S100A14: Novel Modulator of Terminal Differentiation in Esophageal Cancer

Published OnlineFirst October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0317

Molecular Cancer Chromatin, Gene, and RNA Regulation Research

Hongyan Chen1, Jianlin Ma1, Benjamin Sunkel2, Aiping Luo1, Fang Ding1,YiLi1, Huan He1, Shuguang Zhang1, Chengshan Xu1, Qinge Jin1, Qianben Wang1,2, and Zhihua Liu1

Abstract 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 fi – cell growth. Conversely, decreasing S100A14 expression signi cantly 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.

Introduction contribute to the development and progression of ESCCs Ranking eighth in incidence and sixth in cancer-related (3). In addition, the disruption of epithelial differentiation mortality worldwide, esophageal cancer is among the most may be one of the primary mechanisms for ESCC (4). Our aggressive cancers occurring with such high frequency (1). previous studies have clearly shown that a series of genes More than 80% of esophageal cancers occur in developing involved in squamous cell differentiation were coordinately countries, but these malignancies are particularly prevalent downregulated in ESCCs (5). Among them, S100 calcium- in China and other countries in Asian, where esophageal binding proteins have attracted additional attention as they squamous cell carcinoma (ESCC) is most common (1, 2). are implicated in a variety of biologic events closely related to Accumulating evidence shows that a variety of biologic tumorigenesis and cancer progression. abnormalities including altered gene expression, gene muta- Most S100 proteins are clustered at the chromosomal tions, aberrant signaling pathways, and genetic alterations 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 impli- Authors' Afﬁliations: 1State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and cated in cancer as altered expression levels of several S100 Peking Union Medical College, Beijing, China; and 2Department of Molec- proteins have been reported to correlate with tumor differ- ular and Cellular Biochemistry and the Comprehensive Cancer Center, The entiation including ESCCs (7–14). We have recently Ohio State University College of Medicine, Columbus, Ohio reported on the role of the S100 family member, Note: Supplementary data for this article are available at Molecular Cancer S100A14, in driving esophageal carcinogenesis, showing Research Online (http://mcr.aacrjournals.org/). that extracellular S100A14 affects esophageal cancer cell Corresponding Author: Zhihua Liu, State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical proliferation and apoptosis via interaction with RAGE, and Sciences, Beijing 100021, China. Phone: 8610-87788490; Fax: 8610- intracellular S100A14 regulates cell invasion by MMP2 in a 67723789; E-mail: [email protected] p53-dependent manner (15, 16). Moreover, the 461G>A 0 doi: 10.1158/1541-7786.MCR-13-0317 SNP located in the 5 -untranslated region (UTR) of 2013 American Association for Cancer Research. S100A14 is associated with ESCC susceptibility in a Chinese

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The Role of S100A14 in ESCC Differentiation

