Abstract
Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.
Highlights
Major progress in controlling mortality and morbidity from cancer requires an understanding of the molecular mechanisms that are responsible for initiating the disease
TGF-b2 was of direct interest because disruption of the transforming growth factor h (TGF-h) signaling pathway due to mutation is implicated in the pathogenesis of breast cancer [19]
H3K9 deacetylation of p16INK4A, receptors TGF-bR1 and TGF-bR2, and THBS1 was observed during post-selection growth, correlating again with gene suppression. These results show that TGF-b2 and associated pathway members TGF-bR1, TGF-bR2, and THBS1 are concordantly down-regulated in postselection Human mammary epithelial cells (HMEC) by repressive chromatin remodeling associated with H3K9 deacetylation and methylation rather than DNA hypermethylation
Summary
Major progress in controlling mortality and morbidity from cancer requires an understanding of the molecular mechanisms that are responsible for initiating the disease. Analysis of these early aberrant events is complex because by the time the tumor is detected, the cancer progenitor cells have already undergone. Changes in the DNA methylation pattern in cancer include global hypomethylation of CpG dinucleotides in DNA repeated regions [2] in conjunction with hypermethylation of CpG island promoter– associated genes [1, 3]. Repressive chromatin modifications are associated with tumor suppressor gene silencing and hypermethylation of CpG island promoters. Epigenetic changes are a common feature of human cancer cells compared with their normal counterparts, it is not clear at what stage in the transformation process these epigenetic changes occur and whether they occur concurrently or independently
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