Abstract
Background/Aim: We previously reported that expression of melanoma-associated antigen (MAGE)-D4 mRNA was a prognostic factor for esophageal squamous cell carcinoma (ESCC). The aim of this study was to validate the expression of MAGE-D4 in two additional patient cohorts, and to investigate its biological functions. Materials and Methods: The role of MAGE-D4 in cell proliferation, adhesion, and migration was determined by gene knockdown experiments in the KYSE590 cell line. MAGE-D4 protein expression was analyzed in ESCC tissues by immunohistochemistry. A second validation cohort consisted of an ESCC mRNA dataset from The Cancer Genome Atlas. Results: Knockdown of MAGE-D4 significantly decreased cell proliferation and migration. Expression of MAGE-D4 protein was significantly associated with disease-free survival. In the second validation cohort, high MAGE-D4 mRNA expression was associated with significantly shorter overall survival and disease-free survival. Conclusion: MAGE-D4 plays an important role in the malignant behavior of ESCC. MAGE-D4 was validated as a prognostic indicator in two independent ESCC patient cohorts.
Esophageal cancer (EC) is the eighth most common cancer worldwide, with 456,000 new cases per year and about 400,000 deaths per year (1). Esophageal squamous cell carcinoma (ESCC) is more common in Asia and the developing world than in the West. Despite the improved multimodal treatments, there is no currently effective systemic therapy for advanced ESCC, and the prognosis remains poor (2). A better understanding of the mechanisms of ESCC development and progression is necessary to advance the development of novel diagnostic markers and therapeutic targets.
Members of the large melanoma-associated antigen (MAGE) family of proteins, so named because the first member was isolated from a melanoma cell line, have important physiological and pathological functions, and many members have been reported to play important roles in the progression of various malignant tumors (3-7). More than 50 MAGE genes have been identified to date (8), which can be broadly categorized into type I and type II genes based on differences in gene structure and tissue-specific expression (9). Type I MAGEs are expressed during germ cell development and are then silenced in normal mature somatic cells (10, 11). In contrast, relatively little is known about the localization and expression of type II MAGE proteins. Studies on type II MAGE-D4 have reported that its expression in normal tissues is restricted to the brain and ovary; however, it is overexpressed in several human malignancies, including lung, liver, oral, kidney, breast, and colorectal cancers, glioma (12-18), and ESCC, as previously reported by us (19). MAGE-D4 is a prominent tumor-associated antigen and has been shown to contribute to the proliferation, migration, and invasion of breast cancer and oral squamous cell carcinoma cells (14, 16). We have reported that high tumor expression of MAGE-D4 was significantly associated with shorter overall survival (OS) of ESCC patients and that MAGE-D4 overexpression was an independent prognostic factor (19). However, that study had several important limitations, including a relatively small sample size (n=65) from a single institute, no data on protein expression, and no functional analyses to understand the potential oncological roles of MAGE-D4 in ESCC.
The present study was designed to validate and extend our previous findings. To this end, we analyzed the biological functions of MAGE-D4 in ESCC cell lines in vitro and evaluated the relationship between survival and MAGE-D4 expression at both the mRNA and protein levels using two additional patient cohorts.
Sequences of primers and siRNAs.
Materials and Methods
Ethics. This study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects and was approved by the Institutional Review Board of Nagoya University, Japan (approval number 2014-0044) and the Ethics Committee of Akita University School of Medicine (number 1495). Written informed consent for the use of clinical samples and data was obtained from all patients, either directly or through the appropriate database.
Sample collection. Thirteen human ESCC cell lines (NUEC2, TE1, TE2, TE3, TT, TTn, WSSC, KYSE510, KYSE590, KYSE890, KYSE1170, KYSE1260, and KYSE1440) were obtained from the American Type Culture Collection (Manassas, VA, USA) or the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) (20). Cells were stored at −80°C in a cell preservative (Cell Banker; LSI Medience Corporation, Tokyo, Japan) and cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in an atmosphere containing 5% CO2 at 37°C.
