According to the International Agency for Research on Cancer, an estimated 1.998 million new cancer cases and 9.74 million deaths occurred globally in 2022. The majority of cancer-related deaths are attributed to drug resistance that arises before or after cancer therapy (Bray et al., 2024, Zahreddine and Borden, 2013).

Tumor multidrug resistance (MDR) accounts for more than 90 % of deaths among cancer patients receiving conventional chemotherapy or targeted therapies (Bukowski et al., 2020). MDR is a complex phenomenon characterized by various mechanisms that enable cancer cells to survive and proliferate despite chemotherapy. These mechanisms include increased drug efflux, altered drug targets, enhanced DNA repair, evasion of apoptosis, drug inactivation, changes in drug metabolism, remodeling of the tumor microenvironment, epigenetic modifications, autophagy, cancer stem cells (CSCs), and alterations in cell cycle checkpoints (Eslami et al., 2024, Vaidya et al., 2022, Tian et al., 2023, Catalano et al., 2022, Emran et al., 2022). The diverse and interconnected nature of these mechanisms complicates cancer treatment. Understanding these processes is crucial for developing strategies to overcome drug resistance and improve therapeutic outcomes.

By using whole-genome RNAi screening for more than 22,000 genes and cisplatin (CIS)-resistant models, we previously demonstrated that CIS treatment induces protein damage, and that resistance to CIS is correlated with decreased mitochondrial respiratory activity, reduced ATP production, and increased proteasome activity (Shao et al., 2020). Importantly, CIS induces protein damage in a time- and dose-dependent manner. The degradation of ubiquitin-conjugated damaged proteins and cell viability upon CIS exposure are proteasome activity dependent. In the CIS-resistant breast cancer cell line MDA-MB-231-R3, resistance to CIS is unexpectedly accompanied by cross-resistance to more than 40 anticancer drugs, a hallmark of MDR. Since MDA-MB-231-R3 cells exhibit markedly high proteasome activity, treatment with the proteasome inhibitor bortezomib (BTZ) completely reverses MDR. These findings suggest that protein damage is a common driver of MDR. However, several questions remain unanswered, such as how CIS damages proteins, how damaged proteins affect mitochondria, and how cells with mitochondrial damage and reduced respiratory activity gain resistance to CIS. Moreover, since CIS-resistant cells also exhibit cross-resistance to many other drugs, how protein damage caused by different drugs induces mitochondrial stress and subsequently contributes to the development of proteasome activity-related MDR is unknown.

In this study, we found that anticancer drugs and damaged mitochondrial proteins were coimported into the mitochondria, leading to mitochondrial damage and dysfunction. Mass spectrometry analysis of the MTS (mitochondrial targeting sequence)-EGFP interactome after CIS exposure revealed the involvement of the ubiquitin–proteasome system (UPS) in monitoring the import of drug-damaged proteins into mitochondria. We further demonstrated that the import of damaged proteins into mitochondria led to mitochondrial ROS (mtROS) production, the integrity stress response (ISR), the mitochondrial unfolded protein response (UPRmt), and BNIP3-mediated protective mitophagy. Most importantly, the combination of chemical drugs and BTZ triggered extensive mitochondrial protein import, which ultimately induced outer mitochondrial membrane (OMM) collapse, leakage of mitochondrial intermembrane space (IMS) contents, and profound mtROS release, which disabled mitophagy, triggered lysosome membrane permeabilization, and enhanced proteostasis stress-induced cell death.