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ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS
Overexpression of Activin A in Oral Squamous Cell Carcinoma:Association with Poor Prognosis and Tumor Progression
Kai-Ping Chang, MD, PhD1, Huang-Kai Kao, MD2, Ying Liang, MD3, Ming-Hui Cheng, MD, MHA2,
Yu-Liang Chang, DDS, PhD4, Shiau-Chin Liu, BS1, Yu-Chi Lin, MS5, Tzu-Yin Ko, BS1, Yun-Shien Lee, PhD5,6,
Chia-Lung Tsai, PhD5, Tzu-Hao Wang, MD, PhD5, Sheng-Po Hao, MD1, and Chi-Neu Tsai, PhD7
1Department of Otolaryngology—Head & Neck Surgery, Chang Gung Memorial Hospital & Chang Gung University
College of Medicine, Tao-Yuan, Taiwan; 2Department of Plastic & Reconstructive Surgery, Chang Gung Memorial
Hospital, Tao-Yuan, Taiwan; 3Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan;4Department of Oral & Maxillofacial Surgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan; 5Genomic Medicine
Research Core Laboratory, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan; 6Department of Biotechnology, Ming
Chuan University, Tao-Yuan, Taiwan; 7Graduate Institute of Clinical Medical Sciences, Chang Gung University,
Tao-Yuan, Taiwan
ABSTRACT
Background. Both activin A, a member of transforming
growth factor b superfamily, and its inhibitor follistatin
have been shown to be overexpressed in various cancers.
We examined the potential role of activin A and follistatin
in tissue and blood samples from patients with oral squa-
mous cell carcinoma.
Methods. For activin A and follistatin, the expression of
tissue samples from 92 patients was examined by immu-
nohistochemical study, and the serum levels of blood
samples from 111 patients and 91 healthy controls were
measured by enzyme-linked immunosorbent assay.
Results. We found that overexpression of immunohisto-
chemically detected activin A was correlated with positive
N stage, poor histological differentiation, and perineural
invasion (P = 0.029, 0.002, and 0.014, respectively). In
survival analyses, patients with oral squamous cell carci-
noma, whose tumors overexpressed activin A, had a worse
prognosis for overall survival and disease-free survival
(P = 0.009 and 0.007). However, expression of follistatin
in tumor was not correlated with overall survival or dis-
ease-free survival. Serum activin A and follistatin levels in
111 untreated patients were neither significantly different
from those of 91 control samples nor associated with any
clinicopathological manifestations. In vitro suppression
of activin A expression in OC3 cells using specific inter-
fering RNA-attenuated cell proliferation, migration, and
invasiveness.
Conclusions. These findings suggest that activin A over-
expression in oral squamous cell carcinomas is associated
with patients’ survival and may contribute to tumor pro-
gression and metastasis.
Oral squamous cell carcinoma (OSCC) is the most
common cancer of the head and neck and accounts for
approximately 3% of all newly diagnosed cancer cases.1,2
Despite recent advances in surgical, radiotherapy, and
chemotherapy treatment protocols, the long-term survival
of patients with OSCC has remained at approximately
50%–60% for the past three decades.3,4 These unsatisfac-
tory treatment results and lack of improvement despite
advances in treatment modalities may be explained by the
fact that OSCCs frequently present with extensive local
invasion and have a high probability of cervical lymph
node metastasis. OSCC is thought to develop from pre-
cancerous dysplastic lesions through multistep processes
of carcinogenesis involving activation of oncogenes
and loss of the tumor suppressor genes, and exposure of
Electronic supplementary material The online version of thisarticle (doi:10.1245/s10434-010-0926-2) contains supplementarymaterial, which is available to authorized users.
� Society of Surgical Oncology 2010
First Received: 20 May 2009;
Published Online: 23 March 2010
S.-P. Hao, MD
e-mail: shengpo@cgmh.org.tw
C.-N. Tsai, PhD
e-mail: pink7@mail.cgu.edu.tw
Ann Surg Oncol (2010) 17:1945–1956
DOI 10.1245/s10434-010-0926-2
environmental carcinogens such as tobacco, alcohol, and
betel quid chewing.5–8 Detecting carcinogenetic abnor-
malities in OSCCs might provide an important prognostic
indicator of patient prognosis and contribute to the future
development of tailored treatments based on the presence
of specific markers.
Activin A is a member of the transforming growth
factor b (TGF-b) superfamily of secreted signaling mol-
ecules, which mediate cell growth and differentiation.9
This protein was originally purified from ovarian fluid and
identified as a stimulatory factor for pituitary FSH
secretion, but was subsequently found to have diverse
regulatory functions in a wide range of tissues and
organs.10,11 Activin A is a homodimer of 2 disulfide-
linked bA subunits encoded by INHBA gene and has been
shown to be a crucial regulator of organ development and
wound healing.12 In adult mice and humans, abnormal
expression of activin A is associated with various dis-
eases, including different types of cancer.13 Although the
role of activin A in cancer progression remains a topic of
investigation, it has been reported to be overexpressed in
various cancers, including colon, esophageal, ovarian,
prostate, pancreatic, and lung cancers.14–19 Additionally,
high serum levels of activin A have been proposed as a
marker of human breast, hepatocellular, endometrial, and
cervical carcinomas.20–22 Activin A is also reported to
promote cancer cell growth, migration, and invasion in
some in vivo and in vitro studies.23–26 These results
suggest that activin A might be involved in the carcino-
genesis, progression, and metastasis of some types of
cancer. However, other studies have shown that overex-
pression of activin A resulted in antiproliferative and
apoptotic effects in different cancer cells.27–29 Thus, the
exact function of activin A in different human malig-
nancies remains to be elucidated.
