Research ArticleCancer

PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor–positive breast cancer

See allHide authors and affiliations

Science Translational Medicine  15 Apr 2015:
Vol. 7, Issue 283, pp. 283ra51
DOI: 10.1126/scitranslmed.aaa4442
  • Fig. 1. PI3K inhibition promotes ER function.

    (A) MCF7 cells were treated with BYL719 (1 μM) over a period of 48 hours. RNA was isolated at specified time points, and expression microarray analysis was performed. Heat map represents genes whose expression differed significantly across different time points with an FDR ≤1%. Each of the columns under the experimental conditions represents one biological replicate. (B) PI3Kα inhibition leads to modulation of genes containing ER binding sites (ERE). MCF7 cells were treated with BYL719 (1 μM), and gene expression analysis was performed as described in (A). The diagram represents the genes that were differentially regulated upon treatment across all the time points [Significance Analysis of Microarrays (SAM) analysis FDR ≤ 1%] and the percentage of these genes that contained an ER-binding element [defined by ER ChIP (chromatin immunoprecipitation) sequencing (40)]. (C) GSEA was performed to determine which gene sets were enriched in our data set (FDR ≤ 25%). Graph represents enrichment for ER-associated signature as described in (41). ES, enrichment score; NES, normalized enrichment score. (D) MCF7 cells were transfected with 3X-ERE-TATA firefly-luciferase and pRL-TK Renilla luciferase plasmids and treated with vehicle (Ctrl) or BYL719 (BYL) (1 μM) for 16 hours. Results represent firefly luciferase activity measured by luminescence and normalized both to Renilla luciferase luminescence for transfection efficiency and to Ctrl. Two-tailed Student’s unpaired t test was performed to compare Ctrl versus BYL719-treated cells. (E) MCF7 cells were treated with BYL719 (1 μM) over a period of 48 hours, and RNA was isolated at the indicated times. qPCR was performed to detect βACTIN, PGR, GREB1, and IGFBP4 gene expression. The data are presented relative to βACTIN and to expression in vehicle-treated cells (Ctrl). One-way analysis of variance (ANOVA) statistical test was used to compare gene expression between each time point and vehicle-treated cells, applying the Bonferroni method to correct for multiple comparisons. Error bars denote SEM of at least two biological replicates, each with three technical replicates. (F) MCF7 cells were treated with BYL719 (1 μM) or vehicle (Ctrl), and ChIP was performed with anti-ERα antibody or control immunoglobulin G (IgG). Primers to amplify the ER-binding regions of the PGR, GREB1, and IGFBP4 promoters were used in qPCR to determine fold enrichment relative to a noncoding region. Two-tailed Student’s unpaired t test was performed to compare Ctrl versus BYL719-treated cells. Error bars represent SEM of three independent experiments.

  • Fig. 2. PI3K inhibition induces ER expression.

    (A) MCF7 cells were treated with BYL719 (1 μM) over a 48-hour period, and RNA was isolated at the indicated times. qPCR was performed to detect βACTIN and ESR1 expression. The data are presented relative to βACTIN and to expression of ESR1 in vehicle-treated control (Ctrl). One-way ANOVA statistical test was used to compare gene expression between each time point and to vehicle-treated cells, applying the Bonferroni method to correct for multiple comparisons. Error bars denote SEM of at least two biological replicates, each with three technical replicates. (B) MCF7 cells were treated with BYL719 (1 μM) over a period of 48 hours, and total protein was isolated at the indicated times. Immunoblotting was performed to detect expression of ER, phosphorylation of AKT at Ser473 (pAKT S473), and βACTIN. Graph represents the fold change of total ERα with respect to βACTIN and to untreated samples (Ctrl) of two independent experiments. Statistical analysis was done using the one-way ANOVA statistical test with the Bonferroni method to correct for multiple comparisons. (C) 18F-FES uptake in T47D xenograft mouse models treated with vehicle or BYL719 daily. The uptake was measured after a 4-day treatment, 2 hours after the last dose, and is represented as % injected dose per gram of tumor tissue (% ID/g). Statistical analysis to compare 18F-FES uptake between the Ctrl and the BYL719-treated mice was performed by means of a nonparametric Kruskal-Wallis test. (D) Graphical representation of ESR1 transcript abundance in 20 paired breast cancer biopsies before (PRE) and on BYL719 treatment (ON) collected as part of two clinical trials with the p110α inhibitor BYL719. ESR1 was one of the 105 breast cancer–specific genes analyzed using the nCounter platform. (E) Graphical representation of the induction of a luminal A signature upon BYL719 treatment in the tumor samples used in (D). (F) MCF7 cells were treated with vehicle or BYL719 (1 μM) for 2 hours. ChIP was performed with anti-FOXO3A antibody or control IgG. Primers to amplify the FOXO3A-binding regions of the ESR1 promoter were used in qPCR to determine fold enrichment relative to input. Two-tailed Student’s unpaired t test was performed to compare mean signal amplification between vehicle- and BYL719-treated samples. Error bars represent SEM of two independent experiments with three technical replicates each. (G) MCF7 cells were transfected with nontargeted siRNA (Ctrl) or FOXO3A siRNA. Forty-eight hours later, cells were treated with vehicle or BYL719 (1 μM) for 24 hours. mRNA was isolated, and qPCR was performed to detect βACTIN, FOXO3A, and ESR1 expression. The data are presented relative to βACTIN and to expression in the samples treated with Ctrl siRNA and vehicle. One-way ANOVA statistical test was used to compare gene expression between each condition and Ctrl siRNA and vehicle-treated cells, applying the Bonferroni method to correct for multiple comparisons. Error bars denote SEM of two independent experiments with three technical replicates each.

