Tumor Biol. DOI 10.1007/s13277-013-0946-1

RESEARCH ARTICLE

Low expression of cyclic amp response element modulator-1 can increase the migration and invasion of esophageal squamous cell carcinoma Yuchan Wang & Shuhui Zhou & Xiaojing Yang & Hui Shi & Mei Li & Qun Xue & Xianting Huang & Xinxiu Wang & Huijie Wang & Jianguo Zhang

Received: 10 April 2013 / Accepted: 14 June 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Cyclic AMP response element-binding protein (CREB) family can regulate biological functions of various types of cells and has relation with esophageal cancer cell migration and invasion. Cyclic AMP response element modulator-1 (CREM-1) is one member of the family with limited acquaintance. This study was conducted to investigate the effect of CREM-1 on migration and invasion in human esophageal squamous cell carcinoma (ESCC). The expression of CREM-1 protein in ESCC tissues with or without lymph nodes metastasis was determined by western blot. Immunohistochemical analysis of CREM-1 expression were carried out in paraffin-embedded sections of ESCC and correlated with clinical features. The roles of CREM-1 in migration and invasion were studied in TE1 cells through knocking CREM-1 down with siRNA or overexpression of CREM-1 in ECA109 cells. The regulations of CREM-1 on

Yuchan Wang and Shuhui Zhou contributed equally to this work. Y. Wang : X. Yang : X. Huang : X. Wang Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China S. Zhou Department of Radiation Medicine and Protection, Medical College, Soohow University, Suzhou, Jiangsu 215123, People’s Republic of China H. Shi : M. Li : Q. Xue : J. Zhang (*) Department of Pathology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China e-mail: [email protected] H. Wang (*) Department of Medical Oncology, Fudan University Shanghai Cancer Center, No 270 Dongan Road, Shanghai 200032, People’s Republic of China e-mail: [email protected]

invasion and migration were determined by transwell and wounding healing assay. The effect of CREM-1 on chemotherapy drug was analyzed by Cell counting kit-8 assay. We found that the expression of CREM-1 was significantly downregulated in ESCC tissues with lymph nodes metastasis compared with the tissues without lymph nodes metastasis and was correlated with the clinical features of pathological grade, tumor stage and lymph node metastasis. Moreover, knocking CREM-1 down with siRNA increased cell migration and invasion in human ESCC cell lines TE1 while upregulation of CREM-1 inhibited the motility. Our data suggested that CREM-1 might play an important role in the regulation of tumor metastasis and invasion and serve as a tumor suppressor in human ESCC. We proposed that CREM-1 might be used as a potential therapeutic agent for human ESCC. Keywords Esophageal squamous cell carcinoma (ESCC) . CREM-1 . Migration

Introduction Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, occurring mostly in developing countries with marked regional variations in incidence [1]. ESCC is a highly aggressive disease and the 5-year survival rate is approximately 15 % [2]. It is tumor metastasis that mainly leads to the poor prognosis of the ESCC patients. Therefore, any insight into the mechanisms of ESCC cell progression and metastasis may provide important clues for the development of therapeutics [3]. It has been reported that CREB-regulated transcription coactivator (CRTC) played an important role in the migration of esophageal cancer cell [4]. CRTCs are transcription activators

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regulated by cyclic AMP response element-binding protein (CREB) and CRTC entering the nucleus can activate CREB transcription [5]. The CREB family has become more attractive in recent years as CREB is confirmed to play an essential role in regulating the migration and invasion of ESCC. In physiology and pathology conditions, the CREB family can affect cell survival, apoptosis and proliferation [6]. Depending on the exon usage, the CREB family can function as either transcriptional activator or repressor [7, 8]; and family members may have distinct functions under different conditions. Among them, cyclic AMP response element-modulator 1 (CREM-1), also described as cyclic AMP response elementbinding protein-1 (CREB-1) related factor [9], has not been fully understood yet. In this study, we used ESCC cells as models, and it was demonstrated that low expression of CREM-1 promoted TE1 cell survival against cisplatin treatment. Besides the low expression of CREM-1 increased cell metastasis and invasion. Relevant results were verified in ECA109 cell by overexpressing CREM-1. Accordingly, siRNA-mediated knockdown of CREM-1 increased TE1 cell survival. Cisplatin made the TE1 cells interfered of CREM-1 which were harder to apoptosis than the normal TE1 cells. We can see that CREM-1 has a low expression which leads to metastasis and invasion of ESCC.

