Demethylation-mediated upregulation of melanoma-associated antigen-A11 correlates with malignant progression of esophageal squamous cell carcinoma.

BACKGROUND: The expression and methylation status of oncogenes are closely related to the onset and progression of cancer. AIMS: To explore the role and methylation status of melanoma-associated antigen-A11 in the pathogenesis of esophageal squamous cell carcinoma. METHODS: 116 esophageal squamous cell carcinoma patients with tumor tissues and corresponding adjacent normal tissues were obtained. The expression level and methylation status of melanoma-associated antigen-A11 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues were determined respectively. RESULTS: Significant up-regulation of melanoma-associated antigen-A11 was detected in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues. Up-regulation of melanoma-associated antigen-A11 contributed to proliferation and invasion in cancer cells. Hypomethylation of the CpG site was associated with pathological differentiation, clinical stage, tumor size, lymph node metastasis and distant metastasis. Esophageal squamous cell carcinoma patients in stage III and IV, with high expression of melanoma-associated antigen-A11 or hypomethylation of the CpG site within the promoter demonstrated poor survival. CONCLUSION: Melanoma-associated antigen-A11 is up-regulated in esophageal squamous cell carcinoma at least partly by hypomethylation of the CpG site within the promoter and this hypomethylation may affect the prognosis of esophageal squamous cell carcinoma patients.

Expression and prognostic significance of MAGE-A11 and transcription factors (SP1,TFCP2 and ZEB1) in ESCC tissues.

OBJECTIVE: To evaluate the clinical characteristics and prognostic significance of MAGE-A11 and transcription factors (SP1, TFCP2 and ZEB1) in patients with esophageal squamous cell carcinoma (ESCC). METHODS: To assess the expression of MAGE-A11 and transcription factors (SP1, TFCP2 and ZEB1) in 121 ESCC samples were respectively detected by immunohistochemical method. RESULTS: The results showed MAGE-A11 and transcription factors (SP1,TFCP2 and ZEB1) expression were associated with some clinical features in patients, such as pathological differentiation, tumor size, clinical stage, lymph node metastasis and distant metastasis. Kaplan-Meier analysis showed that patients with ESCC having high MAGE-A11 and transcription factors (SP1,TFCP2 and ZEB1) expression had a worse prognosis compared to the patients with low expression. Multivariate Cox proportional hazards regression model revealed that MAGE-A11 expression, TFCP2 expression, lymph node metastasis and distant metastasis were independently associated with ESCC patients' survival. CONCLUSIONS: High expression of MAGE-A11 and transcription factors (SP1,TFCP2 and ZEB1) in ESCC tissues suggests promoting ESCC progression and poor prognosis, co-expression of MAGE-A11 and transcription factors even worse.

Tumor suppressive miR-6775-3p inhibits ESCC progression through forming a positive feedback loop with p53 via MAGE-A family proteins.

Accumulating evidences indicate that microRNAs (miRNAs) play vital roles in multiple diseases, including cancer. In the present study, we showed that miR-6775-3p plays a tumor suppressive role in esophageal squamous cell carcinoma (ESCC). High expression miR-6775-3p is associated with good clinical outcomes of ESCC patients. Over-expression of miR-6775-3p inhibited tumor growth and liver metastasis of ESCC xenograft tumors. Enforced expression of miR-6775-3p inhibited ESCC cell proliferation, migration, and invasion. KEGG pathway analysis revealed that miR-6775-3p was associated with the genes on "pathway in cancer". Mechanically, miR-6775-3p inhibited the expression of tumor antigens MAGE-A family through direct binding the 3'UTR region of MAGE-A mRNAs, and attenuated MAGE-A-inhibited transcriptional activity of tumor suppressor p53. In addition, miR-6775-3p also directly inhibits its host gene SLC7A5 which has been reported to play oncogenic roles in cancer progression. Interestingly, miR-6775-3p and its host gene SLC7A5 were directly transcriptionally induced by p53. Thus, for the first time, our study proposed a novel positive feedback regulation between miR-6775-3p and p53 via MAGE-A family, which plays crucial role in ESCC progression.

miR­92b promotes autophagy and suppresses viability and invasion in breast cancer by targeting EZH2.

