Mitochondria as new therapeutic targets for eradicating cancer stem cells: Quantitative proteomics and functional validation via MCT1/2 inhibition

Abstract

Here, we used quantitative proteomics analysis to identify novel therapeutic targets in cancer stem cells and/or progenitor cells. For this purpose, mammospheres from two ER-positive breast cancer cell lines (MCF7 and T47D) were grown in suspension using low-attachment plates and directly compared to attached monolayer cells grown in parallel. This allowed us to identify a subset of proteins that were selectively over-expressed in mammospheres, relative to epithelial monolayers. We focused on mitochondrial proteins, as they appeared to be highly upregulated in both MCF7 and T47D mammospheres. Key mitochondrial-related enzymes involved in beta-oxidation and ketone metabolism were significantly upregulated in mammospheres, as well as proteins involved in mitochondrial biogenesis, and specific protein inhibitors of autophagy/mitophagy. Overall, we identified >40 “metabolic targets” that were commonly upregulated in both MCF7 and T47D mammospheres. Most of these “metabolic targets” were also transcriptionally upregulated in human breast cancer cells in vivo, validating their clinical relevance. Based on this analysis, we propose that increased mitochondrial biogenesis and decreased mitochondrial degradation could provide a novel mechanism for the accumulation of mitochondrial mass in cancer stem cells. To functionally validate our observations, we utilized a specific MCT1/2 inhibitor (AR-C155858), which blocks the cellular uptake of two types of mitochondrial fuels, namely ketone bodies and L-lactate. Our results indicate that inhibition of MCT1/2 function effectively reduces mammosphere formation, with an IC-50 of ~1 μM, in both ER-positive and ER-negative breast cancer cell lines. Very similar results were obtained with oligomycin A, an inhibitor of the mitochondrial ATP synthase. Thus, the proliferative clonal expansion of cancer stem cells appears to require oxidative mitochondrial metabolism, related to the re-use of monocarboxylic acids, such as ketones or L-lactate. Our findings have important clinical implications for exploiting mitochondrial metabolism to eradicate cancer stem cells and to prevent recurrence, metastasis and drug resistance in cancer patients. Importantly, a related MCT1/2 inhibitor (AZD3965) is currently in phase I clinical trials in patients with advanced cancers: http://clinicaltrials.gov/show/NCT01791595.

A mitochondrial based oncology platform for targeting cancer stem cells (CSCs): MITO-ONC-RX

“…Informatics analysis of the list of up-regulated mitochondrial proteins was consistent with an increase in mitochondrial mass, due either to i) increased mitochondrial biogenesis or ii) a shut down in mitophagy, or both. As such, these results indicated that high mitochondrial mass is a new characteristic feature of the CSC phenotype [13]. These results also suggested the testable hypothesis that CSCs are critically dependent on OXPHOS and/or new mitochondrial biogenesis (protein translation), for their survival and propagation.…”

“…As a result of this molecular comparison, it became clear that over 60 nuclear-encoded mitochondrial proteins were specifically up-regulated in 3D spheroid structures, relative to monolayer cells processed in parallel [13]. Virtually identical results were obtained with two distinct ER(+) breast cancer cell lines (MCF7 and T47D; > 40 overlapping mitochondrial proteins).…”

“…These results also suggested the testable hypothesis that CSCs are critically dependent on OXPHOS and/or new mitochondrial biogenesis (protein translation), for their survival and propagation. In accordance with this hypothesis, 3D spheroid formation was effectively blocked using specific mitochondrial inhibitors, such as oligomycin, which targets mitochondrial Complex V and shuts off ATP synthesis [13]. However, oligomycin is quite toxic, so it cannot be used as an anti-cancer therapeutic.…”

“…In light of the above results, a targeted reduction in mitochondrial mass or OXPHOS should provide a specific Achilles’ heel for the eradication of CSCs [13–15]. …”

A Cancer Stem Cell Potent Cobalt(III)–Cyclam Complex Bearing Two Tolfenamic Acid Moieties

“…Given the implications of CSCs, to provide long-lasting durable responses, cancer therapies must have the ability to remove entire tumor populations, including CSCs. Although vulnerabilities in CSC defenses have been identified-such as overactive organelles [11,12], cell surface markers [13][14][15][16][17], dysregulated signaling pathways [18][19][20], and components of their microenvironment [21,22]-there is still no clinically approved agent (chemical or biological) that can effectively remove CSCs. The development of CSC-potent agents is in its infancy, and most of the drug candidates undergoing preclinical or clinical trials are completely organic in nature [7].…”