Materials and methods Tissue specimens A total of 98 ESCC specimens who underwent surgery between 2005 and 2011 were retrieved from the archival files of the Department of Pathology, affiliated Hospital of Nantong University. None of the patients was treated with such preoperative therapies as radiation, chemotherapy, or immunotherapy. All human tissues were collected using protocols approved by the Ethics Committee of Nantong University Cancer Hospital. Resected specimens were classified according to the International Union Against Cancer TNM classification system [10]. The clinical data were collected after patients obtained informed consent. The mean post-operative follow-up period for the patients was 30.9 months (range, 2.5–48.8 months). Fresh samples were frozen in liquid nitrogen immediately after surgical removal and maintained at −80 °C until used for western blot analysis. Immunohistochemical analyses Serial sections that were 5 μm thick were mounted on glass slides coated with 10 % polylysine. Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 0.3 %

methanolic peroxide for 30 min. Immunoreactivity was enhanced by microwaving by incubating the tissue sections for 10 min in 0.1 M citrate buffer. Immunostaining were performed using the avidin biotin peroxidase complex method and antigen-antibody reactions were visualized with the chromogen diaminobenzadine. The intensity of immunostaining in each tumor section was assessed as strong (3), moderate (2), weak (1), or negative (0); semiquantitatively using the following scale: <5 % of cells (0), 5–25 % (1), 26–50 % (2), 50–75 % (3), and >75 % (4) of cells, and then multiplied these values. This resulted in an overall CREM-1 immunohistochemical score was 0, 1, 2, 3, 4, 6, 9, or 12. CREM-1 expression was considered high when scores were ≥6, and low when scores were ≤4 [11]. E-cadherin antibody stained the membrane intensely and the cytoplasm of cancer cells weakly. Cell cultures and cell lysis The human ESCC cell lines TE1 and EAC109 (Both derived from well-differentiated ESCCs [12]) were purchased from Cell Library, China Academy of Science, and were cultured in RPMI 1640 (GibCo BRL, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillin–streptomycin mixture (GibCo BRL, Grand Island, NY, USA) at 37 °C and 5 % CO2. Cells were lysed in ice-cold RIPA lysis buffer (50 mM Tris HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS, and 1 % sodium deoxycholate). Directly before addition of the lysis buffer, complete protease inhibitors (Roche, Switzerland) and 1 mM PMSF were added. Following the clearing of lysates by centrifugation for 10 min at 4 °C, the lysates were adjusted to the same volume and concentration of total protein as determined by a Non-Interfering Total Protein assay (G Biosciences, USA). Lysates were incubated for 15 min at 90 °C in SDS-PAGE loading buffer supplemented with β-mercaptoethanol. Samples were stored at −20 °C. Western blot analysis and antibodies Fresh or frozen tissues were treated with lysis buffer containing protease inhibitors [50 mM Tris (pH 7.5), 150 mM NaCl, 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 0.15 % SDS, 0.1 mM sodium vanadate, 1 mM dithiothreitol, 20 mM βglycerophosphate, 2 mM EDTA, 1 mg/mL aprotinin, 100 mg/mL phenylmethylsulfonylfluoride, and 10 mg/mL leupeptin]. The lysates were cleared by centrifugation at 14,000 rpm at 4 °C for 10 min. The protein concentrations of lysates were quantified. Samples were denatured at 100 °C for 2 min before SDS-PAGE. Equal amounts (80 μg) of proteins from each sample were used for immunoblotting. After electrophoresis, the gel was blotted onto PVDF membrane. After it was blocked

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in 5 % nonfat milk in Tris–HCl buffered saline, the membrane was incubated with primary antibodies specific for PITSLRE (1:200) for 1–2 h, washed, and then incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibody. An enhanced chemiluminescense kit (ECL Kit) and autoradiography were used to detect immune complexes. ImageJ (NIH) was used to compare the density of bands on western blot. The antibodies used in this study included: anti-CREM-1 (anti- rabbit, 1:500, Santa Cruz Biotechnology), anti-E-cadherin (anti-mouse, 1:1,000, Santa Cruz Biotechnology), anti-Myc (anti-rabbit, 1:1,000, Sigma), and anti-GAPDH (anti-rabbit, 1:1,000, Sigma). Plasmid, siRNA, and transfection The full-length CREM-1 (Genbank Accession No.NM_ 0012675 62.1) was isolated from the human cDNA library and connected to pcDNA3.1-myc. The primers used for CREM-1 were as follows: 5′-CGGAATTCCGATGGCAGTA CCAACTA-3′ (sense) and 3′- CGGGATCCGTAATCTGTT TTGGGTA-5′ (anti-sense). Primer pairs for the CREM-1 siRNA expression vector were target the sequence: 5′-CAG CCAGGTTGTTG TTCAA-3′. For transient transfection, pcDNA3.1-myc, pcDNA3.1-myc-CREM-1, CREM-1 siRNA vector, and the non-specific vector were carried out using lipofectamine 2,000 (Invitrogen) and plus reagent in OptiMEM (Invitrogen) as suggested by the manufacturer. Transfected cells were used for the subsequent experiments 48 h after transfection.

