Aberrant keratinocyte differentiation is a key mechanism in the initiation of cancer. Because activities regulating differentiation exhibit altered or reduced capacity in esophageal cancer cells, it is vital to pinpoint those genes that control epidermal proliferation and terminal differentiation to better understand esophageal carcinogenesis. S100A14 is a member of the S100 calcium-binding protein family and has been suggested to be involved in cell proliferation, apoptosis, and invasion. The present study used immunohistochemistry analysis of S100A14 in clinical specimens of esophageal squamous cell carcinoma (ESCC) to show that decreased S100A14 is strongly correlated with poor differentiation. Furthermore, both 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 a G1-phase cell cycle arrest and promoted calcium-inhibited cell growth. Conversely, decreasing S100A14 expression significantly promoted G1–S transition and prevented the morphologic changes associated with calcium-induced cell differentiation. Molecular investigation demonstrated that S100A14 altered the calcium-induced expression of late markers of differentiation, with the most prominent effect on involucrin (IVL) and filaggrin (FLG). Finally, it was determined that S100A14 is transcriptionally regulated by JunB and that S100A14 and JunB status significantly correlated in ESCC tissue. In summary, these data demonstrate that S100A14 is transcriptionally regulated by JunB and involved in ESCC cell differentiation.

Implications: This study further differentiates the molecular mechanism controlling the development and progression of esophageal cancer. Mol Cancer Res; 11(12); 1542–53. ©2013 AACR.

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). More than 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 biologic abnormalities including altered gene expression, gene mutations, aberrant signaling pathways, and genetic alterations contribute to the development and progression of ESCCs (3). In addition, the disruption of epithelial differentiation may be one of the primary mechanisms for ESCC (4). Our previous studies have clearly shown that a series of genes involved in squamous cell differentiation were coordinately downregulated in ESCCs (5). Among them, S100 calcium-binding proteins have attracted additional attention as they are implicated in a variety of biologic 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; ref. 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 ESCCs (7–14). We have recently reported on the role of the S100 family member, S100A14, in driving esophageal carcinogenesis, showing 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′-untranslated region (UTR) of S100A14 is associated with ESCC susceptibility in a Chinese population (17), providing additional support for the role of S100A14 in tumorigenesis. These findings prompted us to further investigate the functional role of S100A14 and the correlation between S100A14 levels and clinicopathological features in ESCCs.

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 clinicopathologic parameters of ESCCs. Furthermore, we examined the induction of S100A14 upon 12-O-tetra-decanoylphorbol-13-acetate (TPA) and calcium treatment in esophageal cancer cells and investigated the role of S100A14 in calcium-induced esophageal cancer cell morphologic change and differentiation-related gene expression changes. Finally, we provided a preliminary investigation on the underlying mechanism of S100A14-mediated cell differentiation.

Tissue specimens

Tissue samples from 30 patients with ESCCs 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 ESCCs were analyzed by immunohistochemistry (IHC). Patients were recruited at the Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China). 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 histologic examination.

IHC staining

An ESCC tissue microarray including 110 esophageal tumors and the corresponding normal epithelia was constructed with each case represented twice. For IHC staining, the slides were deparaffinized, rehydrated, then immersed in 3% hydrogen peroxide solution for 10 minutes, heated in citrate buffer (pH 6.0) at 95°C for 25 minutes, and cooled at room temperature for 60 minutes. The slides were blocked by 10% normal goat serum at 37°C for 30 minutes 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). 3,3′-Diaminobenzidine (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 2 groups according to the expression score ratio of matched cancer/normal tissue (ratio ≥ 1 was defined as the nonunderexpressed 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; ref. 19). Cells were maintained in RPMI-1640 supplemented with 10% FBS, 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 (25 nmol/L) 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 reverse transcription polymerase chain reaction (qRT-PCR) were performed as previously described (17). Primers used are listed in Supplementary Table S1.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was performed as previously described (21) using anti-JunB (5712-S) antibody from Epitomics and RNA Polymerase II (MA1-10882) antibody from Thermo Scientific Pierce. Primers used are listed in supplementary Table S1.

Western blot analysis

Western blot analyses 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). anti-JunB (5712-S) and anti-JunD (5226-1) were from Epitomics, anti-c-Jun(Sc-1694), anti-c-fos(Sc-52), and anti-Fra-1(Sc-183) were from Santa Cruz Biotechnology.

