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XAF1 is frequently methylated in human esophageal cancer
Xiang-Yu Chen1, Qiao-Yu He, Ming-Zhou Guo
1Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China.
Aim:
To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis.
Methods:
Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BIC1 and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of primary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza-deoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immunohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clinicopathological data was analyzed by logistic regression.
Results:
MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (completely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal cancer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34.70%, χ(2) = 23.5840, P = 0.000). mRNA level of XAF1 measured with semi-quantitative reverse transcription polymerase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue (χ(2)= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal cancer, 24/32 samples were methylated, and 8/32 esophageal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher's exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metastasis with respect to methylation of XAF1 for the 72 tissue samples from patients with esophageal cancer.
Conclusion:
XAF1 is frequently methylated in esophageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.
Insights
Epigenetic silencing of the X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) gene through promoter hypermethylation is frequent in esophageal cancer. This methylation event correlates with decreased XAF1 expression, suggesting its role in carcinogenesis.
Area of Science:
- Epigenetics
- Cancer Biology
- Molecular Oncology
Background:
- Esophageal carcinogenesis involves complex genetic and epigenetic alterations.
- The X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) gene's role in esophageal cancer is not fully understood.
- Epigenetic regulation, particularly DNA methylation, can significantly impact tumor suppressor gene function.
Purpose of the Study:
- To investigate epigenetic modifications, specifically DNA methylation, in the XAF1 gene during esophageal carcinogenesis.
- To determine the correlation between XAF1 methylation status and its expression levels in esophageal cancer.
- To explore the potential role of XAF1 epigenetic silencing in the development of esophageal cancer.
Main Methods:
- Methylation-specific polymerase chain reaction (MSP) was employed to assess XAF1 promoter methylation in cell lines and clinical samples.
- XAF1 gene expression was analyzed using semi-quantitative reverse transcription polymerase chain reaction and Western blotting.
- Demethylation treatment with 5-aza-deoxycytidine (5-aza-dc) was performed to confirm the functional impact of methylation.
- Immunohistochemistry was utilized to evaluate XAF1 protein expression in primary tumors and adjacent tissues.
Main Results:
- XAF1 promoter hypermethylation was detected in a high percentage of esophageal cancer cell lines (75%) and primary tumors (75.00%) compared to normal esophageal mucosa (0%).
- XAF1 expression (mRNA and protein) was significantly reduced or lost in methylated esophageal cancer tissues and cell lines.
- Treatment with a demethylating agent restored XAF1 expression in methylated cell lines.
- XAF1 protein expression was inversely correlated with promoter methylation in primary esophageal cancer tissues (P = 0.004).
Conclusions:
- XAF1 is frequently epigenetically silenced by promoter region hypermethylation in esophageal cancer.
- XAF1 expression is directly regulated by promoter methylation status.
- These findings suggest that XAF1 acts as a tumor suppressor gene in esophageal cancer, and its silencing contributes to carcinogenesis.
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