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. 2004 Jun 2;23(11):2293-303.
doi: 10.1038/sj.emboj.7600231. Epub 2004 May 13.

BRG1/BRM and Prohibitin Are Required for Growth Suppression by Estrogen Antagonists

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Free PMC article

BRG1/BRM and Prohibitin Are Required for Growth Suppression by Estrogen Antagonists

Sheng Wang et al. EMBO J. .
Free PMC article

Abstract

Estrogen antagonists are universally employed in the breast cancer therapy, although antagonist therapy is limited by the inevitable development of cellular resistance. The molecular mechanisms by which these agents inhibit cellular proliferation in breast cancer cells are not fully defined. Recent studies have shown the involvement of the E2F pathway in tamoxifen-induced growth arrest. We show that an E2F repressor, prohibitin, and the chromatin modifiers Brg1/Brm are required for estrogen antagonist-mediated growth suppression through the estrogen receptor, and that their recruitment to native promoter-bound E2F is induced via a JNK1 pathway. In addition, we demonstrate major mechanistic differences among the signaling pathways initiated by estrogen, estrogen deprivation, and estrogen antagonists. Collectively, these findings suggest that the prohibitin/Brg1/Brm node is a major cellular target for estrogen antagonists, and thereby also implicate prohibitin/Brg1/Brm as potentially important targets for breast cancer therapy.

