Unknown,Transcriptomics,Genomics,Proteomics

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Genomic analysis of SRC-2/TIF2 regulated genes in breast cancer cells_

ABSTRACT: We have characterized the global program of transcription in SRC-2-depleted MCF-7 breast cancer cells using short-hairpin RNA technology, and in MCF-7 cells exposed to PKA activating agents. In order to identify genes that may be regulated through PKA-induced downregulation of SRC-2, overlapping transcriptional targets in response to the respective treatments were characterized. MCF-7 human breast cancer cells transduced with shRNA targeting SRC-2 (SRC-2 shRNA) or infected with control shRNA vector (Ctr shRNA) were seeded in 9,2 cm petri dishes, 2,0 x106 cells in each dishes, in DMEM supplemented with 5% charcoaled stripped FBS and 10 nM 17b-estradiol. After 48 hours a selection of the Ctr shRNA cells were treated with cAMP analog (8-CPT-cAMP, 150 µM) and cAMP elevating agents (Forskolin (10 µM) and IBMX (50 µM)) The cells were then incubated for another 24 hour in the cell incubator. Cells were harvested by washing them twice with 8 ml PBS (37 oC), before adding 1,5 ml 0,25 % trypzine. The trypzine reaction was stopped after 5 minutes by adding 2,5 ml DMEM to the cells. Trypzine and cell medium was removed by centrifugating the cell suspensions at 1000 rpm for 3 minutes. The cells were then washed with 400 µl PBS, before addition of 350 µl RLT buffer to the cell pellets. The cells were then homogenized by vortexing for 1 minute and then freezed in -80 oC.

ORGANISM(S): Homo sapiens

SUBMITTER: Thomas Helland

PROVIDER: E-MTAB-1662 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ovarian estrogen (E2) and progesterone (P4) are indispensable for embryo-implantation and endometrial stromal decidualization; however, the molecular mechanisms that underpin these reproductive processes are unclear. Steroid receptor coregulator-2 (SRC-2) belongs to the multifunctional SRC/p160 family which also includes SRC-1 and SRC-3. Sharing strong sequence homology, all three SRCs exert diverse regulatory effects by modulating the transcriptional potency of nuclear receptor family members, including the estrogen and progesterone receptor (ER and PR respectively). Importantly, absence of SRC-2 in PR positive cells in the epithelial, stromal, and myometrial compartments of the murine uterus results in a striking infertility defect. This reproductive phenotype highlights a key role for SRC-2 in uterine function which is not shared with other coregulators. Intriguingly, abrogation of uterine SRC-2 does not block embryo apposition or attachment to the apical surface of luminal epithelial cells of the endometrium but rather prevents P4-dependent local decidualization of the sub-epithelial stroma. Remarkably, epithelial-specific ablation of SRC-2 in the murine uterus does not compromise endometrial functionality, again underscoring the unique importance of stromal derived SRC-2 in uterine function. The stromal decidualization defect resulting from SRC-2 ablation is reflected at the molecular level by a marked attenuation in P4 responsive target genes known to be critical for P4 dependent decidualization (i.e. ERBB receptor feedback inhibitor 1, Follistatin and Fkbp5). Conversely, the induction of E2 or P4 target genes involved in embryo implantation (i.e. leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) respectively) is not affected by SRC-2’s absence. As with mouse studies, decidualization of primary human stromal cells (HESCs) in culture is blocked by SRC-2 knockdown; however, HESC decidualization is unaffected by knockdown of SRC-1 or SRC-3. As a consequence of SRC-2 knockdown, molecular studies disclose a striking decrease in the induction of a subset of P4 target genes (i.e. WNT4 and FKBP5) which are essential for the stromal-epithelioid transformation step, the cellular hallmark of endometrial decidualization. Collectively, these studies not only showcase the evolutionary importance of SRC-2 in endometrial biology but also suggest that deregulation of this coregulator may underpin a spectrum of hormone-dependent uterine pathologies such as endometriosis and endometrial cancer. Microarray analysis was performed on mouse uteri using eighteen SRC-2flox/flox (SRC-2f/f) and eighteen PRCre/+ SRC-2flox/flox (SRC-2d/d) mice. Mice were ovariectomized at 6 weeks and after 2 weeks mice were either treated with sesame oil (vehicle) or 1 mg of P4. RNA from three mice per genotype per treatment were pooled and assigned as one sample (three samples per genotype per treatment). multiple group comparison

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Genomic analysis of SRC-2/TIF2 regulated genes in breast cancer cells_

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