s100a14 is a novel modulator of terminal differentiation ... · 1 s100a14 is a novel modulator of...
TRANSCRIPT
1
S100A14 is a novel modulator of terminal differentiation of esophageal
squamous cell carcinoma
Hongyan Chen1, Jianlin Ma1, Benjamin Sunkel2, Aiping Luo1, Fang Ding1, Yi Li1,
Huan He1, Shuguang Zhang1, Chengshan Xu1, Qinge Jin1, Qianben Wang1,2, Zhihua
Liu1*
1State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing, China and
2Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer
Center, The Ohio State University College of Medicine, Columbus, OH, USA
Requests for reprints:
Zhihua Liu, State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese
Academy of Medical Sciences, Beijing 100021, China. Tel: 8610-87788490, Fax:
8610-67723789; E-mail: [email protected].
Running Title: The role of S100A14 in ESCC differentiation
Keywords: S100A14, ESCC, differentiation, AP-1
Grant support: National Natural Science Foundation of China (81000954) and
Doctoral Fund of Ministry of Education of China (20101106120012).
Conflict interest statement: Non declared
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
2
Abstract
Aberrant keratinocyte differentiation is considered to be a key mechanism in the
initiation of cancer. As activities regulating differentiation exhibit altered or reduced
in esophageal cancer cells, it is vital to identify and characterize the genes controlling
epidermal proliferation and terminal differentiation to better understand esophageal
carcinogenesis. S100A14 is a member of the S100 family of calcium-binding proteins
and was recently reported to be involved in cell proliferation, apoptosis and invasion.
In the present study, we performed immunohistochemistry analysis of S100A14 in
esophageal squamous cell carcinoma (ESCC) and showed that the decreased
expression of S100A14 is strongly correlated with poor differentiation of ESCC.
Furthermore, we demonstrated that the mRNA and protein expression of S100A14
was drastically increased upon 12-O-tetra-decanoylphorbol-13-acetate (TPA) and
calcium-induced esophageal cancer cell differentiation. Overexpression of S100A14
resulted in cell cycle arrest at the G1 phase and promoted the calcium-inhibited cell
growth. Conversely, decreasing S100A14 expression significantly promoted G1/S
transition and prevented the morphological changes of calcium-induced cell
differentiation. Molecular investigation demonstrated that S100A14 affected the
calcium-induced expression of late markers of differentiation, with the most
prominent effect on involucrin (IVL) and filaggrin (FLG). Finally, we showed that
S100A14 is transcriptionally regulated by JunB and that a significant correlation
between S100A14 and JunB is observed in esophageal cancer tissues. In summary,
our data demonstrate that S100A14 is transcriptionally regulated by JunB and
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
3
involved in esophageal cancer cell differentiation, which will help us further
understand the molecular mechanism controlling the development and progression of
esophageal cancer.
Introduction
Ranking eighth in incidence and sixth in cancer-related mortality worldwide,
esophageal cancer is among the most aggressive cancers occurring with such high
frequency [1]. Over 80% of esophageal cancers occur in developing countries, but
these malignancies are particularly prevalent in China and other countries in Asian,
where esophageal squamous cell carcinoma (ESCC) is most common [1,2].
Accumulating evidence shows that a variety of biological abnormalities including
altered gene expression, gene mutations, aberrant signaling pathways and genetic
alterations contribute to the development and progression of ESCC [3]. In addition,
the disruption of epithelial differentiation may be one of the primary mechanisms for
ESCC [4]. Our previous studies have clearly demonstrated that a series of genes
involved in squamous cell differentiation were coordinately downregulated in ESCC
[5]. Among them, S100 calcium-binding proteins have attracted additional attention as
they are implicated in a variety of biological events closely related to tumorigenesis
and cancer progression.
Most S100 proteins are clustered at the chromosomal region 1q21 and constitute
important components of the epidermal differentiation complex (EDC) [6]. S100
proteins are therefore involved in the process of terminal differentiation of human
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
4
epidermis and have been implicated in cancer as altered expression levels of several
S100 proteins have been reported to correlate with tumor differentiation including
ESCC [7-14]. We have recently reported on the role of the S100 family member,
S100A14, in driving esophageal carcinogenesis, demonstrating that extracellular
S100A14 affects esophageal cancer cell proliferation and apoptosis via interaction
with RAGE, and intracellular S100A14 regulates cell invasion by MMP2 in a
p53-dependent manner [15,16]. Moreover, the 461G>A SNP located in the 5’-UTR of
S100A14 is associated with ESCC susceptibility in a Chinese population [17],
providing additional support for the role of S100A14 in driving this disease. These
findings prompted us to further investigate the functional role of S100A14 and the
correlation between S100A14 levels and clinicopathological features in ESCC.
In the present study, we examined the expression of S100A14 in clinical ESCC
samples and their matched normal esophageal epithelia and analyzed the relationships
between S100A14 expression and the clinicopathological parameters of ESCC.
Furthermore, we examined the induction of S100A14 upon TPA and calcium
treatment in esophageal cancer cells and investigated the role of S100A14 in
calcium-induced esophageal cancer cell morphological change and
differentiation-related gene expression changes. Finally, we provided a preliminary
investigation on the underlying mechanism of S100A14-mediated cell differentiation.
Materials and Methods
Tissue specimens. Tissue samples from 30 patients with ESCC were used for
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
5
S100A14 mRNA expression analysis, and these samples were different from those
examined in our previous study [18]. Tissue specimens from 110 patients with ESCC
were analyzed by immunohistochemistry. Patients were recruited at the Chinese
Academy of Medical Sciences Cancer Hospital (Beijing). Patients received no
treatment before surgery and signed informed consent forms for sample collection.
