Epigenetic and genetic silencing of CHFR in esophageal adenocarcinomas

Abstract

Background: The checkpoint with forkhead-associated domain and RING-finger domain (CHFR) is a mitotic checkpoint protein with tumor-suppressor functions. In this study, the authors investigated the epigenetic and genetic mechanisms that regulate CHFR expression in esophageal adenocarcinomas (EACs).

Methods: Quantitative reverse transcriptase polymerase chain reaction analysis demonstrated downregulation of CHFR transcript in 79% of EACs (44 of 56) compared with 41 normal samples (P < .001). Immunohistochemical analysis of CHFR protein expression showed absence or weak immunostaining for CHFR in 75% of EACs (56 of 75) compared with normal tissue samples. The authors next examined the promoter DNA hypermethylation of CHFR by using quantitative bisulfite pyrosequencing technology. They detected significant CHFR promoter DNA hypermethylation in 31% of tumor samples (18 of 58) compared with normal samples (P < .001). Treatment of OE33 cells with 5-Aza-deoxycytidine led to reduction in the promoter DNA methylation levels with restoration of the CHFR mRNA expression, which confirmed promoter DNA methylation as an epigenetic mechanism regulating CHFR expression. However, they identified several EACs where the CHFR mRNA expression was silenced in the absence of notable methylation. Therefore, the authors examined the relative DNA copy number level of CHFR compared with normal samples.

Results: The results confirmed a decrease or absence of the relative CHFR DNA copy number levels in 59% of tumor samples. Nine tumors that showed loss of CHFR mRNA expression, in absence of promoter DNA hypermethylation, demonstrated a significant loss of relative CHFR DNA copy numbers.

Conclusions: Taken together, their findings demonstrated that both epigenetic and genetic mechanisms were involved in silencing CHFR expression in EACs.

Figure 1. Comparison of CHFR mRNA expression levels in Normal vs Tumor

A) The expression of CHFR in normal mucosa (n=41) and EACs (n=56) was determined by Real-time RT-PCR and normalized to the average value of all the normal samples as described in materials and methods. Black boxes and triangles represent normal and tumor samples, respectively. The horizontal bars represent the mean expression fold. The statistical significance (p<0.0001) was determined by t-test. B) Expression of CHFR in 17 representative tumor samples and their corresponding normal samples from the same patients were analyzed side by side for comparison.

Figure 2. Comparison of DNA methylation levels

A) The percentage of promoter DNA methylation of the CHFR gene was determined by quantitative bisulfate pyrosequencing (Biotage). The horizontal bars locate the mean levels of DNA methylation. The statistical analysis of DNA methylation levels were determined by t-test. The tumors (EACs) were compared to normal samples. A p value of 0.05 was considered statistically significant. B) Methylation levels of eight representative matching normal and tumor samples.

Figure 3. A correlation analysis between DNA methylation and gene expression levels

A) The mRNA expression fold is shown for non methylated (Black box) and methylated (Black triangle) samples. The horizontal bars locate the means expression fold. The statistical difference was determined by t-test for paired samples (p<0.05). B) The Spearman correlation analysis between DNA methylation level and mRNA expression fold in CHFR gene. Significant correlation was found for CHFR (p<0.001). C) Treatment of OE33 esophageal adenocarcinoma cells with 5-Aza led to a decrease in DNA methylation and restoration of CHFR expression. The treatment with TSA or DMSO (control) had no effect on DNA methylation or expression levels of CHFR.

Figure 4. Comparison of DNA copy number amplification (variation is better or not?) in Normal vs Tumor samples

Quantification of CHFR DNA copy number in 19 normal samples and 25 EACs was determined using Real-time PCR and normalized to the average value of all the normal samples, as described in materials and methods. Black boxes and triangles represent normal and tumor samples respectively. The horizontal bars represent the mean of DNA copy number.

Figure 5. Immunohistochemistry analysis of CHFR expression

A) Histologically normal esophageal squamous epithelium (arrowheads) demonstrates strong (3+ score) nuclear immunostaining for CHFR. Adjacent poorly differentiated esophageal adenocarcinoma, indicated by arrows, demonstrates weak or absent nuclear immunostaining. B) Barrett's esophagus epithelium (arrowhead) and Barrett's low grade dysplasia (arrows) show moderate nuclear immunostaining (2+). C-D) moderately differentiated (C) and poorly differentiated (D) esophageal adenocarcinoma demonstrate weak (+1) or absent (0) nuclear immunostaining for CHFR, respectively. The upper right corner demonstrates insets of A-D panel at ×100 magnification of normal (N), Barrett's esophagus (BE), low grade dysplasia (LGD) and poorly differentiated adenocarcinoma (Ca). The IHC data are summarized on the lower right corner.