population (17), providing additional support for the role of expression score was determined by multiplying the per- S100A14 in tumorigenesis. These findings prompted us to centage of staining by the staining intensity graded 0 to 3. further investigate the functional role of S100A14 and the The cohort was divided into 2 groups according to the correlation between S100A14 levels and clinicopathological expression score ratio of matched cancer/normal tissue (ratio features in ESCCs. 1wasdefined as the nonunderexpressed group, and ratio < In the present study, we examined the expression of 1 was defined as the underexpressed group). Representative S100A14 in clinical ESCC samples and their matched areas of each section were selected. normal esophageal epithelia and analyzed the relationships between S100A14 expression and the clinicopathologic Cell culture parameters of ESCCs. Furthermore, we examined the induc- Human ESCC cell lines (KYSE series) were gifts from Dr. tion of S100A14 upon 12-O-tetra-decanoylphorbol-13-ace- Y. Shimada of Kyoto University (Kyoto, Japan; ref. 19). Cells tate (TPA) and calcium treatment in esophageal cancer cells were maintained in RPMI-1640 supplemented with 10% and investigated the role of S100A14 in calcium-induced FBS, 100 U/mL streptomycin, and 100 U/mL penicillin. esophageal cancer cell morphologic change and differentiation-related gene expression changes. Finally, we provided a Plasmids preliminary investigation on the underlying mechanism of Full-length cDNA of human S100A14 was cloned into S100A14-mediated cell differentiation. the mammalian expression vector pcDNA3.1. The promoter region of S100A14 (511þ6) was cloned into the Materials and Methods pGL3-basic vector as previously described (17). The result- Tissue specimens ing construct was verified by direct sequencing. C-Jun and Tissue samples from 30 patients with ESCCs were used Fra-1 expression plasmids were generated in our laboratory. for S100A14 mRNA expression analysis, and these samples JunB, JunD, and c-fos expression plasmids were provided by were different from those examined in our previous study Dr. Marta Barbara Wisniewska of University of Warsaw (18). Tissue specimens from 110 patients with ESCCs were (Warsaw, Poland). analyzed by immunohistochemistry (IHC). Patients were recruited at the Chinese Academy of Medical Sciences Transfection and generation of stable cell lines Cancer Hospital (Beijing, China). Patients received no Transfection and establishment of stable cell lines were treatment before surgery and signed informed consent forms performed as previously described (20). for sample collection. This study was approved by the Institutional Review Board of the Chinese Academy of siRNA transfection Medical Sciences Cancer Institute. Representative primary Cells were transfected with siRNAs (25 nmol/L) by tumor regions and the corresponding histologically normal HiperFect (Qiagen) following the manufacturers' protocol. esophageal mucosa from each patient were snap-frozen in The sequences for siRNAs were listed in Supplementary liquid nitrogen and stored at 80C. Additional blocks Table S1. were collected and processed in paraffin for histologic examination. Immunofluorescence The experiment was performed as previously described IHC staining (20). An ESCC tissue microarray including 110 esophageal tumors and the corresponding normal epithelia was con- RNA isolation and PCR analysis structed with each case represented twice. For IHC staining, RNA purification and quantitative reverse transcription the slides were deparaffinized, rehydrated, then immersed in polymerase chain reaction (qRT-PCR) were performed 3% hydrogen peroxide solution for 10 minutes, heated in as previously described (17). Primers used are listed in citrate buffer (pH 6.0) at 95C for 25 minutes, and cooled at Supplementary Table S1. room temperature for 60 minutes. The slides were blocked by 10% normal goat serum at 37C for 30 minutes and then Chromatin immunoprecipitation assay incubated with rabbit polyclonal antibody against S100A14 Chromatin immunoprecipitation (ChIP) was performed at a dilution of 1:500 overnight at 4C. IHC was performed as previously described (21) using anti-JunB (5712-S) anti- using the PV-9000 Polymer Detection System for Immuno- body from Epitomics and RNA Polymerase II (MA1- Histological Staining kit (Beijing Golden Bridge Biotech- 10882) antibody from Thermo Scientific Pierce. Primers nology Company). 3,30-Diaminobenzidine (DAB) was used used are listed in supplementary Table S1. to visualize the reaction, followed by counterstaining with hematoxylin. Visual analysis was performed using Image- Western blot analysis Scope software (Aperio Technologies). The staining inten- Western blot analyses were performed as previously sity was graded from 0 to 3; no staining was scored as 0, weak described (20). Antibodies used were anti-S100A14 (gifts positive staining as 1, positive staining as 2, and strong of Dr. Iver Petersen, University Hospital Charite, Berlin, positive staining as 3. The percentage of staining was and Dr. Youyong Lu,€ Beijing Cancer Hospital and Insti- automatically assessed by ImageScope software, and the tute, Beijing) and anti-b-actin (A5316, Sigma). anti-JunB

