Overexpression of mitochondrial if1 prevents metastatic disease of colorectal cancer by enhancing anoikis and tumor infiltration of NK cells

Increasing evidences show that the ATPase Inhibitory Factor 1 (IF1), the physiological inhibitor of the ATP synthase, is overexpressed in a large number of carcinomas contributing to metabolic reprogramming and cancer progression. Herein, we show that in contrast to the findings in other carcinomas, the overexpression of IF1 in a cohort of colorectal carcinomas (CRC) predicts less chances of disease recurrence, IF1 being an independent predictor of survival. Bioinformatic and gene expression analyses of the transcriptome of colon cancer cells with differential expression of IF1 indicate that cells overexpressing IF1 display a less aggressive behavior than IF1 silenced (shIF1) cells. Proteomic and functional in vitro migration and invasion assays confirmed the higher tumorigenic potential of shIF1 cells. Moreover, shIF1 cells have increased in vivo metastatic potential. The higher metastatic potential of shIF1 cells relies on increased cFLIP-mediated resistance to undergo anoikis after cell detachment. Furthermore, tumor spheroids of shIF1 cells have an increased ability to escape from immune surveillance by NK cells. Altogether, the results reveal that the overexpression of IF1 acts as a tumor suppressor in CRC with an important anti-metastatic role, thus supporting IF1 as a potential therapeutic target in CRCThis research was funded by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2013-41945-R, SAF2016-75916-R and SAF2016-75452-R), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces, Spai

IF1 promotes oligomeric assemblies of sluggish ATP synthase and outlines the heterogeneity of the mitochondrial membrane potential

The coexistence of two pools of ATP synthase in mitochondria has been largely neglected despite in vitro indications for the existence of reversible active/inactive state transitions in the F1-domain of the enzyme. Herein, using cells and mitochondria from mouse tissues, we demonstrate the existence in vivo of two pools of ATP synthase: one active, the other IF1-bound inactive. IF1 is required for oligomerization and inactivation of ATP synthase and for proper cristae formation. Immunoelectron microscopy shows the co-distribution of IF1 and ATP synthase, placing the inactive “sluggish” ATP synthase preferentially at cristae tips. The intramitochondrial distribution of IF1 correlates with cristae microdomains of high membrane potential, partially explaining its heterogeneous distribution. These findings support that IF1 is the in vivo regulator of the active/inactive state transitions of the ATP synthase and suggest that local regulation of IF1-ATP synthase interactions is essential to activate the sluggish ATP synthaseThe work was supported by grants from MINECO (PID2019-108674RB-100), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces, Spain. IRC and PBEM were supported by predoctoral fellowships from FPU-MINECO and La Caixa, respectively. SDZ and CNT were supported by predoctoral fellowships from FPI-MINECO, Fondo Social Europe

Overexpression of the ATPase Inhibitory Factor 1 Favors a Non-metastatic Phenotype in Breast Cancer

Partial suppression of mitochondrial oxidative phosphorylation and the concurrent activation of aerobic glycolysis is a hallmark of proliferating cancer cells. Overexpression of the ATPase inhibitory factor 1 (IF1), an in vivo inhibitor of the mitochondrial ATP synthase, is observed in most prevalent human carcinomas favoring metabolic rewiring to an enhanced glycolysis and cancer progression. Consistently, a high expression of IF1 in hepatocarcinomas and in carcinomas of the lung, bladder, and stomach and in gliomas is a biomarker of bad patient prognosis. In contrast to these findings, we have previously reported that a high expression level of IF1 in breast carcinomas is indicative of less chance to develop metastatic disease. This finding is especially relevant in the bad prognosis group of patients bearing triple-negative breast carcinomas. To investigate the molecular mechanisms that underlie the differential behavior of IF1 in breast cancer progression, we have developed the triple-negative BT549 breast cancer cell line that overexpresses IF1 stably. When compared to controls, IF1-cells partially shut down respiration and enhance aerobic glycolysis. Transcriptomic analysis suggested that migration and invasion were specifically inhibited in IF1-overexpressing breast cancer cells. Analysis of gene expression by qPCR and western blotting indicate that IF1 overexpression supports the maintenance of components of the extracellular matrix (ECM) and E-cadherin concurrently with the downregulation of components and signaling pathways involved in epithelial to mesenchymal transition. The overexpression of IF1 in breast cancer cells has no effect in the rates of cellular proliferation and in the cell death response to staurosporine and hydrogen peroxide. However, the overexpression of IF1 significantly diminishes the ability of the cells to grow in soft agar and to migrate and invade when compared to control cells. Overall, the results indicate that IF1 overexpression despite favoring a metabolic phenotype prone to cancer progression in the specific case of breast cancer cells also promotes the maintenance of the ECM impeding metastatic disease. These findings hence provide a mechanistic explanation to the better prognosis of breast cancer patients bearing tumors with high expression level of IF1.CN-T and IM-R were supported by pre-doctoral FPI-MEC and JAE-CSIC fellowships, respectively. This work was supported by grants from the Ministerio de Economía y Competitividad (SAF2013-41945-R; SAF2016-75916-R), Comunidad Madrid (S2011/BMD-2402), and Fundación Ramón Areces 2015, Spain.Peer reviewedPeer Reviewe

