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. 2012 Jun 15;14(3):R96.
doi: 10.1186/bcr3214.

V体育ios版 - γ-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells

Céline Séveno  1 , Delphine Loussouarn, Sophie Bréchet, Mario Campone, Philippe Juin, Sophie Barillé-Nion
Affiliations

γ-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells

Céline Séveno et al. Breast Cancer Res. .

Abstract

Introduction: Inappropriate Notch signaling, downstream of γ-secretase activity, is understood to have tumor-promoting function and to be associated with poor outcome in cancer, of the breast in particular. The molecular basis of antitumoral effects of its inhibitors, however, remains poorly characterized VSports手机版. Moreover, the effects of their combination with the pro-apoptotic pharmacologic inhibitor of Bcl-2/Bcl-xL, ABT-737, have never been evaluated. In this study, we thus specifically addressed the biologic consequences of targeting γ-secretase and Bcl-2/Bcl-xL, alone or simultaneously, in breast cancer cell lines as well as in a novel human breast cancer ex vivo assay. .

Methods: By using in vitro 2D or 3D cultures of breast cancer cells plus a novel preclinical short-term ex vivo assay that correctly maintains human mammary tissue integrity and preserves tumor microenvironment, we tested the effects of the pharmacologic γ-secretase inhibitor GSIXII used as a single agent or in combination with ABT-737 V体育安卓版. .

Results: We show herein that the γ-secretase inhibitor, GSIXII, efficiently induces apoptosis in breast cancer cell lines by a process that relies on the induction of Noxa, a pro-apoptotic Bcl2-homology 3 domain (BH3)-only protein of the Bcl-2 family that functions as an inhibitor of antiapoptotic Mcl1. GSIXII also targets mammary cancer stem-like cells because it dramatically prevents in vitro mammosphere formation. Moreover, combining GSIXII treatment with ABT-737, a BH3-mimetic inhibitor of additional antiapoptotic proteins, such as Bcl-2 and Bcl-xL, leads to both a synergistic apoptotic response in breast cancer cells and to an inhibitory effect on mammosphere formation V体育ios版. These effects are also found when a Notch transcriptional inhibitor, SAHM1, is used. Finally, we evaluated individual human tumor responses to γ-secretase inhibition alone or in combination with ABT-737 in ex vivo assays. Analysis of a series of 30 consecutive tumors indicated that a majority of tumors are sensitive to apoptosis induction by GSIXII and that association of GSIXII with ABT-737 leads to an enhanced induction of apoptosis in tumor cells. .

Conclusions: We thus provide evidence that γ-secretase, and downstream Notch signaling, are relevant targets in breast cancer. GSIXII, used as single agent or in combination with clinically relevant BH3-mimetics, is a promising innovative proapoptotic strategy to treat mammary tumors. VSports最新版本.

