Abstract
Mitochondrial proteases are key components in mitochondrial stress responses that maintain proteostasis and mitochondrial integrity in harsh environmental conditions, which leads to the acquisition of aggressive phenotypes, including chemoresistance and metastasis. However, the molecular mechanisms and exact role of mitochondrial proteases in cancer remain largely unexplored. Here, we identified functional crosstalk between LONP1 and ClpP, which are two mitochondrial matrix proteases that cooperate to attenuate proteotoxic stress and protect mitochondrial functions for cancer cell survival. LONP1 and ClpP genes closely localized on chromosome 19 and were co-expressed at high levels in most human cancers. Depletion of both genes synergistically attenuated cancer cell growth and induced cell death due to impaired mitochondrial functions and increased oxidative stress. Using mitochondrial matrix proteomic analysis with an engineered peroxidase (APEX)-mediated proximity biotinylation method, we identified the specific target substrates of these proteases, which were crucial components of mitochondrial functions, including oxidative phosphorylation, the TCA cycle, and amino acid and lipid metabolism. Furthermore, we found that LONP1 and ClpP shared many substrates, including serine hydroxymethyltransferase 2 (SHMT2). Inhibition of both LONP1 and ClpP additively increased the amount of unfolded SHMT2 protein and enhanced sensitivity to SHMT2 inhibitor, resulting in significantly reduced cell growth and increased cell death under metabolic stress. Additionally, prostate cancer patients with higher LONP1 and ClpP expression exhibited poorer survival. These results suggest that interventions targeting the mitochondrial proteostasis network via LONP1 and ClpP could be potential therapeutic strategies for cancer.
Highlights
Mitochondria are intracellular organelles that produce the majority of energy in cells by synthesizing ATP via oxidative phosphorylation
LONP1 and caseinolytic peptidase P (ClpP) mRNA expression were strongly positively correlated in prostate cancer patient samples as well as most other cancer samples (Fig. 1D, E; Fig. S2), whereas human mAAA protease AFG3L2 had no correlation with LONP1 and ClpP (Fig. S3)
Successful adaptation to diverse cytotoxic stress provides cancer cells with survival and proliferation advantages, leading to therapeutic resistance, dormancy, and metastasis[4]. In these stress adaptation processes, proteolytic removal of misfolded, denatured, or oxidized proteins is important for the integrity of subcellular organelles, especially mitochondria, which possess their own proteolytic systems that are evolutionarily conserved from bacterial cells[30]
Summary
Mitochondria are intracellular organelles that produce the majority of energy in cells by synthesizing ATP via oxidative phosphorylation. Mitochondria have many other functions including regulation of redox balance, metabolite biosynthesis, regulation of calcium homeostasis, and modulation of cell death pathways[1,2]. As cancer cells are continuously exposed to various cytotoxic stressors in harsh tumor microenvironments, including hypoxia, nutrient deprivation, and oxidative stress, mitochondria must adapt to changes and buffer stress conditions to promote cancer cell proliferation and. Mitochondrial stress responses are linked to alterations in mitochondrial functions required for tumor progression via adaptations to changing metabolic demands, regulation of cell death pathways, and contributions to chemoresistance[4]. Different from the ubiquitin/proteasome system for cytosolic protein homeostasis, mitochondrial chaperone and protease proteins are crucial for mitochondrial proteostasis, are overexpressed in most tumor types, and are involved in metabolic reprogramming that allows evasion of apoptosis and increased survival[14,15,16]. The molecular mechanisms regulating mitochondrial proteostasis and its exact function in cancer remain largely unknown
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