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Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

ABSTRACT: There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.

SUBMITTER: Kastrati I

PROVIDER: S-EPMC3226622 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

Kastrati Irida I Edirisinghe Praneeth D PD Hemachandra L-P-Madhubani P LP Chandrasena Esala R ER Choi Jaewoo J Wang Yue-Ting YT Bolton Judy L JL Thatcher Gregory R J GR

PloS one 20111129 11

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy ...[more]

PMID: 22140478

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Project description:17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.

Project description:Estrogen is a risk factor for breast cancer, yet its mechanism in the initiation of this disease is not clear. Here we use a model of estrogen-mediated malignant transformation of MCF-10F cells to identify the temporal acquisition of changes in genome structure and gene expression that correspond to the progressive transformed phenotype culminating in tumorigenesis. Genomic DNA and total RNA were isolated from MCF10F, trMCF (MCF10F transformed by 70 nM 17-beta estradiol), bsMCF (trMCF selected by Boyden chamber, tumorigenic), bcMCF(clones of bsMCF), and caMCF (cells from tumors of bsMCF grown in SCID mice). The Affymetrix 100k SNP and HG-U133_Plus_2 chips were used for genotyping and gene expression analyses. Changes in chromosomal copy number and loss of heterozygosity were progressive. Gross changes were rarely observed in the trMCF10F cells; the earliest was a gain in chromosome 1p, 1p36.12-1p36.21. In the bsMCF and their sub-clones bcMCF, additional gains were seen in chromosomes 1p, 1p36.12-1pter, 5q, 5q21.1-5q35.3, and 13q, 13q21.32-13q34; losses were detected in chromosomes 4 and 8p, 8p11.1-8p23.1. In the caMCF, additional losses were seen in chromosomes 3p, 3p12.1-3p14.1, 9p, 9p22.1-9p24.3, and 18q, 18q11.2-18q23. Using microarray analysis we were able, in some cases, to narrow the regions of interest and their associated candidate cancer genes. The observation of progressive genomic changes, along with the tumor phenotype of a poorly differentiated adenocarcinoma, indicate an underlying mechanism common to breast cancer development in humans, and adds to the evidence indicating that estrogens are mutagenic and contribute early to the process of breast cancer. Experiment Overall Design: Human MCF-10F cells were transformed by 70nM 17-beta-estradiol. The transformed cells were selected by Boyden chamber. The selected cells were injected into SCID mice and also cloned and expanded. The thus grown tumors were harvested for cell culture. Genomic DNA and total RNA were isolated from cell lines in each step for 100K SNP and gene expression microarray assay.

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Dataset Information

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

Publications

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

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