For the first validation cohort, primary ESCC tissues were collected from 177 patients who underwent esophageal resection for EC at the Department of Thoracic Surgery, Akita University Hospital between 2000 and 2011 (21). The patients had received no treatment before curative surgery. The tissue specimens were embedded in paraffin and a Tissue Microarray (TMA) was constructed at the Pathology Institute (Toyama, Japan) (22-24), and stained as described below. Specimens were classified histologically using the 8th edition of the Union for International Cancer Control (UICC) classification. Relevant clinicopathological parameters were acquired from medical records. For the second validation cohort, an RNA sequencing dataset from 184 ESCC patients was obtained from TCGA via cBioPortal (http://www.cbioportal.org/).
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and PCR array analysis. MAGE-D4 mRNA expression was determined by qRT-PCR. Total RNA isolated using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) from cell lines. Reverse transcription was performed as follows: 10.5 μl 1 μg/μl RNA, 4 μl of 5X first strand buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 μl 100 mM dithiothreitol (Thermo Fisher Scientific, Inc.), 1 μl 10 mM dNTP mix (Promega Corporation, Madison, WI, USA), 1 μl random primers (Roche Diagnostics, Basel, Switzerland), 1 μl 200 U/μl Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific, Inc.) and 0.5 μl RNase inhibitor (Roche Diagnostics) were mixed and incubated for 60 min at 37°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression levels (TaqMan; GAPDH control reagents; Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were quantified, and the data were used to normalize the expression levels. qRT-PCR was performed using the SYBR Green PCR Core Reagents kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) as follows: One cycle at 95°C for 10 min; 40 cycles at 95°C for 5 sec and 60°C for 60 sec without a final extension step. The samples were tested in triplicate, and samples without a template were included in each PCR plate as a negative control. Real-time SYBR Green fluorescence was detected using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) (25) and the 2-ΔΔCq method was used for PCR quantification (26). The expression level of each sample is expressed as the value of the MAGE-D4 amplicon divided by that of GAPDH (27). The PCR primers are listed in Table I. A Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler PCR Array (Qiagen, Hilden, Germany) kit was used to assess expression of 84 key genes that encode transcription factors, ECM proteins as well as proteins involved in EMT, cell differentiation, morphogenesis, growth, proliferation, migration, cytoskeleton and major signalling pathways (27).
Small interfering RNA (siRNA)-mediated knockdown of MAGE-D4. KYSE590 cells were plated at 5×104 cells/ml in 24-well plates and incubated overnight. The cells were then transiently transfected with 30 nM of siRNA (siMAGE-D4 or siControl; sequences given in Table I) using LipoTrust EX Oligo (Hokkaido System Science, Sapporo, Japan). After transfection, cells were cultured in antibiotic-free RPMI-1640 with 10% FBS for 72 h before use in functional assays.
Cell proliferation assays. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells (3×103 cells per well) were seeded into 96-well plates with RPMI-1640 containing 2% FBS. The optical density of the solution in each well was measured 1, 3, and 5 days after seeding, 2 h after the addition of 10 ml of Cell CCK-8 solution (27).
Cell adhesion assay. A CytoSelect 48-Well Cell Adhesion Assay kit (Cell Biolabs, San Diego, CA, USA) was used to measure cell adhesion to the extracellular matrix (ECM) components fibronectin, collagen I, collagen IV, laminin I, and fibrinogen, with bovine serum albumin as a control. Cells (1.5×105 cells per well) were added to each well, and optical density at 560 nm of each well was measured after 1 h (28).
Wound-healing assay. Cell migration was evaluated using wound-healing assays. Cells (2×104 cells per well) were seeded into 12-well plates in RPMI-1640 containing 2% FBS using the ibidi Culture insert method (ibidi GmbH, Martinsried, Germany). After 24 h, the insert was removed, and the wound width was measured in 100-mm intervals (20 measurements per well, 40× magnification) (29).