The protein follistatin (FST), encoded by the FST gene,
is a secreted monomeric protein that is structurally unre-
lated to activins. FST binds to activin A with high affinity
and blocks its interaction with activin receptors.30 As an
inhibitor of activin A, FST also regulates a variety of
biological functions, including cell growth, differentiation,
and apoptosis. A recent study showed that cells transfected
with FST produced fewer metastatic colonies in a multiple-
organ metastasis murine model, predominantly by inhibit-
ing the angiogenesis.31 Furthermore, activin A secreted by
vascular endothelial cells promotes vascular endothelial
growth factor-induced angiogenesis, but these effects are
inhibited by the addition of FST.32 These data suggest that
FST expression in the tumor microenvironment might
influence cancer progression.
Although two previous transcriptomic studies using
cDNA microarray analysis revealed that the gene INHBA
was overexpressed at the mRNA level in the OSCC tissues
compared with matched normal epithelial tissue, to the best
of our knowledge, there are no published reports that fur-
ther address the potential role of activin A and FST in
OSCC.33,34 In the current study, we evaluated the expres-
sion of activin A and FST in human OSCC. Herein, we
provide the first evidence that activin A and FST are both
overexpressed in OSCC tumor cells and that activin A
overexpression is tightly associated with OSCC disease
status and treatment outcome. Finally, specific interfering
RNA (siRNA)-mediated knockdown of INHBA transcripts
in an OSCC tumor cell line suppressed the in vitro pro-
liferation, migration, and invasion ability of OSCC cells.
Based on these results, we discuss the biological signifi-
cance of activin A and FST overexpression in OSCC and
the potential role of these proteins in future targeted
therapies.
MATERIALS AND METHODS
Patient Characteristics and Clinical Specimens
Tumor specimens for immunohistochemical study were
obtained from 92 consecutive patients with OSCC diag-
nosed at the Chang Gung Memorial Hospital (CGMH, Tao-
Yuan, Taiwan) from 2003 to 2006. Serum samples were
collected from 91 healthy controls (88 men and 3 women)
and 111 patients with OSCC (103 men and 8 women) at the
CGMH from March 2005 to November 2006. Patients with
inoperable disease, synchronous cancers, recurrent cancers,
distant metastasis, or a preoperative history of another
malignancy were excluded from the analyses. All patients
provided informed consent prior to study participation.
Patients in the study underwent standard preoperative
work-ups according to institutional guidelines, including a
detailed medical history, complete physical examination,
computed tomography or magnetic resonance imaging
scans of the head and neck, chest radiographs, bone scan,
and abdominal ultrasound. Primary tumors were excised
with adequate margins under frozen section control intra-
operatively. Classic radical or modified neck dissections
(level I–V, about 50–60 lymph nodes) were performed in
patients with clinically positive lymph node disease. Su-
praomohyoid neck dissections (level I–III, about 30–40
lymph nodes) were performed in clinically node-negative
patients.35 Surgical defects were immediately recon-
structed by a free-flap or local-flap by plastic surgeons.
After surgical treatment, the pathological and nodal stage
of all tumors was established according to the AJCC
Cancer Staging Manual (2002). After discharge, all
patients had regular follow-up visits every 2 months for the
first year, every 3 months for the second year, and every
6 months thereafter.
1946 K.-P. Chang et al.
RNA Extraction and Quantitative Real-time RT-PCR
Detection of INHBA and FST
A total of 26 paired OSCC tumor and pericancerous
normal tissues were homogenized in liquid nitrogen with a
pestle and mortar, incubated with RNAzol B reagent (Tel-
Test Inc, Friendwood, TX), and total RNA was extracted
according to the manufacturer’s protocol. Total RNA was
further purified using the RNeasy Mini-Elute cleanup kit
(Qiagen Inc., Valencia, CA) according to the manufac-
turer’s protocol. First-strand cDNA was synthesized from
5 lg of total RNA, and then added to a PCR reaction
mixture consisting of commercially available primers
(INHBA, Hs00171410_m1, FST, Hs 00246256_m1, and
normalization control GAPDH, Hs99999905_m1; Assay-
on-Demand, Applied Biosystems, Foster City, CA),
RNase-free water, and TaqMan Universal PCR Master
Mix. Quantitative real-time RT-PCR was performed and
analyzed by 7900 HT Sequence Detection System and SDS
versions 2 (Applied Biosystems, Foster City, CA),
respectively. All experiments were repeated in triplicate
and mean fold-change was calculated for each sample.