  • Fig. 3. PI3K inhibitor–mediated induction of hormone signaling is dependent on E2 and ER.

    MCF7 cells were treated with BYL719 (1 μM), E2 (10 nM), or the combination after 48 hours in estrogen-free medium. ChIP was performed with anti-ERα antibody or control IgG. (A and B) Primers to amplify the ER-binding regions of the PGR (A) and GREB1 (B) promoters were used in qPCR to determine fold enrichment relative to input. One-way ANOVA statistical test was used to compare mean signal amplification between each treatment and vehicle-treated samples, applying the Bonferroni method to correct for multiple comparisons. Error bars represent SEM of two independent experiments with three technical replicates each. (C) MCF7 cells were preincubated for 48 hours in steroid hormone–depleted medium and subsequently treated with BYL719 (1 μM), E2 (10 nM), or the combination over a period of 16 hours, and RNA was isolated at the indicated times. qPCR was performed to detect βACTIN, PGR, and GREB1 expression. The data are presented relative to βACTIN and to expression at time 0 in the BYL719-treated samples. One-way ANOVA statistical test was used to compare gene expression between each treatment and vehicle-treated cells, applying the Bonferroni method to correct for multiple comparisons. The results presented are for the comparisons at the 16-hour time point. Error bars denote SEM of two independent experiments with three technical replicates each. (D) MCF7 cells, grown under normal serum conditions, were treated with BYL719 (1 μM), alone or in combination with 4-OHT (1 μM) or fulvestrant (FULV) (100 nM) over a period of 16 hours. mRNA was isolated at the indicated times. qPCR was performed to detect βACTIN, PGR, and GREB1 expression. The data are presented relative to βACTIN and to expression at time 0 in the vehicle-treated samples. One-way ANOVA statistical test was used to compare gene expression between each treatment and vehicle-treated cells, applying the Bonferroni method to correct for multiple comparisons. The analysis results presented are for the comparisons at the 16-hour time point. Error bars denote SEM of two independent experiments with three technical replicates each.

  • Fig. 4. Combination of BYL719 and fulvestrant in vivo induces prolonged responses.

