Abstract
FOXA1 functions as a pioneer transcription factor by facilitating the access to chromatin for steroid hormone receptors, such as androgen receptor and estrogen receptor1,2,3,4, but mechanisms regulating its binding to chromatin remain elusive. LSD1 (KDM1A) acts as a transcriptional repressor by demethylating mono/dimethylated histone H3 lysine 4 (H3K4me1/2)5,6, but also acts as a steroid hormone receptor coactivator through mechanisms that are unclear. Here we show, in prostate cancer cells, that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 inhibition globally disrupts FOXA1 chromatin binding. Mechanistically, we demonstrate that LSD1 positively regulates FOXA1 binding by demethylating lysine 270, adjacent to the wing2 region of the FOXA1 DNA-binding domain. Acting through FOXA1, LSD1 inhibition broadly disrupted androgen-receptor binding and its transcriptional output, and dramatically decreased prostate cancer growth alone and in synergy with androgen-receptor antagonist treatment in vivo. These mechanistic insights suggest new therapeutic strategies in steroid-driven cancers.
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Data availability
All data generated or analyzed during this study are included in this published article. The GEO accession for ChIP–seq and RNA-seq data is GSE149007. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to C.C. (changmeng.cai@umb.edu). Source data are provided with this paper.
Code availability
This study did not generate any unpublished code, software or algorithm. All data analyses were performed using cited software with the parameters indicated in the Methods section.
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Acknowledgements
This work is supported by grants from NIH (R00 CA166507 and R01 CA211350 to C.C., U54 CA156734 to C.C. and J.A.M. and P01 CA163227 to S.P.B.), DOD (W81XWH-15-1-0554 and W81XWH-19-1-0777 to S.G., W81XWH-16-1-0445 and W81XWH-19-1-0361 to C.C.), CIHR (142246, 152863, 152864 and 159567 to H.H.H.), Prostate Cancer Canada (RS2016-1022 and TAG2018-2061 to H.H.H.), NSERC (498706 to H.H.H.), Terry Fox Program Project Grants (1093 to H.H.H.) and Princess Margaret Cancer Foundation (to H.H.H.). H.H.H. holds a Joey and Toby Tanenbaum Brazilian Ball Chair in Prostate Cancer Research. We thank J. Asara and M. Yuan from Beth Israel Deaconess Medical Center and J. Lee from Dana-Farber Cancer Institute for the work on mass spectrometry analyses.
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C.C., S.G., H.H.H., Sujun Chen and S.P.B. designed the study. S.G., Sujun Chen, D.H., Z.W., M. Li, W.H., A.B., M. Liu, F.Z., D.B., M.P.L., J.O., Y.L., M.A., J.P., E.C. and Sen Chen performed experiments and analyzed the results. S.P. and J.A.M. performed deep sequencing analyses. C.C., S.G., H.H.H., Sujun Chen and S.P.B. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Extended data
Extended Data Fig. 1 LSD1 promotes FOXA1 chromatin binding in PCa cells.
a, b, FOXA1 ChIP-seq was performed using an anti-FOXA1 antibody (Ab no. 1) in LNCaP cells treated with vehicle or 50 μM S2101 for 4 hours (h) or 48 h. (a) The heatmap view for FOXA1 ChIP-seq peak intensity and (b) the mean of FOXA1 ChIP-seq signals at FOXA1 binding sites (Veh vs S2101-4 h: P = 0.09; Veh vs S2101-48 h: P = 2.5 × 10-5) were shown. c, d, FOXA1 ChIP-seq was performed using an anti-FOXA1 antibody (Ab no. 2) in LNCaP cells treated with vehicle or GSK2879552 (50 μM, 4 h). (c) Heatmap view for FOXA1 ChIP-seq peak intensity and (d) the mean of FOXA1 ChIP-seq signals at FOXA1 binding sites (Veh vs GSK-4 h: P = 7.0 × 10−72) were shown. e, ChIP-qPCR for FOXA1 binding at KLK3/NKX3.1 enhancer site. f, ChIP-qPCR for FOXA1 binding in LNCaP cells treated with vehicle or LSD1 inhibitors for 4 h (S2101, OG-L002 at 50 μM and C12 at 5 μM). g, Immunoblotting for H3K4me2 in LNCaP or CWR22-RV1 cells treated with GSK2879552 at indicated doses for 48 h. h, Immunoblotting for FOXA1 or AR in LNCaP cells treated with 0-50 μM GSK2879552 for 48 h. i, Immunoblotting for FOXA1 or AR in LNCaP cells treated with GSK2879552 (50 μM, 0-48 h). j, Immunoblotting for FOXA1 or AR in CWR22-RV1 cells treated with GSK2879552 (0-5 μM, 48 h). k, Immunoblotting for FOXA1 or AR in CWR22-RV1 control cell line versus LSD1-KO cell line. l, m, LNCaP cells stably overexpressing doxycycline (dox)-regulated LSD1-WT or LSD1-K661A mutant treated with/out doxycycline were subjected to (l) immunoblotting or (m) ChIP-qPCR for FOXA1 binding at indicated sites. Note: Experiments described in this figure were all done under hormone-depleted conditions. We use NS (P > 0.05), ^0.01 < P < 0.05, *0.001 < P < 0.01, and #P < 0.001) to indicate the levels of P value in all Extended Data Figures.
