Limits...
Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity.

Simões BM, O'Brien CS, Eyre R, Silva A, Yu L, Sarmiento-Castro A, Alférez DG, Spence K, Santiago-Gómez A, Chemi F, Acar A, Gandhi A, Howell A, Brennan K, Rydén L, Catalano S, Andó S, Gee J, Ucar A, Sims AH, Marangoni E, Farnie G, Landberg G, Howell SJ, Clarke RB - Cell Rep (2015)

Bottom Line: Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors.In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts.Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Now Research Unit, Institute of Cancer Sciences, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

No MeSH data available.


Related in: MedlinePlus

Tamoxifen or Fulvestrant Treatment Upregulates Notch Target Genes in Patient-Derived Samples and PDXsJAG1-NOTCH4 receptor signaling in ALDH-positive cells drives Notch activity in endocrine-resistant BC.(A and B) Expression of Notch target genes HEY1 and HES1 was assessed by real-time qPCR analysis and compared to control to determine fold change. (A) Metastatic BC patient-derived cells were treated for 7–9 days with ethanol (control), tamoxifen (10−6 M), or fulvestrant (10−7 M) and a correlation between fold change of expression of HEY1 and HES1 and fold change of percentage of ALDH-positive cells is shown. (B) Early (HBCx34) and metastatic (BB3RC31) BC PDXs: the effect of in vivo treatment for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) on HEY1 and HES1.(C) NOTCH4, HES1, and JAG1 protein expression levels determined by western blot in metastatic (Met) (BB3RC31) BC PDX. β-actin was used as a reference for the loading control.(D) NOTCH4, HES1, and JAG1 protein expression levels were determined by western blot in MCF-7 ALDH-negative and ALDH-positive sorted cells. MCF-7 cells were treated with tamoxifen or fulvestrant for 6 days before ALDH sorting.(E) Wild-type MCF-7 cells (filled bars) and a CRISPR clone containing a disruption of NOTCH4 exon 2 (N4EX2 cells, hatched bars) treated in adherence with ethanol (Control, gray bars), 10−6 M tamoxifen (red bars), and 10−7 M fulvestrant (blue bars) for 6 days. N4EX2 cells’ fold change of MFE and ALDH-positive cells after treatments was compared to that of the wild-type cells.(F and G) NICD4 and JAG1 rescue tamoxifen- or fulvestrant-inhibited growth: cell number (using sulforhodamine B [SRB] assay, y axis) of MCF-7 overexpressing (F) NICD4-GFP, (G) JAG1-GFP, or GFP control incubated with tamoxifen or fulvestrant for 1, 3, and 5 days (x axis) compared to the respective cell line treated with control ethanol. p values are for the 5-day treatment.Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figures S2 and S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4594158&req=5

fig2: Tamoxifen or Fulvestrant Treatment Upregulates Notch Target Genes in Patient-Derived Samples and PDXsJAG1-NOTCH4 receptor signaling in ALDH-positive cells drives Notch activity in endocrine-resistant BC.(A and B) Expression of Notch target genes HEY1 and HES1 was assessed by real-time qPCR analysis and compared to control to determine fold change. (A) Metastatic BC patient-derived cells were treated for 7–9 days with ethanol (control), tamoxifen (10−6 M), or fulvestrant (10−7 M) and a correlation between fold change of expression of HEY1 and HES1 and fold change of percentage of ALDH-positive cells is shown. (B) Early (HBCx34) and metastatic (BB3RC31) BC PDXs: the effect of in vivo treatment for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) on HEY1 and HES1.(C) NOTCH4, HES1, and JAG1 protein expression levels determined by western blot in metastatic (Met) (BB3RC31) BC PDX. β-actin was used as a reference for the loading control.(D) NOTCH4, HES1, and JAG1 protein expression levels were determined by western blot in MCF-7 ALDH-negative and ALDH-positive sorted cells. MCF-7 cells were treated with tamoxifen or fulvestrant for 6 days before ALDH sorting.(E) Wild-type MCF-7 cells (filled bars) and a CRISPR clone containing a disruption of NOTCH4 exon 2 (N4EX2 cells, hatched bars) treated in adherence with ethanol (Control, gray bars), 10−6 M tamoxifen (red bars), and 10−7 M fulvestrant (blue bars) for 6 days. N4EX2 cells’ fold change of MFE and ALDH-positive cells after treatments was compared to that of the wild-type cells.(F and G) NICD4 and JAG1 rescue tamoxifen- or fulvestrant-inhibited growth: cell number (using sulforhodamine B [SRB] assay, y axis) of MCF-7 overexpressing (F) NICD4-GFP, (G) JAG1-GFP, or GFP control incubated with tamoxifen or fulvestrant for 1, 3, and 5 days (x axis) compared to the respective cell line treated with control ethanol. p values are for the 5-day treatment.Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figures S2 and S4.

Mentions: We analyzed the patient-derived BC cells that were treated with tamoxifen and fulvestrant in Figures 1B and 1C and found that increased numbers of ALDH-positive cells were strongly correlated to increased expression of Notch target genes (HEY1 and HES1) (Figure 2A). In addition, the BC PDX tumors treated in vivo with tamoxifen or fulvestrant (Figure 1D) for 2 weeks showed increased HEY1 and HES1 expression (Figure 2B), supporting an increased role for the Notch signaling pathway after endocrine therapies.


Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity.