Figures

Figure 1
Figure 1
(A) Estrogen antagonists modulate E2F-driven transcription. MCF7 and ZR75-1 cells were transfected with an E2F-responsive reporter (E2Luc, 12 μg). All cells were co-transfected with 1 μg of pSV-βGal followed by βgal assay as an internal control for transcription efficiency, and the activity of a pSVβgal vector was comparable in all samples. The results shown are statistics of four repeated experiments. Cells were treated with 4HT, ICI182780, or ethanol (vehicle control) for 72 h (initiated 24 h prior to transfection). The relative luciferase activities were calculated relative to the ‘E2Luc', which were arbitrarily assigned a value of ‘100'. The basal activity of the E2Luc reporter was repressed by estrogen antagonists (lanes 3 and 4), similar to the effect observed when a prohibitin expression vector was co-transfected (lane 2). Ectopic expression of prohibitin or treatment with estrogen antagonists did not affect the activity of a non-E2F-responsive reporter pSVECG (data not shown). (B) Suppression of prohibitin by prohibitin SiRNA blocks estrogen antagonist-induced E2F transcriptional repression. MCF7 and ZR75-1 cells were transfected with the E2Luc reporter (12 μg), with or without prohibitin SiRNA or control SiRNA. Cells were treated with 4HT (lanes 3–5), ICI182780 (lanes 6–8), or ethanol (vehicle control) (lane 9) for 72 h (initiated 24 h prior to the transfection). The relative luciferase activities were calculated as described in the legend to Figure 2A. Estrogen antagonist-induced repression of activity of the E2F-driven E2Luc reporter was released by co-transfection of prohibitin SiRNA (lanes 4, 7, and 9). (C) Immunoblot analysis of the protein levels of prohibitin and E2F1 (control) in MCF7 cells and the cells transfected with prohibitin SiRNA or nonsilencing control SiRNA.
Figure 2
Figure 2
Physical association of prohibitin and E2F1 is required for the repressive function of estrogen antagonists. MCF7 cells were stably transfected with pCR3.1 E2F1 (AA304–357) or pCR3.1 E2F1 (AA263–303), both tagged with Gal4. Expression of the transfected E2F peptides was confirmed by immunoblot analysis (C). (A) Whole-cell extracts, or proteins from cell extracts immunoprecipitated using anti-Gal4 (for the E2F mutants) or control (anti-Myc or -E2F) antibodies, were separated electrophoretically, then identified by immunoblotting using an anti-prohibitin antibody, or anti-tubulin antibody as a loading control (upper panel). The experiments were repeated in a reciprocal fashion (lower panel). (B) The E2F peptide-expressing MCF7 cells were transfected with an E2Luc reporter and treated with ethanol (vehicle control) or estrogen antagonists. Cells were harvested for luciferase activity assay and β-galactosidase assay (transfection control). Luciferase activity calculations are normalized for β-galactosidase activity.
Figure 3
Figure 3
Induction of association of prohibitin with Brg1/Brm by estrogen antagonists. (A) MCF7 cells were treated with 4HT or ICI182780 for the indicated time intervals. Cell extracts were immunoprecipitated (IP) by anti-cMyc (control), -Brg1, or -Brm antibodies, followed by immunoblot analysis (IB), using anti-prohibitin or -p38 (as control) antibodies. The reciprocal IP–immunoblot was performed using anti-cMyc or -prohibitin antibodies for the IP, and anti-Brg1, -Brm, or -p38 antibodies for the immunoblot. Increases in associations between prohibitin and Brg1/Brm were evident as early as 20 min after treatment. Immunoblotting with the anti-p38 antibody failed to detect any protein in the immunoprecipitates, indicating the specificity of the prohibitin–Brm/Brg1 associations. (B) The same cell extracts used in panel A were analyzed by immunoblot using anti-Brg1, -Brm, or -prohibitin antibodies. The results shown are representative of experiments that were repeated four times, which yielded identical results.
Figure 4
Figure 4
Estrogen antagonists enhance the transcriptional activity of the prohibitin promoter. MCF7 and ZR75-1 cells were transfected with ProhibitinCAT, a CAT reporter gene driven by a rat prohibitin promoter element spanning −485 to −5 bp. Cells were treated with 4HT or ICI182780 for the indicated time intervals, and harvested for CAT activity assay and β-galactosidase assay (transfection control).
Figure 5
Figure 5
Repression of Brg1 or Brm blocks E2F transcriptional repression induced by estrogen antagonists. (A) MCF7 and ZR75-1 cells were transfected with an E2F-responsive promoter-driven reporter, E2Luc (12 μg/10 cm dish), producing a basal level of luciferase activity (lane 1). Basal luciferase activity was dramatically repressed by treatment of the cells with estrogen antagonists, as indicated (lanes 2 and 5), while treatment with the ethanol vehicle showed no such effect (lane 10). This repression of luciferase activity by estrogen antagonists was reversed by knockdown of Brg1 or Brm using SiRNA (lanes 3, 4, 7 and 8). A 1 μg portion of βGal vector was included in all transfections as a control. βGal values are comparable in all samples. (B) Immunoblot analysis of Brg1, Brm, and tubulin levels in MCF-7 cells or the cells transfected with control SiRNA, or Brg1 or Brm SiRNA.
Figure 6
Figure 6