Extended Data Fig. 2 LSD1 inhibition represses chromatin openning at FOXA1/AR mediated enhancers.
a, qPCR for DHS (DNA hypersensitivity) levels at indicated enhancer sites in LNCaP cells treated with S2101 for 4 h. b, H3K27ac ChIP-seq was performed in LNCaP cells treated with vehicle or GSK2879552 (50 μM, 4 h). The mean values of peak intensities for FOXA1 ChIP-seq (Veh vs GSK-4h: P = 0.03), H3K27ac ChIP-seq (Veh vs GSK-4h: P = 3.9 × 10−12), and ATAC-seq at AR-binding sites (Veh vs GSK-4 h: P = 1.4 × 10−15) were shown. c, ChIP-qPCR for H3K27ac at indicated sites in LNCaP cells treated with vehicle or GSK2879552 (50 μM, 4 h). d, ChIP-qPCR for H3K27ac at indicated sites in CWR22-RV1 cells treated with vehicle or S2101 (50 μM, 4 h). Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 3 LSD1 promotes FOXA1 binding at AR-independent regulatory sites.
a, The heatmap view for FOXA1 ChIP-seq peaks at non-AR binding sites in LNCaP cells. b, ChIP-qPCR for FOXA1 binding at indicated non-AR binding sites (FBS: FOXA1-solo Binding Sites) in LNCaP cells treated with GSK2879552 or S2101 (50 μM, 0-48 h). c, ChIP-qPCR for FOXA1 binding at indicated ER-regulated enhancers in MCF-7 cells treated with GSK2879552 (50 μM, 4 h).
Extended Data Fig. 4 LSD1 enhances AR chromatin binding and activity.
a, AR ChIP-seq analyses were performed in LNCaP cells treated with vehicle only, DHT (10 nM, 4 h), DHT (4 h) with pretreatment of GSK2879552 (50 μM, 0.5 h), or DHT (4 h) with pretreatment of GSK2879552 (50 μM, 48 h). Overlap of AR binding sites in vehicle and DHT treated cells was shown. b, qPCR for DHT-induced DHS (DNase HyperSensitivity) intensity at AR regulated enhancers in LNCaP cells pretreated with S2101 (50 μM, 0.5 h) and then treated with/without DHT (10 nM, 4 h). c, FOXA1, HOXB13, and AR ChIP-seq peaks as well as ATAC-seq peaks at KLK3 locus. d, Identification of the subset of directly AR-regulated genes by BETA (Binding and Expression Target Analysis) using AR ChIP-seq (DHT treated for 4 h) and RNA-seq in LNCaP cells (vehicle versus DHT treated for 24 h). e, RNA-seq analyses in LNCaP cells treated with/without DHT (10 nM, 24 h) and with/without pretreatment of GSK2879552 (50 μM, 24 h) were performed and DHT-induced fold-change of the expression for the identified directly AR-regulated genes was plotted [center: median; box: 25th to 75th interquartile range (IQR); whiskers: 1.5x IQR; outliers: individual data points]. f, RT-qPCR for PSA/NKX3.1 expression in LNCaP cells pretreated with LSD1 inhibitors for 4 h (S2101/OG-L002 at 50 μM and C12 at 5 μM) and then treated with/without DHT (10 nM, 24 h). g, ChIP-qPCR for AR binding in LNCaP stable cells expressing doxycycline-inducible LSD1-WT (LN-LSD1WT) or LSD1-K661A (LN-LSD1K661A). h, RT-qPCR for KLK3 and NKX3.1 mRNA expression in LNCaP cells stably overexpressing LSD1-WT or LSD1-K661A mutant treated with/without DHT (0-1 nM, 24 h). i, LN-LSD1WT cells treated with different doses of GSK2879552 (6 days) and with/without doxycycline were subjected to the measurement of cell density (mean±SD). j, ChIP-qPCR for FOXA1 binding at KLK3 enhancer in LNCaP cells treated with GSK2879552 (1 μM, 4 h). k, GSEA showing top-ranked pathways that were repressed or enhanced by GSK2879552 (1 μM for 2 weeks versus 50 μM for 48 h). Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 5 LSD1 enhances AR-V7 chromatin binding and activity.
