doi: 10.1126/science.aah6893.
PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D
Affiliations
- PMID:28336670
- PMCID: PMC5485411
- DOI: 10.1126/science.aah6893
Item in Clipboard
PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D
Eneda Toska et al. Science..
Display options
Format
Display options
Format
Erratum in
- Erratum for the Report "PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D" by E. Toska, H. U. Osmanbeyoglu, P. Castel, C. Chan, R. C. Hendrickson, M. Elkabets, M. N. Dickler, M. Scaltriti, C. S. Leslie, S. A. Armstrong, J. Baselga.[No authors listed][No authors listed]Science. 2019 Jan 25;363(6425):eaaw7574. doi: 10.1126/science.aaw7574.Science. 2019.PMID:30679344No abstract available.
Abstract
Activating mutations inPIK3CA, the gene encoding phosphoinositide-(3)-kinase α (PI3Kα), are frequently found in estrogen receptor (ER)-positive breast cancer. PI3Kα inhibitors, now in late-stage clinical development, elicit a robust compensatory increase in ER-dependent transcription that limits therapeutic efficacy. We investigated the chromatin-based mechanisms leading to the activation of ER upon PI3Kα inhibition. We found that PI3Kα inhibition mediates an open chromatin state at the ER target loci in breast cancer models and clinical samples. KMT2D, a histone H3 lysine 4 methyltransferase, is required for FOXA1, PBX1, and ER recruitment and activation. AKT binds and phosphorylates KMT2D, attenuating methyltransferase activity and ER function, whereas PI3Kα inhibition enhances KMT2D activity. These findings uncover a mechanism that controls the activation of ER by the posttranslational modification of epigenetic regulators, providing a rationale for epigenetic therapy in ER-positive breast cancer.
Copyright © 2017, American Association for the Advancement of Science.
Figures
(A andB) Volcano plot of ER (A) and FOXA1 (B) ChIP-seq for T47D breast cancer cells treated with dimethyl sulfoxide (DMSO) or BYL719 (1 μM) for 24 hours. Thex axis shows the log fold change (logFC), and they axis shows -log10(P value). The red dots correspond to the ER- or FOXA1-binding sites that are differentially bound upon BYL719 treatment compared with DMSO treatment. Also shown are the top enriched motifs observed at the gained ER- or FOXA1-binding events upon BYL719 treatment. At the gained ER-binding events: ERE, estrogen-responsive element (P = 1 × 10–70); FOXA1, forkhead (P = 1 × 10–27); and homeobox motif (P = 1 × 10–20). At the gained FOXA1-binding events: FOXA1 (P = 1 × 10–260), nuclear receptor class (P = 1 × 10–59), and homeobox motif (P = 1 × 10–29). (C) Example of a binding region of a BYL719-induced ER- and FOXA1-binding event (shown in reads per million).(D) ChIP-qPCR for ER occupancy in the enhancer and promoter regions after FOXA1 was knocked down by two distinct shRNAs in T47D cells upon treatment with BYL719 (1 μM) for 24 hours. IgG, immunoglobulin G. Values are shown as fold enrichment (i.e., the ratio of the mean percentage of input enrichment of the candidate gene to the mean percentage of input enrichment of a control gene). Error bars, SD (n = 3). *P < 0.05, **P < 0.01; Student'st test.(E) mRNA levels measured by reverse transcription (RT)-qPCR in T47D breast cancer cells maintained in estrogen-free media for 3 days, followed by treatment with DMSO, E2 (estrogen; 100 μm), BYL719 (1 μM), or E2 plus BYL719 for 24 hours. Error bars, SD (n = 3). *P < 0.05, **P < 0.01; Student'st test.(F) MCF7 shFOXA1 doxycycline (DOX)-inducible in vivo xenograft treated daily with vehicle or BYL719 (25 mg/kg) (n = 10 per group). The indicated P values were calculated using Student'st test. Error bars, ±SEM.(G) Western blot analysis of lysates of tumors collected at the end of the experiment, 4 hours after the last dose.(H) Tissue ChIP-qPCR assay for ER occupancy in the candidate target genes of randomly collected tumors at the end of the experiment.P values were calculated using Student'st test. Error bars, ±SEM.
(A) Volcano plot of ATAC-seq from T47D breast cancer cells treated with DMSO or BYL719 (1 μM) for 24 hours. The red dots correspond to the accessible sites upon BYL719 treatment.(B) Heat map of accessible sites gained upon BYL719 treatment, shown in a horizontal window of ±3 kb from the peak center. (C) The top enriched motifs of the accessible sites upon BYL719 treatment: ERE (P = 1 × 10–11); FOXA1 (P = 1 × 10–205); and PBX1, homeobox (P = 1 × 10–144). (D) Examples of ChIP-seq ER- and FOXA1-binding regions and ATAC-seq open chromatin regions in T47D breast cancer cells and tumor samples from breast cancer patients treated with BYL719 (in reads per million). The patient samples were collected before the commencement of treatment with BYL719 (“Pre”) and after ∼14 days of treatment (“On”). The latter samples were collected between 2 and 6 hours after administration of the drug.(E) The enriched motifs of the accessible sites gained upon BYL719 treatment. Patient 1 enriched motifs: Esrra, nuclear receptor class (P = 1 × 10–15); FOXA1 (P = 1 × 10–22); and PBX1, homeobox (P = 1 × 10–24). Patient 2 enriched motifs: ERE (P = 1 × 10–6); forkhead class (P = 1 × 10–7), and PBX1, homeobox (P = 1 × 10–5).