This study was approved by the Institutional Review Board of the Chinese Academy
of Medical Sciences Cancer Institute. Representative primary tumor regions and the
corresponding histologically normal esophageal mucosa from each patient were
snap-frozen in liquid nitrogen and stored at -80°C. Additional blocks were collected
and processed in paraffin for histological examination.
Immunohistochemical staining. An ESCC tissue microarray including 110
esophageal tumors and the corresponding normal epithelia was constructed with each
case represented twice. For immunohistochemical staining, the slides were
deparaffinized, rehydrated, then immersed in 3% hydrogen peroxide solution for 10
minutes (min), heated in citrate buffer (pH 6.0) at 95°C for 25min, and cooled at room
temperature for 60 min. The slides were blocked by 10% normal goat serum at 37°C
for 30 min and then incubated with rabbit polyclonal antibody against S100A14 at a
dilution of 1:500 overnight at 4°C. IHC was performed using the PV-9000 Polymer
Detection System for Immuno-Histological Staining kit (Beijing Golden Bridge
Biotechnology Company). DAB was used to visualize the reaction, followed by
counterstaining with Hematoxylin. Visual analysis was performed using
ImageScope software (Aperio Technologies). The staining intensity was graded from
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
6
0 to 3; no staining was scored as 0, weak positive staining as 1, positive staining as 2,
and strong positive staining as 3. The percentage of staining was automatically
assessed by ImageScope software and the expression score was determined by
multiplying the percentage of staining by the staining intensity graded 0 to 3. The
cohort was divided into two groups according to the expression score ratio of matched
cancer/normal tissue (ratio ≥ 1 was defined as the Non-underexpressed group, and
ratio < 1 was defined as the underexpressed group). Representative areas of each
section were selected.
Cell culture. Human ESCC cell lines (KYSE series) were gifts from Dr. Y.
Shimada of Kyoto University (Kyoto, Japan) [19]. Cells were maintained in
RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml streptomycin, and
100 U/ml penicillin.
Plasmids. Full-length cDNA of human S100A14 was cloned into the mammalian
expression vector pcDNA3.1. The promoter region of S100A14 (-511~+6) was cloned
into the pGL3-basic vector as previously described [17]. The resulting construct was
verified by direct sequencing. C-Jun and Fra-1 expression plasmids were generated in
our laboratory. JunB, JunD and c-fos expression plasmids were provided by Dr. Marta
Barbara Wisniewska of University of Warsaw (Warsaw, Poland).
Transfection and generation of stable cell lines. Transfection and
establishment of stable cell lines were performed as previously described [20].
siRNA Transfection. Cells were transfected with siRNAs (25nM) by HiperFect
(Qiagen) following the manufacturers’ protocol. The sequences for siRNAs were
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
7
listed in Supplementary Table S1.
Immunofluorescence. The experiment was performed as previously described
[20].
RNA isolation and PCR analysis. RNA purification and quantitative RT-PCR
were performed as previously described [17]. Primers used are listed in
Supplementary Table S1.
Chromatin immunoprecipitation (ChIP) assay. ChIP was performed as
previously described [21] using anti-JunB (5712-S) antibody from Epitomics
(Burlingame, CA) and RNA Polymerase II (MA1-10882) antibody from Thermo
Scientific Pierce (Rockford, IL). Primers used are listed in supplementary Table S1.
Western blot analysis. Western blots were performed as previously described
[20]. Antibodies used were anti-S100A14 (gifts of Dr. Iver Petersen, University
Hospital Charite, Berlin and Dr. Youyong Lü, Beijing Cancer Hospital and Institute,
Beijing) and anti-β-actin (A5316, Sigma, St. Louis, MO).
Luciferase assay. Luciferase assay was performed as previously described [17].
Cell proliferation assay. Cell proliferation was measured by a direct viable cell
count assay.
Annexin V apoptosis assay. Apoptosis assay was measured using the BD
Annexin V-PE Apoptosis Detection Kit (Becton, Dickinson and company, San Diego,
CA) according to the manufacturer’s protocol. Briefly, cells were incubated with
Annexin V at room temperature for 15 minutes in the dark and then subjected to flow
cytometry analysis.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
8
FACS analysis. Cells were washed in PBS and fixed in methanol overnight.
Subsequently, cells were washed and resuspended in PBS containing 50 mg/ml
propidium iodide, 100 mg/ml RNase and 0.1% Nonidet P-40 for 30 min at 37°C. The
distribution of cells in different phases of the cell cycle was determined by measuring
the nuclear DNA content using a FACS Calibur cell flow cytometer (Becton,
Dickinson and company, San Diego, CA).
Statistical analysis. We statistically evaluated experimental results using
two-tailed paired Student’s t test, two-independent sample t test, and Chi-square test.
All tests of significance were set at p < 0.05.
Results
Confirmation of the reduced expression of S100A14 in ESCC compared with the
matched normal epithelia by qRT-PCR. Our previous study demonstrated that
S100A14 expression is downregulated in ESCC versus adjacent normal tissue by
semiquantitative RT-PCR [18]. To further confirm the differential expression of
S100A14 in ESCC, we performed qRT-PCR analysis in 30 paired ESCC and adjacent
normal epithelial tissues. Consistent with the previous results, S100A14 is
significantly reduced in 21 of 30 ESCC tissues compared with adjacent normal
epithelia (paired t-test, p=0.0118) (Fig.1A). The reduced expression of S100A14 was
further confirmed by Western blot in 11 of 14 cases (Fig.1B). These results clearly
demonstrate that S100A14 is markedly downregulated in ESCC compared with the
matched normal epithelia at both the mRNA and protein levels.
