Methylation silencing of TGF-β receptor type II is involved in malignant transformation of esophageal squamous cell carcinoma

Abstract

Background: Although massive studies have been conducted to investigate the mechanisms of esophageal squamous cell carcinoma (ESCC) carcinogenesis, the understanding of molecular alterations during the malignant transformation of epithelial dysplasia is still lacking, especially regarding epigenetic changes.

Results: To better characterize the methylation changes during the malignant transformation of epithelial dysplasia, a whole-genome bisulfite sequencing analysis was performed on a series of tumor, dysplastic, and non-neoplastic epithelial tissue samples from esophageal squamous cell carcinoma (ESCC) patients. Promoter hypermethylation in TGF-β receptor type II (TGFBR2), an important mediator of TGF-β signaling, was identified. Further, we evaluated the methylation and expression of TGFBR2 in tumor samples through The Cancer Genome Atlas multiplatform data as well as immunohistochemistry. Moreover, treatment of ESCC cell lines with5-Aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, reactivated the expression of TGFBR2. The lentiviral mediating the overexpression of TGFBR2 inhibited the proliferation of ESCC cell line by inducing cell cycle G2/M arrest. Furthermore, the overexpression of TGFBR2 inhibited the tumor growth obviously in vivo.

Conclusions: The characterization of methylation silencing of TGFBR2 in ESCC will enable us to further explore whether this epigenetic change could be considered as a predictor of malignant transformation in esophageal epithelial dysplasia and whether use of a TGFBR2 agonist may lead to a new therapeutic strategy in patients with ESCC.

Keywords: Cancer diagnosis; Esophageal squamous cell carcinoma; Methylation changes; TGFBR2; Treatment; Whole genome bisulfite sequencing.

Fig. 1

Whole genome methylation profiling of ESCC and esophageal dysplasia samples. a Genome-wide methylation level of ESCC, dysplasia, and non-neoplastic samples. b Principal component analysis of whole genome bisulfite sequencing data

Fig. 2

Differential DNA methylation in esophageal dysplasia and ESCC. a Distribution of hypermethylated and hypomethylated CpG sites between different stages. b Proportion of differentially methylated regions in transcribed regions, intergenic regions, and promoters. c Methylation difference in TGFBR2 promoter between dysplastic and tumor stages in two paired samples

Fig. 3

TGFBR2 is hypermethylated and downregulated in TCGA ESCC dataset. a DNA methylation comparison of TGFBR2 promoter-associated CpG sites in normal and tumor samples. b Expression levels of TGFBR2 in normal and ESCC samples. c Correlations of promoter methylation and expression for TGFBR2

Fig. 4

TGFBR2 expression and its relationship with copy number and patients’ outcome. a, b Correlation of TGFBR2 copy number and promoter methylation. c Kaplan-Meier curves of overall survival according to TGFBR2 expression level. d IHC performed on sections from ESCC and adjacent tissues with TGFBR2 antibody

Fig. 5

Treatment of ESCC cells in culture increases the expression of TGFBR2. a RT-qPCR to detect levels of TGFBR2 mRNA in Het-1A and ESCC cell lines using GAPDH as a control gene. b RT-qPCR and western blot analysis performed on RNA and protein isolated from KYSE-150 cells exposed to increasing concentrations of 5-Aza-2′-deoxycytidine (mean ± SD. P < 0.001). c RT-qPCR and western blot analysis performed on RNA and protein isolated from KYSE-30 cells exposed to increasing concentrations of 5-Aza-2′-deoxycytidine (mean ± SD. P < 0.001)

Fig. 6

TGFBR2 overexpression induces ESCC cell cycle arrest but not cell apoptosis. a Western blot analysis of wild-type (WT) cells and lentiviral mediating the overexpression of TGFBR2 (OE) cells in KYSE-150 and KYSE-30 cell lines. b The morphology of WT and OE cells in colony formation assay. c The expression level of phospho-SMAD2 and SMAD2 in WT and OE cells. d Cell cycle distribution in WT and OE cells. Graphic representation of results from cell cycle analysis in WT and OE cells. e Annexin V staining of parental WT and KO cells to detect apoptosis using flow cytometry. Graphic representation of the percentage of apoptotic cells in parental versus OE cells. f Annexin V staining of KYSE-150 cells exposed to increasing concentrations of 5-Aza-2′-deoxycytidine detected with flow cytometry. Graphic representation of the percentage of apoptotic cells with increasing 5-Aza-2′-deoxycytidine concentration

Fig. 7

TGFBR2 overexpression inhibits tumor growth in vivo. a Tumor volume as measured in xenografts over 21 days derived from KYSE-150-vector (WT) and KYSE-150-TGFBR2 (OE) cells. b Image of subcutaneous xenografts derived from the cells indicated after growth in vivo for 3 weeks. c Tumor weight associated with WT- and OE-derived xenografts at 21 days. d IHC reveals the expression of TGFBR2 and CK in WT and OE cell xenografts