Genetic and Epigenetic Alterations of CDH1 Regulatory Regions in Hereditary and Sporadic Gastric Cancer

Abstract

E-cadherin is a key player in gastric cancer (GC) and germline alterations of CDH1, its encoding gene, are responsible for Hereditary Diffuse Gastric Cancer (HDGC) syndrome. This study aimed at elucidating the role of genetic variants and DNA methylation of CDH1 promoter and enhancers in the regulation of gene expression. For this purpose, we analyzed genetic variants of the CDH1 gene through Next-Generation Sequencing (NGS) in a series of GC cell lines (NCI-N87, KATO-III, SNU-1, SNU-5, GK2, AKG, KKP) and the corresponding CDH1 expression levels. By bisulfite genomic sequencing, we analyzed the methylation status of CDH1 regulatory regions in 8 GC cell lines, in a series of 13 sporadic GC tissues and in a group of 20 HDGC CDH1-negative patients and 6 healthy controls. The NGS analysis on CDH1 coding and regulatory regions detected genetic alterations in 3 out of 5 GC cell lines lacking functional E-cadherin. CDH1 regulatory regions showed different methylation patterns in patients and controls, GC cell lines and GC tissues, expressing different E-cadherin levels. Our results showed that alterations in terms of genetic variants and DNA methylation patterns of both promoter and enhancers are associated with CDH1 expression levels and have a role in its regulation.

Keywords: CDH1 gene; DNA methylation; Next-Generation Sequencing; gastric cancer; genetic predisposition; regulatory regions.

Figure 1

Scheme of the CDH1 gene (chr16) with the CpG island represented by the green bar and enhancers by red bars. Blue bars represent the regions tested for methylation in the present work. The lower panels represent H3K27Ac, a chromatin mark associated with transcription activation [26], in normal stomach and B cells, respectively. The figure was generated with pyGenomeTracks [27].

Figure 2

Methylation status of CDH1 promoter and enhancers of eight GC cell lines expressing different levels of CDH1. non-methylated CpG site; partially methylated CpG site; fully methylated CpG site; deletion.

Figure 3

Methylation analysis of CDH1 promoter and enhancers on a selection of DNA samples from peripheral blood of three healthy individuals (in blue) and nine HDGC CDH1-negative patients (six DGC patients in black and three LBC patients in violet). The complete case series is reported in Supplementary Figures S1 and S2. non-methylated CpG site; partially methylated CpG site; fully methylated CpG site.

Figure 4

Methylation analysis of CDH1 promoter and enhancers on DNA samples from gastric normal (N) and tumor (T) tissues from eight IGC patients (GC tissues with CDH1 downregulation in red and GC tissues with CDH1 upregulation in green). non-methylated CpG site; partially methylated CpG site; fully methylated CpG site.