma.
TDO2 expression, n (%)

Age

Sex

T classification

N classification

M classification

Stage

Histological

High

Low

p valuea

≤ 65

26(62)

16

0.586

> 65

27(56)

21

Female

6(43)

8

Male

47(62)

29

T1

15(38)

24

T2/3/4

38(75)

13

N0

20(50)

20

N1/2/3

33(66)

17

M0

53(59)

37

M1

0(-)

0(-)

Stage I/II

27(47)

31

Stage III/IV

26(81)

6

Well/moderately

39(57)

30

Poorly

14(67)

7

Negative

35(51)

33

Positive

18(82)

4

0.185

0.001

0.125

-

0.001

0.408

grade

Recurrence

a

Chi-squared test

0.012

Table 2. Univariate and multivariate Cox regression analysis of TDO2 expression and
survival in esophageal squamous cell carcinoma.
Univariate analysis
Characteristic

HR (95% CI)

Multivariate analysis
p value

HR (95% CI)

p value

Age
≤ 65

1 (Ref.)

> 65

1.26 (0.48-3.32)

0.641

Sex
Female

1 (Ref.)

Male

3.45 (0.50-28.30)

0.2

T grade
T1

1 (Ref.)

T2/T3/T4

4.56 (1.31-15.93)

1 (Ref.)
0.017

0.47 (0.07-3.42)

0.458

N grade
N0

1 (Ref.)

N1/2

17.42 (2.30-131.92)

1 (Ref.)
0.006

17.68 (1.73-180.76)

0.015

Stage
Stage I/II

1 (Ref.)

Stage III/IV

6.01 (2.11-17.16)

1 (Ref.)
0.001

Histologic grade
Well/moderately

1 (Ref.)

Poorly

1.10 (0.38-3.13)

0.864

Postoperative chemotherapy
Received

1 (Ref.)

Did not received

2.13 (0.81-5.62)

0.127

1.78 (0.39-8.15)

0.459

TDO2 expression
Low

1 (Ref.)

High

4.17 (1.20-14.57)

HR: hazard ratio, CI: confidence interval.

1 (Ref.)
0.025

4.30 (0.98-18.76)

0.053

Pham QT Y et al. 26

Table 3. Relationship between TDO2 expression and CD44 expression.
TDO2 expression, n (%)

CD44

Positive

High

Low

p valuea

49(64)

28

0.026

Negative 4(31)

a

Chi-squared test

9

Pham QT Y et al. 27

Fig.1

Pham QT Y et al. 28

Fig.2

Pham QT Y et al. 29

Fig.3

Pham QT Y et al. 30

Fig.4

Pham QT Y et al. 31

Pham QT Y et al. 32

Pham QT Y et al. 33

Pham QT Y et al. 34

Pham QT Y et al. 35