a, ChIP-qPCR for FOXA1 binding at AR-regulated enhancers in LNCaP stable cells expressing doxycycline-inducible ARV7 (LNCaP-tetARV7) treated with/without GSK2879552 (50 μM, 4 h). b, Immunoblotting for V5 (AR-V7) in LNCaP-tetARV7 cells treated with vehicle, doxycycline only, doxycycline plus GSK2879552 (10 μM), doxycycline plus S2101 (10 μM), or doxycycline plus ORY-1001 (2.5 μM) for 48 h. c, RT-qPCR for the expression of AR-V7-regulated genes in CWR22-RV1 cells treated with GSK2879552 (0-2.5 μM for 48 h). d, Immunoblotting for AR-V7 in CWR22-RV1 cells treated with GSK2879552 (0-10 μM, 48 h). e, RT-qPCR for AR-FL/V7-regulated genes in the LSD1-KO line versus the control line with/without 10 nM DHT treatment. Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 6 Identification of methylated lysine 270 in FOXA1 as an LSD1 substrate.
a, FOXA1 immunoprecipitation was performed in LNCaP cells treated with different LSD1 inhibitors (S2101 at 50 μM and C12 at 5 μM), followed by immunoblotting for methyllysine (images cropped from the same blot). b, FOXA1 immunoprecipitation was performed in LN-LSD1WT or LN-LSD1K661A cells, followed by immunoblotting for methyllysine. c, Mass-spectrometry analysis on immunoprecipitated V5-FOXA1 in LNCaP cells stably overexpressing V5-tagged FOXA1. Covered residues are in yellow. Residues with detected post-translational modifications are indicated in green. d, LNCaP stable cells expressing doxycycline-inducible V5-tagged FOXA1-WT (LNCaP-tetFOXA1WT) or FOXA1-K270R (LNCaP-tetFOXA1K270R) were generated. V5-FOXA1 expression induced by doxycycline treatment (0-0.1 μg/ml) was confirmed by immunoblotting. e, ChIP-qPCR for FOXA1-WT or K270R binding (anti-V5) at AR-regulated enhancers in these stable cells (doxycycline supplemented) treated with S2101 (50 μM, 24 h). f, g, CWR22-RV1 cells stably expressing doxycycline-inducible FOXA1-WT (CWR22RV1-tetFOXA1WT) or K270R mutant (CWR22RV1-tetFOXA1K270R) were established. Immunoblotting for LSD1 in those stable cells (doxycycline supplemented) transfected with siRNA against LSD1 (siLSD1) or non-target control (siNTC) (f) and ChIP-qPCR for V5-FOXA1 binding (g) were performed. Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 7 K270R mutation of FOXA1 enhances the chromatin binding of FOXA1 and subsequently stabilizes AR recruitment.
a, LNCaP-tetFOXA1WT cells treated with GSK2879552 (50 μM) were fractionated into the soluble nuclear fraction and the insoluble chromatin-bound fraction, followed by immunoblotting for V5 and histone 3 (H3). b, CWR22RV1-tetFOXA1WT or CWR22RV1-tetFOXA1K270R cells were fractionated into the soluble nuclear fraction and the insoluble chromatin-bound fraction, followed by immunoblotting for V5 and H3. c, LNCaP-tetFOXA1WT or LNCaP-tetFOXA1K270R cells were treated with/without doxycycline for 48 h, and DHT or vehicle for 4 h. ChIP-qPCR for AR binding at AR-regulated enhancers was shown. d, ChIP-qPCR for AR binding in LNCaP-tetFOXA1WT or LNCaP-tetFOXA1K270R cells treated with DHT (10 nM) versus DHT plus enzalutamide (10 μM). e, RT-qPCR for androgen-induced gene expression in response to enzalutamide treatment in CWR22RV1-tetFOXA1WT or CWR22RV1-tetFOXA1K270R cells (in the presence of 10 nM DHT). Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 8 LSD1 inhibition impairs FOXA2 binding in PC-3 cells.