    (A) MCF7 in vivo xenograft was treated with vehicle, BYL719, fulvestrant, or the combination at the indicated doses and schedule. Graph shows the fold change in tumor volume with respect to day 0 of treatment. One-way ANOVA statistical test was performed to compare tumor volume fold change on the last day of treatment between each treatment arm and vehicle, applying the Bonferroni method to correct for multiple comparisons. Error bars represent SEM. (B) ER-positive/PIK3CAmut PDX-bearing mice were randomized to receive treatment with indicated doses and schedules of vehicle, BYL719, and/or fulvestrant. Graph shows the fold change in tumor volume with respect to day 0 of treatment. One-way ANOVA statistical test was performed to compare tumor volume fold change on the last day of treatment between each treatment arm and vehicle, applying the Bonferroni method to correct for multiple comparisons. Error bars represent SEM. (C) Pharmacodynamic study of MCF7 mouse xenograft. Mice were treated with vehicle, BYL719, fulvestrant, or the combination with the same dosing and schedule as in (A) for 4 days. Animals were sacrificed 2 hours after the last dose, and tumors were processed for immunohistochemistry and stained with the indicated antibodies. The figure shows representative images for each of the treatment arms. Scale bars, 50 μm. (D) A parallel pharmacodynamic study was performed with the ER-positive/PIK3CAmut PDX mice, which were treated with either vehicle or BYL719 with the same dosing and schedule as described in (B). Mice were sacrificed, and tumors were obtained on day 4, 2 hours after the last dose, and processed to obtain RNA for microarray gene expression analysis. Graph represents genes whose expression differed significantly across different treatments with an FDR ≤1%. Each of the columns under the experimental conditions represents one biological replicate. Venn diagram represents differentially regulated genes upon treatment with BYL719 (SAM analysis FDR ≤ 1%) and the percentage of these that contained an ER-binding site, defined by ER ChIP-sequencing data available from (40). (E) GSEA analysis was performed to determine which gene sets were enriched in the PDX microarray expression data set obtained in (D). Graph represents enrichment for ER-associated signature (FDR ≤ 25%) as described in (42). (F) Pharmacodynamic studies on the MCF7 xenografts from (A) were performed on day 7 of treatment by means of a punch biopsy in both vehicle- and BYL719-treated mice. A representative number of biopsies (at least two biological replicates per condition) was processed to obtain RNA and submitted for gene expression analysis. GSEA was performed to determine which gene sets were enriched in our data set (FDR ≤ 25%). Graph represents enrichment for ER-associated signature as described in (42).

  • Table 1. Differentially expressed genes upon BYL719 treatment in patients with metastatic breast cancer.

    Paired biopsies of tumors from ER-positive breast cancer patients receiving BYL719 as part of a clinical trial were collected before and during treatment. RNA was extracted, and the expression of 105 breast cancer–specific genes was analyzed using the nCounter platform. Differentially expressed genes (FDR ≤ 25%) between on-treatment and pretreatment biopsies are shown.

    Gene IDScore (d)Fold changeq (%)
    GRB72.1771.2730.000
    BCL22.1501.3630.000
    MDM22.0461.2770.000
    CXXC51.7701.27711.478
    PGR1.5601.35914.667
    ESR11.4521.55414.667
    ACTR3B1.3901.17614.667
    SFRP11.2891.26617.742
    ERBB21.2091.14417.742
    SLC39A61.0341.14019.556
    PHGDH0.9991.25319.556
    FOXA10.9081.10319.556
    FGFR40.8901.26919.556
    GPR1600.8741.13419.556
    FOXC10.6531.15722.564
    MLPH0.6411.08222.564
    MAPT0.6221.12422.564
    BIRC5−2.009−1.21911.478
    MYBL2−1.946−1.22611.478
    EXO1−1.544−1.19011.478
    CENPF−1.424−1.20211.478
    CEP55−1.280−1.17211.478
    TYMS−1.276−1.13011.478
    RRM2−1.265−1.20811.478
    CDH3−1.261−1.31711.478
    MKI67−1.227−1.14811.478
    MELK−1.164−1.14911.478
    CCNE1−1.155−1.12111.478
    UBE2T−1.137−1.13111.478
    KIF2C−1.111−1.16411.478
    KNTC2−1.032−1.10411.478
    CDCA1−0.997−1.13011.478
    ORC6L−0.988−1.13611.478
    CDC6−0.924−1.14311.478
    CDC20−0.886−1.10611.478
    CCNB1−0.871−1.09311.478
    PTTG1−0.759−1.09814.667
    ANLN−0.673−1.09617.742
    MMP11−0.619−1.11417.742

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/283/283ra51/DC1

    Materials and Methods

    Fig. S1. Western blot of MCF7 and T47D cells treated in vitro with BYL719 for a series of time points.

    Fig. S2. T47D transcriptional profile upon p110α inhibition.

    Fig. S3. GSEA for T47D microarray expression data set.

    Fig. S4. Western blot of CAMA1 cells treated with BYL719 or MK2206 for 48 hours.

    Fig. S5. CAMA1 transcriptional profile after AKT inhibition.

    Fig. S6. ER target genes induced by AKT inhibition in ER-positive/PTENmut/null breast cancer cells.

