Targeting mitochondrial proteases CLPP and LONP1 via disruption of mitochondrial redox homeostasis induces proteotoxic stress and suppresses tumor progression

BACKGROUND: Cancer cells, which rely heavily on mitochondria for their energy demands and oncometabolites, have a high mitochondrial load, often associated with an aggressive, invasive, and metastatic phenotype. Mitochondrial ROS (mtROS), which play a causal role in cancer, represent the Achilles' heel of cancer since excessive mtROS causes protein misfolding/aggregation, resulting in cell death via proteotoxic stress. Furthermore, the detailed underlying mechanism(s) of mitochondrial oxidative stress-induced cell death remain obscure. METHODS: Cell growth was estimated by MTT assay, clonogenic assay, live-cell imaging and flow cytometry. Intracellular ROS, mtROS, glutathione and antioxidant levels were studied by spectrophotometry. RNAseq and Western blotting were performed to elucidate the underlying mechanism(s). In vivo efficacy was evaluated using a syngeneic mouse model. RESULTS: We employed a mitochondria-targeted agent to disrupt the mitochondrial redox balance. Among tumors of different origins, such as lung, breast, prostate, bone, skin, cervical and liver, triple-negative breast cancer (TNBC) exhibited the highest sensitivity to mitochondrial oxidative stress. Compared with the parent compound, mitochondria-targeted agent showed 39-fold more effectiveness in killing TNBCs. We observed a possible correlation between the mitochondrial load in different cancer cell lines and their sensitivity to mitochondrial oxidative stress. Transcriptomic analysis revealed an enrichment of biological response to unfolded and/or misfolded proteins, which are regulated by two key proteases, Lon peptidase 1 (LONP1) and Caseinolytic protease P (CLPP), that control mitochondrial proteostasis. Bioinformatics analyses revealed enhanced expression and a strong positive correlation between these proteases in breast cancer patients, with highest expression observed in TNBC. Additionally, an early relapse was observed in breast cancer patients over-expressing both LONP1 and CLPP. Mitochondrial oxidative stress triggered a decrease in the native functional forms and an increase in the aggregated forms of LONP1 and CLPP, thereby disrupting mitochondrial proteostasis. Interestingly, no such changes were observed in normal cells. Mechanistically, excess mtROS induced proteotoxic stress and mitochondrial dysfunction, culminating in growth inhibition both in vitro and in vivo. CONCLUSION: Our studies, for the first time, show that the mitochondrial load and induction of mtROS for concomitant inhibition of LONP1 and CLPP to induce proteotoxic stress, could be novel therapeutic targets for cancer.