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(5712-S) and anti-JunD (5226-1) were from Epitomics, clinicopathologic features, we determined the expression of anti-c-Jun(Sc-1694), anti-c-fos(Sc-52), and anti-Fra-1 S100A14 in a tissue microarray comprised of 110 paired (Sc-183) were from Santa Cruz Biotechnology. esophageal cancer and adjacent normal samples by IHC analysis and evaluated the correlation between S100A14 Luciferase assay protein levels and clinicopathologic parameters in 103 cases. Luciferase assay was performed as previously described The immunostaining results for S100A14 in ESCCs and (17). their corresponding normal epithelia are shown in Fig. 1C. S100A14 showed a clear localization in the plasma mem- Cell proliferation assay brane in normal esophageal epithelia. In contrast, both Cell proliferation was measured by a direct viable cell plasma membrane and cytoplasmic staining were observed count assay. in esophageal cancer tissues. S100A14 exhibited focal, positive immunostaining in certain well-differentiated areas, Annexin V apoptosis assay whereas staining was undetectable in other, less differenti- Apoptosis assay was measured using the BD Annexin V- ated sections. In well-differentiated carcinomas, staining for PE Apoptosis Detection Kit (Becton, Dickinson and Com- S100A14 was positive in keratinized areas at the center of pany) according to the manufacturer's protocol. Briefly, cells tumor foci but was decreased or undetectable in the marginal were incubated with Annexin V at room temperature for 15 areas. However, in moderately and poorly differentiated minutes in the dark and then subjected to flow cytometric carcinomas, the staining was weak or sporadic, occurring analysis. only in the well- or moderately differentiated regions but completely undetectable in other areas. IHC analysis Fluorescence-activated cell sorting analysis showed that S100A14 expression was significantly Cells were washed in PBS and fixed in methanol over- reduced in ESCCs versus matched normal epithelial tissue night. Subsequently, cells were washed and resuspended in in 70 of 103 cases (67.9%). Downregulation of S100A14 PBS containing 50 mg/mL propidium iodide, 100 mg/mL had a significant correlation with ESCC dedifferentiation RNase, and 0.1% Nonidet P-40 for 30 minutes at 37C. (P ¼ 0.005) and clinical stage (P ¼ 0.028) but had no The distribution of cells in different phases of the cell cycle relationship with gender, depth of tumor invasion, or was determined by measuring the nuclear DNA content lymph node metastasis (Table 1). Furthermore, we ana- using a FACS Calibur cell flow cytometer (Becton, Dick- lyzed the correlation between S100A14 expression and inson and Company). differentiation in ESCC cell lines. As shown in Fig. 1D, S100A14 protein exhibited higher expression in well- Statistical analysis differentiated cells such as KYSE30, KYSE180, and We statistically evaluated experimental results using 2- KYSE510 cells than in cells with poor differentiation such tailed paired Student t test, 2-independent sample t test, and as KYSE70 and KYSE410. S100A14 exhibited moderate c2 test. All tests of significance were set at P < 0.05. expression in cells with intermediate differentiation such as KYSE150 (19). These results further confirmed the Results correlation between S100A14 expression and esophageal Confirmation of the reduced expression of S100A14 in cancer differentiation. ESCC compared with the matched normal epithelia by qRT-PCR S100A14 is induced during esophageal cancer cell Our previous study showed that S100A14 expression is differentiation downregulated in ESCCs versus adjacent normal tissue by Our previous study showed that TPA induced the expres- semiquantitative RT-PCR (18). To further confirm the sion of a series of differentiation-associated genes in esoph- differential expression of S100A14 in ESCCs, we per- ageal cancer cells. To further characterize the alteration of formed qRT-PCR analysis in 30 paired ESCCs and S100A14 levels during ESCC differentiation, we treated adjacent normal epithelial tissues. Consistent with the esophageal cancer cell lines KYSE30, KYSE450, and previous results, S100A14 is significantly reduced in 21 KYSE510 with TPA, and mRNA and protein expression of 30 ESCC tissues compared with adjacent normal of S100A14 was determined. We found that TPA treat- epithelia (paired t test, P ¼ 0.0118; Fig. 1A). The reduced ment increased the mRNA and protein levels of S100A14 expression of S100A14 was further confirmed by Western in a time-dependent manner in KYSE450 cells. The blotting in 11 of 14 cases (Fig. 1B). These results clearly induction of S100A14 by TPA occurred at 8 hours, with show that S100A14 is markedly downregulated in ESCCs apeakincreaseofmorethan5-foldby12hours(Fig. compared with the matched normal epithelia at both the 2A). However, the induction of S100A14 was not mRNA and protein levels. observed in KYSE30 and KYSE510 cells (Fig. 2A). To further confirm these results, we treated cells with calci- Downregulation of S100A14 is associated with ESCC um, a commonly used differentiation inducer (22). First, dedifferentiation and clinical stage we investigated the effect of different doses of calcium To further confirm the alteration of S100A14 expression on S100A14 expression in KYSE450 cells by Western in ESCCs and analyze the correlation between S100A14 and blotting and immunofluorescence (Supplementary Fig.

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The Role of S100A14 in ESCC Differentiation

A 5 P = 0.0118 ) t 0

–5

–10 S100A14 relative expression (– D C expression

–15 N T 6 B Normal N T NTNTNT NTNT NT Tumor Figure 1. Reduced expression of 1234567 S100A14 mRNA and protein in S100A14 4 esophageal cancer. A, b-Actin downregulated S100A14 mRNA level was detected in 21 of 30 NTN TN TNTNTNTNT 2 8 9 10 11 12 13 14 tumors (T) compared with normal S100A14 adjacent epithelia (N) by qRT-PCR. b-Actin B, S100A14 protein level was S100A14/ b -actin density ratio 0 1234567 8 9 10 11 12 13 14 reduced in 11 of 14 malignant tissues versus corresponding C N T 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 12 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 was harvested and the lysates were probed with anti-S100A14 34 antibody. b-Actin was used as loading control.

KYSE30KYSE70KYSE140KYSE150KYSE180KYSE410KYSE450KYSE510 S100A14

b-Actin

S1). The results showed that 2.4 mmol/L CaCl2 effec- S100A14: a late differentiation marker of esophageal tivelyinducedS100A14proteinexpressionincellnuclei. cancer cells Subsequent evaluation of calcium-induced S100A14 Upon commitment to terminal differentiation, keratino- expression in KYSE450 and KYSE510 cells showed that cytes undergo several distinct differentiation stages. At each ﬁ 2.4 mmol/L CaCl2 treatment dramatically increased stage, keratinocytes express speci c differentiation-associat- S100A14 mRNA and protein levels in a time-dependent ed genes. In the early stage of terminal differentiation, cells manner (Fig. 2B). In contrast, there is no obvious effect initiate the expression of genes encoding Keratin 1 (KRT1) on S100A14 expression in KYSE30 cells (Fig. 2B). and Keratin 10 (KRT10; ref. 23). At a more advanced stage, Therefore, we selected KYSE450 and KYSE510 cells to cells begin to express ﬁlaggrin (FLG) and other structural performed phenotypic characterization in the following genes, including involucrin (IVL), loricrin (LOR), and small experiments. proline-rich proteins (SPRR; refs. 24, 25). To characterize

