Nutlin-3 sensitizes cancer cells to bortezomib via induction of paraptosis-associated cell death

Abstract

The ubiquitin-proteasome system is a complex and tightly controlled system in charge of degrading 80-90% of proteins, and is central to regulating cellular function and keeping protein homeostasis. Since proteasome inhibition can lead to the accumulation of the misfolded proteins and subsequent proteotoxicity in tumor cells, the proteasome is considered a target for cancer therapies. Bortezomib, the first FAD approved proteasome inhibitor, is now in the clinic to effectively treat multiple myeloma and mantle cell lymphoma, but its efficacy in solid tumors is not satisfactory. In this study, we show that the nutlin-3, a small-molecule inhibitor of murine double minute 2 (MDM2), effectively overcomes the resistance of MDA-MB 435S breast cancer cells to bortezomib. While bortezomib treatment did not markedly alter cellular morphologies, nutlin-3 treatment induced mitochondrial dilation without induction of cell death. Alternatively, combined treatment with bortezomib and nutlin-3 effectively induced cell death, which was accompanied by the simultaneous dilation of mitochondria and the endoplasmic reticulum (ER). These results suggest that nutlin-3 may overcome the resistance of these breast cancer cells to bortezomib via induction of paraptosis-like cell death. We found that the combined treatment yielded in MCU channeled calcium increase. This is supported with inhibition of MCU by Ru360 or knockdown of MCU significantly attenuated the cell death by the combined treatment. We also found that nutlin-3 co-treatment remarkably potentiated the bortezomib-mediated upregulation of ER marker proteins, including ATF4 and CHOP. Knockdown of CHOP effectively inhibited the cellular vacuolation and subsequent cell death by the combined treatment, suggesting that upreugulation of CHOP plays an important role in this process. Taken together, our results suggest that MCU-mediated mitochondrial Ca²⁺ overload and CHOP-mediated ER dilation may critically contribute to paraptosis-like cell death induced by the combination of bortezomib and nutlin-3 in breast cancer cells.