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


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. 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.
Team 8 "Cell survival and tumor escape in breast cancer," UMR 892 INSERM/6299 CNRS/Université de Nantes, Institut de Recherche Thérapeutique de l'Université de Nantes, 8, quai Moncousu, BP 70721, 44007 Nantes Cedex 1, France.
Céline
C
Séveno
Delphine
D
Loussouarn
Sophie
S
Bréchet
Mario
M
Campone
Philippe
P
Juin
Sophie
S
Barillé-Nion
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D017209:Apoptosis / Q000187:drug effects*; D001713:Biphenyl Compounds / Q000494:pharmacology*; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000494:pharmacology*; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols / Q000494:pharmacology*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors; D010879:Piperazines / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D051880:Receptors, Notch / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology*; D059016:Tumor Microenvironment; D051020:bcl-X Protein / Q000037:antagonists & inhibitors
C501332:ABT-737; C494811:BCL2L1 protein, human; C474007:Bax protein (53-86); D001713:Biphenyl Compounds; D004151:Dipeptides; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols; C498702:PMAIP1 protein, human; D010446:Peptide Fragments; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D051880:Receptors, Notch; D013449:Sulfonamides; D051020:bcl-X Protein; C523463:benzyloxycarbonyl-isoleucyl-leucinal; D053829:Amyloid Precursor Protein Secretases