Epigenomic Silencing of the BMP-4 Gene in Pituitary Adenomas: A Potential Target for Epidrug-Induced Re-expression

Abstract

Bone morphogenetic protein (BMP)-4 is a key mediator of anterior pituitary organogenesis. However, through inappropriate expression patterns, BMP-4 is also pathogenic in a pituitary adenoma subtype-specific context. In these cases, increase or decrease in BMP-4 in lactotroph-and corticotroph-derived adenomas, respectively, is consistent with a bifunction role for this protein toward either promotion or inhibition of cell proliferation and hormone secretion. To gain insight into the aberrations responsible for differential expression, we examined BMP-4 transcript and protein expression patterns in the major adenomas subtypes. BMP-4 transcript and protein are differentially expressed and show increase in the majority of prolactinomas relative to normal pituitary, whereas the majority of other adenoma subtypes show reduced expression relative to both prolactinoma and normal pituitaries. Reduced expression of BMP-4 is not associated with change in CpG island methylation status. However, histone tail modifications are apparent, as enrichment for a modification associated with silent genes, H3K27me3, and depletion of a modification associated with active genes, H3K9Ac. In pituitary cell lines, reduced BMP-4 expression is also associated with similar histone tail modifications and contemporaneous increase in CpG island methylation. In these cells, coincubation with the demethylating agent zebularine and histone deacetylase inhibitor, trichostatin A, reversed epigenetic changes and restored expression of BMP-4. These studies show that, in contrast to prolactinomas, other adenoma subtypes show reduced expression of BMP-4 where epidrug induced reexpression, alone or in combination with conventional therapies, may offer new treatment strategies. (Endocrinology 153: 3603-3612, 2012)

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

“…[20] The subtypes comprised, GH-secreting tumours (GH), all of which were grade 2 macroadenomas, Prolactinomas (PRL) were grade 1 microadenomas, and also grade 2 and 3 macroadenomas, Corticotrophinoma (ACTH) were grade 2 macroadenomas, Non-functioning adenomas (NF) were all grade 2 and 3 macroadenomas and their clinical characteristics have been described previously by us. [20] As control we used post-mortem normal pituitaries, acquired within 12 hours of death.…”

“…[20] The subtypes comprised, GH-secreting tumours (GH), all of which were grade 2 macroadenomas, Prolactinomas (PRL) were grade 1 microadenomas, and also grade 2 and 3 macroadenomas, Corticotrophinoma (ACTH) were grade 2 macroadenomas, Non-functioning adenomas (NF) were all grade 2 and 3 macroadenomas and their clinical characteristics have been described previously by us. [20] As control we used post-mortem normal pituitaries, acquired within 12 hours of death. However, a caveat associated with these apparent normal controls and indeed of the normal rat pituitary in the studies employing GH3 cells is that the comparisons represent essentially monoclonal (tumour cells) populations with an admix of cells that comprise the normal gland.…”

“…[20] Briefly, after DNA-Protein crosslinking in 1% formaldehyde (Sigma Aldrich), cells or primary tissues were lysed in ice-cold buffer, supplemented with protease inhibitor cocktail and phenyl methane sulfonyl fluoride (PMSF), after which cells or primary tissues were homogenized to release the nuclei. After resuspension in digestion buffer, supplemented with inhibitor cocktail and an enzymatic shearing cocktail (Active Motif) the samples were centrifuged.…”

“…Relative to normal pituitaries GH3 cells expressed significantly higher levels of hmga1 transcript as determined by RT-qPCR analysis (Figure 2A), however, and as we have shown previously in this cell line, expression of the bmp-4 transcript was significantly reduced relative to normal pituitary counterparts. [20] Stem-loop RT-qPCR showed significantly reduced expression of the rodent homologues of, miR-16, miR-26b, miR-34b miR-196a-2 and let-7a in GH3 cells relative to normal pituitaries ( Figure 2B). …”

“…In these experiments and as we showed previously epidrug challenges (at the doses employed) did not lead to decrease in cell viability. [20] In addition to determining effects of epidrug-induced expression of endogenous miRNAs on the hmga1 transcript we also determined effect on hmga1…”