Immunohistochemical Staining
For immunohistochemistry, formalin-fixed and paraffin-
embedded tissues were sectioned to 4 lm in thickness and
deparaffinized, rehydrated, and prepared for antigen
retrieval. The slides of consecutive sections were then
incubated with an appropriate dilution of antibodies: acti-
vin A (1:20) and follistatin (1:40) (R&D Systems
Minneapolis, MN) at room temperature for 1 hour. After
incubation, these slides were washed with phosphate buf-
fered saline (PBS) 3 times, incubated with horseradish
peroxidase (HRP) polymer antibody (Zymed) at room
temperature for 10 minutes, and developed by addition of
3,30-diaminobenzidine tetrahydrochloride (DAB) reagent
(DAKO) as the chromogen and hematoxylin as a coun-
terstain. Images of the stained slides were obtained using
the ScanScope computed tomography (CT) automated
slide-scanning system (Aperio Technologies, Vista, CA).
Expression of activin A and follistatin was scored using a
combined scoring method that accounts for both the
staining intensity and the percentage of stained cells.36
Strong, moderate, weak, and negative staining intensities
were scored as 3, 2, 1, and 0, respectively. For each of the
intensity scores, the percentage of cells that stained at the
specific level was visually estimated. The resulting com-
bined score was calculated as the sum of the percentage of
stained cells multiplied by the intensity scores. For exam-
ple, a case with 20% weak staining, 30% moderate
staining, and 50% strong staining would be assigned a
score of 230 (20 9 1 ? 30 9 2 ? 50 9 3 = 230) out of a
possible score of 300. All specimens were independently
evaluated by our pathologist (Liang Y.) without prior
knowledge of the clinical origin of the specimen. Immu-
nohistochemical scores 0, 1–100, 101–200, and 201–300
were classified as immunohistochemical levels 0, ?, ??,
and ???, respectively. Levels ?? and ??? were
defined as having protein overexpression.
ELISA Assays of Serum Activin A and FST Levels
Activin A and FST levels in the tested samples were
determined using Quantikine ELISA kits for human activin
A and FST (R&D Systems Minneapolis, MN). Human
recombinant activin A and FST proteins were used as assay
standards. Briefly, 100 lL of serum samples or standard
were added to microtiter plates coated with a murine
monoclonal antibody against human activin A or FST,
incubated for 2 hours at room temperature, and then
washed 3 times with wash buffer. A horseradish peroxi-
dase-conjugated polyclonal antibody was then added to the
individual wells, and the plates were incubated for 2 hours
at room temperature. The plates were then washed, and
hydrogen peroxide and tetramethylbenzidine were added
for color development at room temperature for 30 minutes.
The reaction was stopped by addition of 2 N sulfuric acid.
The color intensity in each well was measured as the
optical density using a microplate reader set to 450 nm. A
standard curve was constructed by plotting the optical
value of the standard and the amounts of Activin A and
FST in the respective samples. Each experiment was per-
formed in duplicate.
Knockdown of Activin A Using RNA Interference
(RNAi)
RNAi specifically targeting human INHBA (No. L-011701-
00-0005, Dharmacon) and scrambled control RNAi
(No. D-001810-10-05, Dharmacon) were purchased from
Thermo Fisher Scientific, Inc. RNAi (final concentration of
RNAi was 400 nM) was mixed with Oligofectamine
reagent (Invitrogen, Carlsbad, CA) and Opti-MEM med-
ium (Invitrogen, Carlsbad, CA) without serum, incubated
for 15 minutes at room temperature, and then added to OC3
cells that were seeded at a density of 2 9 105 cells per well
in six-well plates.37 After incubation for 6 hours at 37�C,
500 lL of 3X concentrated OC3 cell medium (Keratino-
cyte-SFM [Invitrogen, Carlsbad, CA]: Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum = 3:1) was added to each well.
After transfection for 24 hours, cells were harvested for
analysis of cell proliferation, migration, and invasive
capacity.
Activin a as a Prognostic Marker of Oscc 1947
Cell Proliferation Assay
Cell proliferation was measured using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reagent. OC3 cells transfected with specific RNAi,
scrambled RNAi, or PBS were plated at a density of
1 9 104 cells/well in 96-well plates and subsequently
incubated in serum-free medium (supplemented with
DMEM) for 48 hours prior to the addition of MTT for 4
hours. Acidic isopropanol was then added and incubated
overnight. The optical density of color development was
measured at 595 nm using a SpectraMax M5 microplate
reader (Molecular Devices, Sunnyvale, CA). Each data
point was in the result of quadruplicate determinations
from 3 separate experiments.
Cell Migration Assay
Cell migration was evaluated using a chemotaxis
chamber (Corning Inc., Lowell, MA) with a polycarbonate
membrane (pore size of 8 lm) placed between the two
chambers. Transfected OC3 cells (3 9 105) in 300 lL of
culture medium were applied to the upper chamber and 600
lL of medium was added to the lower chamber. Chambers
were incubated at 37�C for 16 hours, and then the mem-
brane was fixed in methanol for 10 minutes and stained
with hematoxylin and eosin. Cells on the upper surface of
the filter were carefully removed with a cotton swab, and
then cells that had migrated through the membrane to the
lower surface of the filter were counted in 9 different visual
fields under a light microscope (magnification: 200 9).
Each migration assay was performed in triplicate during 3
independent experiments.
