Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genomic analyses of breast cancer cells: 17Î²-hydroxysteroid dehydrogenase type 1 induces transcriptional changes in estradiol-dependent and independent manners

ABSTRACT: 17Î²-HSD1 expression modulates T47D transcript profile in in steroid-deprived medium. The enzyme specifically regulates apoptosis and cancer-related genes in T47D cells cultured in steroid-deprived medium. Genes that primarily involved in Cell cycle progression, such as the Cyclin A2 gene, CCNA2, are generally down-regulated whereas most genes involved in apoptosis and cell death, such as the proapoptotic gene XAF1 and FGF12, are on the contrary up-regulated by 17Î²-HSD1 knockdown, and 21% of the modulated genes belong to this latter functional category. This correborates with its role previously shown in increasing breast cancer cell proliferation (Aka et al, 2010) and indicates that in addition to its direct role as cell proliferation via incresing E2 and decreasing DHT, 17Î²-HSD1 may also be involved in oncogenesis by favoring its anti-apoptosis pathway in breast cancer cells 17Î²-HSD1 may also be involved in oncogenesis by favoring anti-apoptosis pathway and/or inhibiting cell survival pathway. We tested the ability of estrogen to induce or repress endogenous genes of T47D by microarray analysis. A total of 131 genes were found to increase or decrease 1.5-fold or higher in response to 17 beta-estradiol (1 nM for 48 h). Besides, the gene regulation occuring in steroid-deprived conditions showed that 17Î²-HSD1 modulates gene expression in steroid-independent manners. Our study thus demonstrates that 17Î²-HSD1 modulates the E2-independent transcription of endogenous genes in T47D cells. The E2-dependent transcription as well as the E2-independent transcription are significantly modulated by 17Î²-HSD1 in breast cancer cells We first report here that breast cancer cell transcript profile is independtly modulated by both 17Î²-HSD1 and its principale product estradiol. Microarrays was used to study the effect of 17Î²-HSD1 gene knockdown on gene expression and on endogenous ERG in T47D cells. The sense and antisense sequences of three 17Î²-HSD1 siRNAs were selected and synthesized. Scramble siRNA was used as control siRNA. Two days before transfection, T47D cells in T75 were cultured in steroid deprived medium. On the transfection day, cells in T75 flasks were trypsined and ressuspended in a fresh charcoal-treated medium. Cells were then reverse-transfected with 17Î²-HSD1 specific siRNAs or with negative control siRNA.Two days (48 hours) after transfection, cell culture media were replaced by fresh charcoal-treated medium containing either the steroid E2 (1 nM) or ethanol as a vehicle control, and cells were incubated for two more days before RNA extraction.

ORGANISM(S): Homo sapiens

SUBMITTER: Ezequiel Calvo

PROVIDER: E-GEOD-77345 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1.

Aka Juliette A JA Calvo Ezequiel-Luis EL Lin Sheng-Xiang SX

Molecular and cellular endocrinology 20160818

17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a steroidal enzyme which, in breast cancer cells, mainly synthesizes 17-beta-estradiol (E2), an estrogenic hormone that stimulates breast cancer cell growth. We previously showed that the enzyme increased breast cancer cell proliferation via a dual effect on E2 and 5α-dihydrotestosterone (DHT) levels and impacted gene expression and protein profile of breast cancer cells cultured in E2-contained medium. Here, we used RNA interference techn ...[more]

PMID: 27544780

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Project description:Despite the success of the aromatase inhibitors (AIs) in treating estrogen receptor positive breast cancer, 15-20% of patients receiving adjuvant AIs will relapse within 5-10 years of treatment initiation. Long term estrogen deprivation (LTED) of breast cancer cells in culture has been successfully used to mimic AI-induced estrogen depletion to dissect mechanisms of AI resistance. However, we hypothesized that a subset of patients receiving AI therapy may maintain low circulating concentrations of estrogens that influence the development of endocrine resistance. We sought to expand established LTED models to account for these observations. MCF-7 cells were grown in medium with charcoal stripped serum supplemented with defined concentrations of E2 or the estrogenic androgen metabolite 3βAdiol. Cells were selected in the EC10 and EC90 of E2 or 3βAdiol, or estrogen deprived. Estrogen-independence was evaluated during selection by assessing cell growth in the absence or presence of E2 or 3βAdiol. Following >7 months of selection, estrogen-independence developed in estrogen-deprived cells and cells maintained in low concentrations of steroid hormone. Functional analyses demonstrated that estrogen-deprived and low-steroid selected cells developed estrogen-independence via unique mechanisms, independent and dependent of ERα, respectively. Estrogen-independent growth in low-steroid selected cells could be blocked by kinase inhibitors. However, these cells were completely resistant to kinase inhibition in the presence of low steroid hormone concentrations. These data offer a novel perspective on the development of resistance to AI therapy, and may yield novel approaches to treat AI-resistant tumors. MCF-7 cells were selected for >7months in IMEM+10% Charcoal Stripped FBS, supplmented with defined steroid hormone conditions. Following outgrowth of estrogen-independent cells, cell lines were grown in estrogen-free conditions and gene expression analysis was performed to identify drivers of estrogen-independent growth. Cells were assessed in biological triplicates.

Project description:Estrogen receptors play critical roles in both the normal physiological, and disease states of numerous tissues, including breast and uterus. Estrogen receptor alpha (ER) can activate or repress the expression of target genes upon estrogen stimulation. In order to better understand the transcriptional network of ER in breast and uterus, we generated genome wide maps ofM-BM- ER binding sites (ERBS) and gene expression profiles in breast cancer cells (MCF7 and T47D) and uterine cancer cells (ECC1 and Ishikawa) through ChIP-Seq and microarray techniques. Surprisingly, we identified large scale differences in the numbers of ERBS between these cell lines when treated with E2 (17-M-NM-2 estradiol). Besides identification of common and unique ERBS between breast and uterine cancer cell types., our data also suggest that both cell types could recruit a large set of common co-operating transcription factors (Co-TFs) and a few unique Co-TFs as well. Besides the genes that are commonly regulatedM-BM- between the different cell lines, there are a number of genes that are differentially regulated in different cell types. Gene pathway analyses of E2 target genes suggest that ER regulates many biological pathways and processes in both tissue-type dependent and independent manners. Our results showed that cell lines derived from same tissue display a greater similarity for both profiles of ERBS and gene expression, and that the differential profiles of ER and preferential recruitment of some Co-TFs are the main determinants for the differential regulation of E2 signaling in breast and uterine cancer cells. In order to explore common and distinctive features of ERM-NM-1 (estrogen receptor alpha) binding profiles between breast and uterus, we generated eight ChIP-Seq libraries for the four cell lines (MCF7, T47D, ECC1 and Ishikawa) under two different treatments (E2, ethanol). In addition, we generated four control libraries for the four cell lines. For all treatment libraries, we generated about 7-12 million unique tags each. ER antibody catalog number is (Santa Cruz,sc-543).

Dataset Information

Genomic analyses of breast cancer cells: 17Î²-hydroxysteroid dehydrogenase type 1 induces transcriptional changes in estradiol-dependent and independent manners

Publications

Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1.

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