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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

NOTCH4 Inhibition using RO4929097 Abrogates Tamoxifen and Fulvestrant Enrichment of CSC Activities(A–C) Early (HBCx34) and metastatic (Met) (BB3RC31) PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) in the presence or absence of the NOTCH4 inhibitor RO4929097 (3 mg/kg/day, oral gavage). (A) MFE (%). (B) Percentage of ALDH-positive cells. (C) Secondary tumor formation. 100,000 cells of metastatic (BB3RC31) PDX were re-implanted subcutaneously in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was determined at day 90 after cell injection.(D and E) MCF-7, T47D, and ZR-75-1 cells were pre-treated in adherence with 10−6 M tamoxifen (red bars) and 10−7 M fulvestrant (blue bars) with RO4929097 (10 μM; hatched bars) or DMSO (filled bars) for 72 hr. (D) MFE and (E) percentage of ALDH-positive cells were assessed after pre-treatments.(F–H) MCF-7 cells were pre-treated in adherence for 6 days in the presence of RO4929097 (10 μM; hatched bars) or DMSO (filled bars). (F) In vivo experiments were carried out in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was assessed at day 60 and is represented as mice positive for growth/mice tested for each cell number tested. ELDA of tumor-initiating cell frequency is shown. (G) Expression of HEY1 and HES1 by real-time PCR was compared to the control. (H) HES1 protein expression levels determined by western blot.Data are represented as mean ± SEM. p values refer to hatched bars compared to filled control bars. ∗p < 0.05; ∗∗p < 0.01.See also Figures S3 and S4.
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fig3: NOTCH4 Inhibition using RO4929097 Abrogates Tamoxifen and Fulvestrant Enrichment of CSC Activities(A–C) Early (HBCx34) and metastatic (Met) (BB3RC31) PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) in the presence or absence of the NOTCH4 inhibitor RO4929097 (3 mg/kg/day, oral gavage). (A) MFE (%). (B) Percentage of ALDH-positive cells. (C) Secondary tumor formation. 100,000 cells of metastatic (BB3RC31) PDX were re-implanted subcutaneously in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was determined at day 90 after cell injection.(D and E) MCF-7, T47D, and ZR-75-1 cells were pre-treated in adherence with 10−6 M tamoxifen (red bars) and 10−7 M fulvestrant (blue bars) with RO4929097 (10 μM; hatched bars) or DMSO (filled bars) for 72 hr. (D) MFE and (E) percentage of ALDH-positive cells were assessed after pre-treatments.(F–H) MCF-7 cells were pre-treated in adherence for 6 days in the presence of RO4929097 (10 μM; hatched bars) or DMSO (filled bars). (F) In vivo experiments were carried out in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was assessed at day 60 and is represented as mice positive for growth/mice tested for each cell number tested. ELDA of tumor-initiating cell frequency is shown. (G) Expression of HEY1 and HES1 by real-time PCR was compared to the control. (H) HES1 protein expression levels determined by western blot.Data are represented as mean ± SEM. p values refer to hatched bars compared to filled control bars. ∗p < 0.05; ∗∗p < 0.01.See also Figures S3 and S4.

Mentions: In order to inhibit NOTCH4 signaling, we used the gamma-secretase inhibitor (GSI) RO4929097, which we found to be effective in reducing levels of the active NOTCH4 ICD in endocrine-resistant models (Figure S4C). RO4929097 inhibited HEY1 and HES1 expression, as well as CBF1-Notch transcriptional activity in TAMR and FULVR cell lines, but not in parental MCF-7 cells (Figure S4D). Therefore, we tested whether RO4929097 would abrogate increases in MFE and ALDH-positive cells induced in vivo by anti-estrogens administered in short-term window treatments, using the same estrogen-dependent ER+ PDX tumors as in Figures 1D–1G. Tamoxifen and fulvestrant treatments reduced tumor growth and proliferation (Figures S3A and S3B) while increasing both MFE and ALDH activity (Figures 3A and 3B). RO4929097 had no impact on growth or proliferation (% Ki67; Figure S3B) but significantly inhibited endocrine-stimulated MFE and ALDH activity (Figures 3A and 3B). The gold standard for functionally determining tumor-initiating cells is xenograft formation in secondary mouse hosts, which we performed using dissociated cells from PDX tumors treated in vivo with anti-estrogens and/or RO4929097. Cells isolated from tumors treated in vivo with RO4929097 had significantly reduced tumor-initiating capacity 90 days post-implantation (Figure 3C). Furthermore, the stimulation of tumorigenicity following in vivo tamoxifen and fulvestrant treatment was completely reversed by RO4929097 (Figure 3C). Overall, these data suggest that BCSCs, measured by tumor-initiating activity and enriched by short-term anti-estrogen treatments, are dependent on NOTCH4 signaling that can be blocked by combination treatment with a NOTCH4 inhibitor.