Simões BM, O'Brien CS, Eyre R, Silva A, Yu L, Sarmiento-Castro A, Alférez DG, Spence K, Santiago-Gómez A, Chemi F, Acar A, Gandhi A, Howell A, Brennan K, Rydén L, Catalano S, Andó S, Gee J, Ucar A, Sims AH, Marangoni E, Farnie G, Landberg G, Howell SJ, Clarke RB - Cell Rep (2015)

Tamoxifen or Fulvestrant Treatment Upregulates Notch Target Genes in Patient-Derived Samples and PDXsJAG1-NOTCH4 receptor signaling in ALDH-positive cells drives Notch activity in endocrine-resistant BC.(A and B) Expression of Notch target genes HEY1 and HES1 was assessed by real-time qPCR analysis and compared to control to determine fold change. (A) Metastatic BC patient-derived cells were treated for 7–9 days with ethanol (control), tamoxifen (10−6 M), or fulvestrant (10−7 M) and a correlation between fold change of expression of HEY1 and HES1 and fold change of percentage of ALDH-positive cells is shown. (B) Early (HBCx34) and metastatic (BB3RC31) BC PDXs: the effect of in vivo treatment for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) on HEY1 and HES1.(C) NOTCH4, HES1, and JAG1 protein expression levels determined by western blot in metastatic (Met) (BB3RC31) BC PDX. β-actin was used as a reference for the loading control.(D) NOTCH4, HES1, and JAG1 protein expression levels were determined by western blot in MCF-7 ALDH-negative and ALDH-positive sorted cells. MCF-7 cells were treated with tamoxifen or fulvestrant for 6 days before ALDH sorting.(E) Wild-type MCF-7 cells (filled bars) and a CRISPR clone containing a disruption of NOTCH4 exon 2 (N4EX2 cells, hatched bars) treated in adherence with ethanol (Control, gray bars), 10−6 M tamoxifen (red bars), and 10−7 M fulvestrant (blue bars) for 6 days. N4EX2 cells’ fold change of MFE and ALDH-positive cells after treatments was compared to that of the wild-type cells.(F and G) NICD4 and JAG1 rescue tamoxifen- or fulvestrant-inhibited growth: cell number (using sulforhodamine B [SRB] assay, y axis) of MCF-7 overexpressing (F) NICD4-GFP, (G) JAG1-GFP, or GFP control incubated with tamoxifen or fulvestrant for 1, 3, and 5 days (x axis) compared to the respective cell line treated with control ethanol. p values are for the 5-day treatment.Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figures S2 and S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594158&req=5

fig2: Tamoxifen or Fulvestrant Treatment Upregulates Notch Target Genes in Patient-Derived Samples and PDXsJAG1-NOTCH4 receptor signaling in ALDH-positive cells drives Notch activity in endocrine-resistant BC.(A and B) Expression of Notch target genes HEY1 and HES1 was assessed by real-time qPCR analysis and compared to control to determine fold change. (A) Metastatic BC patient-derived cells were treated for 7–9 days with ethanol (control), tamoxifen (10−6 M), or fulvestrant (10−7 M) and a correlation between fold change of expression of HEY1 and HES1 and fold change of percentage of ALDH-positive cells is shown. (B) Early (HBCx34) and metastatic (BB3RC31) BC PDXs: the effect of in vivo treatment for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) on HEY1 and HES1.(C) NOTCH4, HES1, and JAG1 protein expression levels determined by western blot in metastatic (Met) (BB3RC31) BC PDX. β-actin was used as a reference for the loading control.(D) NOTCH4, HES1, and JAG1 protein expression levels were determined by western blot in MCF-7 ALDH-negative and ALDH-positive sorted cells. MCF-7 cells were treated with tamoxifen or fulvestrant for 6 days before ALDH sorting.(E) Wild-type MCF-7 cells (filled bars) and a CRISPR clone containing a disruption of NOTCH4 exon 2 (N4EX2 cells, hatched bars) treated in adherence with ethanol (Control, gray bars), 10−6 M tamoxifen (red bars), and 10−7 M fulvestrant (blue bars) for 6 days. N4EX2 cells’ fold change of MFE and ALDH-positive cells after treatments was compared to that of the wild-type cells.(F and G) NICD4 and JAG1 rescue tamoxifen- or fulvestrant-inhibited growth: cell number (using sulforhodamine B [SRB] assay, y axis) of MCF-7 overexpressing (F) NICD4-GFP, (G) JAG1-GFP, or GFP control incubated with tamoxifen or fulvestrant for 1, 3, and 5 days (x axis) compared to the respective cell line treated with control ethanol. p values are for the 5-day treatment.Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figures S2 and S4.
Mentions: We analyzed the patient-derived BC cells that were treated with tamoxifen and fulvestrant in Figures 1B and 1C and found that increased numbers of ALDH-positive cells were strongly correlated to increased expression of Notch target genes (HEY1 and HES1) (Figure 2A). In addition, the BC PDX tumors treated in vivo with tamoxifen or fulvestrant (Figure 1D) for 2 weeks showed increased HEY1 and HES1 expression (Figure 2B), supporting an increased role for the Notch signaling pathway after endocrine therapies.

Bottom Line: Here, we show that short-term treatment with the anti-estrogens tamoxifen or fulvestrant decrease cell proliferation but increase BC stem cell (BCSC) activity through JAG1-NOTCH4 receptor activation both in patient-derived samples and xenograft (PDX) tumors.In support of this mechanism, we demonstrate that high ALDH1 predicts resistance in women treated with tamoxifen and that a NOTCH4/HES/HEY gene signature predicts for a poor response/prognosis in 2 ER+ patient cohorts.Targeting of NOTCH4 reverses the increase in Notch and BCSC activity induced by anti-estrogens.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Now Research Unit, Institute of Cancer Sciences, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

No MeSH data available.


Related in: MedlinePlus