Immunohistochemical (IHC) staining. TMA blocks were sectioned and incubated for 1 h at room temperature (25°C) with a rabbit anti-MAGE-D4b polyclonal antibody (HPA003554, Sigma-Aldrich) diluted 1:1000 in ChemMate antibody diluent (Dako, Carpinteria, CA, USA). For semi-quantification of MAGE-D4 protein expression, two investigators who were blinded to the clinical data evaluated the tissue staining and assigned a score based on the HER2 IHC scoring system (American Society of Clinical Oncology/College of American Pathologists guidelines). Protein expression was scored as 3+ (intense cytoplasmic or nuclear staining in >30% of cells), 2+ (moderate cytoplasmic or nuclear staining in >10% of cells), 1+ (weak staining in <10% of cells), and 0 (no staining, negative) (21).
Statistical analysis. Data are presented as the means±standard deviation (SD) of the indicated number of replicates unless noted. The χ2 test was used to analyze associations between MAGE-D4 expression and clinicopathological parameters. The significance of the difference between two variables was assessed using Spearman's rank correlation coefficient. OS and disease-free survival (DFS) were calculated using the Kaplan–Meier method, and differences were analyzed using the log-rank test. Prognostic factors were identified using Cox proportional hazards models, and variables with p<0.05 in the univariate analysis were entered into the final model. All analyses were performed using JMP 13 software (SAS Institute, Cary, NC, USA). p<0.05 was considered statistically significant.
Results
Coordinated expression of MAGE-D4 and cancer-related genes in ESCC cell lines. In order to investigate the expression and function of MAGE-D4 in ESCC, we first examined the mRNA levels of MAGE-D4 and a panel of 84 EMT-related genes in 13 ESCC cell lines of differentiated (n=6), undifferentiated (n=3), and unknown differentiation (n=4) status. qRT-PCR analysis revealed no significant difference in MAGE-D4 mRNA levels among the ESCC cell lines (Figure 1A). PCR array analysis of 84 EMT-related genes identified strong correlations (Spearman coefficient ≥0.6) between the expression of MAGE-D4 and five EMT-related genes; namely, matrix metalloproteinase 9 (MMP9), transforming growth factor β1 (TGFB1), transmembrane protein 132A (TMEM132A), snail family transcriptional repressor 3 (SNAI3), and versican (VCAN) (Figure 1B, C).
Tumor expression of MAGE-D4 and clinical characteristics of ESCC patients (n=177).
Effect of MAGE-D4 knockdown on the biological activities of ESCC cells. Given the importance of the EMT in the development of malignant behavior, we next evaluated the importance of MAGE-D4 in cell proliferation, migration, and adhesion. The KYSE590 cell line, which expressed the highest levels of MAGE-D4 mRNA, was transfected with a control sequence (siControl) or MAGE-D4-targeting siRNA (siMAGE-D4) and analyzed by qRT-PCR to confirm effective gene silencing (Figure 2A). Next, we evaluated cell proliferation using the CCK-8 assay, interactions with five ECM proteins (fibronectin, collagen I, collagen IV, laminin I, and fibrinogen) using an adhesion assay, and cell migration using a wound-healing assay. Notably, compared with the control cells, MAGE-D4 knockdown significantly decreased the proliferation of KYSE590 cells at 72 h and 120 h after transfection (Figure 2B). Similarly, cell migration at 24 h and 36 h after transfection was significantly suppressed by MAGE-D4 knockdown (Figure 2C). However, MAGE-D4 knockdown had little influence on the ability of KYSE590 cells to adhere to the five ECM proteins. Thus, MAGE-D4 plays a crucial role in ESCC cell proliferation and migration; two fundamental behaviors associated with tumor growth and metastasis.
Analysis of MAGE-D4 expression in 13 ESCC cell lines. (A) qRT-PCR analysis of MAGE-D4 mRNA normalized to GAPDH mRNA levels. (B) PCR array analysis of MAGE-D4 and EMT-related genes. Data were normalized to TE1 mRNA levels. (C) Correlation between MAGE-D4 and MMP9, TGFB1, TMEM132A, SNAI3, and VCAN mRNA levels, using Spearman's rank correlation coefficient.