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

S100A14 expression

Characteristics Nonunderexpresseda (%) Underexpresseda (%) 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 c2 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.

the expression pattern of S100A14 during the course of mmol/L CaCl2 (Fig. 3B, right). Next, we investigated differentiation, we determined the correlation of S100A14 whether the changes in cell growth are due to apoptosis or expression with a series of differentiation stage-specific genes cell-cycle arrest. We performed apoptosis assay using the BD to identify the temporal pattern of S100A14 induction. TPA Annexin V-PE Apoptosis Detection Kit. As shown in Fig. 3C treatment dramatically increased the expression of a series of (left), calcium treatment significantly induced cell apoptosis late differentiation markers but had no effect on the early compared to vehicle-treated cells. However, we failed to differentiation markers KRT1 and KRT10 (Fig. 3A). Inter- observe any increase of apoptotic rate in S100A14-over- estingly, the time line of S100A14 expression overlaps with expressing cells compared to that of empty vector–trans- that of the late differentiation marker SPRR1A (Fig. 3A), fected cells, indicating that overexpression of S100A14 does which is strictly linked to keratinocyte terminal differenti- not increase the sensitivity of KYSE450 cells to calcium- ation (26, 27). Moreover, the expression pattern of S100A14 induced apoptosis. We next measured the cell-cycle status in SPRR1A is also similar to that of during calcium-induced the absence or presence of 2.4 mmol/L CaCl2 at 48 hours. differentiation of esophageal cancer cells. Taken together, Cell-cycle distribution analysis showed that overexpression these data suggest that S100A14 may play a role in esoph- of S100A14 causes an arrest of cells in G1 phase, with an ageal cancer cell terminal differentiation. 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 Effect of S100A14 overexpression on cell cycle, 42.8% 0.3% to 47.5% 0.7% in the presence of calcium, morphology, and calcium-induced cell growth respectively. Furthermore, calcium hampers the cell-cycle inhibition in KYSE450 cells progression by arresting the cells in S-phase. In empty To investigate the functional role of S100A14 in esoph- vector–transfected cells, S-phase was increased from ageal cancer cell differentiation, we selected KYSE450 cells 29.9% 0.9% to 39.6% 0.9%, G2 phase was decreased to perform overexpression experiments as S100A14 exhibits from 32% 2.1% to 17.6% 1.2%, and G1 phase was a moderate-level expression and can be markedly induced in increased from 38.1% 1.2% to 42.8% 0.3%. In this cell line. Western blot analysis showed that S100A14 is contrast, in S100A14-transfected cells, S-phase cells effectively overexpressed (Fig. 3B, left). We firstly examined increased from 27.3% 1.0% to 34.6% 0.4%, and the effect of S100A14 overexpression on cell growth with or consistently G2 phase was decreased from 27.5% 2.2% to without calcium treatment by a direct viable cell count assay. 17.9% 1.1% and G1 phase was increased from 45.1% The results showed that overexpression of S100A14 signif- 1.2% to 47.5% 0.7% (Fig. 3C, right). Taken together, our icantly inhibited cell growth in the absence or presence of 2.4 data strongly suggest that overexpression of S100A14 leads

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The Role of S100A14 in ESCC Differentiation

A TPA (100 ng/mL) KYSE30 cells Time (h) 0246 81224 S100A14

b-Actin TPA (100 ng/mL) 6

5 KYSE30 KYSE450 KYSE450 cells KYSE510 024681224 Figure 2. S100A14 expression is 4 regulated during TPA and calcium- S100A14 3 induced esophageal cancer cell b-Actin differentiation. A, esophageal 2 cancer cells including KYSE30, KYSE450, and KYSE510 cells were 1 cultured in the presence of 100 KYSE510 cells ng/mL TPA. Cells were harvested Relative S100A14 mRNA level 0 0 h 2 h 4 h 6 h 8 h 12 h 24 h 024681224 at indicated time points. Left, S100A14 expression was S100A14 examined by qRT-PCR. Data are b presented as mean SD of the fold -Actin difference; right, S100A14 expression was determined by CaCl (2.4 mmol/L) KYSE30 cells Western blotting. B, esophageal B 2 cancer cells including KYSE30, Time (h) 0 6 12 24 48 72 KYSE450, and KYSE510 cells were S100A14 treated with 2.4 mmol/L CaCl2, cells were harvested at indicated b-Actin CaCl (2.4 mmol/L) time points. Left, qRT-PCR was 12 2 performed to analyze the mRNA KYSE30 10 KYSE450 expression of S100A14; right, KYSE510 KYSE450 cells immunoblots using anti-S100A14 8 0 6 12 24 48 72 antibody to analyze expression of S100A14 S100A14 protein. 6 b-Actin 4

