Eps15 Homology Domain-Containing Protein 3 Hypermethylation as a Prognostic and Predictive Marker for Colorectal Cancer

Abstract

Colorectal cancer (CRC) arises from chromosomal instability, resulting from aberrant hypermethylation in tumor suppressor genes. This study identified hypermethylated genes in CRC and investigated how they affect clinical outcomes. Methylation levels of specific genes were analyzed from The Cancer Genome Atlas dataset and 20 breast cancer, 16 esophageal cancer, 33 lung cancer, 15 uterine cancer, 504 CRC, and 9 colon polyp tissues and 102 CRC plasma samples from a Taiwanese cohort. In the Asian cohort, Eps15 homology domain-containing protein 3 (EHD3) had twofold higher methylation in 44.4% of patients with colonic polyps, 37.3% of plasma from CRC patients, and 72.6% of CRC tissues, which was connected to vascular invasion and high microsatellite instability. Furthermore, EHD3 hypermethylation was detected in other gastrointestinal cancers. In the Asian CRC cohort, low EHD3 mRNA expression was found in 45.1% of patients and was connected to lymph node metastasis. Multivariate Cox proportional-hazards survival analysis revealed that hypermethylation in women and low mRNA expression were associated with overall survival. In the Western CRC cohort, EHD3 hypermethylation was also connected to overall survival and lower chemotherapy and antimetabolite response rates. In conclusion, EHD3 hypermethylation contributes to the development of CRC in both Asian and Western populations.

Keywords: DNA methylation; EHD3; circulating cell-free DNA (ccfDNA); colorectal cancer (CRC); early detection; prognostic marker.

Figure 1

Gene selection and experimental design. (A) Stepwise gene selection flowchart. (B) Gene screening performed using http://www.interactivenn.net/ (accessed on 4 November 2020) [22]. (C) Methylation heatmap of EHD3 in paired colon cancer, liver cancer, pancreatic cancer, esophageal cancer, rectal cancer, and gastric cancer tissues. (D) The experimental design is shown in the flowchart, and sample types and sizes are indicated. BC, breast cancer; EC, esophageal cancer; LC, lung cancer; UC, uterine cancer; CC, colon cancer; RC, rectal cancer; LiC, livesr cancer; GC, gastric cancer; PC, pancreatic cancer; CRC, colorectal cancer; AN, adjacent normal; ccfDNA, circulating cell-free DNA; QMSP, quantitative methylation-specific PCR; qRT-PCR, quantitative reverse-transcription PCR; IHC, immunohistochemistry; 450K, Illumina Infinium HumanMethylation450 BeadChip array.

Figure 2

Different methylation at EHD3 CpG islands in patients with CRC. Methylation levels (average β values) at the differentially methylated loci were identified using an Illumina Methylation 450K array-based assay in (A) 26 patients with CRC in Taiwan and (B) 38 patients with CRC from the TCGA dataset. The scale shows the relative methylation status from 0.00 to 1.00 (yellow: hypomethylation, blue: hypermethylation). Twenty-one CpG sites on EHD3 were detected in 26 paired normal (upper) and CRC (lower) tissues, and array probes 1–21 were sites cg13149833, cg12045528, cg00648955, cg25202298, cg06773122, cg00981472, cg18444347, cg05882522, cg27230038, cg25428398, cg15355118, cg25840208, cg24743639, cg13795465, cg01163837, cg08251399, cg11957382, cg07185119, cg20203365, cg14018959, and cg14613979, respectively.

Figure 3

DNA methylation and mRNA expression analysis of EHD3 from an Asian cohort. * p < 0.05. (A) EHD3 methylation level in nine adjacent normal colon tissues, nine polyps of tubular adenoma, and nine CRC tumors. The Mann–Whitney U test was used to compare adjacent normal colon tissues and tubular adenoma and to compare tubular adenomas and CRC tumors. (B) Spearman’s rank-order correlation was used to estimate the correlation between EHD3 promoter methylation and mRNA expression in the matched normal and tumor tissues. (C) The Kaplan–Meier estimate was used to compute the overall survival of women with and without EHD3 hypermethylation. An EHD3 promoter methylation level in CRC tumors 200-fold higher (90th percentile) than that in adjacent normal colorectal tissues was defined as hypermethylation.

Figure 4

EHD3 DNA methylation, mRNA, and protein expression analysis from TCGA dataset. (A) The Pearson correlation test was used to estimate the correlation between EHD3 DNA methylation and RNA sequencing in 262 patients with CRC in the TCGA dataset. (B) The Kaplan–Meier estimate was used to compute the overall survival of patients with CRC with low and high EHD3 methylation levels. EHD3 was considered hypermethylated at an average β value of >0.445 (75th percentile). (C) The Kaplan–Meier estimate was used to compute the overall survival of patients with late-stage CRC with high and low EHD3 protein expression levels. Image credit: Human Protein Atlas. Image available from https://www.proteinatlas.org/ENSG00000013016-EHD3/pathology/colorectal+cancer#imid_15773055 (accessed on 27 July 2020). (D) The Kaplan–Meier estimate was used to compute the overall survival of female patients with late-stage CRC with high and low EHD3 protein expression levels. Image credit: Human Protein Atlas. Image available from https://www.proteinatlas.org/ENSG00000013016-EHD3/pathology/colorectal+cancer#imid_15773055 (accessed on 27 July 2020).