Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.

Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. Herein, we describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of BNIP3 and BNIP3L. Briefly, we employed a BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS). The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High BNIP3/BNIP3L transcriptional activity was accompanied by increases in i) BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.

In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods.

Although cellular senescence was originally defined as an irreversible form of cell cycle arrest, in therapy-induced senescence models, the emergence of proliferative senescence-escaped cancer cells has been reported by several groups, challenging the definition of senescence. Indeed, senescence-escaped cancer cells may contribute to resistance to cancer treatment. Here, to study senescence escape and isolate senescence-escaped cells, we developed novel flow cytometry-based methods using the proliferation marker Ki-67 and CellTrace CFSE live-staining. We investigated the role of a novel senescence marker (DPP4/CD26) and a senolytic drug (azithromycin) on the senescence-escaping ability of MCF-7 and MDA-MB-231 breast cancer cells. Our results show that the expression of DPP4/CD26 is significantly increased in both senescent MCF-7 and MDA-MB-231 cells. While not essential for senescence induction, DPP4/CD26 contributed to promoting senescence escape in MCF-7 cells but not in MDA-MB-231 cells. Our results also confirmed the potential senolytic effect of azithromycin in senescent cancer cells. Importantly, the combination of azithromycin and a DPP4 inhibitor (sitagliptin) demonstrated a synergistic effect in senescent MCF-7 cells and reduced the number of senescence-escaped cells. Although further research is needed, our results and novel methods could contribute to the investigation of the mechanisms of senescence escape and the identification of potential therapeutic targets. Indeed, DPP4/CD26 could be a promising marker and a novel target to potentially decrease senescence escape in cancer.

FoxO3a Drives the Metabolic Reprogramming in Tamoxifen-Resistant Breast Cancer Cells Restoring Tamoxifen Sensitivity.

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.

Antibiotics that target mitochondria extend lifespan in C. elegans.

Aging is a continuous degenerative process caused by a progressive decline of cell and tissue functions in an organism. It is induced by the accumulation of damage that affects normal cellular processes, ultimately leading to cell death. It has been speculated for many years that mitochondria play a key role in the aging process. In the aim of characterizing the implications of mitochondria in aging, here we used Caenorhabditis elegans (C. elegans) as an organismal model treated a panel of mitochondrial inhibitors and assessed for survival. In our study, we assessed survival by evaluating worm lifespan, and we assessed aging markers by evaluating the pharyngeal muscle contraction, the accumulation of lipofuscin pigment and ATP levels. Our results show that treatment of worms with either doxycycline, azithromycin (inhibitors of the small and the large mitochondrial ribosomes, respectively), or a combination of both, significantly extended median lifespan of C. elegans, enhanced their pharyngeal pumping rate, reduced their lipofuscin content and their energy consumption (ATP levels), as compared to control untreated worms, suggesting an aging-abrogating effect for these drugs. Similarly, DPI, an inhibitor of mitochondrial complex I and II, was capable of prolonging the median lifespan of treated worms. On the other hand, subjecting worms to vitamin C, a pro-oxidant, failed to extend C. elegans lifespan and upregulated its energy consumption, revealing an increase in ATP level. Therefore, our longevity study reveals that mitochondrial inhibitors (i.e., mitochondria-targeting antibiotics) could abrogate aging and extend lifespan in C. elegans.

New insights into cholesterol-mediated ERRα activation in breast cancer progression and pro-tumoral microenvironment orchestration.

Breast cancer remains the greatest cause of cancer-related death in women worldwide. Its aggressiveness and progression derive from intricate processes that occur simultaneously both within the tumour itself and in the neighbouring cells that make up its microenvironment. The aim of the present work was firstly to study how elevated cholesterol levels increase tumour aggressiveness. Herein, we demonstrate that cholesterol, by activating ERRα pathway, promotes epithelium-mesenchymal transition (EMT) in breast cancer cells (MCF-7 and MDA-MB-231) as well as the release of pro-inflammatory factors able to orchestrate the tumour microenvironment. A further objective of this work was to study the close symbiosis between tumour cells and the microenvironment. Our results allow us to highlight, for the first time, that breast cancer cells exposed to high cholesterol levels promote (a) greater macrophages infiltration with induction of an M2 phenotype, (b) angiogenesis and endothelial branching, as well as (c) a cancer-associated fibroblasts (CAFs) phenotype. The effects observed could be due to direct activation of the ERRα pathway by high cholesterol levels, since the simultaneous inhibition of this pathway subverts such effects. Overall, these findings enable us to identify the cholesterol-ERRα synergy as an interesting target for breast cancer treatment.