Estrogen antagonists induce the recruitment of Brg1 and Brm to native E2F-responsive promoters for transcriptional repression. (A) MCF7 cells were cultured in an estrogen-free medium for 3 days. The cells were then treated with tamoxifen alone, tamoxifen plus estradiol, or Estradiol alone, in separate experiments. Cell extracts were collected at the different time points as indicated and analyzed using an in vivo CHIP assay. The DNA recovered from the immunoprecipitates by the indicated antibodies was PCR-amplified, using primers against a region on each of the two E2F-responsive promoters (E2F1 and TK), and one non-E2F-responsive promoter (c-Fos), as a control. Higher levels of amplified products from the Brg1 or Brm antibodies were detected in the CHIP assays of the E2F-responsive promoters in the cells treated with 4HT for more than 40 min. This enhanced recruitment of Brm and Brg1 was not affected by co-treatment with estradiol. Amplified products from CHIP using E2F1 or prohibitin antibodies did not show variations in levels regardless of treatment. Control CHIP assay using p38 antibody, and CHIP assay on the c-Fos promoter using prohibitin, Brg1, Brm, and E2F1 antibodies, failed to generate any product, confirming the specificity of this assay. PCR using DNA directly isolated from the cell extracts produced products in all the lanes tested, serving as a positive control for the PCR reaction (Total). RT–PCR assays demonstrated a relative decrease in the levels of transcripts from the E2F-responsive genes in the tamoxifen-treated cells, but not the estradiol-treated cells, which instead produced increased levels of transcripts from E2F-responsive promoters. CHIP assay using PolII antibody demonstrated the transcriptional repression of E2F-responsive genes by tamoxifen and transcriptional induction by estradiol. (B) Positive control CHIP assay using anti-Brg1 and -Brm antibodies was performed on an estrogen-responsive promoter (pS2). Recruitment of Brm and Brg1 to the pS2 promoter was induced by estradiol. (C) MCF7 cells stably transfected with a vector encoding the prohibitin-binding domain of E2F (304–357) or control peptide (263–303). The expression of the transfected genes was confirmed by immunoblot shown in Figure 2C. A CHIP assay was performed on endogenous E2F1 promoter using Brg1, Brm, E2F1 prohibitin antibodies, and control antibody (Gal4). A significantly lower amount of PCR product associated with Brg1 or Brm was found when E2F (304–357) was present in the cells (left). The induction of Brg1 or Brm recruitment to the promoter by estrogen antagonists was blocked when E2F (304–357) was expressed.
Figure 7
Figure 7
JNK1 mediates estrogen antagonist signaling to the prohibitin/E2F axis. (A) MCF7 cells were transfected with JNK1 or mutant (kinase-deficient) JNK1. Immunoprecipitation/immunoblot analysis (IP/IB), and the reciprocal IP–IB, was performed using the indicated antibodies. Anti-Myc and -tubulin antibodies were used as controls for IP and IB. Equal loading and precipitation are further confirmed by immunoblotting using antibodies for the target proteins (data not shown). JNK1 overexpression induced association between prohibitin and Brg1/Brm, just as tamoxifen does (see Figure 1). (B) The same cell extracts used in panel A were analyzed by immunoblotting using anti-JNK1 antibody or -p38 antibody, confirming the overexpression of the products of the transfected vectors. (C) JNK1 activity was suppressed either by transfection of DNJNK1 or treatment with a JNK1 inhibitor. The cells were co-treated with tamoxifen (‘4HT') or vehicle controls as indicated for 3 h. DMSO was vehicle control for JNK1 inhibitor. Empty vector (‘vector') was transfected as a control for the DNJNK1 vector. Immunoprecipitation/immunoblot analysis (IP/IB), and the reciprocal IP/IB, was performed using the indicated antibodies. Equal loading and precipitation are further confirmed by immunoblotting using antibodies for the target proteins (data not shown). DNJNK1 and JNK1 inhibitor each blocked the enhanced association between prohibitin and Brg1/Brm induced by tamoxifen. (D) In vivo CHIP assay. Left column: MCF7 cells were transfected with empty vector control, JNK1, or mutant (kinase-deficient) JNK1 vectors. Cell extracts were analyzed using an in vivo CHIP assay on the endogenous, native E2F1 promoter, described in Figure 3 (above). Higher levels of amplified products precipitated by the Brg1 or Brm antibodies were detected when JNK1 (but not mutant JNK1) was transfected. This enhanced recruitment of Brm and Brg1 by JNK1 was similar to that induced by tamoxifen treatment, as shown in Figure 3. RT–PCR assays demonstrated a relative decrease in the levels of transcripts from the E2F1 genes when JNK1, but not the mutant JNK1, was transfected. Middle column: MCF7 cells were transfected with control vector or DNJNK1. Cells were treated with ethanol vehicle or tamoxifen as indicated. CHIP assay was performed on the native E2F1 promoter. Transfection of the DNJNK1 blocked the tamoxifen-induced recruitment of BRG1 and BRM to the E2F1 promoter, and reversed the repression of E2F1 transcript levels. Right column: MCF7 cells were treated with ethanol (for tamoxifen vehicle control), DMSO (for JNK1 inhibitor vehicle control), or JNK1 inhibitor. Cells were collected after 3 h of treatment. Extracts were analyzed by CHIP assay. Similar to the effects of DNJNK1 shown in the middle panel, the JNK1 inhibitor also blocked the tamoxifen-induced Brg1/Brm recruitment to the native E2F1 promoter, and the tamoxifen-induced repression of E2F1 transcript levels was reversed by the inhibitor.
Figure 8
Figure 8
Regulation of prohibitin by estrogen antagonist is ER-dependent. (A, B) MDA-MB-231, an ER-negative cell line, was stably transfected with an ER expression vector, or an empty vector as a control. The empty vector-transfected cells and the ER-expressing cells (‘+ER') were treated with vehicle control (‘ETH') or estrogen antagonists (‘4HT' or ‘ICI'), as indicated. Whole-cell extracts (CE), or proteins immunoprecipitated from cell extracts with control antibody (myc) (‘mock IP') or anti-Brg1 or -Brm antibodies, were electrophoretically separated, followed by immunoblotting with an anti-prohibitin antibody (upper panels). The IP:IB analysis was repeated in a reciprocal fashion (lower panels). Equal loading and precipitation were further confirmed by immunoblotting using antibodies for the target proteins (data not shown). (C) Expression of the transfected ER was detected by immunoblot.

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