Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells
Introduction
Estrogens act through their nuclear receptors, estrogen receptor α (ERα; NR2A1) and ERβ (NR3A2) to regulate diverse transcriptional responses that include both stimulation and repression of gene expression [1]. ERs share the evolutionarily conserved functional domains typical of other nuclear receptor family members [2]. These include the amino-terminal activation function-1 (AF1) domain, the centrally located DNA-binding domain (DBD) and the carboxy-terminal ligand-binding domain (LBD), which also contains the ligand-dependent activation function-2 (AF2) region that is important for interaction with coactivators [3]. Ligand binding induces conformational changes in the receptors resulting in homodimerization and subsequent DNA binding. ER homodimers bind to perfect and imperfect palindromic DNA sequences termed estrogen responsive elements (EREs) located in the regulatory region of their target genes. Once bound to DNA, ERs act as nucleation sites for the recruitment of chromatin remodeling complexes and co-regulators (i.e. nuclear receptor coactivator 3 (NCoA3), p300/Creb binding protein), and the assembly of basal transcriptional machinery [4]. ERs also regulate transcription via half-site EREs and by a tethering mechanism that involves protein:protein interactions with other DNA-bound transcription factors, including activating protein 1 and stimulating protein 1 [4].
Estrogens are critical for the growth, development and differentiation of the mammary gland. ER expression in breast cancer is associated with improved responsiveness to endocrine targeted therapies, such as treatment with the anti-estrogen tamoxifen to reduce ER activity or treatment with aromatase inhibitors to deplete estrogen levels [5]. In addition to increasing the expression of genes that regulate cell proliferation and cancer development, ERα has also been reported to regulate the expression of many drug metabolizing enzymes, including cytochrome P450 1A1 (CYP1A1), CYP1B1, and CYP2A6 [6], [7], [8], many of which are involved in the metabolism of estrogens. Two recent studies have reported higher CYP2B6 expression in ERα-positive compared to ERα-negative breast tumors [9], [10], implicating CYP2B6 as an ERα target gene. ER-dependent regulation of the aforementioned drug metabolizing enzymes may contribute to alterations in endogenous steroid levels as well as increased metabolism or bioactivation of therapeutic agents and xenobiotics.
Hepatic CYP2B genes are the most inducible CYP isoforms by phenobarbital (PB)-type inducers. CYP2B6 is expressed in the human liver, where it constitutes approximately 6% of total microsomal P450s [11], but it is also expressed in extra-hepatic tissues including the intestine, kidney, lung, skin, brain and mammary gland [12], [13], [14]. CYP2B6 metabolizes a wide variety of pharmaceutical agents including cyclophosphamide, buproprion and tamoxifen; environmental contaminants such as aflatoxin B and dibenzanthracene; nicotine and methylenedioxymethamphetamine (MDMA “ecstasy”) [15], [16]. CYP2B6 expression is regulated through the constitutive androstane receptor (CAR; NR1I3), a member of the nuclear receptor superfamily of transcription factors [17]. Upon nuclear translocation, CAR associates with its dimerization partner retinoid X receptor (RXR; NR2B1). The CAR-RXR complex binds the phenobarbital-responsive enhancer module (PBREM) and recruits co-regulator proteins to modulate target gene expression, such as CYP2B6 [18]. Although CAR has long been recognized as a key regulator of CYP2B, emerging evidence suggests that other nuclear receptors such as pregnane X receptor (PXR; NR1I2) [19] and glucocorticoid receptor (GR; NR3C1) [20], and liver enriched transcription factors are also involved in the regulation of this gene [21]. Interestingly, many of the potent inducers of CYP2B6 expression are ligands for PXR but not CAR [22]. Putative glucocorticoid response elements (GREs) present in the upstream regulatory regions of mouse Cyp2b10 and rat CYP2B1/2 are also responsive to the synthetic glucocorticoid, dexamethasone (DEX) [23], [24]. DEX treatment has been reported to be required for maximum induction of Cyp2b10 expression by activators of CAR [23]. It is unclear if GR is involved in the regulation of CYP2B6, since the CYP2B6 promoter region does not contain a GRE. However, activation of GR has been reported to increase the expression of PXR and CAR in human hepatocytes [25]. Estrogens have also been implicated in the regulation of Cyp2b10 with one study reporting that high doses (μM) of 17β-estradiol (E2) activate mouse but not human CAR [26]. However, direct regulation of CYP2B6 by ERs has not been determined.
Recent studies using chromatin immunoprecipitation (ChIP) combined with microarrays (ChIP-chip) have identified several ERα-bound regions across the genome [27], [28]. A number of breast cancer cell lines have been used in in vitro studies of estrogen responses, but most of the data, including genome-wide analyses of ERα binding sites, have come from experiments using MCF-7 cells [27], [28], [29], [30]. MCF-7 cells express higher ERα levels than T-47D human breast cancer cells, while both cell lines expressing low, but similar, levels of ERβ [31], [32]. Although MCF-7 and T-47D cells exhibit similar global gene expression profiles in response to E2 treatment [33], significant differences in the expression of many genes have been reported [10]. Determining ERα binding profiles in other ERα-positive breast cancer cell lines, such as T-47D, will ensure that any cell line-specific differences in ERα action are not overlooked. In the present study, we performed ChIP-chip to identify ERα-bound genomic regions in T-47D human breast cancer cells. One of the ERα-bound regions identified was located in the 5′-regulatory region of CYP2B6. Reporter gene assays, mRNA expression and protein expression analysis provide evidence for ER-dependent regulation of CYP2B6.
Section snippets
Chemicals
Dimethyl sulfoxide (DMSO), 17β-estradiol (E2) and ICI 182,780 were purchased from Sigma (St. Louis, MO). Primers for quantitative real-time polymerase chain reaction (qPCR) were purchased from Integrated DNA Technology (Coralville, Iowa, USA). Cell culture media, fetal bovine serum (FBS) and trypsin were purchased from Wisent (St. Bruno, Canada). All other chemicals and biochemicals were of the highest quality available from commercial vendors.
Plasmids
To generate pGL3p-2B7P, the ChIP-chip isolated
Estrogen receptor binding sites in T-47D cells
To identify the ERα binding sites in T-47D human breast cancer cells, we performed ChIP-chip assays on cells plated in medium containing 10% FBS and cultured for 48 h to reach 85% confluency. ERα was active under these conditions due to residual levels of estrogens in the serum [38]. ELISA assays showed that the E2 concentration in our medium was approximately 0.1 nM (data not shown). Therefore due to the level of residual estrogens in the serum-containing medium, exogenous E2 was not added to
Discussion
The regulation of CYP2B6 is important in drug metabolism and xenochemical toxicity [15]. Most studies on CYP2B6 regulation have focused on the transcriptional induction mediated by xenobiotics, where the main focus of the studies was the transcriptional regulation of CYP2B enzymes by CAR [16]. However CAR does not operate alone in the transactivation of P450 genes, but rather works in concert with several transcription factors, nuclear receptors, and coactivators to control signaling pathways
Acknowledgements
We would like to thank Dr. Rachel Tyndale (University of Toronto, Toronto, ON) and Prof. Jan-Åke Gustafsson (University of Houston, TX, USA) for providing the materials used in this study. JM is the recipient of a Canadian Institute of Health Research New Investigator Award. This work was supported by the CIHR operating grant MOP-82715 to JM.
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