SOX2-high cancer cells exhibit an aggressive phenotype, with increases in stemness, proliferation and invasion, as well as higher metabolic activity and ATP production.

Cancer stem cells (CSCs) are responsible for cancer recurrence, treatment failure and metastatic dissemination. As such, the elimination of CSCs represents one of the most important approaches for the future of cancer treatment. Among other properties, CSCs show the activation of particular cell signalling pathways and the over-expression of certain transcription factors, such as SOX2. Herein, we describe a new model system to isolate stem-like cancer cells, based on the functional transcriptional activity of SOX2. Briefly, we employed a SOX2-enhancer-GFP-reporter system to isolate cancer cells with high SOX2 transcriptional activity by FACS sorting. The over-expression of SOX2 in this sub-population was validated by Western blot analysis and flow cytometry. SOX2-high cancer cells showed CSCs features, such as greater mammosphere forming ability, validating that this sub-population was enriched in CSCs. To further explore the model, we analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, corroborating that SOX2-high cells were more metabolically active, proliferative, migratory, invasive, and drug-resistant. SOX2-high MDA-MB-231 cells also showed a loss of E-cadherin expression, and increased Vimentin expression, consistent with an epithelial-mesenchymal transition (EMT). Therefore, endogenous SOX2 transcriptional activity and protein levels are mechanistically linked to aggressive phenotypic behaviours and energy production in CSCs.

Identification of natural products and FDA-approved drugs for targeting cancer stem cell (CSC) propagation.

Here, we report the identification of key compounds that effectively inhibit the anchorage-independent growth and propagation of cancer stem cells (CSCs), as determined via screening using MCF7 cells, a human breast adenocarcinoma cell line. More specifically, we employed the mammosphere assay as an experimental format, which involves the generation of 3D spheroid cultures, using low-attachment plates. These positive hit compounds can be divided into 5 categories: 1) dietary supplements (quercetin and glucosamine); 2) FDA-approved drugs (carvedilol and ciprofloxacin); 3) natural products (aloe emodin, aloin, tannic acid, chlorophyllin copper salt, azelaic acid and adipic acid); 4) flavours (citral and limonene); and 5) vitamins (nicotinamide and nicotinic acid). In addition, for the compounds quercetin, glucosamine and carvedilol, we further assessed their metabolic action, using the Seahorse to conduct metabolic flux analysis. Our results indicate that these treatments can affect glycolytic flux and suppress oxidative mitochondrial metabolism (OXPHOS). Therefore, quercetin, glucosamine and carvedilol can reprogram the metabolic phenotype of breast cancer cells. Despite having diverse chemical structures, these compounds all interfere with mitochondrial metabolism. As these compounds halt CSCs propagation, ultimately, they may have therapeutic potential.

High ATP Production Fuels Cancer Drug Resistance and Metastasis: Implications for Mitochondrial ATP Depletion Therapy.

Recently, we presented evidence that high mitochondrial ATP production is a new therapeutic target for cancer treatment. Using ATP as a biomarker, we isolated the "metabolically fittest" cancer cells from the total cell population. Importantly, ATP-high cancer cells were phenotypically the most aggressive, with enhanced stem-like properties, showing multi-drug resistance and an increased capacity for cell migration, invasion and spontaneous metastasis. In support of these observations, ATP-high cells demonstrated the up-regulation of both mitochondrial proteins and other protein biomarkers, specifically associated with stemness and metastasis. Therefore, we propose that the "energetically fittest" cancer cells would be better able to resist the selection pressure provided by i) a hostile micro-environment and/or ii) conventional chemotherapy, allowing them to be naturally-selected for survival, based on their high ATP content, ultimately driving tumor recurrence and distant metastasis. In accordance with this energetic hypothesis, ATP-high MDA-MB-231 breast cancer cells showed a dramatic increase in their ability to metastasize in a pre-clinical model in vivo. Conversely, metastasis was largely prevented by treatment with an FDA-approved drug (Bedaquiline), which binds to and inhibits the mitochondrial ATP-synthase, leading to ATP depletion. Clinically, these new therapeutic approaches could have important implications for preventing treatment failure and avoiding cancer cell dormancy, by employing ATP-depletion therapy, to target even the fittest cancer cells.