Cell Invasion Assay
The Cell Invasion Assay Kit (Chemicon, Temecula, CA)
was used to measure the invasiveness of the cancer cell
lines. Briefly, polycarbonate membranes (8 lm pore size)
in the upper compartments of the provided Transwell cul-
ture chambers were coated with the provided ECMatrix.
Cells (1 9 105) were suspended in 100 lL serum-free
medium and placed in the upper compartments. The lower
compartments were filled with 500 lL of cell growth
medium containing 10% FBS. Cells were incubated at
37�C for 48 hours, and then the membranes were fixed in
methanol and stained with hematoxylin and eosin. Cells on
the upper surface of each filter were carefully removed
with a cotton swab, and the cells that had migrated through
the membrane to the lower surface of the filter were
counted in 9 different fields under a light microscope
(magnification: 200 9). Each invasion assay was per-
formed twice in 3 independent experiments.
Statistical Analysis
All statistical data are expressed as the mean ± SD. The
Wilcoxon signed ranks test was used for comparison of the
relative signal intensity of quantitative real-time PCR and
immunohistochemical staining scores between the paired
tumor and pericancerous normal mucosa samples. Serum
protein levels, cell proliferation, cell growth, and migration
experiments were compared using unpaired t tests. The
associations between the various characteristics of patients
and activin A and FST overexpression were evaluated
using the chi-square test. For analyzing the correlation of
expression level of INHBA and FST transcripts, the average
Ct of GAPDH was subtracted from the raw Ct value to
obtain d-Ct (dCt). The experimentally normalized dCt
values were converted to 39 – Ct used to estimate the
expression level of mRNA transcripts. All statistical anal-
yses were performed using SPSS and SAS software. All
patients received follow-up evaluations at our outpatient
clinic until November 2008 or death. Survival analysis was
plotted using the Kaplan-Meyer method and differences
were evaluated using the log-rank test. Univariate and
multivariate regression analyses were used to define spe-
cific risk factors for overall survival and disease-free
survival. All P values were 2-sided, and statistical signifi-
cance was accepted when P \ .05.
RESULTS
Overexpression of Activin A and FST in Tumor Cells
of OSCC Tissues
To distinguish the specific cell types in the tumor mass
that expressed activin A and FST, consecutive tissue
sections from 92 patients were subjected to immunohisto-
chemical staining. Both Activin A and FST were highly
expressed in the cytoplasm of tumor cells without nuclear
or membranous staining. Both expression was largely
absent or faintly stained in the tumor-infiltrating lympho-
cytes and interstitial tissues except that activin A was also
expressed in some macrophages in the interstitial tissues
(Fig. 1). Moreover, the paired normal oral epithelium
samples showed either low or no expression of activin A
and FST (Fig. 1). Expression of INHBA and FST were
examined by quantitative real-time RT-PCR in 26 paired
OSCC tumor and adjacent normal tissues. Transcripts
for both INHBA and FST were significantly elevated in
OSCC tumor specimens compared with adjacent normal
tissue (INHBA: 20,100 ± 3160 vs. 2876 ± 585.0, P \.0001; FST: 1490 ± 1677 vs. 660.8 ± 221.1, P = .0003)
(Fig. 2a). Statistical analysis of the 80 paired samples
available from these 92 OSCC patients revealed that
immunohistochemical scores of both activin A and FST
1948 K.-P. Chang et al.
were significantly higher in tumor tissue than in nontumor
tissue (activin A: 141.5 ± 50.2 vs. 9.1 ± 28.0, P \ .0001;
FST: 126.5 ± 49.1 vs. 6.7 ± 22.7, P \ .0001) (Fig. 2b).
These quantitative real-time RT-PCR and immunohisto-
chemistry results demonstrated that both activin A and FST
are overexpressed in OSCC tumor cells, but not in nontu-
mor cells within the tumor specimen or in adjacent normal
tissue. Furthermore, activin A and FST mRNA expression
in OSCC tissues was analyzed by normalizing to GAPDH
expression. Activin A and FST protein expression was
performed using immunohistochemical scores from the
consecutive sections. Statistical significant correlations
between activin A and FST were observed in both mRNA
and immunohistochemical expression (Pearson’s correla-
tion r = 0.507, P = .008 and r = 0.354 and P = .0005,
respectively; see Fig. 2c).
Association of Activin A and FST Expression
with Various Clinicopathological Manifestations
We next evaluated whether increased expression of
activin A or FST was correlated with various clinicopath-
ological characteristics in 92 OSCC patients. Over-
expression of activin A was significantly correlated with
positive N stage, poorer OSCC histological differentiation,
and perineural invasion (P = .029, .002, and .014,
respectively). Nevertheless, there were no correlations
related to patient age, T stage, or overall tumor stage
(Table 1). Of the various clinicopathological characteris-
tics evaluated, overexpression of FST was only
significantly correlated with overall tumor stage (P = .03;
Table 1). There was also no association between overex-
pression of activin A or FST in OSCC tumors and the
habitual behaviors of betel nut chewing, smoking, or
alcohol consumption (Table 1).