Downregulation of S100A14 is associated with ESCC dedifferentiation and
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
9
clinical stage. To further confirm the alteration of S100A14 expression in ESCC and
analyze the correlation between S100A14 and clinicopathological features, we
determined the expression of S100A14 in a tissue microarray comprised of 110 paired
esophageal cancer and adjacent normal samples by immunohistochemical analysis
and evaluated the correlation between S100A14 protein levels and clinicopathological
parameters in 103 cases. The immunostaining results for S100A14 in ESCC and their
corresponding normal epithelia are shown in Fig.1C. S100A14 showed a clear
localization in the plasma membrane in normal esophageal epithelia. In contrast, both
plasma membrane and cytoplasmic staining were observed in esophageal cancer
tissues. The majority of tumors showed focal, positive immunostaining in certain
well-differentiated areas while staining was undetectable in other, less differentiated
sections. In well-differentiated carcinomas, staining for S100A14 was positive in
keratinized areas at the center of tumor foci but was decreased or undetectable in the
marginal areas. However, in moderately and poorly differentiated carcinomas, the
staining was weak or sporadic, occurring only in the well- or
moderately-differentiated regions but completely undetectable in other areas.
Immunohistochemical analysis demonstrated that S100A14 expression was
significantly reduced in ESCC versus matched normal epithelial tissue in 70 of 103
cases (67.9%). Downregulation of S100A14 had a significant correlation with ESCC
dedifferentiation (P = 0.005) and clinical stage (P = 0.028), but had no relationship
with gender, depth of tumor invasion, or lymph node metastasis (Table 1).
Furthermore, we analyzed the correlation between S100A14 expression and
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
10
differentiation in ESCC cell lines. As shown in Fig.1D, S100A14 protein exhibited
higher expression in well-differentiated cells such as KYSE30, KYSE180 and
KYSE510 cells than in cells with poor differentiation such as KYSE70, KYSE410.
S100A14 exhibited moderate expression in cells with intermediate differentiation
such as KYSE150 [19]. These results further confirmed the correlation between
S100A14 expression and esophageal cancer differentiation.
S100A14 is induced during esophageal cancer cell differentiation. Our previous
study showed that TPA induced the expression of a series of differentiation-associated
genes in esophageal cancer cells. To further characterize the alteration of S100A14
levels during ESCC differentiation, we treated esophageal cancer cell lines KYSE30,
KYSE450, and KYSE510 with TPA, and mRNA and protein expression of S100A14
was determined. We found that TPA treatment increased the mRNA and protein levels
of S100A14 in a time-dependent manner in KYSE450 cells. The induction of
S100A14 by TPA occurred at 8 hours (h), with a peak increase of more than 5-fold by
12 h (Fig.2A). However, the induction of S100A14 was not observed in KYSE30 and
KYSE510 cells (Fig.2A). To further confirm these results, we treated cells with
calcium, a commonly used differentiation inducer [22]. Firstly, we investigated the
effect of different doses of calcium on S100A14 expression in KYSE450 cells by
Western blot and immunofluorescence (Fig.S1). The results demonstrated that 2.4
mM CaCl2 effectively induced S100A14 protein expression in cell nuclei. Subsequent
evaluation of calcium-induced S100A14 expression in KYSE450 and KYSE510 cells
showed that 2.4 mM CaCl2 treatment dramatically increased S100A14 mRNA and
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
11
protein levels in a time-dependent manner (Fig.2B). In contrast, there is no obvious
effect on S100A14 expression in KYSE30 cells (Fig.2B). Therefore, we selected
KYSE450 and KYSE510 cells to perform phenotypic characterization in the
following experiments.
S100A14: a late differentiation marker of esophageal cancer cells. Upon
commitment to terminal differentiation, keratinocytes undergo several distinct
differentiation stages. At each stage, keratinocytes express specific
differentiation-associated genes. In the early stage of terminal differentiation, cells
initiate the expression of genes encoding Keratin 1 (KRT1) and Keratin 10 (KRT10)
[23]. At a more advanced stage, cells begin to express Filaggrin (FLG) and other
structural genes, including Involucrin (IVL), Loricrin (LOR) and small proline rich
proteins (SPRRs) [24,25]. To characterize the expression pattern of S100A14 during
the course of differentiation, we determined the correlation of S100A14 expression
with a series of differentiation stage-specific genes to identify the temporal pattern of
S100A14 induction. TPA treatment dramatically increased the expression of a series
of late differentiation markers but had no effect on the early differentiation markers
KRT1 and KRT10 (Fig.3A). Interestingly, the time line of S100A14 expression
overlaps with that of the late differentiation marker SPRR1A (Fig. 3A), which is
strictly linked to keratinocyte terminal differentiation [26,27]. Moreover, the
expression pattern of S100A14 is also similar to that of SPRR1A during
calcium-induced differentiation of esophageal cancer cells. Taken together, these data
suggest that S100A14 may play a role in esophageal cancer cell terminal
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
12
differentiation.
Effect of S100A14 overexpression on cell cycle, morphology, and
calcium-induced cell growth inhibition in KYSE450 cells. To investigate the
functional role of S100A14 in esophageal cancer cell differentiation, we selected
KYSE450 cells to perform overexpression experiments since S100A14 exhibits a
moderate-level expression and can be markedly induced in this cell line. Western blot
analysis demonstrated that S100A14 is effectively overexpressed (Fig.3B, left panel).
