Subtypes of Barrett's oesophagus and oesophageal adenocarcinoma based on genome-wide methylation analysis

Abstract

Objective: To identify and characterise DNA methylation subtypes in oesophageal adenocarcinoma (EAC) and its precursor Barrett's oesophagus (BE).

Design: We performed genome-wide DNA methylation profiling on samples of non-dysplastic BE from cancer-free patients (n=59), EAC (n=23), normal squamous oesophagus (n=33) and normal fundus (n=9), and identified methylation subtypes using a recursively partitioned mixture model. We assessed genomic alterations for 9 BE and 22 EAC samples with massively parallel sequencing of 243 EAC-associated genes, and we conducted integrative analyses with transcriptome data to identify epigenetically repressed genes. We also carried out in vitro experiments treating EAC cell lines with 5-Aza-2'-Deoxycytidine (5-Aza-dC), short hairpin RNA knockdown and anticancer therapies.

Results: We identified and validated four methylation subtypes of EAC and BE. The high methylator subtype (HM) of EAC had the greatest number of activating events in ERBB2 (p<0.05, Student's t-test) and the highest global mutation load (p<0.05, Fisher's exact test). PTPN13 was silenced by aberrant methylation in the HM subtype preferentially and in 57% of EACs overall. In EAC cell lines, 5-Aza-dC treatment restored PTPN13 expression and significantly decreased its promoter methylation in HM cell lines (p<0.05, Welch's t-test). Inhibition of PTPN13 expression in the SK-GT-4 EAC cell line promoted proliferation, colony formation and migration, and increased phosphorylation in ERBB2/EGFR/Src kinase pathways. Finally, EAC cell lines showed subtype-specific responses to topotecan, SN-38 and palbociclib treatment.

Conclusions: We identified and characterised methylator subtypes in BE and EAC. We further demonstrated the biological and clinical relevance of EAC methylator subtypes, which may ultimately help guide clinical management of patients with EAC.

Keywords: dysplasia; gastrointestinal cancer; methylation.

Figure 1

Heatmaps of methylated CpGs in EAC and BE cases show distinct clusters based on methylation patterns. The clusters were identified with recursive partition modeling using β-value methylation at the 1515 most variable CpG probes (MVPs). (A). MVP heatmap of BETRNet samples, including EACs used for MVP discovery (N = 23), NDBE from cancer-free patients (N = 59), a representative subset of normal SQ tissues from BE patients (N = 11), and normal fundus (N = 9) from EAC patients. A comprehensive heatmap including all normal SQ samples (N =33) is shown in Supplemental Figure 3. (B). MVP heatmap of independent validation samples from TCGA, including EACs (N = 87) and matched normal tissues (N = 11). Methylation is measured as β-values from unmethylated or 0 (dark blue) to methylated or 1 (yellow). Methylation subtype annotation: high methylator (HM, yellow); intermediate methylator (IM, coral); low methylator (LM, grey) and minimal methylator (MM, blue)

Figure 2

Characterization of methylation subtypes in EAC (A) Mutation loads (Mut/Mb) calculated using MutSig, and results of comparing HM to other subtypes (two-tailed Student’s t test, P < 0.05 is considered as statistically significant). (B) BETRNet EAC sample gene alterations detected using sequencing of a panel of genes functionally important in EAC. (C) Venn diagram of comparing epigenetically repressed genes across subtypes. (D) Venn diagram comparing differentially expressed miRNAs (DEmiRs) in pairwise subtype comparisons. (E) Volcano plots of miR and DEmiR expression and differential expression test result (t-test, p-unadj) in three subtype comparisons (HM vs. IM, LM, and MM, respectively). X-axis is difference in mean expression between subtypes (HM minus IM, LM, or MM mean, respectively), y-axis is the -log₁₀(p-unadj) from t-tests.

Figure 3

Aberrant methylation of PTPN13 in a subset of EAC led to its transcriptional repression. (A) The relative methylation of PTPN13 (normalized to C-LESS control) in normal esophagus tissues (N=7) and EAC biopsy samples (N=16) was determined using qMS-PCR. Average relative methylation, depicted as a line, is significantly elevated in EAC than normal esophagus (Welch’s t test, P <0.05). The red dots indicate EAC samples with methylated PTPN13 (See Supplemental Methods). (B) The relative DNA methylation level (normalized to C-LESS control) (Y-axis) and mRNA expression (normalized to GUSB) of PTPN13 (X-axis) for 14 EAC biopsy samples with available RNA. The mRNA expression was determined using qRT-PCR with GUSB as control gene (See Supplemental Methods). The red dots indicate EAC samples with methylated PTPN13. (C-D) A significant transcriptional re-expression of the methylated PTPN13 following 5-Aza treatment in OE33 and ESO26 cell lines. Cells were treated with 5 and 10 µM 5-Aza for 72 hrs. *, Student t test P < 0.05. (E-F) A significant decrease in the relative methylation level of PTPN13 following 5-Aza treatment in OE33 and ESO26 cell lines. *, Student t test P < 0.05.

Figure 4

Inhibition of PTPN13 promotes proliferation, colony formation, migration and key kinase phosphorylation n EAC cells. (A) The relative DNA methylation level (normalized to C-LESS control) (Y-axis) and mRNA expression (normalized to GUSB) of PTPN13 (X-axis) in 7 EAC cell lines. The PTPN13 mRNA level in SK-GT-4 cells is higher than other lines. (B) Daily proliferation assays in SKGT4-PTPN13 shRNA cells vs. non-specific scrambled control (NSC) or parental cells. **, Student t test P < 0.01. (C) Colony formation assays in NSC control cells (2 wells at left) versus PTPN13-shRNA (2 wells at right) at 50 cells or 100 cells seeded per well (original magnification 10×). (D) Compared to mock or NSC control cells, transfection with PTPN13-shRNA promoted migration. *, Student t test P < .05) in SK-GT-4 cells. (E) Western blotting revealed enhanced tyrosine phosphorylation in a panel of key kinases including ERBB2-Tyr1268, EGFR-Tyr1068 and SRC-Tyr416 at basal level, and further upon EGF stimulation, in PTPN13-shRNA SK-GT-4 cells compared to NSC control cells.

Figure 5

Treatment response in EAC cell lines representative of different methylation groups. (A) DNA methylation subtypes in EAC cell lines were determined using the HM450 array data. In vitro drug sensitivity curves for JH-EsoAd1 and OE33 (HM cell lines) are compared to SK-GT-4 (LM) and OE19 (MM) after 72-h exposure to a topoisomerase inhibitor, topotecan (B) and SN-38, the active metabolite of irinotecan (C). Cell survival rate (%) is expressed as the percentage of growth relative to DMSO-treated control cells (± s.d. for three experiments).