Mitochondrial Matrix Protease ClpP Agonists Inhibit Cancer Stem Cell Function in Breast Cancer Cells by Disrupting Mitochondrial Homeostasis

Abstract

Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.

Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer

“…CSCs also utilize metabolites generated from tumor stromal cells in what is called a “Reverse Warburg effect” [ 37 , 50 , 51 , 52 , 53 ] ( Figure 1 , left). OxPhos is shown to be critical for breast CSCs function [ 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ]. For instance, inhibition of OxPhos by depleting mtDNA reduced an anchorage-independent survival and propagation of breast CSC-like cells [ 55 , 56 , 57 , 58 ].…”

“…OxPhos is shown to be critical for breast CSCs function [ 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ]. For instance, inhibition of OxPhos by depleting mtDNA reduced an anchorage-independent survival and propagation of breast CSC-like cells [ 55 , 56 , 57 , 58 ]. MtDNA in exosomes mediates endocrine therapy resistance [ 59 ].…”

“…The role of ClpP as a master regulator of mitochondrial protein quality control and homeostasis is well established. Many mitochondrial proteins are identified as substrates of ClpXP, including proteins involved in ETC, the TCA cycle, mitochondrial ribosome, mitochondrial gene transcription and translation, glutamine metabolism, and folate metabolism [ 58 , 247 , 248 , 249 ]. By degrading misfolded or damaged proteins, ClpXP maintains the integrity of mitochondrial functions.…”

The regulatory mechanisms of proline and hydroxyproline metabolism: Recent advances in perspective

“…Whether this effect is parallel to the reactivation of cancer stem-like cells has to be shown. A paper recently showed that knock down of PYCR1 and 2 effectively blocked the formation of mammospheres ( 12 ) in contradistinction to inhibitors of glutaminase which had little effect on mammosphere formation. This finding clearly defines that although glutamine serves as the main delivery source of degradation products of various amino acids and proteins, it is the synthesis of proline which is critical for forming mammospheres, an accepted criteria of renewal of cancer stem-like cells ( 24 ).…”

“…Although it would take a voluminous review to cover them all, I have taken the liberty of emphasizing a number of new and somewhat unexpected discoveries. These include: 1) The transitional pluripotency mediated by proline metabolism in embryonic stem cells (ESC) ( 11 ), and 2) also in cancer stemlike cells (CSC) ( 12 ); 3) The interaction between proline metabolism and hydroxyproline metabolism ( 13 ) 4) the effect of hydroxyproline with HIF-1α ( 13 ); 5) the hydroxylation of collagen and its modulation of demethylation in TET and Jmj ( 8 ); 6) In CAR-T Lymphocytes, screened by gain-of-function CRISPR screens, the discovery that PRODH2 (hydroxyproline dehydrogenase) was the only gene which significantly activated the tumorlytic effect of CAR-T cells ( 14 ). We will discuss these subjects in order and attempt to explain the mechanisms and their implications using state-of-the-art insights into proline metabolism and cancer ( 11 ).…”