Correlation of Activin A or FST Overexpression
and Patient Overall Survival
Using data from the 92 patients in the prospective
cohort, we further evaluated whether overexpression of
activin A or FST was correlated with patient overall sur-
vival (OS). Based on survival analysis on 88 patients
undergoing complete standardized treatment and regular
follow-up, we found that the long-term OS for patient
subgroups stratified by the absence or presence of activin A
overexpression were 91.3% and 55.5%, respectively. This
difference in OS was statistically significant when com-
pared using a log-rank test (P = .009) (Fig. 3a).
Conversely, long-term OS shown by Kaplan-Meier plots of
patients stratified by the absence or presence of FST
overexpression was not significantly different (72.3% and
60.1%, respectively; P = 0.179) (Fig. 3b). Besides, activin
A overexpression was also a significant predictor of OS in
univariate analysis of Cox proportional regression models
(Table 2), but FST overexpression was not. To determine
whether activin A overexpression is an independent pre-
dictor of OS, a multivariate analysis was carried out using
tumor stage, nodal stage, overall stage, perineural invasion,
tumor differentiation, and activin A overexpression as
parameters. The results indicated that only patients with
positive nodal stages had a significantly lower OS than
patients without cervical metastasis (hazard ratio: 3.234,
P = .034, Table 2), suggesting that nodal stage is an
independent predictor of OS in patients with OSCC.
Although activin A overexpression was not a key
FIG. 1 Overexpression of activin A and follistatin (FST) in OSCC
cells. Immunohistochemical staining of activin A and FST in paired
pericancerous adjacent normal epithelium (AN) and tumor tissues
from two representative cases (scale bar = 100 lm). Expression
(brown staining) of activin A and FST indicated that the proteins were
localized in the cytoplasm of OSCC tumor cells. Both expressions
were largely absent or faintly stained in the tumor infiltrating
lymphocytes and interstitial tissues
Activin a as a Prognostic Marker of Oscc 1949
independent factor of OS in our multivariate analysis, we
suggested that activin A might be associated with nodal
metastatic processes in OSCC patients according to the
results of Table 1.
Activin A and FST Expression and Disease-Free
Survival
As shown by Kaplan-Meier plots, long-term disease-free
survival (DFS) for patients stratified by the absence or
presence of activin A overexpression was 86.5% and
49.5%, respectively. This difference in DFS was statisti-
cally significant using a log-rank test (P = .007) (Fig. 3c).
Long-term DFS for patients stratified by the absence
or presence of FST overexpression was not significantly
different using a log-rank test (70.0% and 52.4%,
respectively; P = .098) (Fig. 3d). Besides, activin A
overexpression was also a significant predictor of DFS in
univariate analysis of Cox proportional regression models
(Table 3), but FST overexpression was not. To determine
whether activin A overexpression is an independent pre-
dictor of DFS, a multivariate analysis was carried out using
tumor stage, nodal stage, overall stage, perineural invasion,
tumor differentiation, and activin A overexpression as
parameters. The results indicated that tumor stage, nodal
stage, and tumor differentiation are independent predictors
of DFS (P = .010, .033, and .002, respectively; Table 3),
but activin A is not (P = .070, Table 3). Although activin
A overexpression was also not a key independent factor of
DFS in our multivariate analysis, it still also associated
with nodal metastatic processes and tumor differentiation
in OSCC patients according to the results of Table 1.
a
25,000
20,000
15,000
10,000
5,000
Copy per106 GAPDH
Normal Tumor
Activin A
p < 0.0001
Activin A
p < 0.0001
b
300
250
200
150
50
100
ImmunohistochemicalScore
Normal Tumor
FST
p < 0.0001
300
250
200
150
50
100
ImmunohistochemicalScore
Normal Tumor
c
18
16
14
12
Activin AQuantitative Real-Time RT-PCRr = 0.507p = 0.008
6 8 10 12 14 16FST
2,000
1,500
1,000
500
Copy per106 GAPDH
Normal Tumor
FST
p = 0.0003
300
200
100
Activin AImmunohistochemistryr = 0.354p = 0.0005
0 50 100 150 200 250FST
FIG. 2 Expression and
correlation of INHBA and
follistatin (FST) in OSCC cells.
a The overexpression of INHBAand FST transcripts (P \ .0001
and P = .0003, respectively) in
OSCC tumor tissues was
demonstrated using quantitative
real-time RT-PCR. Expression of
both genes was normalized using
endogenous GAPDH expression.
b Box plot analysis of the
immunohistochemical staining
scores for activin A and FST
expression in the paired
pericancerous AN and tumor
tissues. Both activin A and FST
were highly overexpressed in
OSCC tissues (P \ .0001 and
P \ .0001, respectively).
c Correlation between activin A
and FST in OSCC tissues using
quantitative real-time RT-PCR
and immunohistochemical
analyses. Pearson’s correlation
coefficient and P values for
individual analysis are shown in
the inserts
1950 K.-P. Chang et al.
No Correlation between Serum Activin A and FST
Levels and Disease Status
Since activin A and FST are secretory proteins, we
further evaluated whether serum protein levels might serve
as biomarkers for OSCC. Serum activin A and FST levels
in untreated OSCC patients (n = 111) were 744.3 ±
2118 pg/mL and 695.9 ± 343.5 pg/mL, respectively, and
were not significantly different from control samples
(n = 91) (468.4 ± 1246 pg/mL and 760.3 ± 769.0 pg/
mL; P = .251 and P = .487; Fig. 4). Similarly, no corre-
lation was found between the serum levels of activin A and
FST and various clinicopathological characteristics of 111
patients with OSCC, including age, gender, T stage, N
stage, overall stage, or histological differentiation. These
data may indicate that overexpression of both proteins is a
local change within OSCC tumors rather than a systemic
change detectable in the circulation.