MicroRNAs (miRs) are a small non-coding RNA family with a length of 18-22 nucleotides. They are able to regulate gene expression by either triggering target messenger RNA degradation or by inhibiting mRNA translation. Enhancer of zeste homolog 2 (EZH2) is the core enzymatic subunit of polycomb repressor complex 2 and is responsible for the trimethylation of histone 3 on lysine 27 (H3K27me3); it is also able to silence a bundle of tumor suppressor genes through promoter binding. However, little is known regarding the effect of miR­92b on cell autophagy, viability and invasion as well as how it interacts with EZH2. The present study investigated the major role of miR­92b in the autophagy, viability and invasion of breast cancer. It was revealed that in MCF­7 and MDA­MB­453 cells, the expression of miR­92b promoted autophagy induced by starvation and rapamycin treatment. The results of in vitro experiments results demonstrated that miR­92b inhibited breast cancer cell viability, invasion and migration. To further elucidate the regulatory mechanisms of miR­92b in autophagy, a dual luciferase reporter assay was performed to determine whether miR­92b targeted the EZH2 gene. The expression of miR­92b was negatively correlated to EZH2 mRNA expression in breast cancer. Depletion of EZH2 induced phenocopied effects on miR­92b overexpression, thereby demonstrating its importance in autophagy. These results indicated that miR­92b may serve an important role in breast cancer in controlling autophagy, viability and invasion. The present study indicated that miR­92b and EZH2 may serve as potential biomarkers for cancer detection and highlighted their possible therapeutic implications.

Circular RNA ciRS-7 accelerates ESCC progression through acting as a miR-876-5p sponge to enhance MAGE-A family expression.

As the most well-known circular RNA, ciRS-7 (also termed CDR1as) has been reported to act as a miR-7 sponge, resulting in reduced miR-7 activity and increased miR-7-targeted transcripts. Here, we showed that ciRS-7 is up-regulated in esophageal squamous cell carcinoma (ESCC), and is associated with the poor clinicopathological parameters of ESCC patients. Moreover, over-expression of ciRS-7 increased the proliferation, migration and invasion of ESCC cells. Mechanistic studies revealed that ciRS-7 contains nineteen miR-876-5p binding sites and acts as a miR-876-5p sponge. Over-expression of ciRS-7 resulted in the reduced tumor-repressive function of miR-876-5p on its downstream target MAGE-A family. In animal experiments, enforced ciRS-7 increased ESCC tumor growth and metastasis through targeting miR-876-5p/MAGE-A family axis. Collectively, our study provided novel evidence that ciRS-7 accelerates ESCC progression by acting as a miR-876-5p sponge to enhance MAGE-A family expression.

MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates ESCC tumor growth.

Recently, we have reported that the product of Melanoma Antigens Genes (MAGE) family member MAGE-A11 is an independent poor prognostic marker for esophageal squamous cell carcinoma (ESCC). However, the reason how MAGE-A11 is activated in ESCC progression still remains unclear. In the current study, we demonstrated that DNA methylation and the subsequent histone posttranslational modifications play crucial roles in the regulation of MAGE-A11 in ESCC progression. We found that the methylation rate of TFCP2/ZEB1 binding site on MAGE-A11 promoter in ESCC tissues and cells is higher than the normal esophageal epithelial tissues and cells. Transcription factors TFCP2 and ZEB1 directly bind MAGE-A11 promoter and regulate the endogenous MAGE-A11 expression in a methylation-dependent manner in ESCC cells. Following MAGE-A11 promoter methylation, the methyl-CpG-binding protein MeCP2 was found to bind the methylated MAGE-A11 promoter to mediate histone deactylation by recruiting HDAC1 and HDAC2. Simultaneously, histone inactivation marks including H3K27me3 as well as H3K9me3 were increased, whereas histone activation mark H3K4me3 was decreased. HDAC inhibitor Trichostatin A (TSA) increased DNA methylase inhibitor Decitabine (DAC)-induced MAGE-A11 expression. siRNA-mediated knockdown of histone methltransferase EZH2 or DZNep (a EZH2 inhibitor) treatment increased DAC-induced MAGE-A11 expression. Our results indicate that MAGE-A11 is activated through DNA demethylation, histone acetylation and histone methylation in ESCC, and its activation promotes ESCC tumor growth.

MAGE-A gene expression in peripheral blood serves as a poor prognostic marker for patients with lung cancer.