2 KYSE510 cells Relative S100A14 mRNA level 0 0 h 6 h 12 h 24 h 48 h 72 h 0 6 12 24 48 72 S100A14

b-Actin

to an arrest of cells in G1 phase, and calcium further hampers Effect of S100A14 knockdown on cell-cycle progression cells in S-phase. Arrested cells were unable to proceed into and morphology in KYSE510 cells – the G2 M phase thereby leading to the inhibition of cell To further determine the role of S100A14 in calcium- growth. However, S100A14-overexpressing cells did not induced phenotypic changes, we selected KYSE510 cells to exhibit morphologic changes compared with control cells. perform the knockdown experiments as S100A14 exhibits Moreover, there is no significant difference in the differen- high levels of expression that can be effectively inhibited in tiation-associated morphological phenotype induced by cal- this cell line. Western blot analysis showed that S100A14 cium, suggesting that the variation of S100A14 expression expression is efficiently diminished in S100A14-shRNA– alone is not sufficient to alter the differentiation phenotype transfected cells (Fig. 4A). Cell-cycle analysis showed that (Supplementary Fig. S2). To characterize the effect of S100A14 silencing significantly decreased the proportion of S100A14 overexpression on cell differentiation at the molec- G1 phase cells (Fig. 4A). To examine the effect of S100A14 ular level, we examined the expression of differentiation- knockdown on KYSE510 cell differentiation, cells were associated genes. qRT-PCR analysis showed that S100A14 treated with calcium for 4 days. Calcium treatment in overexpression resulted in a 2-fold increase of IVL and 3.4- shControl-transfected cells induced a dramatic change in fold upregulation of FLG in calcium-treated cells (Fig. 3D). cell–cell contact. Distinct spaces between cells became much These data indicate that S100A14 overexpression interferes less apparent and cells stratified within 2 days. These with calcium-induced cell growth inhibition and affects the morphologic changes occurred at day 2 of differentiation expression of differentiation-associated genes in terminally of KYSE510 cells, the time point at which S100A14 expres- differentiating esophageal cancer cells. sion was induced (Figs. 4B and 2A). Knockdown of

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TPA (100 ng/mL) CaCl (2.4 mmol/L) A 2 50 S100A14 30 S100A14 IVL IVL 40 FLG FLG LOR LOR 30 SPRR1A SPRR1A SPRR3 20 SPRR3 20 KRT1 KRT1 KRT4 KRT10 KRT4 10 KRT10 4 Figure 3. S100A14 acts as a late 5 terminal differentiation modulator and regulates esophageal cancer Relative mRNA level Relative mRNA level cell differentiation. A, qRT-PCR 0 0 analysis was performed to analyze 0 h 2 h 4 h 6 h 8 h 12 h 24 h 0 h 6 h 12 h 24 h 48 h mRNA expression of a selected 25 Cell counting group of terminal differentiation B Vector vehicle genes in KYSE450 cells treated by S100A14 vehicle TPA (left) and 2.4 mmol/L CaCl2

) 20 5 Vector CaCl 2 (right). B, KYSE450 cells were S100A14 CaCl Vector S100A14 2 transfected with pcDNA3.1 and S100A14 15 pcDNA3.1-S100A14 vectors, stable cells were established by b-Actin 10 Geneticin (G418) selection for about 2 weeks. Left, cells were 5 harvested and Western blotting Number of cells (1×10 was performed to measure the protein expression of S100A14. 0 Vector vehicle S100A14 vehicle Right, decreased cell growth of C 0 2 4 Day S100A14-transfected KYSE450 cells compared with empty vector– 120 transfected KYSE450 cells with or without calcium treatment [mean 100 (n ¼ 2) SD; two-sided t test; , P < 0.05]. C, empty vector– 80 transfected and S100A14- G 1 overexpressed KYSE450 cells 60 S 5 Vector CaCl2 S100A14 CaCl2 were seeded at 1 10 cells per % Cells G –M 40 2 well in conventional medium with or without 2.4 mmol/L CaCl2 on 6- 20 well plates, cells were stained with Annexin V-PE (AV-PE) and 7-AAD 0 Vector S100A14 Vector S100A14 (left) or propidium iodide (PI; right) ﬂ vehicle vehicle CaCl2 CaCl2 and analyzed by ow cytometry at 48 hours. D, S100A14 regulates differentiation-associated genes 8 IVL D FLG expression. Cells were treated with CaCl2 (2.4 mmol/L) for 48 hours. 6 The cells were harvested, total RNA was isolated, and mRNA 4 expression of IVL and FLG genes was examined by qRT-PCR. Relative mRNA expression level expression 2

0 ) ) 2 2

Vector (CaCl Vector (vehicle) S100A14 (CaCl S100A14 (vehicle)