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Figures

Figure 1
Figure 1
GSIXII induced apoptosis in ER+ or ER-/HER2- breast cancer cell lines. (A) MDAMB231 cells were treated with increasing GSIXII doses and analyzed with flow cytometry after Apo2.7 immunostaining. Data represent (percentage of Apo2.7-positive cells) and the means ± standard errors (SEM) of three independent experiments. (B) Three ER+ human breast cancer cell lines (ZR75.1, T47D, and MCF7) and three ER-/HER2- (BT549, Cal51, and MDAMB231) were treated with GSIXII, 15 μM, for 48 hours and then analyzed with flow cytometry after Apo2.7 immunostaining. Represented data are the means ± SEM of three independent experiments.
Figure 2
Figure 2
GSIXII inhibited the Notch signaling pathway in breast cancer cell lines. (A) Breast cancer cell lines were evaluated for Notch1-ICD expression with immunoblot analysis after treatment by GSIXII 15 μM (+) or mock (-) for 48 hours, by using actin as loading control. (B) Notch activity was evaluated with CBF1- or mutated CBF1-luciferase promoter assay after treatment by GSIXII, 15 μM, for 24 hours, compared with mock-treated cells. Represented data are means of CBF1/mutated CBF1 ratios ± SEM of three independent experiments. (C) Cells were infected with control-GFP (white) or N1ICD-GFP (black) lentivectors and treated with 15 μM GSIXII for 48 hours. Apoptosis of GFP-positive cells was assessed with Apo 2.7 staining followed by flow-cytometry analysis. Represented data are means ± SEM of three independent experiments.
Figure 3
Figure 3
GSIXII-induced cell death involved a canonic intrinsic apoptotic pathway. Cell death triggered by GSIXII depended on caspase activity. Breast cancer cells were pretreated with the pan-caspase inhibitor QVD-OPH (20 μM) before GSIXII treatment at 15 μM for 48 hours and analyzed for Apo2.7 or Annexin-V staining (more suitable for the MCF7 caspase-3 deficient cell line). Data are the means of positive cells ± SEM; n = 3. (B) Bax siRNA protected breast cancer cells from GSIXII-induced apoptosis. SiRNA (control (Ct) or Bax)-transfected cells were treated with 15 μM GSIXII before Apo2.7 immunostaining and flow-cytometry analysis. Represented data are the means of positive cells ± SEM, from three independent experiments.
Figure 4
Figure 4
Noxa induction triggered GSIXII-induced apoptosis. (A) Breast cancer cells were first transfected by siRNA targeting Bim, Puma, or Noxa, or control siRNA (siCt), and then treated or not with GSIXII for 48 hours and analyzed with flow cytometry after Apo2.7 immunostaining. Represented data are the means of positive cells ± SEM, from three independent experiments. SiRNA effects were compared with corresponding controls. (B) Expression of Noxa protein was assessed with immunoblot after 48 hours of treatment of GSIXII, 15 μM, in indicated breast cancer cell lines. (C) Noxa mRNA is induced by a GSIXII treatment. Quantitative PCR was performed on cell lines after 48 hours of treatment with mock (white) or GSIXII (black) and quantified as arbitrary units (au) compared with the mock-treated condition. Represented data are the means of positive cells ± SEM, from three independent experiments.
Figure 5
Figure 5
Notch inhibition decreased mammosphere formation. (A) MCF7 and BT549 cell lines were evaluated for their mammosphere-formation capacity with GSIXII treatment with indicated concentrations. Represented data are the means of MFU% compared with the mock-treated condition ± SEM, from three independent experiments. (B) The capacity of MCF7 and BT549 cells to form mammospheres at the first (1st), second (2nd), and third (3rd) generations was assessed in the presence or not of 8 μM GSIXII added at the beginning of the MFU assay. Represented data are from three independent experiments. Statistical analysis compared mammosphere formation in each serial generation with its mock-treated control. (C) SiRNA targeting Noxa (black bar) significantly rescued mammosphere formation in BT549 and MCF7 cells on GSIXII or mock treatment compared with siRNA control (white bar).
Figure 6
Figure 6
GSIXII synergized with ABT-737 to trigger apoptosis in breast cancer cells. Breast cancer cell lines were incubated for 48 hours with 10 μM GSIXII or DMSO (Ct) in combination or not with ABT-737, 1 μM. Then apoptosis was evaluated with Apo2.7 or Annexin-V staining and flow-cytometry analysis. Represented data are the means of positive cells ± SEM, from three independent experiments. (A) Suboptimal concentrations of GSIXII (5 μM) and 1 μM ABT-737 were used alone or in combination in MFU assay in MCF7 and BT549 cell lines. Results were obtained from three independent experiments and compared with mock-treated condition. (B) The 20 μM SAHM1 was used alone or in combination in MFU assay in MCF7 and BT549 cell lines. Results were obtained from three independent experiments and compared with the mock-treated condition.
Figure 7
Figure 7
GSIXII induced apoptosis in primary breast tumor cells in ex vivo assay. Active caspase-3 immunostaining analyzed with immunohistochemistry in one of the human breast tumors 48 hours after 15 μM GSIXII treatment or untreated (Ct). Magnification, ×200 and ×400. (A) 23 ER+ (Δ) or 7 ER- (▲) primary human tumor samples were cultured 48 hours with 15 μM GSIXII or not treated (Ct), as described in Materials and methods, and then analyzed for active caspase-3 with immunohistochemistry. Data are represented as percentage of tumoral cells positive for active caspase-3 immunostaining in each specimen. (B) Noxa expression was induced in breast tumor samples after 48-hour GSIXII treatment compared with the untreated condition, as shown in two samples assessed with immunoblot analysis. (C) The GSIXII and ABT-737 combination enhanced apoptosis triggering in breast cancer tumors. Six human primary tumor samples (three ER+ and three ER-) were cultured for 48 hours with or without 15 μM GSIXII in combination with 1 μM ABT 737 or not for 48 hours, in ex vivo assay. The percentage of active caspase-3-positive tumoral cells was then established with immunohistochemistry. A one-way ANOVA test performed on the presented cohort of tumors indicates that the combination (GSIXII+ABT-737) was significantly better than either GSIXII or ABT-737 single treatment (** and ***, respectively).
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References

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