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) 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 cytometric analysis.

Fluorescence-activated cell sorting 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 minutes 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).

Statistical analysis

We statistically evaluated experimental results using 2-tailed paired Student t test, 2-independent sample t test, and χ2 test. All tests of significance were set at P < 0.05.

Confirmation of the reduced expression of S100A14 in ESCC compared with the matched normal epithelia by qRT-PCR

Our previous study showed that S100A14 expression is downregulated in ESCCs versus adjacent normal tissue by semiquantitative RT-PCR (18). To further confirm the differential expression of S100A14 in ESCCs, we performed qRT-PCR analysis in 30 paired ESCCs 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 blotting in 11 of 14 cases (Fig. 1B). These results clearly show that S100A14 is markedly downregulated in ESCCs compared with the matched normal epithelia at both the mRNA and protein levels.

Figure 1.

Reduced expression of S100A14 mRNA and protein in esophageal cancer. A, downregulated 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 IHC staining on the tissue microarray. There were 3 normal tissues and 4 cancer tissues in each case. Representative pictures of S100A14 in normal esophageal epithelium and well- , moderately , and poorly differentiated carcinoma tissues were shown. D, a series of esophageal cancer cells was harvested and the lysates were probed with anti-S100A14 antibody. β-Actin was used as loading control.

Figure 1.

Reduced expression of S100A14 mRNA and protein in esophageal cancer. A, downregulated 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 IHC staining on the tissue microarray. There were 3 normal tissues and 4 cancer tissues in each case. Representative pictures of S100A14 in normal esophageal epithelium and well- , moderately , and poorly differentiated carcinoma tissues were shown. D, a series of esophageal cancer cells was harvested and the lysates were probed with anti-S100A14 antibody. β-Actin was used as loading control.

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Downregulation of S100A14 is associated with ESCC dedifferentiation and clinical stage

To further confirm the alteration of S100A14 expression in ESCCs and analyze the correlation between S100A14 and clinicopathologic features, we determined the expression of S100A14 in a tissue microarray comprised of 110 paired esophageal cancer and adjacent normal samples by IHC analysis and evaluated the correlation between S100A14 protein levels and clinicopathologic parameters in 103 cases. The immunostaining results for S100A14 in ESCCs 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. S100A14 exhibited focal, positive immunostaining in certain well-differentiated areas, whereas 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. IHC analysis showed that S100A14 expression was significantly reduced in ESCCs 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 and 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.

Table 1.

The correlation between S100A14 underexpression in ESCCs and clinicopathologic features

S100A14 expression
CharacteristicsNonunderexpresseda (%)Underexpresseda (%)TotalP
Overall 33 70 103  
TNM classification 
 pT     
  pT1 0 (0) 2 (100) 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) 0.028 
  II 27 (39.7) 41 (60.3) 68  
  III 3 (8.3) 23 (91.7) 36  
  IV  
 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  
S100A14 expression
CharacteristicsNonunderexpresseda (%)Underexpresseda (%)TotalP
Overall 33 70 103  
TNM classification 
 pT     
  pT1 0 (0) 2 (100) 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) 0.028 
  II 27 (39.7) 41 (60.3) 68  
  III 3 (8.3) 23 (91.7) 36  
  IV  
 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 χ2 test. P values with significance are shown as superscripts.

aFor S100A14 expression levels, a matched cancer/normal ratio ≥ 1 was defined as the nonunderexpressed group, and a ratio <1 was defined as the underexpressed group.

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, with a peak increase of more than 5-fold by 12 hours (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). First, we investigated the effect of different doses of calcium on S100A14 expression in KYSE450 cells by Western blotting and immunofluorescence (Supplementary Fig. S1). The results showed that 2.4 mmol/L 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 mmol/L 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 performed phenotypic characterization in the following experiments.

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 100 ng/mL TPA. Cells were harvested at indicated time points. Left, S100A14 expression was examined by qRT-PCR. Data are presented as mean ± SD of the fold difference; right, S100A14 expression was determined by Western blotting. B, esophageal cancer cells including KYSE30, KYSE450, and KYSE510 cells were treated with 2.4 mmol/L CaCl2, cells were harvested at indicated time points. Left, qRT-PCR was performed to analyze the mRNA expression of S100A14; right, immunoblots using anti-S100A14 antibody to analyze expression of S100A14 protein.