examined under a microscope. Cells in at least six random microscopic fields (200×) were counted. Cell counting kit-8 assay Cells transfected and non-transfected siCREM-1 or MycCREM-1 (5×103/well in 100 μl/well) were seeded into a 96-well plate and cultured in RPMI 1640 medium containing 10 % FBS for 8 h then cultured in no serum medium. After incubation at 37 °C in an atmosphere containing 5 % CO2 for 72 h, the number of living cells was calculated by Cell Counting Kit-8 (CCK8) assay. Briefly, a CCK8 solution was added into each well and the cells were incubated for an additional 2 h. The number of living cells was then counted by measuring the absorbance at 490 nm. Statistical analysis Statistical analysis was performed using the PASW statistics 18 software package. CREM-1 expression was studied using the Spearman rank correlation test because the data were not normally distributed. For analysis of survival data, Kaplan– Meier curves were constructed, and the log-rank test was performed. Multivariate analysis was performed using Cox’s proportional hazards model, with P<0.05 considered statistically significant. The results are expressed as the mean±SE.

Wound healing assay

Results

TE1 cells were transfected with either CREM-1 siRNA vector or Myc-CREM-1 and scrambled negative control. When cell confluence reached about 80 % at 48 h post-transfection, wounds were created in confluent cells using a 100-μl pipette tip. The cells were then rinsed with medium to remove any free-floating cells and debris. Medium was then added, and culture plates were incubated at 37 °C. Wound healing was observed at different time points within the scrape line, and representative scrape lines were photographed. Duplicate wells for each condition were examined, and each experiment was repeated three times.

CREM-1 expression and its correlation with clinicopathologic variables in ESCC

Transwell invasion assay Cell invasion assays were performed using 24-well transwells (8 mm pore size, Corning Life Sciences) coated with matrigel (1 mg/ml, BD Sciences). Cells (104/well) were seeded in the upper chambers of the wells in 200 μl FBS-free medium, and the lower chambers were filled with 500 μl 10 % FBS medium to induce cell migration. Following incubation for 72 h, the cells on the filter surface were fixed with 4 % formaldehyde, stained with 0.5 % crystal violet, and

To reveal the role CREM-1 plays in ESCC, immunohistochemistry was performed to measure CREM-1 and Ecadherin expression in ESCC tissues (Fig. 1). In each case in which CREM-1 immunoreactivity was observed, the immunoreactivity was localized to the nucleus of tumor cells. CREM-1 was high expressed in the tissues without lymph nodes metastasis, and low expressed in lymph nodes metastasizing tissues, agreeing with E-cadherin expression. Ninetyeight tumor-banked specimens from 2005 to 2011 were available for review. Median age of the patients was 51.3 (range 31–85) at the cancer diagnosis. The presences of CREM-1 in different pathological grade, size, lymph node metastasis, tumor invasion, and E-cadherin were summarized in Table 1. The percentage of CREM-1-positive tumor cells ranged from 4 to 91, the mean percentages was 59.31. There is no statistical relationship between the presence of CREM-1, age, gender, or size status. But CREM-1 expression correlates significantly with tumor metastasis (P<0.001), tumor invasion (P=0.018), and E-cadherin (P=0.022; Table 1).

Tumor Biol.

