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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 of ER+ Patient-Derived Samples and PDXs Selectively Enriches for Cells with CSC PropertiesHigh BCSC frequency is associated with worse outcomes for tamoxifen-treated BC patients.(A) Mammosphere self-renewal of freshly isolated ER+ early and metastatic patient-derived samples. Primary mammospheres cultured in the presence of ethanol (Control) or 10−6 M 4-hydroxy-tamoxifen (Tamoxifen) were dissociated and re-plated in secondary mammosphere suspension culture for a further 7–9 days to measure self-renewal of mammosphere-initiating cells treated in the first generation. p value was calculated with Wilcoxon signed-rank test.(B) Representative micrographs of metastatic BC cells before fluorescence-activated cell sorting (FACS) analysis of ALDH1 enzymatic activity (ALDEFLUOR assay). ALDH-positive cells were discriminated from ALDH-negative cells using the ALDH inhibitor DEAB.(C) Percentage of ALDH-positive cells in nine ER+ metastatic BC patient-derived samples. Cells were grown in adherence with ethanol (Control), tamoxifen (10−6 M), or fulvestrant (10−7 M) for 7–9 days. Arrows indicate fold change greater than 20% compared to control.(D–G) Early (HBCx34) and metastatic (BB3RC31) BC estrogen-dependent PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage; red bars) or fulvestrant (200 mg/kg/week, subcutaneous injection; blue bars). Gray bars correspond to vehicle control. FFPE, formalin-fixed paraffin-embedded. (E) Representative micrographs and quantification of Ki67 expression determined by immunohistochemistry (IHC). (F) Percentage of MFE. (G) ALDH-positive cells (%) determined using the ALDEFLUOR assay.(H) ALDH1 expression was assessed by immunohistochemistry in breast tumor epithelial cells, and the percentage of positive cells was scored. Representative micrographs of ALDH-high (ALDHhi) and -low (ALDHlo) epithelial expression are shown. Kaplan-Meier curves represent cumulative survival for the ALDHlo population and ALDHhi population of a cohort of 322 pre-menopausal ER+ BC patients who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment versus no systemic treatment (control). Vertical bars on survival curves indicate censored cases. p values are based on a log-rank (Mantel-Cox) test of equality of survival distributions.Scale bars, 100 μm. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figure S1.
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fig1: Tamoxifen or Fulvestrant Treatment of ER+ Patient-Derived Samples and PDXs Selectively Enriches for Cells with CSC PropertiesHigh BCSC frequency is associated with worse outcomes for tamoxifen-treated BC patients.(A) Mammosphere self-renewal of freshly isolated ER+ early and metastatic patient-derived samples. Primary mammospheres cultured in the presence of ethanol (Control) or 10−6 M 4-hydroxy-tamoxifen (Tamoxifen) were dissociated and re-plated in secondary mammosphere suspension culture for a further 7–9 days to measure self-renewal of mammosphere-initiating cells treated in the first generation. p value was calculated with Wilcoxon signed-rank test.(B) Representative micrographs of metastatic BC cells before fluorescence-activated cell sorting (FACS) analysis of ALDH1 enzymatic activity (ALDEFLUOR assay). ALDH-positive cells were discriminated from ALDH-negative cells using the ALDH inhibitor DEAB.(C) Percentage of ALDH-positive cells in nine ER+ metastatic BC patient-derived samples. Cells were grown in adherence with ethanol (Control), tamoxifen (10−6 M), or fulvestrant (10−7 M) for 7–9 days. Arrows indicate fold change greater than 20% compared to control.(D–G) Early (HBCx34) and metastatic (BB3RC31) BC estrogen-dependent PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage; red bars) or fulvestrant (200 mg/kg/week, subcutaneous injection; blue bars). Gray bars correspond to vehicle control. FFPE, formalin-fixed paraffin-embedded. (E) Representative micrographs and quantification of Ki67 expression determined by immunohistochemistry (IHC). (F) Percentage of MFE. (G) ALDH-positive cells (%) determined using the ALDEFLUOR assay.(H) ALDH1 expression was assessed by immunohistochemistry in breast tumor epithelial cells, and the percentage of positive cells was scored. Representative micrographs of ALDH-high (ALDHhi) and -low (ALDHlo) epithelial expression are shown. Kaplan-Meier curves represent cumulative survival for the ALDHlo population and ALDHhi population of a cohort of 322 pre-menopausal ER+ BC patients who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment versus no systemic treatment (control). Vertical bars on survival curves indicate censored cases. p values are based on a log-rank (Mantel-Cox) test of equality of survival distributions.Scale bars, 100 μm. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figure S1.

Mentions: We tested the effect of the anti-estrogen tamoxifen on the mammosphere-forming efficiency (MFE) of patient-derived ER+ tumor cells and found that tamoxifen increases mammosphere self-renewal by about 2-fold (Figures 1A, S1A, and S1B). Next, we investigated ALDH activity, another functional assay for CSCs, in nine patient samples treated with tamoxifen or fulvestrant and showed significant increases in ALDH enzymatic activity in seven patients (Figures 1B and 1C). These data suggest that endocrine therapies, given for a period of a few days, enrich for stem cell activity.