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)

NOTCH4 Inhibition using RO4929097 Abrogates Tamoxifen and Fulvestrant Enrichment of CSC Activities(A–C) Early (HBCx34) and metastatic (Met) (BB3RC31) PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) in the presence or absence of the NOTCH4 inhibitor RO4929097 (3 mg/kg/day, oral gavage). (A) MFE (%). (B) Percentage of ALDH-positive cells. (C) Secondary tumor formation. 100,000 cells of metastatic (BB3RC31) PDX were re-implanted subcutaneously in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was determined at day 90 after cell injection.(D and E) MCF-7, T47D, and ZR-75-1 cells were pre-treated in adherence with 10−6 M tamoxifen (red bars) and 10−7 M fulvestrant (blue bars) with RO4929097 (10 μM; hatched bars) or DMSO (filled bars) for 72 hr. (D) MFE and (E) percentage of ALDH-positive cells were assessed after pre-treatments.(F–H) MCF-7 cells were pre-treated in adherence for 6 days in the presence of RO4929097 (10 μM; hatched bars) or DMSO (filled bars). (F) In vivo experiments were carried out in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was assessed at day 60 and is represented as mice positive for growth/mice tested for each cell number tested. ELDA of tumor-initiating cell frequency is shown. (G) Expression of HEY1 and HES1 by real-time PCR was compared to the control. (H) HES1 protein expression levels determined by western blot.Data are represented as mean ± SEM. p values refer to hatched bars compared to filled control bars. ∗p < 0.05; ∗∗p < 0.01.See also Figures S3 and S4.
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fig3: NOTCH4 Inhibition using RO4929097 Abrogates Tamoxifen and Fulvestrant Enrichment of CSC Activities(A–C) Early (HBCx34) and metastatic (Met) (BB3RC31) PDX tumors treated in vivo for 14 days with tamoxifen (10 mg/kg/day, oral gavage) or fulvestrant (200 mg/kg/week, subcutaneous injection) in the presence or absence of the NOTCH4 inhibitor RO4929097 (3 mg/kg/day, oral gavage). (A) MFE (%). (B) Percentage of ALDH-positive cells. (C) Secondary tumor formation. 100,000 cells of metastatic (BB3RC31) PDX were re-implanted subcutaneously in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was determined at day 90 after cell injection.(D and E) MCF-7, T47D, and ZR-75-1 cells were pre-treated in adherence with 10−6 M tamoxifen (red bars) and 10−7 M fulvestrant (blue bars) with RO4929097 (10 μM; hatched bars) or DMSO (filled bars) for 72 hr. (D) MFE and (E) percentage of ALDH-positive cells were assessed after pre-treatments.(F–H) MCF-7 cells were pre-treated in adherence for 6 days in the presence of RO4929097 (10 μM; hatched bars) or DMSO (filled bars). (F) In vivo experiments were carried out in NSG mice with 90-day slow-release estrogen pellets. Tumor growth (>100 mm3) was assessed at day 60 and is represented as mice positive for growth/mice tested for each cell number tested. ELDA of tumor-initiating cell frequency is shown. (G) Expression of HEY1 and HES1 by real-time PCR was compared to the control. (H) HES1 protein expression levels determined by western blot.Data are represented as mean ± SEM. p values refer to hatched bars compared to filled control bars. ∗p < 0.05; ∗∗p < 0.01.See also Figures S3 and S4.
Mentions: In order to inhibit NOTCH4 signaling, we used the gamma-secretase inhibitor (GSI) RO4929097, which we found to be effective in reducing levels of the active NOTCH4 ICD in endocrine-resistant models (Figure S4C). RO4929097 inhibited HEY1 and HES1 expression, as well as CBF1-Notch transcriptional activity in TAMR and FULVR cell lines, but not in parental MCF-7 cells (Figure S4D). Therefore, we tested whether RO4929097 would abrogate increases in MFE and ALDH-positive cells induced in vivo by anti-estrogens administered in short-term window treatments, using the same estrogen-dependent ER+ PDX tumors as in Figures 1D–1G. Tamoxifen and fulvestrant treatments reduced tumor growth and proliferation (Figures S3A and S3B) while increasing both MFE and ALDH activity (Figures 3A and 3B). RO4929097 had no impact on growth or proliferation (% Ki67; Figure S3B) but significantly inhibited endocrine-stimulated MFE and ALDH activity (Figures 3A and 3B). The gold standard for functionally determining tumor-initiating cells is xenograft formation in secondary mouse hosts, which we performed using dissociated cells from PDX tumors treated in vivo with anti-estrogens and/or RO4929097. Cells isolated from tumors treated in vivo with RO4929097 had significantly reduced tumor-initiating capacity 90 days post-implantation (Figure 3C). Furthermore, the stimulation of tumorigenicity following in vivo tamoxifen and fulvestrant treatment was completely reversed by RO4929097 (Figure 3C). Overall, these data suggest that BCSCs, measured by tumor-initiating activity and enriched by short-term anti-estrogen treatments, are dependent on NOTCH4 signaling that can be blocked by combination treatment with a NOTCH4 inhibitor.

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