Abstract
17-β-estradiol (estrogen) is a steroid hormone important to human development; however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.
Highlights
Estrogens are members of the family of steroid hormones that stimulate the development and maintenance of female characteristics and sexual reproduction. 17-β-estradiol (E2) is the most abundant estrogen in the human body [1,2]
The MCF-7 (ER positive) and MDAMB-231 (ER negative) human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin
Once normal cell growth was established in DMEM, both cell types were moved to Heavy or Light SILAC media supplemented with 10% dialyzed fetal bovine serum and grown for at least six passages before the start of experiments to allow for complete incorporation of the labeled amino acids into the entire proteome
Summary
Estrogens are members of the family of steroid hormones that stimulate the development and maintenance of female characteristics and sexual reproduction. 17-β-estradiol (E2) is the most abundant estrogen in the human body [1,2]. Estrogen circulates through the bloodstream and targets organs containing estrogen receptors (ER). Higher levels of estrogen exposure are associated with increased breast cancer risk [3] and the expression of estrogen receptor varies in breast cancer subtypes. Once E2 binds to ERα located mainly in the cytoplasm, the activated receptor complex moves to the nucleus where it dimerizes and binds to specific DNA sequences called estrogen response elements (ERE) via the DNA binding domain of ER [4]. The activated EREs are able to regulate the expression of specific genes located downstream of the sequence [5] and with the help of other activators, impact transcription of genes involved in cell proliferation and differentiation. The ligand-bound ERα binds to DNA indirectly through protein-protein interactions with other transcription factors such as AP-1 and Sp1 [6,7]
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