Fig. 1 Immunohistochemical staining reveales CREM-1 and Ecadherin expression in paraffin-emedded ESCC tissues. a, b, e, f Cancer tissues with no lymph node metastasis showed high CREM-1 and Ecadherin expression (×200 and ×400). c, d, g, h Cancer tissues with

To explore the significance of CREM-1 in ESCC progression and to further study the relations between CREM-1 and E-cadherin, we investigated their abundance by western blot analysis. The result is shown in Fig. 2. The expression of CREM-1 was high in tissues without lymph nodes metastasis, and low in tissues with lymph nodes metastasis. Amount of GAPDH, a housekeeping protein, was demonstrated to be rather constant among the samples. Next we took an interest in investing the function of CREM-1 in cell level. E-cadherin is one of the markers of epithelial cells. The results showed that the expression level of CREM-1 was high in TE1 cells, while it was low in ECA109 cells. Prognostic significance of CREM-1 expression We carried out Kaplan–Meier analysis to study the correlation between CREM-1 expression and patient survival. Survival analysis was restricted to ninety-eight patients with follow-up data and results of CREM-1 by immunohistochemistry. By using the Kaplan–Meier analysis, patients with low expression of CREM-1 were significantly associated with short overall survival (P=0.002; Fig. 3). Multivariate analysis using the Cox proportional hazards model showed that CREM-1 (P=0.001), tumor metastasis (P=0.007), invasion (P=0.001), as well as E-cadherin (P=0.008) were independent prognostic indicators of overall survival (Table 2). Effect of downregulated CREM-1 expression on the migration and invasion of TE1 cells Underexpression of a tumor suppressor is crucial for the development of tumors as it can promote strong migration

lymph nodes metastasis showed low CREM-1 and E-cadherin expression (×200 and ×400). Details of the experiments were given in the “Materials and methods” section

Table 1 CREM-1 expression and clinicopathological parameters in 98 ESCC specimens Parameters

P

Total CREM-1 Score≤4, n (%) Score≥6, n (%)

Age (years) <60 37 ≥60 61 Gender Male 71 Female 27 Pathological grading Well 17 Moderately 47 Poorly 34 Metastasis Presence Absence Tumor size (cm) <5 ≥5 Tumor invasion T1 T2 T3 T4 E-cadherin Low expression High expression

15 (40.5) 29 (47.5)

22 (59.5) 32 (52.5)

0.321

32 (45.1) 12 (44.4)

39 (54.9) 15 (55.6)

0.569

4 (23.5) 21 (44.7) 19 (55.9)

13 (76.5) 26 (55.3) 15 (44.1)

0.091

68 30

17 (25.0) 27 (90.0)

51 (75.0) 3 (10.0)

0.000*

75 23

28 (37.3) 16 (69.6)

47 (62.7) 7 (30.4)

0.255

11 15 25 47

2 (18.2) 3 (20.0) 12 (48.0) 27 (57.4)

9 (81.8) 12 (80.0) 13 (52.0) 20 (42.6)

0.018*

61 37

40 (65.6) 4 (10.8)

21 (34.4) 33 (89.2)

0.022*

Statistical analyses were performed by Pearson χ2 test *P<0.05 was considered significant

Tumor Biol. Table 2 Contribution of various potential prognostic factors to survival by Cox regression analysis in 98 specimens

Age (years) Gender Tumor size Metastasis Pathological grading Tumor invasion CREM-1 E-cadherin

Relative ratio

95 % Confidence interval

P

1.417 0.789 1.044 2.271 0.997 2.152 0.336 2.069

0.758∼2.648 0.408∼1.562 0.522∼2.087 1.247∼4.137 0.735∼1.353 1.369∼3.382 0.173∼0.652 1.314∼3.625

0.275 0.511 0.903 0.007* 0.986 0.001* 0.001* 0.008*

Statistical analyses were performed by Cox test *P<0.05 was considered significant

Fig. 2 CREM-1 and E-cadherin expression in human ESCC by western blot analysis. a CREM-1 protein levels in human ESCC tissues (n=8 patients) determined by western blot analysis. b CREM-1, Ecadherin, and GAPDH protein in ECA109 and TE1 ESCC cell lines analyzed by western blot analysis. c Phase-contrast micrographs of two ESCC cell lines (×400)

and invasion of tumor cells. As TE1 cells have high invasion capability [13], we chose TE1 cells as a model. To determine the effect of CREM-1 expression on the growth of TE1 cells, we constructed siRNA expression vectors specific to CREM-1 transcripts and transfected them into TE1 cells. The knockdown efficiency was confirmed by western blot