We firstly examined the effect of S100A14 overexpression on cell growth with or
without calcium treatment by a direct viable cell count assay. The results showed that
overexpression of S100A14 significantly inhibited cell growth in the absence or
presence of 2.4mM CaCl2 (Fig.3B, right panel). Next, we investigated whether the
changes in cell growth are due to apoptosis or cell cycle arrest. We performed
apoptosis assay using the BD Annexin V-PE Apoptosis Detection Kit. As shown in
Fig.3C (left panel), calcium treatment significantly induced cell apoptosis compared
to vehicle treated cells. However, we failed to observe any increase of apoptotic rate
in S100A14-overexpressing cells compared to that of empty vector-transfected cells,
indicating that overexpression of S100A14 does not increase the sensitivity of
KYSE450 cells to calcium-induced apoptosis. We next measured the cell cycle status
in the absence or presence of 2.4mM CaCl2 at 48 h. Cell cycle distribution analysis
showed that overexpression of S100A14 causes an arrest of cells in G1 phase, with an
increase in the percentage of cells in G1 phase from 38.1±1.2% to 45.1±1.2% in the
absence of calcium or from 42.8±0.3% to 47.5±0.7% in the presence of calcium,
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
13
respectively. Furthermore, calcium hampers the cell cycle progression by arresting the
cells in S-phase. In empty vector-transfected cells, S-phase was increased from
29.9±0.9% to 39.6±0.9%, G2 phase was decreased from 32±2.1% to 17.6±1.2%, and
G1 phase was increased from 38.1±1.2% to 42.8±0.3%. In contrast, in
S100A14-transfected cells, S-phase cells increased from 27.3±1.0% to 34.6±0.4%,
and consistently G2 phase was decreased from 27.5±2.2% to 17.9±1.1%, and G1
phase was increased from 45.1±1.2% to 47.5±0.7% (Fig.3C, right panel). Taken
together, our data strongly suggest that overexpression of S100A14 leads to an arrest
of cells in G1 phase, and calcium further hampers cells in S phase. Arrested cells were
unable to proceed into the G2/M phase thereby leading to the inhibition of cell growth.
However, S100A14-overexpressing cells did not exhibit morphological changes
compared with control cells. Moreover, there is no significant difference in the
differentiation-associated morphological phenotype induced by calcium, suggesting
that the variation of S100A14 expression alone is not sufficient to alter the
differentiation phenotype (Fig.S2). To characterize the effect of S100A14
overexpression on cell differentiation at the molecular level, we examined the
expression of differentiation-associated genes. QRT-PCR analysis showed that
S100A14 overexpression resulted in a 2 fold increase of IVL and 3.4 fold
up-regulation of FLG in calcium-treated cells (Fig.3D). These data indicate that
S100A14 overexpression interferes with calcium-induced cell growth inhibition and
affects the expression of differentiation-associated genes in terminally differentiating
esophageal cancer cells.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
14
Effect of S100A14 knockdown on cell cycle progression and morphology in
KYSE510 cells. To further determine the role of S100A14 in calcium-induced
phenotypic changes, we selected KYSE510 cells to perform the knockdown
experiments since S100A14 exhibits high levels of expression that can be effectively
inhibited in this cell line. Western blot analysis showed that S100A14 expression is
efficiently diminished in S100A14-shRNA-transfected cells (Fig.4A). Cell cycle
analysis showed that S100A14 silencing significantly decreased the proportion of
G1-phase cells (Fig.4A). To examine the effect of S100A14 knockdown on KYSE510
cell differentiation, cells were treated with calcium for 4 days. Calcium treatment in
shControl-transfected cells induced a dramatic change in cell-cell contact. Distinct
spaces between cells became much less apparent and cells stratified within 2 days.
These morphological changes occurred at day 2 of differentiation of KYSE510 cells,
the time point at which S100A14 expression was induced (Fig.4B and Fig.2A).
Knockdown of S100A14 markedly inhibited these calcium-induced morphological
changes. However, calcium treatment of S100A14-silenced KYSE510 cells did not
induce FLG or IVL mRNA expression, whereas S100A14 overexpression resulted in
FLG and IVL mRNA up-regulation in KYSE450 cells, silencing of S100A14 in
KYSE510 cells had no significant effect on expression of these genes (data not
shown). The discrepancy may be due to cell-type differences. Taken together, these
data demonstrate that S100A14 knockdown interferes with cell cycle progression and
affects the esophageal cancer cell terminal differentiation program.
The underlying mechanism of S100A14-mediated esophageal cancer cell
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
15
differentiation. One of the mechanisms of terminal differentiation of keratinocytes
involves the MAP kinase pathway that leads to induction of AP-1, a transcription
factor comprised of members of the Jun and Fos protein families [28]. Our previous
study demonstrated that among the Jun family of transcription factors, c-Jun/AP-1
could bind and activate the expression of a series of differentiation-associated genes
in esophageal cancer cells [29]. Therefore, we speculated that transcriptional
regulation by AP-1 might contribute to the underlying mechanism of S100A14
involved in esophageal cancer cell differentiation. KYSE450 cells were transiently
transfected with a series of AP-1 expression plasmids including JunB, JunD, c-Jun,
c-fos, and Fra-1, and 48 h later, Western blot was performed. As shown in Fig.5A,
ectopic expression of JunB drastically increased S100A14 expression compared with
the empty vector control. In contrast, a slight effect on S100A14 expression was
observed in c-Jun and c-fos overexpressing cells, and overexpression of JunD and
Fra-1 only marginally influenced S100A14 expression. Next, we tested whether JunB,
c-Jun, and c-fos could drive the transcriptional activity of S100A14 in KYSE450 cells.
