BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis.

BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.

NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation.

The nongenetic mechanisms required to sustain malignant tumor state are poorly understood. During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation and acquire invasive features. Using transcriptional profiling of cancer stem cells from benign tumors and malignant skin squamous cell carcinoma (SCC), we identified the nuclear receptor NR2F2 as uniquely expressed in malignant SCC. Using genetic gain of function and loss of function in vivo, we show that NR2F2 is essential for promoting the malignant tumor state by controlling tumor stemness and maintenance in mouse and human SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, epithelial-mesenchymal transition and invasive features, while repressing tumor differentiation and immune cell infiltration by regulating a common transcriptional program in mouse and human SCCs. Altogether, we identify NR2F2 as a key regulator of malignant cancer stem cell functions that promotes tumor renewal and restricts differentiation to sustain a malignant tumor state.

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte's death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A 1H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

A Comparative Study of Rat Urine 1H-NMR Metabolome Changes Presumably Arising from Isoproterenol-Induced Heart Necrosis Versus Clarithromycin-Induced QT Interval Prolongation.

Cardiotoxicity remains a challenging concern both in drug development and in the management of various clinical situations. There are a lot of examples of drugs withdrawn from the market or stopped during clinical trials due to unpredicted cardiac adverse events. Obviously, current conventional methods for cardiotoxicity assessment suffer from a lack of predictivity and sensitivity. Therefore, there is a need for developing new tools to better identify and characterize any cardiotoxicity that can occur during the pre-clinical and clinical phases of drug development as well as after marketing in exposed patients. In this study, isoproterenol and clarithromycin were used as prototypical cardiotoxic agents in rats in order to evaluate potential biomarkers of heart toxicity at very early stages using 1H-NMR-based metabonomics. While isoproterenol is known to cause heart necrosis, clarithromycin may induce QT interval prolongation. Heart necrosis and QT prolongation were validated by histological analysis, serum measurement of lactate dehydrogenase/creatine phosphate kinase and QTc measurement by electrocardiogram (ECG). Urine samples were collected before and repeatedly during daily exposure to the drugs for 1H-NMR based-metabonomics investigations. Specific metabolic signatures, characteristic of each tested drug, were obtained from which potential predictive biomarkers for drug-induced heart necrosis and drug-induced QT prolongation were retrieved. Isoproterenol-induced heart necrosis was characterized by higher levels of taurine, creatine, glucose and by lower levels of Krebs cycle intermediates, creatinine, betaine/trimethylamine N-oxide (TMAO), dimethylamine (DMA)/sarcosine. Clarithromycin-induced QT prolongation was characterized by higher levels of creatinine, taurine, betaine/TMAO and DMA/sarcosine and by lower levels of Krebs cycle intermediates, glucose and hippurate.

Metabolic Reprogramming in Metastatic Melanoma with Acquired Resistance to Targeted Therapies: Integrative Metabolomic and Proteomic Analysis.

Treatments of metastatic melanoma underwent an impressive development over the past few years, with the emergence of small molecule inhibitors targeting mutated proteins, such as BRAF, NRAS, or cKIT. However, since a significant proportion of patients acquire resistance to these therapies, new strategies are currently being considered to overcome this issue. For this purpose, melanoma cell lines with mutant BRAF, NRAS, or cKIT and with acquired resistances to BRAF, MEK, or cKIT inhibitors, respectively, were investigated using both 1H-NMR-based metabonomic and protein microarrays. The 1H-NMR profiles highlighted a similar go and return pattern in the metabolism of the BRAF, NRAS, and cKIT mutated cell lines. Indeed, melanoma cells exposed to mutation-specific inhibitors underwent metabolic disruptions following acute exposure but partially recovered their basal metabolism in long-term exposure, most likely acquiring resistance skills. The protein microarrays inquired about the potential cellular mechanisms used by the resistant cells to escape drug treatment, by showing decreased levels of proteins linked to the drug efficacy, especially in the downstream part of the MAPK signaling pathway. Integrating metabonomic and proteomic findings revealed some metabolic pathways (i.e., glutaminolysis, choline metabolism, glutathione production, glycolysis, oxidative phosphorylation) and key proteins (i.e., EPHA2, DUSP4, and HIF-1A) as potential targets to discard drug resistance.

New Insights About Doxorubicin-Induced Toxicity to Cardiomyoblast-Derived H9C2 Cells and Dexrazoxane Cytoprotective Effect: Contribution of In Vitro 1H-NMR Metabonomics.

Doxorubicin (DOX) is an anticancer drug widely used in oncology. The main limitation to DOX treatments though is due to the cumulative dose that may lead to cardiotoxicity. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure consecutive to a progressive loss in cardiomyocytes due to cell necrosis and apoptosis induced by DOX. For many years, the cardiac oxidative stress caused by DOX was considered as its main toxic mechanism. Therefore, several clinical trials were carried out to assess the efficacy of various antioxidants as a cardioprotective strategy. Only dexrazoxane (DEX), did significantly reduce DOX cardiotoxicity. However, since other antioxidants used later on to counteract DOX cardiotoxicity were not as successful as DEX, DOX-induced oxidative stress and DEX antioxidant activity are not considered as the main feature anymore and this led the scientific world to suspect other involved mechanisms which are still unknown. The objective of the present work was to study from a metabolic point of view the side effects of DOX and the protective properties of DEX. In vitro 1H-NMR metabonomics was applied to the rat cardiomyoblastic H9C2 cell line. This strategy was used with the hope of unveiling possible new targets to cope with DOX cardiotoxicity. Another underlying goal was the validation of H9C2 in vitro model for metabolic investigations of DOX and DEX effects. For this purpose, several parameters, including oxidative stress, cell mortality, and apoptosis, were measured to assess the effects of DOX and DEX alone or in combination. The metabonomic study was carried out on cellular fluids collected after either 4 or 24 hours of DOX-exposure. Under such experimental conditions, both the major adverse effects reported in patients exposed to DOX and the protective effect of DEX were demonstrated in vitro, suggesting that the H9C2 in vitro model is relevant to investigate both DOX cardiotoxicity and putative cardioprotective strategies. In addition, the metabonomics findings highlighted several metabolic pathways involved in DOX cardiotoxicity and DEX cardioprotective effects as potential metabolic targets for cardioprotection: energy metabolism, redox balance, as well as phospholipids and proteins metabolism.

A New Classification Method of Metastatic Cancers Using a 1H-NMR-Based Approach: A Study Case of Melanoma, Breast, and Prostate Cancer Cell Lines.

In this study, metastatic melanoma, breast, and prostate cancer cell lines were analyzed using a 1H-NMR-based approach in order to investigate common features and differences of aggressive cancers metabolomes. For that purpose, 1H-NMR spectra of both cellular extracts and culture media were combined with multivariate data analysis, bringing to light no less than 20 discriminant metabolites able to separate the metastatic metabolomes. The supervised approach succeeded in classifying the metastatic cell lines depending on their glucose metabolism, more glycolysis-oriented in the BRAF proto-oncogene mutated cell lines compared to the others. Other adaptive metabolic features also contributed to the classification, such as the increased total choline content (tCho), UDP-GlcNAc detection, and various changes in the glucose-related metabolites tree, giving additional information about the metastatic metabolome status and direction. Finally, common metabolic features detected via 1H-NMR in the studied cancer cell lines are discussed, identifying the glycolytic pathway, Kennedy's pathway, and the glutaminolysis as potential and common targets in metastasis, opening up new avenues to cure cancer.