ANK2 Hypermethylation in Canine Mammary Tumors and Human Breast Cancer

Abstract

Canine mammary tumors (CMT) constitute the most common tumor types found in female dogs. Understanding this cancer through extensive research is important not only for clinical veterinary applications, but also in the scope of comparative oncology. The use of DNA methylation as a biomarker has been noted for numerous cancers in the form of both tissue and liquid biopsies, yet the study of methylation in CMT has been limited. By analyzing our canine methyl-binding domain sequencing (MBD-seq) data, we identified intron regions of canine ANK2 and EPAS1 as differentially methylated regions (DMGs) in CMT. Subsequently, we established quantitative methylation specific PCR (qMSP) of ANK2 and EPAS1 to validate the target hypermethylation in CMT tissue, as well as cell free DNA (cfDNA) from CMT plasma. Both ANK2 and EPAS1 were hypermethylated in CMT and highlighted as potential tissue biomarkers in CMT. ANK2 additionally showed significant hypermethylation in the plasma cfDNA of CMT, indicating that it could be a potential liquid biopsy biomarker as well. A similar trend towards hypermethylation was indicated in HBC at a specific CpG of the ANK2 target on the orthologous human region, which validates the comparative approach using aberrant methylation in CMT.

Keywords: CMT; HBC; biomarker; cfDNA; hypermethylation.

Figure 1

Selection of ANK2 and EPAS1 as canine mammary tumor (CMT) hypermethylated targets. (A) Schematic overview of target selection using MBD-seq and RNA-seq. Each step indicates the number of identified genes at the end of that step. (B) RNA-seq gene expression in FPKM of top 20 hypermethylated genes; blue dots = normal, red dots = CMT, * = p-value < 0.05. Fold change (log2) for each target gene indicated with grey bar graph. (C) IGV peak calling for ANK2 and EPAS1 in both normal and cancer. Differential methylation assigned via LMM with both 5% and 10% threshold, and CpG island presence is indicated. (D) Overall methylation levels of ANK2 and EPAS1 from MBD-seq data for 11 paired CMT and adjacent normal samples. EdgeR. ** = p-value < 0.01, **** = p-value < 0.0001. (E) ANK2 and (F) EPAS1 correlation plots between expression (FPKM) and methylation of 10 paired CMT (red) and normal (blue) samples with matching density plots for both FPKM and methylation. Pearson correlation |r| value and p value indicated. Fold change graphs are also depicted for 10 CMT samples indicating expression (green) and methylation (orange).

Figure 2

Bisulfite Sequencing PCR (BSP) of ANK2 and EPAS1. Between 9 and 10 colonies were sequenced for three paired normal and CMT samples (separated by grey dotted line) for (A) ANK2 and (B) EPAS1. Methylation of a particular CpG is indicated by a black circle, and non-methylation is indicated by a white circle. The CpGs that were included in the forward and reverse MSP primers for each respective target with a black box.

Figure 3

Quantitative Methylation Specific PCR (qMSP) validation of ANK2 and EPAS1 hypermethylation. (A) Schemes for both ANK2 and EPAS1 indicating the target region in terms of its position on the CpG island of its respective intron. BSP primers (grey) are shown to flank the regions targeted by the MSP primers (yellow). (B) Methylation Indexes for both ANK2 and EPAS1. CMT samples are shown in red and paired normal samples are shown in blue. (C) Overall methylation indexes for both ANK2 and EPAS1 are shown. CMT in red, normal in blue. * = p-value < 0.05, ** = p-value < 0.01. (D) ROC curve analyses for ANK2 and EPAS1.

Figure 4

Canine Mammary Tumor (CMT) hypermethylation analysis in cfDNA. Overall methylation index for (A) ANK2 and (B) EPAS1 in CMT (red) and normal (blue) cfDNA. * = p-value < 0.05.

Figure 5

Hypermethylation of orthologous human target ANK2 and EPAS1 regions. Mean Methylation (beta value) plots for orthologous human (A) ANK2 and (D) EPAS1 regions for normal (blue) and invasive breast cancer patient(red) data obtained from TCGA. Specific methylation probes, annotated at the bottom of the graph are indicated to be in a CpG island when written in green (only seen for EPAS1), and designated as significant (*) with an adjusted p-value < 0.05. Three significantly hypermethylated CpGs from ANK2 are highlighted in light blue to indicate that they are conserved in the dog genome. (B) ANK2 and (E) EPAS1 log2 (normalized rsem +1) expression data from TCGA for normal (blue) and invasive breast cancer patients (red). Wilcoxon p-values are indicated. (C) ANK2 and (F) EPAS1 Kaplan-Meier plots indicting relapse-free survival with high and low expressions each. (G) ANK2 sequencing results for cfDNA from normal and HBC patients. Between 10 and 11 colonies were sequenced for 6 individual HBC cfDNA samples (separated by grey dotted lines) and between 10 and 12 colonies were sequenced from 3 separate normal cfDNA samples. Methylation of a specific CpG is indicated by a black circle and non-methylation by a white circle. The CpGs at positions 2, 3, and 5 are highlighted in light blue as these are the CpGs that correlate with the hypermethylated CpG probes indicated in (A) and the respective percentage methylation for these CpGs in the HBC and normal human (HN) cfDNA samples, as well as in canine CMT and normal (CN) tissue (Figure 2A), are indicated in the inserted table.