Quantitative genome-wide methylation analysis of high-grade non-muscle invasive bladder cancer

Abstract

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P<0.05), and similarly significant increases in mean levels of methylation in high-grade tumors for the ATP5G2, VAX2, INSRR, PRDM14, VSX1, TFAP2b, PRRX1, and HIST1H4F genes (P<0.05). Although inappropriate promoter methylation was not invariantly associated with reduced transcript expression, a significant association was apparent for the ARHGEF4, PON3, STAT5a, and VAX2 gene transcripts (P<0.05). Herein, we present the first genome-wide DNA methylation analysis in a unique HG-NMIBC cohort, showing extensive and discrete methylation changes relative to normal bladder and low-intermediate-grade tumors. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination, with more conventional therapeutic options in the treatment of this clinically unpredictable disease.

Keywords: Epigenetics; HumanMethylation450 BeadChip Array; gene expression; high-grade non-muscle invasive bladder cancer; methylation.

Figure 1.

Array filtering steps. Summary of the steps implemented for the identification of CpGs hypermethylated in HG-NMIBC. The initial filtering steps (*) included exclusion of non-significant probe data, probes with missing data and probes located on allosomes. RefSeq (National Center for Biotechnology Information Reference Sequence Database). CpG island based upon the UCSC genome browser definition from Gardiner-Garden and Frommer.

Figure 2.

Unsupervised hierarchical clustering analysis of the 1,057 gene promoter-associated hypermethylated CpGs in HG-NMIBC. Heatmap and dendrogram of differentially methylated gene promoter-associated CpG sites identified by array analysis. The dendrogram above the heatmap separates normal bladder (green bar, n = 3) and high-grade-NMIBC bladder tumors (red bar, n = 21). Each row represents an individual CpG locus, and each column represents a normal control or tumor sample (listed beneath the heatmap). The color scale beneath the heatmap represents methylation status: unmethylated is yellow (β-value = 0.0), and fully methylated is blue (β-value = 1.0).

Figure 3.

Heatmap for 25 hypermethylated gene promoter-associated CpG islands. Pyrosequencing validation of 25 gene promoter-associated CpG islands, identified as frequently differentially methylated in high-grade tumors by 450 K BeadChip-array analysis. As indicated above the heatmap, the four normal bladder controls are presented to the left-side of the heatmap, followed by 18 low-intermediate-grade tumors, and 51 high-grade tumors (the combined discovery and investigation cohorts). Each row represents the promoter-associated CpG island of the indicated gene, and each color block the mean level of methylation across the island. The color scale beneath the heatmap represents methylation status: unmethylated is green (0.0% methylation), and fully methylated is red (100.0% methylated).

Figure 4.

Mean levels of methylation in high-grade tumors relative to low-intermediate-grade tumors and normal bladder. Top ten genes showing an increase in mean level of methylation (solid red bar) in high-grade tumors (HG, n = 51) relative to low-intermediate-grade tumors (LG, n = 18) and in comparison to normal bladder controls (C, n = 4). Each individual control or tumor sample is shown as an unfilled blue circle. Significant differences in the mean levels of methylation between the low-intermediate- and high-grade tumors, or between control and low-intermediate-grade tumors, are indicated by *, P<0.05, or **, P<0 .005 (Student's T-test).

Figure 5.

Association of methylation with gene transcript expression in HG-NMIBC. Tumor transcript expression in unmethylated (UM, unfilled circles) and methylated (M, filled circles) high-grade tumors, relative to normal bladder control (C, unfilled triangles) for the 4 genes showing significant Spearman's correlation coefficients between promoter methylation and gene expression (PON3, STAT5a, VAX2 and ARHGEF4; P = 0.0006, P = 0.005, P = 0.013 and P = 0.0007, respectively). The double-headed arrow represents the threshold for 3-fold reduced expression relative to the mean of the normal controls (solid blue bar); expression at or below this threshold signifies reduced expression in tumor samples.