Expression and prognostic value of MAGE-D4 protein in the first validation cohort. To investigate the expression of MAGE-D4 protein and its relationship to clinicopathological characteristics we performed IHC staining on TMA of tumor and normal tissue samples collected from 177 patients undergoing curative surgery for ESCC. The population was composed of 153 males and 24 females (66±8.2 years, mean±SD; range=38-82 years) with pathologically diagnosed differentiated (n=120) and undifferentiated (n=57) ESCC. Based on the UICC classification (eighth edition), 10, 44, and 123 patients had stage I, II, and III ESCC, respectively.
IHC staining of MAGE-D4 protein was evaluated using a semiquantitative scoring system that evaluates the localization, intensity, and extent of protein expression. Representative photomicrographs of specimens with negative (0) and positive (3+, 2+, 1+) expression are shown in Figure 3A. Of the 177 patient samples, 83 (46.9%) scored 0, 61 (34.5%) scored 1+, 31 (17.5%) scored 2+, and 2 (1.1%) scored 3+. For evaluation of the prognostic value of MAGE-D4 protein expression, patients were dichotomized into a MAGE-D4-positive group (scores 1+, 2+, or 3+; n=83) and a MAGE-D4-negative group (score 0; n=94), and OS and DFS were compared using the Kaplan–Meier method. We found that the 5-year OS rate of the MAGE-D4-positive group was significantly smaller than that of the MAGE-D4-negative group (37% vs. 65%, p=0.0001), and the same outcome was obtained when the 5-year DFS rates were compared (42% vs. 66%, p=0.0015; Figure 3B).
Effects of siRNA-mediated knockdown of MAGE-D4 on ESCC cell function. (A) qRT-PCR analysis of MAGE-D4 expression in untransfected or siRNA-transfected KYSE590 cells. *p<0.05. (B) CCK-8 cell proliferation assay of untransfected or siRNA-transfected KYSE590 cells. Mean±SD. *p<0.05. (c) Representative images of migration and the mean migration distance from wound-healing assays of untransfected or siRNA-transfected KYSE590 cells. Mean±SD. *p<0.05.
We examined the potential correlation between MAGE-D4 protein expression and patient clinicopathological characteristics (Table II) and found no significant differences between the MAGE-D4-positive and -negative patient groups with respect to age, sex, Brinkman index, alcohol consumption, tumor location, tumor depth, differentiation, lymphatic involvement, vascular invasion, lymph node metastasis, or UICC Stage. However, univariate analysis identified age ≥70 years, undifferentiated tumor, lymphatic involvement, lymph node metastasis, stage III disease, and positive tumor expression of MAGE-D4 protein as significant indicators of poor OS (Table III). In multivariate analysis, age ≥70 [hazard ratio (HR)=1.65, 95% confidence interval (95%CI)=1.06-2.53; p<0.025] and MAGE-D4 protein expression (HR=2.36, 95%CI=1.52-3.75; p<0.001) remained the only independent prognostic factors.
Expression and prognostic value of MAGE-D4 mRNA in the second validation cohort. To confirm these results, we analyzed a second validation cohort consisting of a TCGA RNA sequencing dataset from 184 patients with ESCC. The patients were dichotomized into low (n=152) and high (n=32) MAGE-D4 mRNA expression groups using a cut-off value of 0.7 (log). Patients with high MAGE-D4 mRNA expression had significantly shorter OS and DFS than the patients with low tumor mRNA levels (p=0.0082 and p=0.0327, respectively; Figure 3C), consistent with the findings from the first validation cohort.
Discussion
In this study, we sought to increase our understanding of the functions of MAGE-D4 in ESCC and to determine the prognostic value of MAGE-D4 mRNA and protein expression in two independent patient cohorts, thereby validating the results of our previous study. In the in vitro analyses, we found that expression of MAGE-D4 correlated positively with that of five EMT-related genes (MMP9, TGFB1, TMEM132A, SNAI3, and VCAN) and that silencing of MAGE-D4 expression significantly decreased the proliferation and migration abilities of ESCC cells. In the clinical study, MAGE-D4 protein expression was an independent prognostic factor for OS in ESCC.