MitoTracker Deep Red (MTDR) Is a Metabolic Inhibitor for Targeting Mitochondria and Eradicating Cancer Stem Cells (CSCs), With Anti-Tumor and Anti-Metastatic Activity In Vivo.

MitoTracker Deep Red (MTDR) is a relatively non-toxic, carbocyanine-based, far-red, fluorescent probe that is routinely used to chemically mark and visualize mitochondria in living cells. Previously, we used MTDR at low nano-molar concentrations to stain and metabolically fractionate breast cancer cells into Mito-high and Mito-low cell sub-populations, by flow-cytometry. Functionally, the Mito-high cell population was specifically enriched in cancer stem cell (CSC) activity, i) showing increased levels of ESA cell surface expression and ALDH activity, ii) elevated 3D anchorage-independent growth, iii) larger overall cell size (>12-µm) and iv) Paclitaxel-resistance. The Mito-high cell population also showed enhanced tumor-initiating activity, in an in vivo preclinical animal model. Here, we explored the hypothesis that higher nano-molar concentrations of MTDR could also be used to therapeutically target and eradicate CSCs. For this purpose, we employed an ER(+) cell line (MCF7) and two triple negative cell lines (MDA-MB-231 and MDA-MB-468), as model systems. Remarkably, MTDR inhibited 3D mammosphere formation in MCF7 and MDA-MB-468 cells, with an IC-50 between 50 to 100 nM; similar results were obtained in MDA-MB-231 cells. In addition, we now show that MTDR exhibited near complete inhibition of mitochondrial oxygen consumption rates (OCR) and ATP production, in all three breast cancer cell lines tested, at a level of 500 nM. However, basal glycolytic rates in MCF7 and MDA-MB-468 cells remained unaffected at levels of MTDR of up to 1 µM. We conclude that MTDR can be used to specifically target and eradicate CSCs, by selectively interfering with mitochondrial metabolism, by employing nano-molar concentrations of this chemical entity. In further support of this notion, MTDR significantly inhibited tumor growth and prevented metastasis in vivo, in a xenograft model employing MDA-MB-231 cells, with little or no toxicity observed. In contrast, Abemaciclib, an FDA-approved CDK4/6 inhibitor, failed to inhibit metastasis. Therefore, in the future, MTDR could be modified and optimized via medicinal chemistry, to further increase its potency and efficacy, for its ultimate clinical use in the metabolic targeting of CSCs for their eradication.

Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase.

Here, we provide evidence that high ATP production by the mitochondrial ATP-synthase is a new therapeutic target for anticancer therapy, especially for preventing tumor progression. More specifically, we isolated a subpopulation of ATP-high cancer cells which are phenotypically aggressive and demonstrate increases in proliferation, stemness, anchorage-independence, cell migration, invasion and multi-drug resistance, as well as high antioxidant capacity. Clinically, these findings have important implications for understanding treatment failure and cancer cell dormancy. Using bioinformatic analysis of patient samples, we defined a mitochondrial-related gene signature for metastasis, which features the gamma-subunit of the mitochondrial ATP-synthase (ATP5F1C). The relationship between ATP5F1C protein expression and metastasis was indeed confirmed by immunohistochemistry. Next, we used MDA-MB-231 cells as a model system to functionally validate these findings. Importantly, ATP-high MDA-MB-231 cells showed a nearly fivefold increase in metastatic capacity in vivo. Consistent with these observations, ATP-high cells overexpressed (i) components of mitochondrial complexes I-V, including ATP5F1C, and (ii) markers associated with circulating tumor cells (CTCs) and metastasis, such as EpCAM and VCAM1. Knockdown of ATP5F1C expression significantly reduced ATP-production, anchorage-independent growth, and cell migration, as predicted. Similarly, therapeutic administration of the FDA-approved drug, Bedaquiline, downregulated ATP5F1C expression in vitro and prevented spontaneous metastasis in vivo. In contrast, Bedaquiline had no effect on the growth of non-tumorigenic mammary epithelial cells (MCF10A) or primary tumors in vivo. Taken together, our results suggest that mitochondrial ATP depletion is a new therapeutic strategy for metastasis prophylaxis, to avoid treatment failure. In summary, we conclude that mitochondrial ATP5F1C is a promising new biomarker and molecular target for future drug development, for the prevention of metastatic disease progression.