Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

Abstract

Background: The DACT (Dishevelled-associated antagonist of β-catenin) family of scaffold proteins may play important roles in tumorigenesis. However, the epigenetic changes of DACT1, 2, 3 and their effect on esophageal squamous cell carcinoma (ESCC) have not been investigated so far. The aim of this study was to investigate the promoter methylation and expression of DACT family, in order to elucidate more information on the role of DACT with regard to the progression and prognosis of ESCC.

Methods: MSP and BGS methods were respectively applied to examine the methylation status of DACT; RT-PCR, Western blot and immunohistochemistry methods were respectively used to determine the mRNA and protein expression of DACT; MTT, Colony-formation and Wound-healing assay were performed to assess the effect of DACT1 and DACT2 on proliferation and migration of esophageal cancer cells.

Results: Frequent reduced expression of DACT1, DACT2 and DACT3 were found in esophageal cancer cell lines and the expression levels of DACT1 and DACT2 were reversed by 5-Aza-Dc. Decreased mRNA and protein expression of DACT1 and DACT2 were observed in ESCC tumor tissues and were associated with the methylation status of transcription start site (TSS) region. The hypermethylation of CpG islands (CGI) shore region in DACT1 was observed both in tumor and corresponding adjacent tissues but wasn't related to the transcriptional inhibition of DACT1. The methylation status of TSS region in DACT1 and DACT2 and the protein expression of DACT2 were independently associated with ESCC patients' prognosis.

Conclusions: The TSS region hypermethylation may be one of the main mechanisms for reduced expression of DACT1 and DACT2 in ESCC. The simultaneous methylation of DACT1 and DACT2 may play important roles in progression of ESCC and may serve as prognostic methylation biomarkers for ESCC patients.

Keywords: DACT gene; Esophageal squamous cell carcinoma; Methylation; Prognosis.

Fig. 1

The mRNA expression and methylation analysis of DACT1, DACT2 and DACT3 in esophageal cancer cell lines. a, b, c Schematic structure of CpG islands in DACT1, DACT2 and DACT3 genes. The MSP and BGS-regions were shown in the schematic. The transcription start point was as +1. d The changes of DACT1, 2, 3 mRNA expression in treated or untreated cells with 5-Aza-Dc or TSA by RT-PCR method; e, f, g Relative mRNA expression of DACT1, 2, 3 in treated or untreated cells with 5-Aza-Dc or TSA by Quantitative realtime RT-PCR method. h The methylation status of DACT1, 2, 3 in four cell lines with(+) or without(−) 5-Aza-Dc treatment detected by MSP method. M, methylated gene; U, unmethylated gene. i The methylation status of every CpG site in two regions of DACT1, one region of DACT2 and one region of DACT3 in four tumor cell lines by BGS method. The color of circles for each CpG site represents the percentage of methylation. The “F” and “R” represent forward and reverse primers for MSP, respectively

Fig. 2

The effect on cell proliferation, colony formation and immigration abilities in TE1 and TE13 cell lines after treated with 5-Aza-dC or stable transfected of DACT1 /DACT2. a The protein expression of DACT1 and DACT2 in TE1 or TE13 cells after stable transfected with DACT1/DACT2 plasmid or treated with 5-Aza-dC using western-blot method. b The results of MTT assay in treated or untreated TE1/TE13 cells with 5-Aza-dC or the stable transfected TE1/TE13 cells; **Compared with untreated cell line, P < 0.01; c The results of colony formation assays in treated or untreated TE1/TE13 cells with 5-Aza-dC or the stable transfected TE1/TE13 cells; **Compared with untreated cell line, P < 0.01; d The relative scratch width of treated or untreated TE1/TE13 cells or the stable transfected TE1/TE13 cells detected by Wound healing analysis. The initial width of the scratch was as 1

Fig. 3

The expression and methylation analysis of DACT1, DACT2 and DACT3 in ESCC and corresponding adjacent non-cancerous tissues. a Representative RT-PCR results of DACT1, 2, 3 in 5 matched pairs (case1-case5) of ESCC tissues (T) and non-cancerous tissues (N). GAPDH was used as an endogenous control. b Relative mRNA expression of DACT1, 2, 3 in ESCC and adjacent tissues. c Representative immunohistochemical staining of DACT1, 2, 3 in ESCC tumor tissues (SP × 200). a: the positive expression of DACT1; b: the positive expression of DACT2; c: the positive expression of DACT3; d Positive expression frequency of DACT1, 2, 3 proteins in ESCC and corresponding adjacent tissues. e, f Relative mRNA expression of DACT1 and DACT2 in different subgroups of ESCC cases, which was expressed as the mean ± standard deviation. ^* P < 0.05, ^** P < 0.01. g Representative MSP results of DACT1, DACT2 and DACT3 in 3 matched pairs (case1-case3) of tumor tissue (T) and non-cancerous tissues (N). M, Methylation; U, Unmethylation; PC, positive control; NC, negative control. h The methylation rate of DACT1, 2, 3 in ESCC tissues and non-cancerous tissues. ^** P < 0.01. i The relative mRNA expression of DACT1 and DACT2 in methylated or unmethylated groups. The mRNA expression of DACT1 and DACT2 were all associated with methylation status of their TSS-regions in ESCC. ^** P < 0.01

Fig. 4

Kaplan–Meier univariate survival analysis of DACT1 / DACT2 expression and methylation status in ESCC. a, b, c Kaplan–Meier curves for cumulative survival stratified by DACT1 and DACT2 expression: showing a direct correlation between DACT1 or DACT2 expression and ESCC patient’s survival. d, e, f, g Kaplan–Meier curves for cumulative survival stratified by the methylation status of two regions of DACT1 and one region of DACT2: There was not correlation between the region 1 methylation status of DACT1 and the ESCC patient’s survival. The hypermethylation of TSS-region in DACT1 and DACT2 were associated with the poor patient survival of ESCC cases; Simultaneous methylation of DACT1 and DACT2 showed worst prognosis. (H) Kaplan–Meier curves for cumulative survival stratified by methylation status and UGIC family history: ESCC patients with positive UGIC family history and DACT1 or DACT2 methylation showed poor patient survival. (I) Kaplan–Meier curves for cumulative survival stratified by methylation status and TNM stages: Stage III and IV ESCC patients with DACT1 or DACT2 methylation showed the poor patient survival