BACKGROUND: MAGE-A genes belong to the cancer/testis antigens family. The prognostic significance of MAGE-A expression in the peripheral blood of patients with lung cancer is unknown. Therefore, this study evaluated the expression and possible prognostic significance of MAGE-A in the peripheral blood of patients with lung cancer. METHODS: In this study, we detected MAGE-A gene expression in the peripheral blood of 150 patients with lung cancer and 30 healthy donors using multiplex semi-nested PCR and analyzed their correlation with clinicopathological risk factors. RESULTS: MAGE-A expression was associated with factors indicating poor prognosis. The expression of MAGE-A and each individual MAGE-A gene were also associated with low overall survival in patients with lung cancer. CONCLUSION: The expression of MAGE-A genes in peripheral blood may act as a poor prognostic marker in patients with lung cancer.

miR-92b suppresses growth and invasion of esophageal cancer Eca109 cells by regulating EZH2 gene expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the regulating effects of miR-92b on the expression of EZH2 (enhancer of zeste homolog 2) gene and the proliferation and invasion abilities of esophageal cancer (EC) cells. Methods: Fifteen cases of esophageal cancer tissues that preserved in the research center of the Fourth HospitalAffiliated to Heibei Medical University from January 2016 to January 2017 were selected for this study. The bioinformatics tool was used to predict the possible miRNAs that might target EZH2. The mimics of predicted miRNAs were transfected into human esophageal carcinoma cell lines Eca109, respectively. Then the regulation effect of miRNAs on EZH2 gene expression was validated by real-time PCR, Western blotting and dual luciferase reporter experiment. In the meanwhile, EZH2 over-expression plasmids were co-transfected into esophageal carcinoma Eca109 cells, and the effects of miRNAs and EZH2 expression changes on the proliferation, apoptosis , invasion and migration of esophageal carcinoma cells were detected by CCK-8 method, Flow Cytometry, Transwell Invasion and migration assay, respectively. Results: Bioinformatics analysis showed that miR-92b, let7a and miR-25 could combine with potential binding sites at 3’-terminal non-translation region of EZH2 gene. Real-time PCR results showed that only miR-92b was able to regulate the expression of EZH2, and miR-92b was negatively correlated to EZH2 in esophageal cancer (P<0.01). Compared with mimic-NC, the expression of EZH2 mRNA, protein and luciferase activity in Eca109 cells after miR-92b mimic transfection was significantly down-regulated (both P<0.01). However, miR-92b mimic transfection had no effect on the apoptosis of Eca109 cells. Moreover, the proliferation, invasion and migration of Eca109 cells were significantly inhibited after transfection with miR-92b-mimic (P<0.01). In addition, after co-transfection with EZH2 over-expression plasmids, the effects of miR-92b-mimic on the proliferation, invasion and migration of Eca109 cells were significantly weakened (P<0.01). Conclusion: miR-92b can inhibit the proliferation,invasionandmigrationofesophagealcarcinomacells,anditsmechanismmayberelatedtoitstargetregulationofEZH2. ··

Expression of circular RNA ciRS-7 in tissues of sophageal squamous cell carcinoma and its effect on cell biological characteristics of TE1 cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the expression of ciRS-7 in esophageal squamous cell carcinoma (ESCC) and its effect on the cellular proliferation, migration and invasion. Methods: The cancer tissues and paired adjacent normal tissues from 60 ESCC patients treated in the Fourth Hospital of Hebei Medical University between May, 2016 andApril, 2017 were selected for this study. The expressions of ciRS-7 were detected by qRT-PCR. After over-expressing or silencing of ciRS-7, the proliferation of ESCC cell line TE1 was measured by CCK-8 assay; and the migration and invasion were tested by wound healing assay and Transwell invasion assay，respectively. Finally, the effect was validated via animal experiment. Results: CiRS-7 was highly expressed in ESCC tissues (P<0.05), and its expression level was closely related to pathological grade and lymph node metastasis (P<0.05). Over-expression of ciRS-7 significantly increased the proliferation, migration and invasion (all P<0.05) of TE1 cells; while silencing of ciRS-7 remarkably suppressed the proliferation, migration and invasion (all P<0.05). Conclusion: CiRS-7 was up-regulated in ESCC and could enhance ESCC cell proliferation, migration and invasion, suggesting that ciRS-7 could be used as a potential target for the diagnosis and treatment of ESCC.

Expression and clinical significance of melanoma antigen A gene family in peripheral blood of esophageal carcinoma patients / 中国肿瘤生物治疗杂志

