Abstract P2-09-02: Epigenetic Regulation of histone variants - a role in hormone therapy resistant breast cancer?
Authors: SR Nayak, S Oesterreich, T Pathiraja
Journal: Cancer Research (2012)

Abstract OBJECTIVE To utilize a genome wide approach to characterize epigenetic changes associated with resistance to estrogen deprivation, thus mimicking resistant to AIs. BACKGROUND Mechanisms of endocrine resistance include the activation of growth factor receptor pathways, overexpression of ER co-activators, and metabolic resistance due to polymorphisms in metabolizing enzymes. Recently, epigenetic mechanisms have been investigated to explain endocrine resistance. The majority of mechanisms for acquired resistance have been described following tamoxifen therapy, but the mechanisms for resistance to aromatase inhibitors (AI) remain less well known. HYPOTHESIS Estrogen deprivation results in altered methylation and altered expression of critical target genes in breast cancer cells, which helps the cell to survive in the absence of estrogen, contributing to acquired AI resistance in breast cancer patients. MATERIALS AND METHODS We utilized derivatives of the ER_ MCF-7 cell line which were isolated after being cultured in estrogen-free media for 9 months (C4-12 cell line) and 18–24 months (LTED, Long term estrogen deprivation cell line). A genome-wide methylation screen was performed using Methyl CpG binding Domain (MBD) pull down assay followed by hybridization into Affymetrix Human Promoter 1.0R Array. Altered DNA methylation was validated by bisulfite genomic sequencing assays while gene expression of candidate genes was studied by qRT-PCR and Western blotting. RESULTS There were a significant amount of differentially methylated genes in C4-12 and LTED when compared to MCF7. Intriguingly, among the genes which were significantly hypomethylated in both endocrine resistant cell lines were a number of histone genes, with the homomorphic variant HIST1H2BE showing the strongest hypomethylation. Canonical histones have both homomorphic and heteromorphic variants, which differ in their timing of expression and similarity to the canonical histone protein's amino acid sequence. Overexpression of various heteromorphic histone variants have been implicated in carcinogenesis, but to date, there have been no studies on homomorphic variants, and specifically H2B variants, in regards to their role in tumorigenesis. We show that hypomethylation of HIST1H2BE was associated with its upregulation in C4-12 and LTED cells. Furthermore, knockdown of of HIST2BE resulted in decreased growth in the endocrine resistant cells, but not in the parental MCF-7 cell lines. CONCLUSION &amp; FUTURE DIRECTIONS The study is novel in two ways; first, this is the first study to show a potential role for histone variants in endocrine resistance. Second, this is one of very few examples where hypomethylation (instead of hypermethylation) results in altered expression of genes critical in tumorigenesis. Current studies include the detailed characterization of the phenotype following over-expression and knockdown of HIST1H2BE, the examination of the timing of expression of this variant in the S phase, and finally we are measuring methylation and expression of HIST2BE in a large collection of tumors from AI-treated breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-09-02.