Activin A Promotes OSCC Cell Proliferation,
Migration, and Invasion In Vitro
To evaluate the biological significance of activin A
overexpression using an in vitro system, OSCC cell
endogenous expression of activin A was knocked down
using specific RNAi. The effects of RNAi were deter-
mined using quantitative real-time RT-PCR in OC3 cells
transfected with either INHBA-specific RNAi (si-INHBA)
TABLE 1 Clinicopathological characteristics related to the expression of activin A and follistatin in 92 samples of OSCCs
Activin A overexp (%) P value Follistatin overexp (%) P value
No Yes No Yes
Age
Range 30.1–72.0 30.7–82.5 31.0–82.5 30.1–72.1
Mean ± SD 47.4 ± 11.7 52.0 ± 11.4 .088 50.4 ± 13.0 51.0 ± 10.7 .812
T stage
T1–T2 13 (28) 34 (72) .914 20 (43) 27 (57) .363
T3–T4 12 (27) 33 (73) 15 (33) 30 (67)
N stage
N = 0 20 (35) 37 (65) .029a 25 (35) 32 (65) .142
N [ 0 5 (14) 30 (86) 10 (29) 25 (71)
Overall stage
I–II 11 (34) 21 (66) .257 17 (53) 15 (47) .030a
III–IV 14 (23) 46 (77) 18 (30) 42 (70)
Differentiation
W-D 16 (47) 18 (53) .002a 16 (47) 18 (53) .176
M-D 9 (16) 41 (82) 18 (36) 32 (64)
P-D 0 (0) 8 (100) 1 (12) 7 (88)
Perineural invasion
Negative 22 (35) 41 (65) .014a 28 (44) 35 (56) .062
Positive 3 (10) 26 (90) 7 (24) 22 (76)
Alcohol consumption
No 11 (29) 27 (71) .748 17 (45) 21 (55) .267
Yes 14 (26) 40 (74) 18 (33) 36 (67)
Betel nut chewing
No 4 (36) 7 (66) .482 6 (55) 5 (45) .229
Yes 21 (26) 60 (74) 29 (29) 52 (52)
Smoking
No 2 (18) 9 (82) .721 4 (36) 7 (64) .902
Yes 23 (28) 58 (72) 31 (38) 50 (62)
Total 25 67 35 57
overexp overexpression, W-D well-differentiated squamous cell carcinoma, M-D moderately differentiated squamous cell carcinoma, P-D poorly
differentiated squamous cell carcinomaa Statistically significant
Activin a as a Prognostic Marker of Oscc 1951
or a scrambled sequence control RNAi. As shown in
Fig. 5a, expression of endogenous INHBA was signifi-
cantly reduced (approximately 32-fold reduction) in
si-INHBA-transfected cells compared with control RNAi
transfectants.
Control and si-INHBA-transfected cells were further
analyzed for cell proliferation, migration, and invasiveness.
As shown in Fig. 5b, the cell proliferation ability in OC3
cell proliferation was also significantly reduced by si-IN-
HBA (37% reduction, P \ 0.0001). Also, both cell
migration and cell invasiveness were attenuated by addi-
tion of si-INHBA (63% and 67% reduction, P \ 0.001 and
P \ .0001, respectively; Fig. 5c, d). Similar decreases cell
proliferation, migration, and invasiveness were observed
when SCC4 cells, another OSCC cell line, were transfected
with si-INHBA (data not shown). Collectively, these
1.0
0.8
0.6
0.4
0.2
0 60Months
30 4010 20 50
0 60Months
30 4010 20 50
Activin A (0, +)Activin A (++, +++)
p = 0.009
OverallSurvival
a
1.0
0.8
0.6
0.4
0.2
Activin A (0, +)Activin A (++, +++)
p = 0.007
Disease-FreeSurvival
c
1.0
0.8
0.6
0.4
0.2
0 60Months
30 4010 20 50
0 60Months
30 4010 20 50
FST (0, +)FST (++, +++)
p = 0.179
OverallSurvival
b
1.0
0.8
0.6
0.4
0.2
FST (0, +)FST (++, +++)
p = 0.098
Disease-FreeSurvival
d
FIG. 3 Overexpression of
activin A was associated with a
poorer prognosis with regard to
OSCC patient overall survival
and disease-free survival.
a, b Kaplan-Meier plot for
overall survival indicated that the
4-year overall survival for
patient subgroups stratified by
overexpression of activin A and
FST was 91.3% versus 55.5%
(P = .009) and 72.3% versus
60.1% (P = .179), respectively.
