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Inhibition of apoptosis by Notch signalling in breast epithelial cells
Breast Cancer Research volume 10, Article number: P20 (2008)
Aberrant Notch signalling has been shown to be involved in many cancers. We have recently observed accumulation of the Notch intracellular domain (NICD) in breast cancer cell lines and tissue samples in comparison with normal cell lines and tissues. Moreover, Notch activation has been shown to inhibit apoptosis induced by chemotherapeutics that activate the p53 pathway. We are thus investigating the role of Notch signalling in breast cancer by studying the molecular mechanisms that underlie its suppression of apoptosis.
Notch signalling was activated in the normal breast epithelial cells MCF10A by expression of NICD or a fusion protein CBF1-VP16. To inhibit Notch signalling in MCF7, BT474 and Hs578t cancer cells we expressed NUMB, a natural inhibitor of the pathway or a dominant-negative Mastermind protein. Signalling through apoptotic pathways and extent of cell death were monitored by western blot analysis and chromatin condensation by Hoechst staining, respectively. Apoptosis was induced by melphalan treatment (100 μM). Pretreatment with SH6 (10 μM) or SP600125 (10 μM) was used to inhibit AKT pathways or JNK activity, respectively. Nutlin-3 (10 μM) was used to inhibit p53–MDM2 interaction.
Activation of Notch signalling in MCF10A cells caused resistance to p53-dependent apoptosis induced by DNA damage. Conversely, inhibiting Notch signalling in MCF7, BT474 or Hs578t cancer cell lines led to a sensitization to apoptosis. We further showed that AKT was phosphorylated on serine 473 and that the AKT targets ASK1 and MDM2 were phosphorylated on serine 83 and 166, respectively, in Notch-activated MCF10A cells. Furthermore, AKT inhibition by treatment with SH6 restored sensitivity of NICD-expressing or CBF1-VP16-expressing cells to DNA-damage-induced apoptosis, showing that AKT activation is sufficient to confer resistance to apoptosis. We thus investigated the role of AKT-mediated MDM2 phosphorylation. Inhibition of p53–MDM2 interaction by treatment with Nultin-3 restored neither NOXA or PUMA accumulation nor sensitivity of NICD-expressing or CBF-1-VP16-expressing MCF10A cells following DNA-damaging agent treatment. We next showed that, following DNA-damaging agent treatment, Notch activation prevented JNK phosphorylation and PUMA and NOXA accumulation. Furthermore, JNK activation in NICD-expressing or CBF1-VP16-expressing cells is sufficient to induce cell death, and inhibition of JNK signalling by treatment with SP600125 is sufficient to prevent cell death in normal MCF10A cells.
Notch activation of the AKT pathway inhibits DNA damage-induced apoptosis by inhibition of JNK via ASK1 phosphorylation.