Analysis of MAGE-D4 protein expression in ESCC and correlation with patient survival. (A) Representative images of MAGE-D4 immunohistochemical staining in a TMA from the first validation cohort. Examples of negative (0) and positive (1+, 2+, and 3+) expression are shown (100× magnification). (B) Overall survival and disease-free survival of the first validation cohort (n=177) stratified by positive (n=83) or negative (n=94) MAGE-D4 protein expression. (C) Overall survival and disease-free survival periods of the second validation cohort (TCGA, n=184) stratified by high (n=32) or low (n=152) MAGE-D4 mRNA expression.
Zhili reported that microRNA-539 could inhabit EMT in TE3 cells through TWIST1-mediated regulation of MAGE-A4 (30). To our knowledge, this is the first report of an association between MAGE-D4 and the EMT. Because the EMT is the process through which cells switch their differentiation and behavior from an adhesive to an invasive/migratory phenotype, it is a crucial event in the progression of cancer and is highly regulated at the transcriptional level (31, 32). In this study, we found that expression of the EMT-related genes MMP9, TGFB1, TMEM132A, SNAI3, and VCAN correlated strongly with that of MAGE-D4 in ESCC cells. MMPs have established roles in the development of human malignancies through remodeling of the ECM. Degradation of collagen IV in the basement membrane and ECM facilitates tumor invasion, metastasis, growth, and angiogenesis, thereby promoting tumor progression (33). TGF-β is a multifunctional cytokine that controls the proliferation and differentiation of many cell types, among other functions. TGF-β secreted within the tumor microenvironment is one of several factors known to promote the EMT (34). TMEM132A is a poorly characterized protein, but it is thought to promote neuronal cell survival by regulating stress-related genes (35). The Snail family protein SNAI3 is a transcriptional repressor implicated in embryogenesis and carcinogenesis. During development, SNAI3 represses transcription of the adhesion molecule E-cadherin and regulates the EMT (36). The role of VCAN in cell adhesion, migration, and proliferation has been extensively studied. An increase in VCAN expression is detected in many tumors, including cancers of the breast, brain, ovary, gastrointestinal tract, and prostate, as well as melanoma, sarcoma, and peritoneal mesothelioma (37). Our data suggest that, in ESCC, MAGE-D4 participates in the EMT through its coordinated expression with other EMT-regulating molecules. However, further work will be necessary to fully understand the involvement of MAGE-D4 in the EMT in ESCC and other cancers.
Prognostic factors for overall survival in ESCC patients (n=177).
In the present study, we showed that knockdown of MAGE-D4 expression reduced the proliferation and migration abilities of KYSE590 ESCC cells, which is indicative of an active role of MAGE-D4 in the malignant behavior of cells and is consistent with our finding that high MAGE-D4 expression is associated with poor prognosis. Our results have some important translational implications for ESCC. For example, patients with positive MAGE-D4 expression detected in endoscopic biopsies or surgical specimens might benefit from intensive postoperative surveillance and perioperative systemic treatment to anticipate early recurrence and subsequent adverse prognosis.
Several limitations of this study should be acknowledged. First, experimental analysis of MAGE-D4 expression was performed retrospectively on tissues collected from a relatively small number of patients. Second, pathway analyses of the MAGE-D4 network would strengthen the results and further our understanding of the functions of this gene in ESCC biology. Finally, in vivo experiments using mouse xenograft models would be helpful in clarifying the role of MAGE-D4 in ESCC pathogenesis.
In conclusion, our findings support an important role for MAGE-D4 in the malignant behavior of ESCC. Our study of two independent cohorts totaling 361 patients also robustly confirms the validity of our previous finding that MAGE-D4 expression is an independent prognostic factor in ESCC.
Acknowledgements
The Authors would like to thank Anne M. O'Rourke, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Footnotes
Authors' Contributions
YU, MKa, and SU performed the experiments and the data analysis. YU, MKa, YS, DS, SU, NH, MH, CT, DK, SY GN, SM, MKo and YK collected the cases and the clinical data. YU and MKa conceived and designed the study and prepared the initial manuscript. YK supervised the project. All Authors contributed to the final manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare no competing financial interests regarding this study.
- Received September 25, 2019.
- Revision received October 8, 2019.
- Accepted October 9, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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