Supplementary Material for: The EFEMP1 Gene: A Frequent Target for Epigenetic Silencing in Multiple Human Pituitary Adenoma Subtypes

Background/Aims: In a genome-wide investigation we recently identified the EGF-containing fibulin-like extracellular matrix protein 1 gene, EFEMP1, as hypermethylated in growth hormone-secreting adenoma. Methods: In an independent cohort we determined expression of EFEMP1, CpG island methylation and histone tail modification status. The causal consequences of epigenetic modification were determined through epidrug-induced reversal and enforced EFEMP1 expression in GH3 cells. Results: The majority of adenomas, irrespective of subtype, show reduced EFEMP1 expression. However, epigenetic change, as determined by CpG island methylation, was not invariantly associated with decreased EFEMP1 expression. Conversely, chromatin immunoprecipitation assays revealed enrichment for modifications associated with either active or silenced genes in adenoma that did or did not express EFEMP1 respectively. In AtT-20 and GH3 cells a causal relationship between epigenetic silencing and expression of EFEMP1 was established where co-incubation with the epidrugs zebularine and TSA induced expression of EFEMP1 and concomitant histone tail modifications toward those associated with expressed genes. Enforced expression of EFEMP1 in GH3 cells was without effect on cell proliferation or apoptotic end-points, however inhibition of endogenous matrix metalloproteinase (MMP)-2 expression was apparent. Primary adenomas did not show this relationship, however a positive correlation was apparent with the MMP7 transcript and perhaps reflects cell or species differences. Conclusions: The protein product of the EFEMP1 gene, fibulin-3, is reported to impact on multiple pathways in a cell-specific context. Subtype-independent loss of EFEMP1 expression in the majority of primary adenomas should prompt more detailed investigation in this tumour type.