a, The amino acid sequences at the wing2 region of FOXA1 and FOXA2 were aligned. The red arrow indicates the K270 of FOXA1 and K265 of FOXA2. b, Immunoblotting for FOXA2 in the indicated PCa cell lines. c, d, FOXA2 ChIP-seq was performed in PC-3 cells treated with vehicle or GSK2879552 (50 μM, 4 h). (c) Heatmap view for FOXA2 ChIP-seq peak intensity and (d) the mean of FOXA2 ChIP-seq signals at FOXA2 binding sites (Veh vs GSK-4 h: P = 1.6 × 10−6) were shown. e, HOXB13 ChIP-seq was performed in LNCaP cells treated with vehicle or GSK2879552 (50 μM, 4 h). The heatmap view for HOXB13 ChIP-seq peak intensity was shown. f, ChIP-qPCR for HOXB13 binding at indicated enhancer sites in LNCaP cells treated with/without GSK2879552 (50 μM, 4 h). Note: Experiments described in this figure were all done under hormone-depleted conditions.
Extended Data Fig. 9 LSD1 inhibition suppresses tumor growth in the CWR22-RV1 CRPC model.
a, The correlation between LSD1 and FOXA1 mRNA expression levels in TCGA PCa cohort. b, LSD1 and FOXA1 expressions in normal prostate versus PCa tumors using TCGA PCa dataset (center: median; box: 25th to 75th IQR; whiskers: 1.5x IQR; outliers: individual data points). c, FOXA1 mRNA expression levels in cell lines derived from different diseases using data from cancer cell line encyclopedia, CCLE (center: median; box: 25th to 75th IQR; whiskers: 1.5x IQR; outliers: individual data points). d, CWR22-RV1 cells (under hormone depleted condition) were treated with different doses of GSK2879552, and cell density was measured after 2 days of treatments (mean±SD). e, Castrated SCID male mice bearing CWR22-RV1 xenograft tumors received DMSO or GSK2879552 treatment and the body weight was measured at the endpoint. f, Immunoblotting for H3K4me2 in the vehicle versus GSK2879552 treated group.
Extended Data Fig. 10 LSD1 inhibition suppresses tumor growth of FOXA1-positive CRPC patient-derived xenograft models.
a, The mRNA expression of AR-V7 was examined in tumor samples from vehicle-treated xenograft tumors in comparison with C4-2 (AR-V7 negative) derived xenograft tumors. b, c, LuCaP35CR tumors were established in castrated male SCID mice and treated with LSD1 inhibitors. Immunoblotting for H3K4me2 in LuCaP35CR treated with vehicle versus GSK2879552 (b) or vehicle versus ORY-1001 (c). d, Bodyweight for mice bearing LuCaP35CR treated with vehicle versus ORY-1001. e, f, Castrated SCID male mice bearing LuCaP35CR xenograft tumors received daily DMSO or ORY-1001 (0.06 mg/kg) via intraperitoneal injection (n = 7 independent tumors). (e) The tumor volume was measured at the indicated time. (f) After the mice were sacrificed, tumor samples were subjected to RT-PCR analysis for indicated AR-FL/V7 regulated genes. g, LuCaP77CR tumors were established in castrated male SCID mice and treated with LSD1 inhibitors. Immunoblotting for H3K4me2 in LuCaP77CR tumors treated with vehicle versus GSK2879552. h, LNCaP and DU145 cell lines were treated with 0-200 μM GSK2879552 for 4 days and the cell density was measured (mean±SD). i, CWR22-RV1 cells (under hormone depleted condition) were treated with DMSO, GSK2879552 (5 μM), enzalutamide (10 μM), or combination of GSK2879552 and enzalutamide for 2 days, and then cell density was measured.
Supplementary information
Supplementary Table 1
List of oligonucleotide sequences
Source data
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Gao, S., Chen, S., Han, D. et al. Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer. Nat Genet 52, 1011–1017 (2020). https://doi.org/10.1038/s41588-020-0681-7
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DOI: https://doi.org/10.1038/s41588-020-0681-7
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