(A) ChlP-qPCR assay of ER, F0XA1, PBX1, and control IgG binding at the candidate target genes, after KMT2D was knocked down by two distinct shRNAs, in T47D breast cancer cells upon treatment with BYL719 (1 μM) for 24 hours. GFP, green fluorescent protein. Error bars, SD (n = 3). *P < 0.05, **P < 0.01; Student'st test. (B) RT-qPCR in estrogen-depleted T47D cells for 3 days, followed by treatment with DMSO, E2 (100 nM), BYL719 (1 μM), or E2 plus BYL719 for 24 hours. Values are the average of 3 replicates. *P < 0.05, **P < 0.01; Student'st test. (C) MCF7 shKMT2D doxycycline-inducible in vivo xenograft treated daily with vehicle or BYL719 (25 mg/kg) (n = 10 per group). The indicatedP values were calculated using Student'st test. Error bars, ±SEM. (D) Western blot analysis of lysates of tumors collected at the end of the experiment. RFP, red fluorescent protein. (E) Tissue ChlP-qPCR analysis of ER, F0XA1, and PBX1 binding at the candidate target genes of tumors collected at the end of the experiment, 4 hours after the last BYL719 dose.P values were calculated using Student'st test. Error bars, ±SEM. (F) ChlP-qPCR for KMT2D, H3K4mel, H3K4me2, and IgG control in T47D breast cancer cells treated with BYL719 (1 μM) for 2,4,8,12, and 24 hours. Error bars, SD (n = 3). *P < 0.05, **P < 0.01; Student'st test. (G) H3K4 methyltransferase activity of T47D nuclear extracts after the cells were treated with BYL719 (1 μM) for 4,8, and 24 hours.The substrate was a synthetic H3 peptide, and the activity was measured using a H3K4 methyltransferase kit. Error bars, SD (n = 3). *P < 0.05; Student'st test. (H) Immunoblot analysis of H3K4mel, H3K4me2, H3, and actin in T47D cells after treatment with BYL719 (1 μM) for 2, 4, 8, and 24 hours.
In silico analysis of AGC phosphorylation motifs RXRXX(S/T) ‘R, arginine; X, any amino acid; (S/T), phosphorylatable serine or threonine’. Rl and R3 are highlighted in blue, and phosphorylatable S is indicated by the black triangle. Alignment was performed with CustalW2, using KMT2D protein sequences from chimpanzee (Pan troglodytes), mouse (Mus musculus), frog (Xenopus laevis), and zebrafish (Danio rerio). G, glycine; A, alanine; L, leucine; K, lysine. (B) Co-immunoprecipitation (IP) assay in 293Tcells transfected with the indicated plasmids and probed with HA and V5 antibodies. WCL, whole cell lysate.(C) Coimmunoprecipitation of endogenous KMT2D and AKT in T47D breast cancer cells and immunoblotted with KMT2D and AKT antibodies. (D) Schematic representation of different truncated fragments of KMT2D used for coimmunoprecipitation assays (FL, full length). Also shown are the key domains of the KMT2D protein: PHD, plant homeodomain; HMG, high mobility group; FYRN, FY-rich N-terminal; FYRC, FY-rich C-terminal; SET, Su(var)3-9, enhancer-of-zeste, trithorax domain; LXXLL, nuclear receptor recognition motif.(E) Coimmunoprecipitation assays in 293T cells transfected with yellow fluorescent protein (YFP)-AKTl and each of the indicated V5-tagged KMT2D fragments.(F) In vitro kinase assay using recombinant His-AKTand wild-type (WT) KMT2D or S1331A KMT2D immunoprecipitated from 293Tcells as a substrate, treated with MK2206 (2 (μM) for 1 hour. EV, empty vector.(G) H3K4 methyltransferase activity of IgG control, WT, S1331A, and S1331D KMT2D immunoprecipitated from 293Tcells. The substrate was a synthetic H3 substrate, and the activity was measured using a H3K4 methyltransferase kit. D, aspartic acid. Error bars, SD (n = 3). *P < 0.05, **P < 0.01; Student'st test.(H) Immunoblot analysis of the indicated histone methylation marks in 293Tcells transfected with control, WT, S1331A, and S1331D KMT2D vectors.(I) ChlP-qPCR of ER, F0XA1, PBX1, and IgG control binding in ER target genes in T47D cells expressing WT, S1331A, and S1331D KMT2D. Error bars, SD (n = 3). *P < 0.05; Student'st test.
Comment in
- Tackling Resistance to PI3K Inhibition by Targeting the Epigenome.Koren S, Bentires-Alj M.Koren S, et al.Cancer Cell. 2017 May 8;31(5):616-618. doi: 10.1016/j.ccell.2017.04.010.Cancer Cell. 2017.PMID:28486103
References
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Miscellaneous