Identification of methylated genes associated with aggressive bladder cancer

Abstract

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.

Figure 1. Methylation bead array profiles distinguish non-invasive and invasive bladder tumors.

(A) Scatterplot of average methylation beta values in non-invasive bladder tumors (x-axis) and average methylation beta values in invasive tumors (y-axis) in series 1 (n = 73) and series 2 (n = 264) samples. (B) Recursive partition mixture models of each series result in 4 classes separated by red vertical lines, with the width of the classes corresponding to the number of samples in each class. Loci are represented as rows with the mean methylation for the class depicted. Above each class is the prevalence of invasive tumors (n invasive/total n) within the class. Methylation profiles significantly distinguish bladder tumor from non-diseased bladder epithelium (P<0.00001).

Figure 2. HOXB2 is re-expressed following treatment of HTB-9 bladder cancer cells with 5-aza-2′deoxycytidine.

Quantitative RT-PCR analysis was used to determine the gene expression of HOXB2 in human bladder carcinoma cell line HTB-9, following a 5-day treatment with 1 or 2 µM 5-aza-2′-deoxycytidine or mock treatment. Bars represent the mean fold change in expression compared to mock treatment for 4 or 6 replicate experiments, and error bars denote the standard errors.

Figure 3. Bisulfite pyrosequencing reveals a greater extent of methylation in invasive bladder cancer.

The mean (filled circle) and 95% confidence intervals of the extent of methylation of the promoter regions of (A) FRZB, (B) KRT13, and (C) HOXB2 are depicted (y-axis) comparing non-diseased bladder epithelium, non-invasive tumors and invasive tumors (x-axis). The differences in the extent of methylation across these groups is significantly different (Kruskal-Wallis test), for HOXB2 (P<0.00001) and KRT13 (P<0.04), and FRZB (P<0.003). Comparisons specifically of noninvasive compared to invasive disease also revealed significant differences for HOXB2 *P<0.00001 and KRT13 **P<0.02.

Figure 4. Bisulfite pyrosequencing demonstrates that extent of methylation is associated with tumor grade in non-invasive tumors.

The mean (filled circle) and 95% confidence intervals of the extent of methylation of the promoter regions of (A) FRZB, (B) KRT13, and (C) HOXB2 are depicted (y-axis) comparing low (1,2) to high (3+) grade tumors (x-axis). P-values resulting from the Wilcoxon Rank Sums demonstrate that these differences are significant for HOXB2 (P<0.01), KRT13 (P<0.01) and FRZB (P = 0.0001).