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Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells.

ABSTRACT: Estrogen receptor alpha (ERalpha) mediates the biological actions of estrogens and also contributes to the development and progression of breast cancer. To gain a more comprehensive understanding of ERalpha-mediated transcription, we used chromatin immunoprecipitation and promoter focused microarrays (ChIP-chip) to identify ERalpha binding sites in T-47D human breast cancer cells. Transcription factor binding site analysis revealed that the estrogen response element (ERE) was significantly over-represented and was found in 50% of the 243 ERalpha-bound regions identified. Interestingly, multiple ERalpha-bound regions were detected in the upstream regulatory sequences of the CYP2B gene cluster. Because ERalpha has been reported to regulate the expression of other cytochrome P450 enzymes and CYP2B6 is highly expressed in ERalpha-positive breast tumors, we focused on characterizing the ERalpha-dependent regulation of CYP2B6. Reporter gene assays revealed that ERalpha and ERbeta increased CYP2B6-regulated gene expression through a functional ERE located at -1669 to -1657 in the upstream regulatory region of CYP2B6. E2 increased ERalpha and nuclear receptor coactivator 3 (NCoA3) recruitment to the 5'-flanking region of CYP2B6, and increased CYP2B6 mRNA levels in T-47D but not in MCF-7 human breast cancer cells. RNAi-mediated knockdown of ERalpha in the T-47D cells resulted in a significant decrease in CYP2B6 mRNA levels. Taken together, our study provides evidence for cell-type specific transcriptional regulation of the CYP2B6 gene by ERs.

SUBMITTER: Lo R

PROVIDER: S-EPMC5153331 | biostudies-literature | 2010 May-Jun

REPOSITORIES: biostudies-literature

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Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells.

Lo Raymond R Burgoon Lyle L Macpherson Laura L Ahmed Shaimaa S Matthews Jason J

Biochimica et biophysica acta 20100114 5-6

Estrogen receptor alpha (ERalpha) mediates the biological actions of estrogens and also contributes to the development and progression of breast cancer. To gain a more comprehensive understanding of ERalpha-mediated transcription, we used chromatin immunoprecipitation and promoter focused microarrays (ChIP-chip) to identify ERalpha binding sites in T-47D human breast cancer cells. Transcription factor binding site analysis revealed that the estrogen response element (ERE) was significantly over- ...[more]

PMID: 20079471

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Project description:BACKGROUND:Breast cancer (BC) is highly heterogeneous with ~?60-70% of estrogen receptor positive BC patient's response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERR? in breast cancer. METHODS:Estrogen related receptor ? (ERR?) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERR? is a direct target of ER?, we investigated the expression of ERR? in short hairpin ribonucleic acid knockdown of ER? breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ER? by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERR? in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERR? with survival in breast cancer patients. RESULTS:Tissue microarray (TMA) analysis showed that ERR? is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER?+?ve breast tumors and cell lines showed a significant expression of ERR? compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERR? and it was ER? dependent. Mechanistic analyses indicate that ER? directly targets ERR? through estrogen response element and ERR? also mediates cell cycle regulation through p18, p21cip and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERR? promoter activity in ectopically co-expressed ER? and ERR? breast cancer cell lines. Fluorescence-activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERR? overexpressed MCF7 cells. Furthermore, ERR? expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERR? in breast cancer cells which provide a potential avenue to target ERR? signaling pathway in breast cancer. CONCLUSION:Our results indicate that ERR? is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERR? could be therapeutic target for the treatment of breast cancer.

Project description:BACKGROUND:Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear. METHODS:Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry. RESULTS:ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation. CONCLUSIONS:These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

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Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells.

Publications

Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells.

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