Abstract
BACKGROUND: Despite histologically benign, pituitary tumors (PT) may invade important adjacent neurovascular structures which can incur in significant comorbidities preventing a complete surgical resection and contributing to resistance to medical treatment. DNA methylation clearly stratified PT based on their functional status i.e. nonfunctioning PTs (NFPTs) from functioning PT (FPTs). However associations of methylation aberrations with invasive behavior is less clear. MATERIAL AND METHODS: In order to evaluate whether DNA methylation alterations in regulatory regions other than promoter and coding regions are associated with invasive behavior we performed a meta-analysis of the genome-wide methylome of three public available PT cohorts plus our own (Illumina HumanMethylation platforms- 450K/EPIC). Pituitary specimens comprised of 43 invasive pituitary tumors (InvPT) and 37 noninvasive (NInvPT); 12 FPT and 68 NFPTs, in addition to 20 non-tumor pituitaries. RNA-seq data were available for one cohort (n=23, 12 InvPT,11NInvPT) and integrated with DNA methylation. Invasiveness criteria was based on Knosp grade >= 2 and/or sphenoid or dural invasion. RESULTS: Wilcoxon Rank-sum test; Δβ=0.15; p-value <0.001 identified 58 differentially methylated CpG sites in InvPT that were mainly hypomethylated (95%) in relation to NInvPT. NInvPT methylation profile was similar to non-tumor specimens, despite its heterogeneity. Thirty-four percent (n=20) of the differentially methylated CpG sites were located within predicted enhancer regions distributed in intronic (40%), intergenic (40%) and promoter (20%) regions. Predicted enhancer-target genes were enriched for actin filament cell movement, response to starvation, growth factor stimulus and protein autophosporilation pathways. Among them, ZNF625 and INO80E were found mostly negative correlated among methylation and expression data (-0.50 and -0.48, respectively), besides DOC2A found to be one potentially differentially expressed gene under enhancer control (log2FC > 0.2, pvalue <0.05). CONCLUSION: Our results suggest that methylation alterations in predicted regulatory regions, such as enhancers, annotated in non-promoter regions (introns and intergenic) may contribute to the invasive behavior of PT.