c, d Kaplan-Meier plot for
disease-free survival indicated
that the 4-year disease-free
survival for patient subgroups
stratified by overexpression of
activin A and FST was 86.5%
versus 54.0% (P = .007) and
70.0% versus 57.5% (P = .098),
respectively
TABLE 2 Cox proportional hazard models on overall survival
Univariate crude HR (95% CI) P value Multivariate adjusted HR (95% CI) P value
T stage
T3–T4 vs. T1–T2
1.632 (0.784–3.394) .190 1.807 (0.718–4.546) .208
N stage
N [ 0 vs. N = 0
4.262 (1.971–9.216) \.001a 3.234 (1.090–9.595) .034a
Overall stage
III–IV vs. I–II
3.312 (1.262–8.691) .015a 0.689 (0.146–3.263) .639
Differentiation
P-D vs. W-D ? M-D
6.308 (1.905–20.892) .002a 3.421 (0.940–12.456) .062
Perineural invasion
Positive vs. negative
2.686 (1.288–5.598) .008a 1.357 (0.628–2.934) .437
Activin A overexpression
Yes vs. no
5.533 (1.315–23.281) .019a 2.610 (0.579–11.760) .211
HR hazard ratio, CI confidence interval, W-D well-differentiated squamous cell carcinoma, M-D moderately differentiated squamous cell
carcinoma, P-D poorly differentiated squamous cell carcinomaa Statistically significant
1952 K.-P. Chang et al.
findings indicate that overexpression of activin A in vitro
can mediate cell proliferation, migration, and invasion
processes in OSCC cells.
DISCUSSION
Overexpression of activin A has been reported for var-
ious cancers, but its mechanistic roles in cancer cells and
contributions to the carcinogenic process have remained to
be elucidated. The results of the present study strongly
suggest that overexpression of activin A in OSCC is
associated with poorer cell differentiation, positive cervical
nodal status, and perineural invasion of OSCC tumors and
therefore may be a risk factor for OSCC tumor progression.
These three different clinicopathological factors have all
been suggested as poor prognostic factors for locoregional
control in OSCC and may explain why activin A expres-
sion has a vital impact on long-term OS and DFS.38 These
findings are in agreement with the previous reports
that indicated that activin A overexpression was associated
with advanced stages of colon cancer and lymph node
metastasis and poor prognosis of esophageal squamous cell
carcinomas.14,15 Although activin A and FST are secretory
proteins and serum levels of activin A have been proposed
as a potential tumor marker in some cancers, our results
clearly demonstrated that overexpression of activin A was
only found in cancer tissues.20–22 In the present patient
cohort, neither activin A nor FST serum levels could be
used to differentiate OSCC patients from healthy controls
or as a stratification marker of the clinicopathological
manifestations of OSCC patients. In other words, activin A
secreted from OSCC cells might have pivotal roles in
tumor biology through autocrine or paracrine mechanisms,
but it does not have systemic effects and is not a potential
serum marker for OSCC.
In vivo and in vitro studies of various types of malig-
nancies have demonstrated that activin A promotes cancer
cell growth, migration, and invasion.23–26 In addition,
studies of prostate and lung cancers revealed that inhibition
of activin A expression by its specific RNAi or antibody
neutralization attenuated cancer cell proliferation and cell
migration, respectively, which was similar to the present
TABLE 3 Cox proportional hazard models on disease-free survival
Univariate crude HR (95% CI) P value Multivariate adjusted HR (95% CI) P value
T stage
T3–T4 vs. T1–T2
2.701 (1.309–5.573) .007a 4.219 (1.404–12.673) .010a
N stage
N [ 0 vs. N = 0
3.651 (1.805–7.381) \.001a 2.639 (1.080–6.449) .033a
Overall stage
III–IV vs. I–II
5.254 (1.844–14.966) .002a 0.893 (0.188–4.242) .886
Differentiation
P-D vs. W-D ? M-D
4.970 (2.118–11.663) \.001a 6.038 (1.886–19.334) .002a
Perineural invasion
Positive vs. negative
2.225 (1.112–4.452) .024a 0.921 (0.393–2.158) .849
Activin A overexpression
Yes vs. no
4.336 (1.322–14.218) .015a 3.058 (0.911–10.260) .070
HR hazard ratio, CI confidence interval, W-D well-differentiated squamous cell carcinoma, M-D moderately differentiated squamous cell
carcinoma, P-D poorly differentiated squamous cell carcinomaa Statistically significant
Activin A
p = 0.251
a
14,000
12,000
10,000
250
500
Concentration(pg/ml)
OSCC Control
FST
p = 0.487
b
7,000
6,000
5,000
1,000
2,000
3,000
4,000
Concentration(pg/ml)
OSCC Control
FIG. 4 Serum activin A and
FST levels in untreated OSCC
patients (n = 111) were
744.3 ± 2118 pg/mL and
695.9 ± 343.5 pg/mL,
respectively, and were not
significantly different from
control samples (n = 91)
(468.4 ± 1246 pg/mL and
760.3 ± 769.0 pg/mL; P = .251
and P = .487)
Activin a as a Prognostic Marker of Oscc 1953
study using OSCC cells.19,39 Furthermore, RNAi targeting
of an activin A downstream effector, Smad3, reduced cell
proliferation and invasion.40 Thus, these results highlight
the role of activin A and its downstream signaling on
cancer cell malignancies. Another possible mechanism by
which activin A overexpression can promote malignant
characteristics of various cancers might be through stimu-
lation of vascular endothelial growth factor (VEGF)
expression.41,42 Because VEGF is a key regulator of
angiogenesis and its overexpression is associated with poor
prognosis of cancer patients, several therapeutic agents,
such as bevacizumab, that inhibit the activity of VEGF or
its receptors have been developed for the treatment of solid
malignancies.43,44 Therefore, inhibition of activin A and its
downstream signaling effectors might be of clinical rele-
vance for OSCC treatment in the future.