Expression plasmids for JunB, c-Jun, or c-fos were co-transfected with a S100A14
promoter (-511~+6bp from the transcription start site) reporter plasmid into KYSE450
cells, and 48 h later, luciferase activity was measured. JunB exhibited a greater ability
than c-Jun to stimulate reporter activity (Fig.5B). In contrast, no increase in reporter
activity was observed when the c-fos expression vector was co-transfected. We
performed a chromatin immunoprecipitation (ChIP) assay to ask whether JunB binds
directly to the S100A14 promoter in esophageal cancer cells. The results show that
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
16
JunB is significantly enriched at this regulatory region compared to the IgG control in
KYSE450 cells. As expected, a significant enrichment of the Pol II with the promoter
region of S100A14 gene is also observed (Fig.5C). To ask whether JunB binding leads
to activation of endogenous S100A14, we used siRNAs targeting JunB (two
independent siRNAs) to deplete the endogenous JunB to examine the effect of JunB
on S100A14 expression in KYSE450 cells. As shown in Fig.5D (left panel), both
siRNAs dramatically reduced cellular JunB levels, and effectively decreased
S100A14 protein levels. Meanwhile, we examined the effect of JunB silencing on a
series of differentiation-associated genes mRNA expression levels. As shown in
Fig.5D (right panel), among the ten genes examined by qRT-PCR, silencing of JunB
markedly decreased S100A14, IVL, FLG, LOR, SPRR1A, SPRR3, KRT1, and KRT4
but no KRT10 expression levels. Finally, to assess the correlation between S100A14
and JunB in esophageal cancer tissues, we simultaneously examined the mRNA
expression level of S100A14 and JunB in 30 esophageal cancer tissues and calculated
the Pearson’s correlation coefficient. The term –△Ct (Ctβ-actin – CtS100A14 or CtJunB) was
used to describe the expression of S100A14 and JunB. Statistical analysis indicated
that S100A14 mRNA expression was significantly associated with JunB mRNA
expression in esophageal cancer specimens (Pearson correlation coefficient R=0.582,
P=0.001) (Fig.5E). Collectively, these results suggest a role for JunB in the
transcriptional regulation of S100A14 and provide a molecular mechanism whereby
S100A14 contributes to esophageal cell differentiation.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
17
Discussion
Squamous cell differentiation is a multistep process that requires the coordinated
activation and repression of squamous cell-specific genes, and disruption of
differentiation is an important characteristic of malignant tumors [30,31]. Human
esophageal cancer exhibits a reduced degree of differentiation and defects in the
terminal differentiation pathway [32,33]. A better understanding of the mechanisms
regulating differentiation would offer the basis for identification of tumor biomarkers.
Our previous study showed that S100A14 belongs to a subset of genes that are
down-regulated in esophageal cancers, and as one of many differentiation-associated
genes, reduced S100A14 expression might contribute to esophageal carcinogenesis
[17,18]. Furthermore, our study demonstrated that S100A14 regulated cell
proliferation and apoptosis in a dose-dependent manner via interaction with RAGE in
ESCC [15]. However, information is limited regarding the possible biological
significance of the altered expression of S100A14 during ESCC development. In this
study, we revealed the marked down-regulation of S100A14 expression in the
majority of ESCCs and a significant correlation between S100A14 expression level
and differentiation and clinical stage of ESCC. Well-differentiated or
moderately-differentiated ESCC clinical samples showed higher S100A14 expression
than poorly-differentiated cases, consistent with previous findings that
down-regulation of S100A14 is associated with poor differentiation in colon cancer
[10]. Protein translocation between different subcellular compartments is crucial for
protein function [34]. Concordantly, in this study, we found that S100A14 exhibited
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
18
plasma membrane localization in normal esophageal epithelial tissues but plasma
membrane and cytoplasmic localization in esophageal cancer tissues. Previously,
S100A14 was identified as a plasma membrane-associated protein in breast cancer
cell lines and exhibited an increased expression in breast cancer tissues versus
matched normal tissues. Accordingly, S100A14 exhibited different patterns of
subcellular distribution, typified by plasma membrane localization in breast cancer
tissues but cytosolic expression in non-tumor breast epithelial cells [35]. Previous
studies showed that some members of the S100 family of proteins exhibit
calcium-dependent translocation [36,37], and the translocation of S100A14 is
regulated in a calcium-dependent manner through interaction with nucleobindin,
which has strong association with Gα proteins [38]. We also found that calcium
treatment induced S100A14 expression in cell nuclei in esophageal cancer cell lines,
further suggesting that calcium plays a role in the induction and translocation of
S100A14. These data suggest the difference in subcellular distribution of S100A14
may be regulated by tissue-type-specific factors in a calcium-dependent manner,
which might play an important role in determining the functions of S100A14 in
tumorigenesis and progression.
In addition, we showed that TPA and calcium, known inducers of terminal
differentiation, markedly induced S100A14 expression. S100A14 overexpression and
silencing experiments further substantiated the role of S100A14 in terminal
differentiation of esophageal cancer cells. S100A14 overexpression in KYSE450 cells
inhibited cell growth in the absence or presence of calcium although the
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
19
overexpression of S100A14 alone was not sufficient to induce the morphological
changes associated with terminal differentiation. Importantly, our data demonstrated
that S100A14 can exert anti-cancer function during the process of ESCC
differentiation by blocking the cell cycle in G1 and S phases in the presence of
calcium. In contrast, S100A14 knockdown in KYSE510 cells led to a notable
impairment of differentiation, as was evident morphologically (Fig.4B). Molecular
investigations further supported the morphological findings as altered expression of
S100A14 positively correlated with changes in expression levels of late differentiation
markers such as IVL and FLG, which are major components of the cornified envelope
and are considered to be appropriate markers for terminal differentiation [39,40].
Since S100A14 does not bind to DNA and contains no nuclear localization sequence,
the mechanisms by which S100A14 regulates these terminal differentiation-associated
genes may involve the intermediary activities of S100A14 partner proteins. It will
therefore be of great importance to identify and characterize the proteins with which
S100A14 interacts in future studies. To further identify the effect of S100A14 on the
pathways regulating differentiation, gene expression profiling analysis needs to be
performed for further study.
We also demonstrated that the transcription factor AP-1 is involved in the
transcriptional regulation of S100A14. This is in line with our previous study showing
that AP-1 could transcriptionally regulate a series of differentiation-associated genes
[29]. Here, we have added another gene into the AP-1-regulated network involved in
esophageal cancer cell differentiation. Whereas most AP-1 factors have no significant
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
20
effect on S100A14 expression, we demonstrated that S100A14 is a direct target gene
of JunB, which regulates transcription by directly binding to the proximal S100A14
promoter. This result is consistent with previous reports that different members of the
AP-1 transcriptional complex exhibited varying degrees of importance in regulating
the expression of specific differentiation-related genes. For instance, JunB, JunD and
Fra-1 were identified as major regulators of involucrin expression [41]. Additionally,
most AP-1 factors efficiently bind to the SPRR1A minimal promoter region in
proliferating keratinocytes. Following induction of terminal differentiation, altered
ability of AP-1 factors to bind this sequence, notably JunB and JunD, is observed [27].