(Fig. 4e); the CREM-1/RNAi reduced the level of CREM-1 protein expression by >60 % compared with the negative control (CREM-1/NC) and the mock TE1 cells (Fig. 4f). Next, we examined the impact of CREM-1 expression on the migration of TE1 cells by a wound healing assay, shown in Fig. 4a. Following incubation of physically-wounded cells for 72 h, siCREM-1 promoted the migration of TE1 cells (Fig. 4b). To analyze invasiveness, another important feature of malignant cells, we performed transwell invasion assays using cell culture inserts covered by extracellular matrix components. CREM-1/RNAi cells had strong invasive abilities resulted in a massive addition in invasion (Figs. 4c, d). The wound healing and invasion assays indicated that downregulation of CREM-1 expression increased the migration and invasion of TE1 cells. High expression of CREM-1 decreased TE1 cells motility As ECA109 cells had a low expression of CREM-1 (Fig. 2b), we studied overexpression of CREM-1 in ECA109 cells by Myc-CREM-1. Then, the transfection efficiency was verified by western blot (Fig. 5a). We also found that the upregulation of CREM-1 caused an increase in the expression of epithelial marker E-cadherin (Fig. 5a). In addition, Myc-CREM-1 inhibited the migration of ECA109 cells by wound healing assays (Figs. 5b, c) and accordingly reduced the invasion (Figs. 5d, e). In summary, these results showed that the overexpression of CREM-1 could decrease the motility of ECA109 cells. Knockdown of CREM-1 decreased cisplatin sensitivity in TE1 cells

Fig. 3 Cumulative survival curves according to CREM-1 expression. On the basis of score of CREM-1, patients were divided into high CREM-1 expressers (score ≥6) and low CREM-1 expressers (score ≤4). Patients in the low- expression CREM-1 group had significantly shorter overall survival

We speculated that underexpression of CREM-1 could reduce chemosensitivity in ESCC. Therefore, we tested whether siCREM-1 affected the response to cisplatin treatment in

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Fig. 4 Inhibiting CREM-1 expression facilitates the migration and invasion of TE1 cells. Migration and invasion of cells treated with siCREM-1 (or negative control) were analyzed at 48 h post-infection. a Wound healing assays with CREM-1/RNAi and CREM-1/NC cells. Migration of the cells to the wound was visualized at 0, 36, and 72 h with an inverted Leica phase-contrast microscope (×200 magnification). b Histogram showed the relative migration distance of cells. The data are means±SEM (n=3, *P<0.01 by t test, compared with

CREM-1/NC and mock cells). c CREM-1 expression inhibited cell invasion by transwell assays. CREM-1/RNAi cells showed higher penetration rate through the membrane compared with CREM-1/NC and mock cells. d Number of cells that invaded through the member was counted in 10 fields under ×20 objective lens. Bars, SD. *P<0.01 by t test. e CREM-1 interference efficiency was certified by western blot. f A related histogram showed CREM-1 protein expression. Data represent mean±SEM from four independent experiments; *P<0.01 by t test

ESCC cell line TE1. TE1 cells were transfected with siCREM1, and they were treated with various concentrations of cisplatin for 48 h. The result of the CCK8 assay indicated that the transfection of siCREM-1 significantly increased cell viability compared with mock transfection. At concentrations higher than 10 μmol/L, the survival fraction of the cells that were transfected with siCREM-1 was significantly higher than that of the cells that were transfected with mock and negative control (Fig. 6a). As stated above, the underexpression CREM-1 was insensitive to cisplatin in TE1 cells. Epithelial marker E-cadherin was seen in these mock and siCREM1 transfected TE1 cells no matter treated with cisplatin or not

(Fig. 6b). It was a low expression of E-cadherin in TE1 which underexpression of CREM-1. In conclusion, CREM-1 was closely related to the metastasis and invasion of TE1 cells. CREM-1 could be used as one of the prognostic factors of ESCC.

Discussion Clinically defined, metastasis is the major cause of lethality among human cancer patients, including those with ESCC [14, 15]. To develop novel treatments and cures, it is

Tumor Biol. Fig. 5 Exogenously expressed CREM-1 protein inhibits ECA109 cells migration and invasion ability. a The transfection efficiency was verified and western blot analysis of E-cadherin. b Wound healing assays with pcDNA3.1-myc- vector and pcDNA3.1-myc-CREM-1 cells. Migration of the cells to the wound was visualized at 0 and 72 h with an inverted Leica phase-contrast microscope (×200 magnification). c Histogram showed the relative migration distance of cells. The data are means±SEM (n=3, *P<0.01 by t test, compared with mock and control cells). d Overexpression of CREM-1 inhibited cell invasion by transwell assays. e Number of cells that invaded through the member was counted in 10 fields under ×40 objective lens. Bars, SD. *P<0.01 by t test

imperative to find out the factors underlying tumorigenesis, invasion and metastasis. Our results showed that CREM-1 expression correlated a lot with tumor metastasis (P<0.001), tumor invasion (P=0.018), and E-cadherin