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 100 ng/mL TPA. Cells were harvested at indicated time points. Left, S100A14 expression was examined by qRT-PCR. Data are presented as mean ± SD of the fold difference; right, S100A14 expression was determined by Western blotting. B, esophageal cancer cells including KYSE30, KYSE450, and KYSE510 cells were treated with 2.4 mmol/L CaCl2, cells were harvested at indicated time points. Left, qRT-PCR was performed to analyze the mRNA expression of S100A14; right, immunoblots using anti-S100A14 antibody to analyze expression of S100A14 protein.

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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; ref. 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 (SPRR; refs. 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.

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) and 2.4 mmol/L CaCl2 (right). B, KYSE450 cells were transfected with pcDNA3.1 and pcDNA3.1-S100A14 vectors, stable cells were established by Geneticin (G418) selection for about 2 weeks. Left, cells were harvested and Western blotting was performed to measure the protein expression of S100A14. Right, decreased cell growth of S100A14-transfected KYSE450 cells compared with empty vector–transfected KYSE450 cells with or without calcium treatment [mean (n = 2) ± SD; two-sided t test; *, P < 0.05]. C, empty vector–transfected and S100A14-overexpressed KYSE450 cells were seeded at 1 × 105 cells per well in conventional medium with or without 2.4 mmol/L CaCl2 on 6-well plates, cells were stained with Annexin V-PE (AV-PE) and 7-AAD (left) or propidium iodide (PI; right) and analyzed by flow cytometry at 48 hours. D, S100A14 regulates differentiation-associated genes expression. Cells were treated with CaCl2 (2.4 mmol/L) for 48 hours. The cells were harvested, total RNA was isolated, and mRNA expression of IVL and FLG genes was examined by qRT-PCR.

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) and 2.4 mmol/L CaCl2 (right). B, KYSE450 cells were transfected with pcDNA3.1 and pcDNA3.1-S100A14 vectors, stable cells were established by Geneticin (G418) selection for about 2 weeks. Left, cells were harvested and Western blotting was performed to measure the protein expression of S100A14. Right, decreased cell growth of S100A14-transfected KYSE450 cells compared with empty vector–transfected KYSE450 cells with or without calcium treatment [mean (n = 2) ± SD; two-sided t test; *, P < 0.05]. C, empty vector–transfected and S100A14-overexpressed KYSE450 cells were seeded at 1 × 105 cells per well in conventional medium with or without 2.4 mmol/L CaCl2 on 6-well plates, cells were stained with Annexin V-PE (AV-PE) and 7-AAD (left) or propidium iodide (PI; right) and analyzed by flow cytometry at 48 hours. D, S100A14 regulates differentiation-associated genes expression. Cells were treated with CaCl2 (2.4 mmol/L) for 48 hours. The cells were harvested, total RNA was isolated, and mRNA expression of IVL and FLG genes was examined by qRT-PCR.

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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 as S100A14 exhibits a moderate-level expression and can be markedly induced in this cell line. Western blot analysis showed that S100A14 is effectively overexpressed (Fig. 3B, left). 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 2.4 mmol/L CaCl2 (Fig. 3B, right). 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), 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 2.4 mmol/L CaCl2 at 48 hours. 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). 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 morphologic 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 (Supplementary 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 upregulation 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 as 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 morphologic changes occurred at day 2 of differentiation of KYSE510 cells, the time point at which S100A14 expression was induced (Figs. 4B and 2A). Knockdown of S100A14 markedly inhibited these calcium-induced morphologic 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 upregulation 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 show that S100A14 knockdown interferes with cell-cycle progression and affects the esophageal cancer cell terminal differentiation program.

Figure 4.

Depletion of S100A14 inhibits calcium-induced cell differentiation. A, KYSE510 cells were transfected with control shRNA and 2 different S100A14 shRNAs (shRNA-1 and shRNA-2), and stable cells were obtained by G418 selection for about 2 weeks. Cells were harvested and Western blotting 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) ± SD; 2-sided t test; *, P < 0.05]. B, morphologic studies at different time points in KYSE510 cell differentiation. S100A14-silenced cells and corresponding control cells were cultivated in conventional medium supplemented with 2.4 mmol/L CaCl2 at the indicated time points, phase-contrast photomicrographs were taken.

