Presentation Authors: Jenna Buckwalter*, Vasty Osei Amponsa, Lauren Shuman, Vonn Walter, Joshua Warrick, Hershey, PA, Xue-Ru Wu, New York, NY, Jay Raman, David DeGraff, Hershey, PA
Introduction: Bladder cancer (BC) is the second most common urologic malignancy and has notable molecular and morphologic heterogeneity that is associated with clinical outcome. Transcription factor forkhead box A1 (FOXA1) is required for maintenance of urothelial differentiation, and FOXA1 loss is associated with the development of basal-squamous BC which responds differentially to therapeutic intervention. Thus, manipulating FOXA1 expression to control subtype specification is an attractive concept. The aim of this study was to determine the mechanism(s) responsible for loss of FOXA1 expression in BC.
Methods: Computational analysis of publically available data through the TCGA bladder study was used to examine the relationship between FOXA1 mutational status, copy number, methylation status and gene expression. In tandem, the UCSC genome browser was used to identify CpG islands in the FOXA1 promoter as potential sites of promoter methylation. A PCR-based system for detecting methylated CpG islands was used to determine the methylation status of FOXA1 in a panel of human BC cell lines.
Results: Analysis of RNA-seq data confirms decreased FOXA1 expression is common in human BC and associated with the basal-squamous molecular subtype. However, FOXA1 expression was not correlated with mutational status and/or copy number alterations, suggesting a role for epigenetic silencing. Three CpG islands were identified in the FOXA1 promoter and gene body, which include islands 99, 123, and 143. Statistical analysis of TCGA DNA methylation data identified methylation at CpG island 99 is significantly associated with reduced FOXA1 expression (p < 0.0009; Wilcoxon rank Sum), while island 143 was unmethylated. We next extended our methylation analysis for CpG islands to human BC cell lines with defined molecular subtypes. CpG island 99 was methylated specifically in basal BC cell lines which fail to express FOXA1, while CpG island 143 was unmethylated. Although CpG island 123 was methylated in all ten cell lines, we failed to detect any association between methylation status and FOXA1 expression or molecular subtype.
Conclusions: Our data indicates that unlike mutational status or copy number, methylation patterns of specific CpG islands are significant contributors to the regulation of FOXA1 expression in human BC. Specifically, methylation of CpG island 99 is found in advanced disease and human BC cell lines. Our study additionally shows the importance of examining promoter methylation in association with gene expression to determine the functional consequences of epigenetic alterations.
Source of Funding: National Cancer Institute grants 1K99CA172122 and R00CA172122 (D.J.D.)