Finally, the significant correlation between mRNA expression levels of S100A14 and
JunB further confirmed the regulation of S100A14 by JunB in esophageal cancer
tissues. As our previous study showed that Krüppel-like factor 4 (KLF4) plays an
important role in the transcriptional regulation of differentiation-related genes in
ESCC [5], we cannot exclude the potential contributions of other transcription factors
such as KLF4 in regulating S100A14 during cell differentiation.
In summary, we have characterized the role of S100A14 as a novel and pivotal
modulator of esophageal cancer cell differentiation. Further research on S100A14
should focus on the identification of S100A14 partner proteins and the elucidation of
the molecular mechanisms whereby S100A14 modulates esophageal cancer cell
differentiation.
Acknowledgments
We thank Dr. Iver Petersen and Dr. Youyong Lü for providing the S100A14
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
21
antibodies, and we thank Dr. Marta Barbara Wisniewska for the generous gifts of the
JunB, JunD and c-fos expression plasmids.
References
1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of
worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:
2893-2917.
2. Ke L. Mortality and incidence trends from esophagus cancer in selected
geographic areas of China circa 1970-90. Int J Cancer 2002;102: 271-274.
3. Denlinger CE, Thompson RK. Molecular basis of esophageal cancer development
and progression. Surg Clin North Am 2012;92: 1089-1103.
4. Watanabe S, Ichikawa E, Takahashi H, Otsuka F. Changes of cytokeratin and
involucrin expression in squamous cell carcinomas of the skin during progression
to malignancy. Br J Dermatol 1995;132: 730-739.
5. Luo A, Kong J, Hu G, Liew CC, Xiong M, Wang X, et al. Discovery of
Ca2+-relevant and differentiation-associated genes downregulated in esophageal
squamous cell carcinoma using cDNA microarray. Oncogene 2004; 23:
1291-1299.
6. Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Genes encoding structural
proteins of epidermal cornification and S100 calcium-binding proteins form a
gene complex ("epidermal differentiation complex") on human chromosome 1q21.
J Invest Dermatol 1996;106: 989-992.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
22
7. Cao LY, Yin Y, Li H, Jiang Y, Zhang HF. Expression and clinical significance of
S100A2 and p63 in esophageal carcinoma. World J Gastroenterol 2009;15:
4183-4188.
8. Zhang HY, Zheng XZ, Wang XH, Xuan XY, Wang F, Li SS. S100A4 mediated
cell invasion and metastasis of esophageal squamous cell carcinoma via the
regulation of MMP-2 and E-cadherin activity. Mol Biol Rep 2012;39: 199-208.
9. Hua Z, Chen J, Sun B, Zhao G, Zhang Y, Fong Y, et al. Specific expression of
osteopontin and S100A6 in hepatocellular carcinoma. Surgery 2011;149: 783-791.
10. Wang HY, Zhang JY, Cui JT, Tan XH, Li WM, Gu J, et al. Expression status of
S100A14 and S100A4 correlates with metastatic potential and clinical outcome in
colorectal cancer after surgery. Oncol Rep 2010;23: 45-52.
11. Ohuchida K, Mizumoto K, Miyasaka Y, Yu J, Cui L, Yamaguchi H, et al.
Over-expression of S100A2 in pancreatic cancer correlates with progression and
poor prognosis. J Pathol 2007;216: 275-282.
12. Rosty C, Ueki T, Argani P, Jansen M, Yeo CJ, Cameron JL, et al. Overexpression
of S100A4 in pancreatic ductal adenocarcinomas is associated with poor
differentiation and DNA hypomethylation. Am J Pathol 2002;160: 45-50.
13. Kong JP, Ding F, Zhou CN, Wang XQ, Miao XP, Wu M, et al. Loss of
myeloid-related proteins 8 and myeloid-related proteins 14 expression in human
esophageal squamous cell carcinoma correlates with poor differentiation. World J
Gastroenterol 2004;10: 1093-1097.
14. Xiao MB, Jiang F, Ni WK, Chen BY, Lu CH, Li XY, et al. High expression of
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
23
S100A11 in pancreatic adenocarcinoma is an unfavorable prognostic marker. Med
Oncol 2012;29: 1886-1891.
15. Jin Q, Chen H, Luo A, Ding F, Liu Z. S100A14 stimulates cell proliferation and
induces cell apoptosis at different concentrations via receptor for advanced
glycation end products (RAGE). PLoS One 2011;6: e19375.
16. Chen H, Yuan Y, Zhang C, Luo A, Ding F, Ma J, et al. Involvement of S100A14
protein in cell invasion by affecting expression and function of matrix
metalloproteinase (MMP)-2 via p53-dependent transcriptional regulation. J Biol
Chem 2012;287: 17109-17119.
17. Chen H, Yu D, Luo A, Tan W, Zhang C, Zhao D, et al. Functional role of S100A14
genetic variants and their association with esophageal squamous cell carcinoma.
Cancer Res 2009; 69: 3451-3457.
18. Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, et al. Differential expression of S100
gene family in human esophageal squamous cell carcinoma. J Cancer Res Clin
Oncol 2004;130: 480-486.
19. Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T. Characterization of 21
newly established esophageal cancer cell lines. Cancer 1992;69: 277-284.
20. Zhang C, Zhu C, Chen H, Li L, Guo L, Jiang W, et al. Kif18A is involved in
human breast carcinogenesis. Carcinogenesis 2010;31: 1676-1684.