S100A14 markedly inhibited these calcium-induced mor- together, these data show that S100A14 knockdown inter- phologic changes. However, calcium treatment of S100A14- feres with cell-cycle progression and affects the esophageal silenced KYSE510 cells did not induce FLG or IVL mRNA cancer cell terminal differentiation program. expression, whereas S100A14 overexpression resulted in FLG and IVL mRNA upregulation in KYSE450 cells, The underlying mechanism of S100A14-mediated silencing of S100A14 in KYSE510 cells had no signiﬁcant esophageal cancer cell differentiation effect on expression of these genes (data not shown). The One of the mechanisms of terminal differentiation of discrepancy may be due to cell-type differences. Taken keratinocytes involves the mitogen-activated protein (MAP)

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Flow cytometry A 50 shCon * * shS100A14-1 40 shS100A14-2 shCon shS100A14-1shS100A14-2 * Figure 4. Depletion of S100A14 S100A14 30 * inhibits calcium-induced cell * differentiation. A, KYSE510 cells b-Actin * were transfected with control 20 5 shRNA and 2 different S100A14 phase % of Cells in each 0 shRNAs (shRNA-1 and shRNA-2), G1 SG2–M and stable cells were obtained by G418 selection for about 2 weeks. B Cells were harvested and Western CaCl2 (2.4 mmol/L) KYSE510 cells blotting was performed using anti- S100A14 antibody. b-Actin was Time (d) 024 used as loading control. Cell-cycle distribution was analyzed by FACS, and a signiﬁcant G1 phase decrease was observed in shCon S100A14-silenced cells compared with control shRNA-transfected cells. [mean (n ¼ 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 shS100A14-1 cultivated in conventional medium supplemented with 2.4 mmol/L CaCl2 at the indicated time points, phase-contrast photomicrographs were taken.

shS100A14-2

kinase pathway that leads to induction of AP-1, a transcrip- promoter (511þ6 bp from the transcription start site) tion factor composed of members of the Jun and Fos protein reporter plasmid into KYSE450 cells, and 48 hours later, families (28). Our previous study showed that among the luciferase activity was measured. JunB exhibited a greater Jun family of transcription factors, c-Jun/AP-1 could bind ability than c-Jun to stimulate reporter activity (Fig. 5B). In and activate the expression of a series of differentiation- contrast, no increase in reporter activity was observed when associated genes in esophageal cancer cells (29). Therefore, the c-fos expression vector was cotransfected. We performed we speculated that transcriptional regulation by AP-1 might a ChIP assay to ask whether JunB binds directly to the contribute to the underlying mechanism of S100A14 S100A14 promoter in esophageal cancer cells. The results involved in esophageal cancer cell differentiation. KYSE450 show that JunB is significantly enriched at this regulatory cells were transiently transfected with a series of AP-1 region compared to the IgG control in KYSE450 cells. As expression plasmids including JunB, JunD, c-Jun, c-fos, expected, a significant enrichment of the Pol II with the and Fra-1, and 48 hours later, Western blotting was per- promoter region of S100A14 gene is also observed (Fig. 5C). formed. As shown in Fig. 5A, ectopic expression of JunB To ask whether JunB binding leads to activation of endog- drastically increased S100A14 expression compared with the enous S100A14, we used siRNAs targeting JunB (2 inde- empty vector control. In contrast, a slight effect on S100A14 pendent siRNAs) to deplete the endogenous JunB to exam- expression was observed in c-Jun and c-fos–overexpressing ine the effect of JunB on S100A14 expression in KYSE450 cells, and overexpression of JunD and Fra-1 only marginally cells. As shown in Fig. 5D (left), both siRNAs dramatically influenced S100A14 expression. Next, we tested whether reduced cellular JunB levels and effectively decreased JunB, c-Jun, and c-fos could drive the transcriptional activity S100A14 protein levels. Meanwhile, we examined the of S100A14 in KYSE450 cells. Expression plasmids for effect of JunB silencing on a series of differentiation- JunB, c-Jun, or c-fos were cotransfected with a S100A14 associated genes mRNA expression levels. As shown

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A pCG JunB pCG JunD pCMV c-Jun pCMV c-fos pCMV Fra-1 JunB JunD c-Jun c-fos Fra-1 Figure 5. AP-1 is involved in the b-Actin b b-Actin -Actin b-Actin b-Actin transcriptional regulation of S100A14. A, KYSE450 cells were transiently transfected with a series pCG JunB JunD pCMV C-Jun c-fos Fra-1 of AP-1 family expression vectors S100A14 including JunB, JunD, c-Jun, c-fos, b-Actin and Fra-1; 48 hours later, cells were harvested and Western blotting was performed using anti- B C 70 20 b 3.5 S100A14 antibody. -Actin was used as loading control. B, 3.0 60 15 S100A14 promoter construct was 2.5 50 cotransfected with the indicated 2.0 40 constructs into KYSE450 cells; 48 10 1.5 hours later, reporter activity was 30 1.0 then determined. Data are Fold over IgG Fold over 20 IgG Fold over 5 presented as mean SEM of the 0.5 10 fold difference. C, ChIP assay Relative luciferase activity Relative luciferase 0.0 showed that JunB and Pol II were pS100A14 +++++ 0 0 enriched in the promoter region of pCG ––––+ IgG IgG JunB Pol II the S100A14 gene. KYSE450 cells pCG-JunB –––+– were harvested, ChIP assay was pCMV-myc ––+–– siControl performed with anti-JunB, anti-Pol pCMV-c-Jun –+––– 1.2 siJunB1# siJunB2# II antibodies, and anti-Rabbit IgG pCMV-c-fos +–––– antibody was used as a negative 1.0 control. D, JunB directly regulates the expression of target genes D 0.8 involved in differentiation. Two 0.6 independent siRNAs targeting siControlsiJunB1# siJunB2# JunB and control siRNAs were JunB 0.4 transfected into KYSE450 cells; 72