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 of ER+ Patient-Derived Samples and PDXs Selectively Enriches for Cells with CSC PropertiesHigh BCSC frequency is associated with worse outcomes for tamoxifen-treated BC patients.(A) Mammosphere self-renewal of freshly isolated ER+ early and metastatic patient-derived samples. Primary mammospheres cultured in the presence of ethanol (Control) or 10−6 M 4-hydroxy-tamoxifen (Tamoxifen) were dissociated and re-plated in secondary mammosphere suspension culture for a further 7–9 days to measure self-renewal of mammosphere-initiating cells treated in the first generation. p value was calculated with Wilcoxon signed-rank test.(B) Representative micrographs of metastatic BC cells before fluorescence-activated cell sorting (FACS) analysis of ALDH1 enzymatic activity (ALDEFLUOR assay). ALDH-positive cells were discriminated from ALDH-negative cells using the ALDH inhibitor DEAB.(C) Percentage of ALDH-positive cells in nine ER+ metastatic BC patient-derived samples. Cells were grown in adherence with ethanol (Control), tamoxifen (10−6 M), or fulvestrant (10−7 M) for 7–9 days. Arrows indicate fold change greater than 20% compared to control.(D–G) Early (HBCx34) and metastatic (BB3RC31) BC estrogen-dependent PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage; red bars) or fulvestrant (200 mg/kg/week, subcutaneous injection; blue bars). Gray bars correspond to vehicle control. FFPE, formalin-fixed paraffin-embedded. (E) Representative micrographs and quantification of Ki67 expression determined by immunohistochemistry (IHC). (F) Percentage of MFE. (G) ALDH-positive cells (%) determined using the ALDEFLUOR assay.(H) ALDH1 expression was assessed by immunohistochemistry in breast tumor epithelial cells, and the percentage of positive cells was scored. Representative micrographs of ALDH-high (ALDHhi) and -low (ALDHlo) epithelial expression are shown. Kaplan-Meier curves represent cumulative survival for the ALDHlo population and ALDHhi population of a cohort of 322 pre-menopausal ER+ BC patients who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment versus no systemic treatment (control). Vertical bars on survival curves indicate censored cases. p values are based on a log-rank (Mantel-Cox) test of equality of survival distributions.Scale bars, 100 μm. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figure S1.
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fig1: Tamoxifen or Fulvestrant Treatment of ER+ Patient-Derived Samples and PDXs Selectively Enriches for Cells with CSC PropertiesHigh BCSC frequency is associated with worse outcomes for tamoxifen-treated BC patients.(A) Mammosphere self-renewal of freshly isolated ER+ early and metastatic patient-derived samples. Primary mammospheres cultured in the presence of ethanol (Control) or 10−6 M 4-hydroxy-tamoxifen (Tamoxifen) were dissociated and re-plated in secondary mammosphere suspension culture for a further 7–9 days to measure self-renewal of mammosphere-initiating cells treated in the first generation. p value was calculated with Wilcoxon signed-rank test.(B) Representative micrographs of metastatic BC cells before fluorescence-activated cell sorting (FACS) analysis of ALDH1 enzymatic activity (ALDEFLUOR assay). ALDH-positive cells were discriminated from ALDH-negative cells using the ALDH inhibitor DEAB.(C) Percentage of ALDH-positive cells in nine ER+ metastatic BC patient-derived samples. Cells were grown in adherence with ethanol (Control), tamoxifen (10−6 M), or fulvestrant (10−7 M) for 7–9 days. Arrows indicate fold change greater than 20% compared to control.(D–G) Early (HBCx34) and metastatic (BB3RC31) BC estrogen-dependent PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage; red bars) or fulvestrant (200 mg/kg/week, subcutaneous injection; blue bars). Gray bars correspond to vehicle control. FFPE, formalin-fixed paraffin-embedded. (E) Representative micrographs and quantification of Ki67 expression determined by immunohistochemistry (IHC). (F) Percentage of MFE. (G) ALDH-positive cells (%) determined using the ALDEFLUOR assay.(H) ALDH1 expression was assessed by immunohistochemistry in breast tumor epithelial cells, and the percentage of positive cells was scored. Representative micrographs of ALDH-high (ALDHhi) and -low (ALDHlo) epithelial expression are shown. Kaplan-Meier curves represent cumulative survival for the ALDHlo population and ALDHhi population of a cohort of 322 pre-menopausal ER+ BC patients who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment versus no systemic treatment (control). Vertical bars on survival curves indicate censored cases. p values are based on a log-rank (Mantel-Cox) test of equality of survival distributions.Scale bars, 100 μm. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.See also Figure S1.
Mentions: We tested the effect of the anti-estrogen tamoxifen on the mammosphere-forming efficiency (MFE) of patient-derived ER+ tumor cells and found that tamoxifen increases mammosphere self-renewal by about 2-fold (Figures 1A, S1A, and S1B). Next, we investigated ALDH activity, another functional assay for CSCs, in nine patient samples treated with tamoxifen or fulvestrant and showed significant increases in ALDH enzymatic activity in seven patients (Figures 1B and 1C). These data suggest that endocrine therapies, given for a period of a few days, enrich for stem cell activity.

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