Figure 4.

Depletion of S100A14 inhibits calcium-induced cell differentiation. A, KYSE510 cells were transfected with control shRNA and 2 different S100A14 shRNAs (shRNA-1 and shRNA-2), and stable cells were obtained by G418 selection for about 2 weeks. Cells were harvested and Western blotting 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) ± SD; 2-sided t test; *, P < 0.05]. B, morphologic studies at different time points in KYSE510 cell differentiation. S100A14-silenced cells and corresponding control cells were cultivated in conventional medium supplemented with 2.4 mmol/L CaCl2 at the indicated time points, phase-contrast photomicrographs were taken.

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The underlying mechanism of S100A14-mediated esophageal cancer cell differentiation

One of the mechanisms of terminal differentiation of keratinocytes involves the mitogen-activated protein (MAP) kinase pathway that leads to induction of AP-1, a transcription factor composed of members of the Jun and Fos protein families (28). Our previous study showed 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 hours later, Western blotting 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 cotransfected with a S100A14 promoter (−511∼+6 bp from the transcription start site) reporter plasmid into KYSE450 cells, and 48 hours 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 cotransfected. We performed a 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 (2 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), 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), among the 10 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 correlation coefficient. The term −ΔCt (Ct_β-actinCt_S100A14 or Ct_JunB) 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.

Figure 5.

AP-1 is involved in the transcriptional regulation of S100A14. A, KYSE450 cells were transiently transfected with a series of AP-1 family expression vectors including JunB, JunD, c-Jun, c-fos, and Fra-1; 48 hours later, cells were harvested and Western blotting 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 hours later, reporter activity was then determined. Data are presented as mean ± SEM of the fold difference. C, ChIP assay showed 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 hours later, cells were harvested. Left, Western blotting was performed using anti-JunB and anti-S100A14 antibodies. β-Actin was used as loading control. Right, 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 ESCCs. 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.

Figure 5.

AP-1 is involved in the transcriptional regulation of S100A14. A, KYSE450 cells were transiently transfected with a series of AP-1 family expression vectors including JunB, JunD, c-Jun, c-fos, and Fra-1; 48 hours later, cells were harvested and Western blotting 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 hours later, reporter activity was then determined. Data are presented as mean ± SEM of the fold difference. C, ChIP assay showed 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 hours later, cells were harvested. Left, Western blotting was performed using anti-JunB and anti-S100A14 antibodies. β-Actin was used as loading control. Right, 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 ESCCs. 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.

Close modal

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 downregulated in esophageal cancers, and as one of many differentiation-associated genes, reduced S100A14 expression might contribute to esophageal carcinogenesis (17, 18). Furthermore, our study showed that S100A14 regulated cell proliferation and apoptosis in a dose-dependent manner via interaction with RAGE in ESCC (15). However, information is limited about the possible biologic significance of the altered expression of S100A14 during ESCC development. In this study, we revealed the marked downregulation of S100A14 expression in the majority of ESCCs and a significant correlation between S100A14 expression level and differentiation and clinical stage of ESCCs. Well-differentiated or moderately differentiated ESCC clinical samples showed higher S100A14 expression than poorly differentiated cases, consistent with previous findings that downregulation 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 nontumor 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 morphologic changes associated with terminal differentiation. Importantly, our data showed 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 morphologic 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). As 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 showed 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 showed 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). In addition, 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 ESCCs (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.

No potential conflicts of interest were disclosed.

Conception and design: H. Chen, B. Sunkel, Q. Wang, Z.-H. Liu

Development of methodology: H. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Chen, J. Ma, A. Luo, F. Ding, Z.-H. Liu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Chen, J. Ma, A. Luo, Y. Li, H. He, S. Zhang, C. Xu, Q. Jin

Writing, review, and/or revision of the manuscript: H. Chen, B. Sunkel, Q. Wang, Z.-H. Liu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Li

Study supervision: Z.-H. Liu

The authors thank Dr. Iver Petersen and Dr. Youyong Lü for providing the S100A14 antibodies and Dr. Marta Barbara Wisniewska for the generous gifts of the junB, junD, and c-fos expression plasmids.

The study was funded by National Natural Science Foundation of China (81000954) and Doctoral Fund of Ministry of Education of China (20101106120012).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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