21. Wang Q, Li W, Liu XS, Carroll JS, Janne OA, Keeton EK, et al. hierarchical
network of transcription factors governs androgen receptor-dependent prostate
cancer growth. Mol Cell 2007;27: 380-392.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
24
22. Xie Z, Singleton PA, Bourguignon LY, Bikle DD. Calcium-induced human
keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol
3-kinase-dependent activation of phospholipase C-gamma1. Mol Biol Cell
2005;16: 3236-3246.
23. Fuchs E, Green H. Changes in keratin gene expression during terminal
differentiation of the keratinocyte. Cell 1980;19: 1033-1042.
24. Steven AC, Steinert PM. Protein composition of cornified cell envelopes of
epidermal keratinocytes. J Cell Sci 1994;107 ( Pt 2): 693-700.
25. Kalinin A, Marekov LN, Steinert PM. Assembly of the epidermal cornified cell
envelope. J Cell Sci 2001;114: 3069-3070.
26. Kartasova T, van Muijen GN, van Pelt-Heerschap H, van de Putte P. Novel protein
in human epidermal keratinocytes: regulation of expression during differentiation.
Mol Cell Biol 1988;8: 2204-2210.
27. Sark MW, Fischer DF, de Meijer E, van de Putte P, Backendorf C. AP-1 and ets
transcription factors regulate the expression of the human SPRR1A keratinocyte
terminal differentiation marker. J Biol Chem 1998 ;273: 24683-24692.
28. Angel P, Szabowski A, Schorpp-Kistner M. Function and regulation of AP-1
subunits in skin physiology and pathology. Oncogene 2001;20: 2413-2423.
29. Yu X, Luo A, Zhou C, Ding F, Wu M, Zhan Q, et al. Differentiation-associated
genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in
KYSE450 cells. Biochem Biophys Res Commun 2006;342: 286-292.
30. Simon M, Green H. Participation of membrane-associated proteins in the
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
25
formation of the cross-linked envelope of the keratinocyte. Cell 1984;36: 827-834.
31. Tenen DG. Disruption of differentiation in human cancer: AML shows the way.
Nat Rev Cancer 2003;3: 89-101.
32. Banks-Schlegel SP, Quintero J. Growth and differentiation of human esophageal
carcinoma cell lines. Cancer Res 1986;46: 250-258.
33. Helm J, Enkemann SA, Coppola D, Barthel JS, Kelly ST, Yeatman TJ.
Dedifferentiation precedes invasion in the progression from Barrett’s metaplasia
to esophageal adenocarcinoma. Clin Cancer Res 2005;11:2478–2485.
34. Cross BC, Sinning I, Luirink J, High S. Delivering proteins for export from the
cytosol. Nat Rev Mol Cell Biol 2009; 10:255-264.
35. Adam PJ, Boyd R, Tyson KL, Fletcher GC, Stamps A, Hudson L, et al.
Comprehensive proteomic analysis of breast cancer cell membranes reveals
unique proteins with potential roles in clinical cancer. J Biol Chem 2005;
278:6482-6489.
36. Stradal TB, Gimona M. Ca (2+)-dependent association of S100A6 (Calcyclin)
with the plasma membrane and the nuclear envelope. J Biol Chem. 1999; 274:
31593-31596.
37. Davey GE, Murmann P, Hoechli M, Tanaka T, Heizmann CW. Calcium-dependent
translocation of S100A11 requires tubulin filaments. Biochim Biophys Acta 2000;
1498: 220-232.
38. Lin P, Fischer T, Weiss T, Farquhar MG. Calnuc, an EF-hand Ca(2+) binding
protein, specifically interacts with the C-terminal alpha5-helix of G(alpha)i3. Proc
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
26
Natl Acad Sci U S A 2000; 97: 674-679.
39. Rice RH, Green H. Presence in human epidermal cells of a soluble protein
precursor of the cross-linked envelope: activation of the cross-linking by calcium
ions. Cell 1979; 18: 681-694.
40. Sandilands A, Sutherland C, Irvine AD, McLean WH. Filaggrin in the frontline:
role in skin barrier function and disease. J Cell Sci 2009; 122: 1285-1294.
41. Welter JF, Crish JF, Agarwal C, Eckert RL. Fos-related antigen (Fra-1), junB, and
junD activate human involucrin promoter transcription by binding to proximal and
distal AP1 sites to mediate phorbol ester effects on promoter activity. J Biol Chem
1995; 270: 12614-12622
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
27
Table 1.The correlation between S100A14 underexpression in ESCC and clinicopathologic features
Characteristics
S100A14 expression
Total P Non-underexpressed* underexpressed* (%) (%)
Overall 33 70 103
TNM classification
pT
pT1 0 (0) 2 (100) 2 0.615
pT2 12 (33.3) 24 (66.7) 36
pT3 21 (32.3) 44 (67.7) 65
N
N0 22 (31.4) 48 (68.6) 70 0.847
N1 11(33.3) 22 (66.7) 33
Clinical stage
I 3 (42.9) 4 (57.1) 7 0.028
II 27 (39.7) 41 (60.3) 68
III 3 (8.3) 23 (91.7) 36
IV 0 0 0
Differentiation 0.005
Well 18 (46.2) 21 (53.8) 39
Moderately 15 (30.6) 34 (69.4) 49
Poorly 0 (0) 15 (100) 15
NOTE: These results were analyzed by the Pearson X2 test. P values with significance
are shown as superscripts.
