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, 44 (18), 8655-8670

LSD1 Engages a Corepressor Complex for the Activation of the Estrogen Receptor α by Estrogen and cAMP

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LSD1 Engages a Corepressor Complex for the Activation of the Estrogen Receptor α by Estrogen and cAMP

Marcela A Bennesch et al. Nucleic Acids Res.

Abstract

The estrogen receptor α (ERα) is a transcription factor that can be directly activated by estrogen or indirectly by other signaling pathways. We previously reported that activation of the unliganded ERα by cAMP is mediated by phosphorylation of the transcriptional coactivator CARM1 by protein kinase A (PKA), allowing CARM1 to bind ERα directly. This being insufficient by itself to activate ERα, we looked for additional factors and identified the histone H3 demethylase LSD1 as a substrate of PKA and an important mediator of this signaling crosstalk as well as of the response to estrogen. Surprisingly, ERα engages not only LSD1, but its partners of the CoREST corepressor complex and the molecular chaperone Hsp90. The recruitment of Hsp90 to promote ERα transcriptional activity runs against the steroid receptor paradigm and suggests that it might be involved as an assembly factor or scaffold. In a breast cancer cell line, which is resistant to the anti-estrogen tamoxifen because of constitutively activated PKA, some interactions are constitutive and drug combinations partially rescue tamoxifen sensitivity. In ERα-positive breast cancer patients, high expression of the genes encoding some of these factors correlates with poor prognosis. Thus, these mechanisms might contribute to ERα-driven breast cancer.

Figures

Figure 1.
Figure 1.
The phosphorylation of LSD1 by PKA promotes its activation of ERα. (A) LSD1 is a substrate of PKA in vivo. MDA-MB-134 cells transfected with vectors for wild-type LSD1 or phosphothreonine/serine mutant T110A/S111A were stimulated with F/I for 2 h before immunoprecipitation (IP) and immunoblotting with indicated antibodies. NT, not transfected; F/I, two-compound mixture increasing cAMP levels. The asterisk points out which input extract was used in parallel for the control IP. (B and C) Overexpression of wild-type and phosphoserine mimics of LSD1 and CARM1 increases ERα activation by cAMP. Luciferase reporter assays in Hela cells with exogenously expressed ERα. The bar graphs show averages of three replicate experiments with triplicates, standardized to the values obtained with the control transfections with vector and treated with the solvent (DMSO), and the error bars represent the standard error of the mean.
Figure 2.
Figure 2.
LSD1 interacts with both ERα and components of the CoREST transactivation complex. (A) Endogenous ERα co-immunoprecipitates with overexpressed LSD1 and endogenous Hsp90β and CARM1. MDA-MB134 cells transfected to express Flag-tagged LSD1 were stimulated with F/I or E2 or a combination thereof for 2 h before cell lysis and immunoprecipitation with an antibody against ERα or a control antibody (IgG). coIP, co-immunoprecipitation; NI, non-induced. (B) LSD1 co-immunoprecipitates with both ERα and CoREST complex proteins. Co-immunoprecipitation experiment with an antibody against endogenous LSD1 or control IgG with extracts from MDA-MB-134 cells treated as indicated. (C) Co-immunoprecipitation experiment indicating constitutive interactions of ERα with LSD1 and other factors in OHT-resistant LCC2 cells. (D) Schematic representation of interactions. The arrows indicate both previously published and new interactions. Note that interactions assessed by co-immunoprecipitation may not be direct and that the exact arrangement of the hexagons is mostly arbitrary. (E) Analysis of interaction of LSD1 truncations with ERα. Indicated proteins were overexpressed together with ERα in 293T cells. FL, Flag tag; NFR, N-terminal flexible region; SWIRM, SWIRM domain; AOL, Amine-oxidase-like domain; Tower, Tower domain; C, C-terminal region. Co-immunoprecipitated proteins were displayed with an anti-Flag antibody.
Figure 3.
Figure 3.
The Hsp90-HDAC6-CoREST complexes are required for ERα transcriptional activity. (A-F) Luciferase reporter gene assays for ERα activity in transfected MDA-MB 134 cells with stable shRNA-mediated knockdowns of LSD1, CoREST, HDAC1, HDAC6, REST and Hsp90β, respectively. Two different shRNA sequences were used for each target. (G) Overexpression of LSD1 and HDACs increases cAMP-induced ERα transcriptional activity. MDA-MB-134 cells were transiently transfected and the activity of ERα was assessed with a luciferase reporter (left panel). The immunoblot on the right confirms the overexpression of the indicated proteins relative to the loading control GAPDH. These data points are averages of three experiments with triplicates and the error bars represent the standard error of the mean. Relevant statistically significant values are highlighted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 4.
Figure 4.
The core components of the ERα-Hsp90-HDAC6-CoREST complex are recruited to endogenous ERα target genes. (A-G) ChIP experiments with MDA-MB-134 cells stimulated with F/I or E2 for 45 min. The data points are averages of three independent experiments and the error bars represent the standard error of the mean.
Figure 5.
Figure 5.
The LSD1 demethylase and HDAC deacetylase activities are required to stimulate ERα-dependent transcription and breast cancer cell proliferation. (A and B) The inhibition of HDAC and LSD1 decreases ERα activation. Reporter gene assays with MDA-MB-134 cells treated with the pan-HDAC inhibitor trichostatin A (TSA) or the LSD1 inhibitor GSK-LSD1, respectively, were stimulated with F/I, E2 or OHT for 24 h. (C) HDAC inhibition decreases ERα binding to endogenous target genes. MDA-MB 134 cells were treated overnight with TSA before stimulation for 45 min and processing for a ChIP assay with an antibody against ERα. (D) Drug inhibition of Hsp90 and HDACs abolishes the assembly of the ERα transactivation complex. Co-immunoprecipation experiment with MDA-MB-134 cells were treated for 2 h with vehicle (DMSO) or the indicated inhibitors. GA, geldanamycin. (E) Drug inhibition of LSD1, HDAC and Hsp90 increases inhibitory effects of OHT in ER-positive OHT-resistant LCC2 breast cancer cells. The proliferation of LCC2 cells was measured by MTT assay after 96 h of culture. The data points are averages of three independent experiments made in triplicates and the error bars represent the standard error of the mean. Relevant statistically significant values are highlighted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 6.
Figure 6.
High expression levels of the gene set encoding LSD1-HDAC-Hsp90β correlate with poor breast cancer outcome. (A) Merged gene set used for the Kaplan–Meier survival analyses of different breast cancer types. The prognosis-linked gene expression data were obtained using the tool GOBO. (B) Relapse-free survival (RFS) analysis of patients with ERα-positive (ER+) versus ERα-negative (ER−) breast tumors. (C) RFS analysis of patients with positive lymph nodes (LN+), ERα-positive grade 3 tumors and tamoxifen-treated (TAM) tumors. (D) Analysis of overall survival (OS) of patients with ERα-positive tumors.
Figure 7.
Figure 7.
Comparative model of transcriptional activation of ERα by cognate ligand versus cAMP signaling in the absence of estrogen. Note that in the estrogen-dependent pathway the recruitment of CARM1 to ERα is indirect and mediated by Grip1 whereas upon cAMP signaling and phosphorylation of CARM1 and LSD1 by PKA, at least CARM1 is directly recruited to the unliganded ERα. This signaling crosstalk may favor OHT resistance by rendering some steps OHT-insensitive. Additional AF2-directed coactivators are likely to be involved.

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