S100A14 Relative mRNA level hours later, cells were harvested. 0.2 Left, Western blotting was b -Actin performed using anti-JunB and 0.0 anti-S100A14 antibodies. b-Actin IVL FLG LOR KRT1 KRT4 was used as loading control. Right, SPRR3 KRT10 E The correlation between S100A14 and S100A14 SPRR1A total RNA was isolated and mRNA JunB expression in ESCC 15.00 expression of differentiation- associated genes was examined by qRT-PCR. E, the mRNA expression of S100A14 is 10.00 correlated with the mRNA expression of JunB in ESCCs. The correlation between the mRNA S100A14 y S100A14 expression of ( -axis) and 5.00 JunB (x-axis) in tumor is analyzed in ESCC specimens. Correlation coefﬁcient is 0.582 and P is 0.001. .00 .00 2.50 5.00 7.50 10.00 12.50 JunB

in Fig. 5D (right), among the 10 genes examined by qRT- sion was significantly associated with JunB mRNA expres- PCR, silencing of JunB markedly decreased S100A14, sion in esophageal cancer specimens (Pearson correlation IVL, FLG, LOR, SPRR1A, SPRR3, KRT1,andKRT4 but coefficient, R ¼ 0.582, P ¼ 0.001; Fig. 5E). Collectively, no KRT10 expression levels. Finally, to assess the corre- these results suggest a role for JunB in the transcriptional lation between S100A14 and JunB in esophageal cancer regulation of S100A14 and provide a molecular mecha- tissues, we simultaneously examined the mRNA expres- nism whereby S100A14 contributes to esophageal cell sion level of S100A14 and JunB in 30 esophageal cancer differentiation. tissues and calculated the Pearson correlation coefficient. DC C C C Discussion The term t ( t_b-actin t_S100A14 or t_JunB)was used to describe the expression of S100A14 and JunB. Squamous cell differentiation is a multistep process Statistical analysis indicated that S100A14 mRNA expres- that requires the coordinated activation and repression of

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The Role of S100A14 in ESCC Differentiation