@# Objective: To evaluate the expression of melanoma antigen A family（MAGE-As）in the peripheral blood of patients with esophageal carcinoma (EC), and to analyze its correlations to the clinicopathological features and the prognosis of EC patients. Methods: mRNA expression of MAGE-As in peripheral blood from 153 EC patients and 30 healthy donors was detected using multiplex semi-nested PCR. In addition, restriction endonuclease treatment was used to determine the expression of MAGE-As family members, including MAGE-A1, A2, A3, A4 and A6. Results: The positive expression of MAGE-As was observed in 30 of 153 EC patients (19.61%) in peripheral blood. The positive expression rate of MAGE-A1, A2, A3, A4, A6 was 10.46% (16/153), 16.34%(25/153), 9.8% (15/153), 11.11% (17/153) and 18.30%(28/153), respectively. Additionally, the expression of MAGE-As was positively associated with clinical stage, lymphatic metastasis and distant metastasis (all P<0.05). The positive expressions of MAGE-As and its sub-type genes were all associated with low 5-year overall survival of ES patients (all P<0.05). Expression of MAGE-As, tumor volume, lymphatic metastasis and distant metastasis can be used as independent prognostic factors for the survival of EC patients (all P<0.01). Conclusion: The expression of MAGE-As in peripheral blood of EC patients was associated with the prognosis of EC, and may be used as an important indicator for the prognosis of esophageal carcinoma.

miR-124 regulates autophagy to inhibit invasion and migration of esophageal cancer KYSE170 cells by targeting BECN1 / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of miR-124 on the invasion and migration of esophageal cancer KYSE170 cells by regulating autophagy. Methods: miR-124 mimic was transfected into esophageal cancer KYSE170 cells. Transwell assay was used to detect the change of invasion and migration ability of cells. Dual luciferase reporter gene assay was used to verify the targeted regulation of BECN1 (Beclin1) by miR-124, and Western blotting was used to analyze the expressions of BECN1, P62 and LC3 protein. siRNA targeting BECN1 was transfeted into KYSE170 cells, and then the cell invasion and migration ability was calculated by Transwell assay. The expressions of BECN1, P62 and LC3 protein were detected by Western blotting. miR-124 mimic and BECN1 over-expression plasmid were co-transfected into KYSE170 cells, and then Transwell assay was used to detect the changes of cell invasion and migration ability, and Western blotting to examine the expression levels of autophagy-related gene. Results: The invasion and migration ability of KYSE170 cells were significantly inhibited after transfection with miR-124 mimic (All P<0.05). The expression of autophagyrelated protein P62 was increased, and the expression of BECN1 and LC3 was significantly decreased (All P<0.01); in addition, the activity of luciferase reporter gene was also significantly reduced (P<0.01). Silencing BECN1 expression inhibited the invasion and migration of esophageal cancer KYSE170 cells (P<0.01). However, after co-transfection with BECN1 over-expression plasmids, the effects of miR-124 mimic on the autophagy, invasion and migration of esophageal carcinoma KYSE170 cells were significantly weakened (P<0.01), it was also accompanied with lower P62 expression, and higher LC3 expression (P<0.01). Conclusion: miR-124 mimic can inhibit the invasion and migration of esophageal carcinoma cells. The mechanism may be related to the autophagy-related gene BECN1 expression.

MAGE-A family is involved in gastric cancer progression and indicates poor prognosis of gastric cancer patients.

BACKGROUND: As the best characterized CTA family members, melanoma-associated antigens (MAGE) have been reported to express in various malignant tumors. However, the expression pattern of MAGE-A family in gastric cancer (GC) specimens and their prognostic and therapeutic significance for GC patients is still unclear. MATERIALS AND METHODS: Tissue microarray - based immunohistochemistry analysis was used to examine the expression of MAGE-A family members (including MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12) in 86 cases of GC specimens, 20 cases of the corresponding adjacent normal gastric specimens, and 9 cases of intraepithelial neoplasia specimens. The association between MAGE-A expression and the clinicopathological parameters as well as the 5-year overall survival of GC patients was analyzed. RESULTS: 54.7% of GC specimens showed positive MAGE-A expression. In the adjacent normal gastric specimens, MAGE-A was not expressed in the normal surface mucous cells, but expressed in some normal fundic glands. In addition, MAGE-A expression was also detected in intraepithelial neoplasia specimens. In GC specimens, MAGE-A expression was associated with lymph node metastasis, poor differentiation, and high clinical TNM stage. MAGE-A expression was also correlated with the poor 5-year overall survival of GC patients. However, MAGE-A expression is not an independent prognostic factor for GC patients. CONCLUSIONS: MAGE-A family may be involved in the gastric cancer progression, and their expression could be considered to improve the prognostic evaluation for GC patients.

Zebularine Treatment Induces MAGE-A11 Expression and Improves CTL Cytotoxicity Using a Novel Identified HLA-A2-restricted MAGE-A11 Peptide.

