The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma, Thereby Determining Bortezomib Sensitivity Independent of Prosurvival MCL1 Expression

Bernhard Meister,Andreas Villunger,Martin Bodner,Verena Porto,Judith Hagenbuchner,Reinhard David,Michael J. Ausserlechner,Petra Obexer,Kathrin Geiger

doi:10.1074/jbc.m109.038331

Bernhard Meister, Andreas Villunger + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m109.038331

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Abstract

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Highlights

The proteasome is a large, multicatalytic enzyme that degrades proteins that are labeled by ubiquitin chains at specific sites
Cell lines varied in their sensitivity to these concentrations of the proteasome inhibitor: Whereas 85% of SH-EP cells became apoptotic after 48 h of treatment with 100 nM bortezomib, in other cell lines such as STA-NB1 only about 35% of cells exposed to as much as 100 nM bortezomib underwent programmed cell death
In this report we analyzed the molecular basis of bortezomibinduced apoptosis in neuroblastoma cells and identified Noxa and Bcl-xL as bortezomib-engaged BCL2 family members that are critical for apoptosis initiation

Summary

Introduction

The proteasome is a large, multicatalytic enzyme that degrades proteins that are labeled by ubiquitin chains at specific sites. In different cancer cell lines bortezomib treatment was associated with the induction of the BH3-only proteins Noxa, Bim, or BIK [7,8,9], whereas the expression of pro-survival BCL2 and Bcl-xL was not affected. Activation of caspase-8 was detectable 24 h after bortezomib treatment (Fig. 2C), death receptor signaling is not critical for apoptosis.

Results

Conclusion