squamous cell–specific genes, and disruption of differen- cells. S100A14 overexpression in KYSE450 cells inhibited tiation is an important characteristic of malignant tumors cell growth in the absence or presence of calcium, (30, 31). Human esophageal cancer exhibits a reduced although the overexpression of S100A14 alone was not degree of differentiation and defects in the terminal sufficient to induce the morphologic changes associated differentiation pathway (32, 33). A better understanding with terminal differentiation. Importantly, our data of the mechanisms regulating differentiation would offer showed that S100A14 can exert anti-cancer function the basis for identification of tumor biomarkers. Our during the process of ESCC differentiation by blocking previous study showed that S100A14 belongs to a subset the cell cycle in G1 and S-phases in the presence of of genes that are downregulated in esophageal cancers, calcium. In contrast, S100A14 knockdown in KYSE510 and as one of many differentiation-associated genes, cells led to a notable impairment of differentiation, as was reduced S100A14 expression might contribute to esoph- evident morphologically (Fig. 4B). Molecular investiga- ageal carcinogenesis (17, 18). Furthermore, our study tions further supported the morphologic findings as showed that S100A14 regulated cell proliferation and altered expression of S100A14 positively correlated with apoptosis in a dose-dependent manner via interaction changes in expression levels of late differentiation markers with RAGE in ESCC (15). However, information is such as IVL and FLG, which are major components of limited about the possible biologic significance of the the cornified envelope and are considered to be appro- altered expression of S100A14 during ESCC develop- priate markers for terminal differentiation (39, 40). As ment.Inthisstudy,werevealedthemarkeddownregula- S100A14 does not bind to DNA and contains no nuclear tion of S100A14 expression in the majority of ESCCs localization sequence, the mechanisms by which and a significant correlation between S100A14 expression S100A14 regulates these terminal differentiation–associ- level and differentiation and clinical stage of ESCCs. ated genes may involve the intermediary activities of Well-differentiated or moderately differentiated ESCC S100A14 partner proteins. It will therefore be of great clinical samples showed higher S100A14 expression than importance to identify and characterize the proteins with poorly differentiated cases, consistent with previous find- which S100A14 interacts in future studies. To further ings that downregulation of S100A14 is associated with identify the effect of S100A14 on the pathways regulating poor differentiation in colon cancer (10). Protein trans- differentiation, gene expression profiling analysis needs to location between different subcellular compartments is be performed for further study. crucial for protein function (34). Concordantly, in this We also showed that the transcription factor AP-1 is study, we found that S100A14 exhibited plasma mem- involved in the transcriptional regulation of S100A14. This brane localization in normal esophageal epithelial tissues is in line with our previous study showing that AP-1 could but plasma membrane and cytoplasmic localization in transcriptionally regulate a series of differentiation-associat- esophageal cancer tissues. Previously, S100A14 was iden- ed genes (29). Here, we have added another gene into the tified as a plasma membrane–associated protein in breast AP-1–regulated network involved in esophageal cancer cell cancer cell lines and exhibited an increased expression in differentiation. Whereas most AP-1 factors have no signif- breast cancer tissues versus matched normal tissues. icant effect on S100A14 expression, we showed that Accordingly, S100A14 exhibited different patterns of S100A14 is a direct target gene of JunB, which regulates subcellular distribution, typified by plasma membrane transcription by directly binding to the proximal S100A14 localization in breast cancer tissues but cytosolic expres- promoter. This result is consistent with previous reports that sion in nontumor breast epithelial cells (35). Previous different members of the AP-1 transcriptional complex studies showed that some members of the S100 family of exhibited varying degrees of importance in regulating the proteins exhibit calcium-dependent translocation (36, expression of specific differentiation-related genes. For 37), and the translocation of S100A14 is regulated in instance, JunB, JunD, and Fra-1 were identified as major a calcium-dependent manner through interaction with regulators of involucrin expression (41). In addition, most nucleobindin, which has strong association with Ga AP-1 factors efficiently bind to the SPRR1A minimal pro- proteins (38). We also found that calcium treatment moter region in proliferating keratinocytes. Following induced S100A14 expression in cell nuclei in esophageal induction of terminal differentiation, altered ability of cancer cell lines, further suggesting that calcium plays a AP-1 factors to bind this sequence, notably JunB and JunD, role in the induction and translocation of S100A14. is observed (27). Finally, the significant correlation between These data suggest the difference in subcellular distribu- mRNA expression levels of S100A14 and JunB further tion of S100A14 may be regulated by tissue-type–specific confirmed the regulation of S100A14 by JunB in esophageal factors in a calcium-dependent manner, which might play cancer tissues. As our previous study showed that Kruppel-€ an important role in determining the functions of like factor 4 (KLF4) plays an important role in the tran- S100A14 in tumorigenesis and progression. scriptional regulation of differentiation-related genes in In addition, we showed that TPA and calcium, known ESCCs (5), we cannot exclude the potential contributions inducers of terminal differentiation, markedly induced of other transcription factors such as KLF4 in regulating S100A14 expression. S100A14 overexpression and silenc- S100A14 during cell differentiation. ing experiments further substantiated the role of In summary, we have characterized the role of S100A14 as S100A14 in terminal differentiation of esophageal cancer a novel and pivotal modulator of esophageal cancer cell

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Chen et al.

differentiation. Further research on S100A14 should focus Administrative, technical, or material support (i.e., reporting or organizing data, fi constructing databases): Y. Li on the identi cation of S100A14 partner proteins and the Study supervision: Z.-H. Liu elucidation of the molecular mechanisms whereby S100A14 modulates esophageal cancer cell differentiation. Acknowledgments The authors thank Dr. Iver Petersen and Dr. Youyong Lu€ for providing the S100A14 antibodies and Dr. Marta Barbara Wisniewska for the generous gifts of the Disclosure of Potential Conflicts of Interest junB, junD, and c-fos expression plasmids. No potential conflicts of interest were disclosed. Grant Support Authors' Contributions The study was funded by National Natural Science Foundation of China Conception and design: H. Chen, B. Sunkel, Q. Wang, Z.-H. Liu (81000954) and Doctoral Fund of Ministry of Education of China Development of methodology: H. Chen (20101106120012). The costs of publication of this article were defrayed in part by the payment of page Acquisition of data (provided animals, acquired and managed patients, provided advertisement facilities, etc.): H. Chen, J. Ma, A. Luo, F. Ding, Z.-H. Liu charges. This article must therefore be hereby marked in accordance with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- 18 U.S.C. Section 1734 solely to indicate this fact. tational 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, Received June 13, 2013; revised September 9, 2013; accepted September 24, 2013; Z.-H. Liu published OnlineFirst October 9, 2013.

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S100A14: Novel Modulator of Terminal Differentiation in Esophageal Cancer

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

Mol Cancer Res 2013;11:1542-1553. Published OnlineFirst October 9, 2013.

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