    Fig. S7. ESR1 expression induced by PI3Kα inhibition in ER-positive/PIK3CAmut breast cancer cells.

    Fig. S8. ESR1 transcription increased by PI3Kα inhibition.

    Fig. S9. Induction of ESR1 and its target genes by different PI3K inhibitors.

    Fig. S10. Comparison of induction of ESR1 and its target genes between BYL719 and the mTORC1 allosteric inhibitor rapamycin.

    Fig. S11. Decreased expression of ER target genes after anti-ER therapy, with no effect on ESR1 mRNA.

    Fig. S12. Up-regulation of ER target genes reversed by combining BYL719 with anti-ER treatment.

    Fig. S13. Better tumor control in vivo after combining BYL719 with fulvestrant.

    Fig. S14. Analysis of the effect of PI3Kα inhibition alone or with anti-ER therapy on the cell cycle.

    Table S1. GSEA to assess ER-dependent signatures enriched in MCF7 cells treated with BYL719.

    Table S2. GSEA to assess ER-dependent signatures enriched in CAMA1 cells treated with MK2206.

    Table S3. Clinical and pathologic features corresponding to paired pretreatment and BYL719-treated tumor samples.

    Table S4. Raw data (provided as an Excel file).

    References (4345)

  • Supplementary Material for:

    PI3K inhibition results in enhanced estrogen receptor function and dependence in hormone receptor–positive breast cancer

    Ana Bosch, Zhiqiang Li, Anna Bergamaschi, Haley Ellis, Eneda Toska, Aleix Prat, Jessica J. Tao, Daniel E. Spratt, Nerissa T. Viola-Villegas, Pau Castel, Gerard Minuesa, Natasha Morse, Jordi Rodón, Yasir Ibrahim, Javier Cortes, Jose Perez-Garcia, Patricia Galvan, Judit Grueso, Marta Guzman, John A. Katzenellenbogen, Michael Kharas, Jason S. Lewis, Maura Dickler, Violeta Serra, Neal Rosen, Sarat Chandarlapaty,* Maurizio Scaltriti,* José Baselga*

    *Corresponding author. E-mail: chandars{at}mskcc.org (S.C.); scaltrim{at}mskcc.org (M.S.); baselgaj{at}mskcc.org (J.B.)

    Published 15 April 2015, Sci. Transl. Med. 7, 283ra51 (2015)
    DOI: 10.1126/scitranslmed.aaa4442

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Western blot of MCF7 and T47D cells treated in vitro with BYL719 for a series of time points.
    • Fig. S2. T47D transcriptional profile upon p110α inhibition.
    • Fig. S3. GSEA for T47D microarray expression data set.
    • Fig. S4. Western blot of CAMA1 cells treated with BYL719 or MK2206 for 48 hours.
    • Fig. S5. CAMA1 transcriptional profile after AKT inhibition.
    • Fig. S6. ER target genes induced by AKT inhibition in ER-positive/PTENmut/null breast cancer cells.
    • Fig. S7. ESR1 expression induced by PI3Kα inhibition in ER-positive/PIK3CAmut breast cancer cells.
    • Fig. S8. ESR1 transcription increased by PI3Kα inhibition.
    • Fig. S9. Induction of ESR1 and its target genes by different PI3K inhibitors.
    • Fig. S10. Comparison of induction of ESR1 and its target genes between BYL719 and the mTORC1 allosteric inhibitor rapamycin.
    • Fig. S11. Decreased expression of ER target genes after anti-ER therapy, with no effect on ESR1 mRNA.
    • Fig. S12. Up-regulation of ER target genes reversed by combining BYL719 with anti-ER treatment.
    • Fig. S13. Better tumor control in vivo after combining BYL719 with fulvestrant.
    • Fig. S14. Analysis of the effect of PI3Kα inhibition alone or with anti-ER therapy on the cell cycle.
    • Table S1. GSEA to assess ER-dependent signatures enriched in MCF7 cells treated with BYL719.
    • Table S2. GSEA to assess ER-dependent signatures enriched in CAMA1 cells treated with MK2206.
    • Table S3. Clinical and pathologic features corresponding to paired pretreatment and BYL719-treated tumor samples.
    • References (4345)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Raw data (provided as an Excel file).

    [Download Table S4]

Stay Connected to Science Translational Medicine

Navigate This Article