S100A14 Is a Novel Modulator of Terminal Differentiation of Esophageal

Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

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

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

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

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

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.

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

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

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

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

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,

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.

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

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

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.

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

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

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

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

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.

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

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.

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

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

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

Table 1.The correlation between S100A14 underexpression in ESCC and clinicopathologic features S100A14 expression

Characteristics Non-underexpressed* underexpressed* Total P (%) (%) 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.

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.

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

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

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.

S100A14 is a novel modulator of terminal differentiation of esophageal squamous cell carcinoma

Hongyan Chen, Jianlin Ma, Benjamin Sunkel, et al.

Mol Cancer Res Published OnlineFirst October 9, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-13-0317

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2021/03/16/1541-7786.MCR-13-0317.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/early/2013/10/09/1541-7786.MCR-13-0317. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.