In the current study, we observed statistically significant
correlations between activin A and FST mRNA and protein
expression. Although FST is an antagonist of activin A, it
is also overexpressed in OSCC. Other negative regulators
of activin A, including another binding antagonist, FLRG,
and cell membrane antagonistic coreceptors, such as Cripto
or BAMBI, have also been discovered.45 The interaction
between activin A and its antagonists in different tumor
microenvironment will need to be further evaluated in the
future studies. To the best of our knowledge, the correla-
tion between activin A and FST has not been well
addressed to date. In a previous study of hepatocellular
carcinoma, deregulation of the FST-activin A system was
reported.27 FST overexpression was observed in both of
human and rat liver tumors, stimulating DNA synthesis
preferentially in preneoplastic rat hepatocytes.27 Moreover,
another study demonstrated that differential antagonism of
activin A occurred through binding with wild-type or
mutant FST.46 These findings might help explain why FST
overexpression was also associated with poor stages of
MigrationAssay
p < 0.001
c
e
1,500
1,000
500
Cells(No. /Field)
Scramble si-INHBA
Scramble
CellMigration
CellInvasion
si-INHBA
InvasionAssay
p < 0.0001
d
250
200
150
50
100
Cells(No. /Field)
Scramble si-INHBA
MTT Assay
b
20
0
–20
–40
Cell Growth(Percent)
PBS Scramble
50,000
40,000
30,000
20,000
10,000
0 40Cycle
15 205 10 25 30 35
Scramble-GAPDHsi-INHBA-GAPDHScramble-INHBAsi-INHBA-INHBA
Fluorescence(dR)
a
si-INHBA
FIG. 5 OSCC cell proliferation,
migration, and invasiveness was
attenuated by INHBA-specific
RNAi. a A plot of the threshold
PCR cycle showing expression
of endogenous GAPDH and
INHBA in OC3 cells transfected
with si-INHBA or a control
scrambled RNAi shown in a log
scale. The relative transcripts of
INHBA in si-INHBA (as orangeline shown) and scramble
transfected cells (as green lineshown) were normalized using
GAPDH endogenous expression
levels (as blue and light orangelines indicated). INHBAtranscripts were reduced about
32-fold in si-INHBA-transfected
OSCC cells as comparing
scramble transfectant. b MTT
assays showed that OC3 cell
proliferation was significantly
decreased following si-INHBA
transfection (37%, P \ 0.0001).
c, d Both OC3 cell migration and
invasiveness were attenuated by
INHBA-specific RNAi (63% and
67%, P \ 0.001 and P \ 0.0001,
respectively). e Representative
example images of cell
migration and invasion assays
are shown for control RNAi and
si-INHBA-treated groups. Data
are the mean ± SD of 3
independent experiments
1954 K.-P. Chang et al.
OSCC patients. Nevertheless, the true mechanism remains
to be elucidated and awaits further investigation in the
future.
In the current study, when endogenous activin A
expression was inhibited by specific RNAi, the prolifera-
tive, migratory, and invasive capabilities of OSCC cells
were significantly reduced. To understand the role of
activin A in carcinogenesis, many previous studies have
reported their in vitro findings illustrating potential mech-
anisms. Activin A has been proposed to induce a RhoA-
ROCK-MEKK1-JNK pathway and a MEKK1-p38 path-
way as Smad-independent mechanisms controlling actin
cytoskeleton reorganization and promoting the migratory
and invasive potential in human epithelial cells during
epithelial-to-mesenchymal transitions.47 Also, activin A
induction of N-cadherin and MMP-7 overexpression are
reported to be associated with the invasiveness of esoph-
ageal cancer cells.25,48 Recently, overexpression of activin
A has been suggested to mediate ovarian carcinogenesis
through activation of Akt and repression of GSK, resulting
in increased cell proliferation.49 Collectively, inhibition of
activin A might be a potential method for attenuating
OSCC tumor progression and aggressiveness.
In conclusion, this study provides the first evidence that
activin A is aberrantly overexpressed in OSCC cells rela-
tive to histologically noncancerous tissue. In addition,
overexpression of activin A is associated with several
important clinicopathological factors and patient prognosis
in survival analyses for OSCC, suggesting its potential as
an immunohistochemical marker of OSCC. Furthermore,
inhibition of activin A expression can attenuate OSCC cell
proliferation, migration, and invasiveness, supporting
activin A as potential target of future OSCC therapeutic
agents.
ACKNOWLEDGMENT This study was supported by Chang
Gung Memorial Hospital (Grant No. CMRPG360212 and
CMRPG340481) and National Science Council (Grant No. NSC96-
2314-B-182A-109-MY3), Taiwan.
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