Melanoma-associated antigen-A11 (MAGE-A11) is frequently expressed in breast cancer and is associated with poor prognosis. Therefore, MAGE-A11 is a potential immunotherapy target in breast cancer. MAGE-A11 expression, however, is downregulated in many patients, thus constraining the application of immunotherapy. The induction of MAGE-A11 expression is crucial for the recognition and killing of breast cancer cells by cytotoxic T lymphocytes (CTL). In this study, a series of HLA-A2-restricted candidate MAGE-A11 peptides were predicted, synthesized, and tested. Of the selected peptides, p350 (FLFGEPKRL) elicited peptide-specific CTLs from healthy HLA-A*0201-positive donors. The induced CTLs can lyse MAGE-A11-expressing breast cancer cells but not MAGE-A11-negative tumor cells. To improve antitumor immune response, zebularine, a DNA methyltransferase inhibitor, was used to induce MAGE-A11 expression and upregulate the cytotoxicity of antigen-specific T cells in breast cancer cell lines and primary breast cancer cells. The present findings suggested that peptide p350 induces peptide-specific cytolytic activity and is thus a potential candidate for tumor vaccination or T-cell therapy. Epigenetic modulation by zebularine can induce MAGE-A11 expression in breast cancer cells and facilitate cytotoxicity via MAGE-A11-specific CTL.

MAGE-A family expression is correlated with poor survival of patients with lung adenocarcinoma: a retrospective clinical study based on tissue microarray.

OBJECTIVES: As the best characterised cancer/testis antigen family members, melanoma-associated antigens (MAGE) have been reported to be expressed in various malignant tumours. However, the expression pattern of MAGE-A family in lung adenocarcinoma (LAC) specimens and their prognostic and therapeutic significance for patients with LAC is still unclear. MATERIALS AND METHODS: Tissue microarray-based immunohistochemistry analysis was used to examine the expression of MAGE-A family members (including MAGE-A1, A2, A3, A4, A6, A10, A11 and A12) in 105 paired LAC specimens and the corresponding pericarcinoma specimens. The association between MAGE-A expression and the clinicopathological parameters, and the 10-year overall survival of patients with LAC were analysed. In addition, the association between MAGE-A expression and the epithelial growth factor receptor (EGFR) amplification and ALK-EML4 rearrangements of patients with LAC were also analysed. RESULTS: The immunohistochemical evaluation revealed that MAGE-A family was expressed in 46.66% of LAC specimens, but not in the corresponding pericarcinoma specimens. MAGE-A expression was not associated with the clinicopathological factors but with worse 10-year survival, and was a poor prognostic marker for patients with LAC. MAGE-A expression was not correlated with EGFR amplification and ALK rearrangements. Interestingly, MAGE-A expression can affect the overall survival of patients with LAC without EGFR amplification or ALK rearrangements, but not affect the overall survival of patients with LAC and EGFR amplification or ALK rearrangements. CONCLUSIONS: Molecular assessment of MAGE-A family members could be considered to improve the prognostic evaluation and to provide a new potential therapeutic strategy for patients with LAC.

Prognostic Significance of MAGE-A11 in Esophageal Squamous Cell Carcinoma and Identification of Related Genes Based on DNA Microarray.

BACKGROUND AND AIMS: This study aims to investigate the expression pattern of melanoma-associated antigen-A11 (MAGE-A11) in esophageal squamous cell carcinoma (ESCC) specimens and analyze its prognostic significance for ESCC patients. In addition, the purpose of our study was also to explore the biological function of MAGE-A11 in ESCC cells based on DNA microarray. METHODS: Immunohistochemistry was used to detect the expression of MAGE-A11 in ESCC specimens, and its prognostic significance was analyzed by statistical analysis. DNA microarray and quantitative RT-PCR were used to explore the different expression of MAGE-A11 downstream genes in ESCC cells. Cell invasion assay and MTT assay were used to detect the effect of MAGE-A11 cDNA on the invasion and proliferation of ESCC cells. RESULTS: Of the ESCC specimens, 59.3% showed positive MAGE-A11 expression. MAGE-A11 expression in ESCC specimens was positively associated with distant lymph node metastasis. Overall survival of ESCC patients with positive MAGE-A11 expression was shorter than in patients with negative MAGE-A11 expression. Multivariate Cox regression analysis showed MAGE-A11 expression is an independent poor prognostic factor for ESCC patients. Overexpression of MAGE-A11 changed a variety of gene expressions, which was associated with various cell functions such as protein ubiquitination, cell proliferation and apoptosis, tumor invasion and metastasis. Overexpression of MAGE-A11 directly increased the invasion and proliferation of ESCC cells. CONCLUSIONS: MAGE-A11 is an independent poor prognostic marker for ESCC patients. MAGE-A11 regulates various cell functions and directly increases the invasion and proliferation of ESCC cells.