(P=0.022). Furthermore, patients were divided into two groups: high CREM-1 expressers (score ≥6) and low CREM-1 expressers (score ≤4). The latter had a poor prognosis. As a multivariate Cox proportional hazard

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Fig. 6 Interference of CREM-1 affected TE1 cells insensitive to chemotherapy drugs. a Treat TE1 cells with cisplatin for 48 h at 5, 10, and 20 μmol/L, respectively after interference of CREM-1/RNAi or CREM-1/NC for 48 h. The data are mean±SEM (n=3, *P<0.01 by t test, compared with CREM-1/NC and mock cells). b Western blot analyses TE1 cells when interference of CREM-1 and with or without treatment of cisplatin

model was constructed, CREM-1, tumor invasion and metastasis were the strong independent predictors of survival. Tumor cells generally disseminate from the site of the primary tumors and establish secondary tumors in distant organs by metastasis, which is one of the six distinct hallmarks of cancer [16, 17]. In this study, we identified and functionally characterized CREM-1 as an important factor in ESCC progression. The current studies first illustrated the expression of CREM-1 in ESCC tissues, followed by demonstrating the association between the CREM-1 expression and clinicopathologic parameters, and finally discovered the role of CREM-1 in ESCC prognosis in 98 samples. Differential expression of CREM-1 had been detected among primary ESCC tissues with or without lymph nodes metastasis. CREM-1 expression was much higher in ESCC tissues with no lymph nodes metastasis compared with the tissues with lymph nodes metastasis. Immunohistochemistry results showed that high expression of CREM-1 was correlated with high E-cadherin expression, while low expression of CREM-1 was correlated with low E-cadherin expression in the same ESCC specimen. Western blot analysis presented the similar results. As a result, we supposed that CREM-1 was involved in tumor migration and invasion. In

this report, there was a statistically significant close relation between survival rate and expression of CREM-1. As a result, low expression of CREM-1 correlated significantly with a poor prognosis. The survival rate of patients with low expression of CREM-1 was lower than that of other patients, which suggested that the degree of expression of CREM-1 might have an effect on the survival rate. Further experiments presented here suggested TE1 cells interference of CREM-1 had a higher migration and invasion speed than that of normal which were verified by wound healing and transwell assay. Overexpression of CREM-1 in ECA109 cells resulted in the similar consequence. After treated with different concentrations of cisplatin, we found that CREM-1/RNAi TE1 cells were harder to apoptosis than mock and CREM-1/NC cells. CREM-1 negatively regulated ESCC cells migration: the loss of CREM-1 promoted cell migration whereas CREM-1 overexpression had repressive effects. Future gene expression and mutational studies of the CREM-1 gene in a larger set of patient samples, correlated with the information of cancers at various clinical stages, would elucidate the extent of aberrant CREM-1 expression in ESCC and critical events of cancer development that CREM-1 might be involved in. Among the CREB family members, CREM-1 plays an essential role in several types of cells [18–21]. Although CREM-1 is shown to regulate cell growth, survival and apoptosis, the exact role of CREM-1 in the initiation and progression of ESCC is unclear [6]. Our studies provided strong evidence that CREM-1 loss had a pro-metastatic effect on cell-based assays, because CREM-1 expression negatively regulated cell migration and invasion. Epithelialmesenchymal- transition (EMT) is implicated in tumor progression and metastasis. A basic mechanism involved in progression of EMT is upregulation of the mesenchymal marker Vimentin and downregulation of the epithelial marker E-cadherin—the main transmembrane adhesion molecule responsible for cell-to-cell interactions and tissue organization in epithelial cells [22]. As CREM-1 related to E-cadherin, we speculated that CREM-1 downexpression might reduce E-cadherin expression which led to EMT in ESCC. These data suggested that CREM-1 probably played a role in regulating cancer metastasis and invasion, which indicated that CREM-1 downregulation possibly contributed to the highly invasive properties of human ESCC. To summarize, this study, for the first time, indicated that CREM-1 expression provided important prognostic information in ESCC, and CREM-1 expression was associated with ESCC cells migration and invasion and the sensitivity to cisplatin. CREM-1 might serve as a novel molecular target for the detection and treatment of ESCC. Further study is required to elucidate how CREM-1 acts as a useful marker for ESCC and other human cancers.

Tumor Biol. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81201858) and Natural Scientific Foundation of Jiangsu Province Grant (No. BK2012231); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

11.

Conflicts of interest None

12.

13.

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