Integrative analysis of the epigenetic basis of muscle-invasive urothelial carcinoma - PubMed (original) (raw)

Integrative analysis of the epigenetic basis of muscle-invasive urothelial carcinoma

Thomas Sanford et al. Clin Epigenetics. 2018.

Abstract

Background: Elucidation of epigenetic alterations in bladder cancer will lead to further understanding of the biology of the disease and hopefully improved therapies. Our aim was to perform an integrative epigenetic analysis of invasive urothelial carcinoma of the bladder to identify the epigenetic abnormalities involved in the development and progression of this cancer.

Methods: Pre-processed methylation data and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and processed using the R package TCGA-Assembler. An R package MethylMix was used to perform an analysis incorporating both methylation and gene expression data on all samples, as well as a subset analysis comparing patients surviving less than 2 years and patients surviving more than 2 years. Genes associated with poor prognosis were individually queried. Pathway analysis was performed on statistically significant genes identified by MethylMix criteria using ConsensusPathDB. Validation was performed using flow cytometry on bladder cancer cell lines.

Results: A total of 408 patients met all inclusion criteria. There were a total of 240 genes differentially methylated by MethylMix criteria. Review of individual genes specific to poor-prognosis patients revealed the majority to be candidate tumor suppressors in other cancer types. Pathway analysis showed increase in methylation of genes involved in antioxidant pathways including glutathione and NRF2. Genes involved in estrogen metabolism were also hypermethylated while genes involved in the EGFR pathway were found to be hypomethylated. EGFR expression was confirmed to be elevated in six bladder cancer cell lines.

Conclusions: In patients with invasive urothelial carcinoma, we found differential methylation in patients with better and worse prognosis after cystectomy. Differentially methylated genes are involved in many relevant oncologic pathways, including EGFR and antioxidant pathways, that may be a target for therapy or chemoprevention.

Keywords: Epigenetics; Integrative analyses; Urothelial carcinoma.

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Conflict of interest statement

Consent for participation for all patients was obtained through The Cancer Genome Atlas Project.N/A.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Fig. 1

Summary of top hypermethylated and top hypomethylated genes. The red line demarcates the distribution of methylation in tumor samples and normal samples. The histogram (below red line) demonstrates the distribution of methylation in tumor samples (denoted as beta values where higher beta values represent greater methylation). The horizontal black bar above the red line represents the distribution of methylation values in the normal samples

Fig. 2

Fig. 2

Of the 220 statistically significant genes for patients who died within 2 years, 32% were hypermethylated. Of the 266 statistically significant genes for patients who survived more than 2 years after cystectomy, 26% were hypermethylated and the remainder of the genes were hypomethylated. For both hypermethylated and hypomethylated genes, the majority of genes were shared by the better and worse prognosis groups. However, there were some genes with unique methylation status between the two groups, and there were more genes uniquely hypermethylated in the group that did not survive 2 years compared with the group that survived at least 2 years (p = 0.02). Identities of all genes significant by MethylMix are included in Additional file 1: Table S1

Fig. 3

Fig. 3

Heatmap of methylation values (beta values) for 24 genes uniquely hypermethylated in patients who survived less than 2 years after cystectomy. The gray bar histogram represents the mean beta value across all genes for each patient

Fig. 4

Fig. 4

Select pathways enriched for genes hypermethylated and hypomethylated by MethylMix criteria in the analysis including all patients in the TCGA cohort. The full list of pathways is included in Additional file 2: Table S2

Fig. 5

Fig. 5

Flow cytometry analysis demonstrating expression of EGFR on the cell surface of various cell lines. The curve shaded gray represents binding of rat monoclonal antibody to human EGFR tagged with phycoerythrin (PE). The curve without shading (white) represents binding of an isotype control, which in the case was a PE-tagged monoclonal rat IgG2a kappa. The vertically written numbers next to each peak represent median fluorescence intensity for isotype control and anti-EGFR PE. For all cell lines tested (UMUC1, UMUC5, Scaber, HT1376, SW780, and 253 J), there was substantial EGFR expression compared with isotype control

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