*For S100A14 expression levels, a matched cancer/normal ratio ≥ 1 was defined as
the Non-underexpressed group, and a ratio < 1 was defined as the underexpressed
group.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
28
Figure Legends
Figure 1. Reduced expression of S100A14 mRNA and protein in esophageal
cancer. (A) Down-regulated S100A14 mRNA level was detected in 21 of 30 tumors
(T) compared with normal adjacent epithelia (N) by qRT-PCR. (B) S100A14 protein
level was reduced in 11 of 14 malignant tissues versus corresponding normal epithelia
by Western blot analysis. (C) Example case showing that S100A14 is underexpressed
in esophageal tumors by immunohistochemical staining on the tissue microarray.
There were three normal tissues and four cancer tissues in each case. Representative
pictures of S100A14 in normal esophageal epithelium ① and well- ②, moderately-
③ and poorly-differentiated ④ carcinoma tissues were shown. (D) A series of
esophageal cancer cells were harvested and the lysates were probed with
anti-S100A14 antibody, β-actin was used as loading control.
Figure 2. S100A14 expression is regulated during TPA and calcium-induced
esophageal cancer cell differentiation. (A) Esophageal cancer cells including
KYSE30, KYSE450 and KYSE510 cells were cultured in the presence of 100ng/ml
TPA, cells were harvested at indicated time points. Left panel: S100A14 expression
was examined by qRT-PCR. Data are presented as mean±S.D. of the fold difference;
Right panel, S100A14 expression was determined by Western blot. (B) Esophageal
cancer cells including KYSE30, KYSE450 and KYSE510 cells were treated with
2.4mM CaCl2, cells were harvested at indicated time points. Left panel: qRT-PCR was
performed to analyze the mRNA expression of S100A14; Right panel: Immunoblots
using anti-S100A14 antibody to analyze expression of S100A14 protein.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
29
Figure 3. S100A14 acts as a late terminal differentiation modulator and regulates
esophageal cancer cell differentiation. (A) qRT-PCR analysis was performed to
analyze mRNA expression of a selected group of terminal differentiation genes in
KYSE450 cells treated by TPA (left panel) and 2.4mM CaCl2 (right panel). (B)
KYSE450 cells were transfected with pcDNA3.1 and pcDNA3.1-S100A14 vectors,
stable cells were established by Geneticin (G418) selection for about two weeks. Left
panel: cells were harvested and Western blot was performed to measure the protein
expression of S100A14. Right panel: Decreased cell growth of S100A14-transfected
KYSE450 cells compared with empty vector-transfected KYSE450 cells with or
without calcium treatment (mean (n=2)±S.D.) (two-sided t-test, *P<0.05). (C) Empty
vector-transfected and S100A14-overexpressed KYSE450 cells were seeded at 1×105
cells/well in conventional medium with or without 2.4mM CaCl2 on 6-well plates,
cells were stained with Annexin V-PE (AV-PE) and 7-AAD (left panel) or propidium
iodide (PI) (right panel) , and analyzed by flow cytometry at 48 h. (D) S100A14
regulates differentiation-associated genes expression. Cells were treated with CaCl2
(2.4mM) for 48h. The cells were harvested, total RNA was isolated and mRNA
expression of IVL and FLG genes was examined by qRT-PCR.
Figure 4. Depletion of S100A14 inhibits calcium-induced cell differentiation. (A)
KYSE510 cells were transfected with control shRNA and two different S100A14
shRNAs (shRNA-1 and shRNA-2), and stable cells were obtained by G418 selection
for about two weeks. Cells were harvested and Western blot was performed using
anti-S100A14 antibody, β-actin was used as loading control. Cell cycle distribution
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
30
was analyzed by FACS, and a significant G1-phase decrease was observed in
S100A14-silenced cells compared with control shRNA-transfected cells. (mean
(n=2)±S.D.) (two-sided t-test, *P<0.05). (B) Morphological studies at different time
points in KYSE510 cell differentiation. S100A14-silenced cells and corresponding
control cells were cultivated in conventional medium supplemented with 2.4mM
CaCl2 at the indicated time points, phase-contrast photomicrographs were taken.
Figure 5. AP-1 is involved in the transcriptional regulation of S100A14. (A)
KYSE450 cells were transiently transfected with a series of AP-1family expression
vectors including JunB, JunD, c-Jun, c-fos, and Fra-1, 48 h later, cells were harvested
and Western blot was performed using anti-S100A14 antibody, β-actin was used as
loading control. (B) S100A14 promoter construct was cotransfected with the indicated
constructs into KYSE450 cells, 48 h later, reporter activity was then determined. Data
are presented as mean±SEM of the fold difference. (C) ChIP assay demonstrated that
JunB and Pol II were enriched in the promoter region of the S100A14 gene. KYSE450
cells were harvested, ChIP assay was performed with anti-JunB, anti-Pol II antibodies,
and anti-Rabbit IgG antibody was used as a negative control. (D) JunB directly
regulates the expression of target genes involved in differentiation. Two independent
siRNAs targeting JunB and Control siRNAs were transfected into KYSE450 cells, 72
h later, cells were harvested. Left panel: Western blot was performed using anti-JunB
and anti-S100A14 antibodies, β-actin was used as loading control. Right panel: Total
RNA was isolated and mRNA expression of differentiation-associated genes was
examined by qRT-PCR. (E) The mRNA expression of S100A14 is correlated with the
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
31
mRNA expression of JunB in ESCC. The correlation between the mRNA expression
of S100A14 (y axis) and JunB (x axis) in tumor is analyzed in ESCC specimens.
Correlation coefficient is 0.582 and P is 0.001.
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317
Published OnlineFirst October 9, 2013.Mol Cancer Res Hongyan Chen, Jianlin Ma, Benjamin Sunkel, et al. esophageal squamous cell carcinomaS100A14 is a novel modulator of terminal differentiation of
Updated version
10.1158/1541-7786.MCR-13-0317doi:
Access the most recent version of this article at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://mcr.aacrjournals.org/content/early/2013/10/09/1541-7786.MCR-13-0317To request permission to re-use all or part of this article, use this link
on April 20, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317