Dysfunctional oxidative phosphorylation shunts branched-chain amino acid catabolism onto lipogenesis in skeletal muscle

It is controversial whether mitochondrial dysfunction in skeletal muscle is the cause or consequence of metabolic disorders. Herein, we demonstrate that in vivo inhibition of mitochondrial ATP synthase in muscle alters whole-body lipid homeostasis. Mice with restrained mitochondrial ATP synthase activity presented intrafiber lipid droplets, dysregulation of acyl-glycerides, and higher visceral adipose tissue deposits, poising these animals to insulin resistance. This mitochondrial energy crisis increases lactate production, prevents fatty acid b-oxidation, and forces the catabolism of branched-chain amino acids (BCAA) to provide acetyl-CoA for de novo lipid synthesis. In turn, muscle accumulation of acetyl-CoA leads to acetylation-dependent inhibition of mitochondrial respiratory complex II enhancing oxidative phosphorylation dysfunction which results in augmented ROS production. By screening 702 FDA-approved drugs, we identified edaravone as a potent mitochondrial antioxidant and enhancer. Edaravone administration restored ROS and lipid homeostasis in skeletal muscle and reinstated insulin sensitivity. Our results suggest that muscular mitochondrial perturbations are causative of metabolic disorders and that edaravone is a potential treatment for these diseasesThis work was ts from Ministerio de Economía, Industria y Competitividad, MINECO, Spain (SAF2016-76028-R and SAF2016-75916-R) and Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Raras, Spain (CB06/07/0017

Analysis of the metabolic proteome of lung adenocarcinomas by reverse-phase protein arrays (RPPA) emphasizes mitochondria as targets for therapy

Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patientsThe work was supported by grants from MINECO (SAF2016–75916-R and PID2019–108674RB-100), CIBERER-ISCIII (CB06/07/0017) and Fundación Ramón Areces, Spai

Regulación de la OXPHOS mediada por IF1 y su potencial como diana terapéutica en cáncer

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-04-2019Esta tesis tiene embargado el acceso al texto completo hasta el 26-10-2020La mitocondria es el orgánulo encargado de generar ATP mediante el proceso de fosforilación oxidativa (OXPHOS). En este proceso destaca la ATP sintasa, cuello de botella de la OXPHOS y responsable de catalizar la síntesis de ATP. El inhibidor fisiológico de la ATP sintasa (IF1) es una proteína mitocondrial cuya función es inhibir las actividades sintética e hidrolítica de la ATP sintasa. Estudiando la regulación de la actividad de IF1 hemos demostrado que la fosforilación en su residuo S39 impide la unión del inhibidor a la ATP sintasa. Por lo tanto, es la forma defosforilada de IF1 quien inhibe la enzima. Esta fosforilación estaría mediada por la actividad de una PKA mitocondrial que regula la respiración aumentando su eficiencia cuando dicha quinasa está activa. En un cribado de inhibidores de quinasas, también encontramos la quinasa SGK-1 como posible candidata en la fosforilación de IF1. SGK-1 colocaliza parcialmente con la mitocondria y su inhibición química o silenciamiento causan la defosforilación de IF1 inhibiendo la producción de ATP mitocondrial. Otro mecanismo de regulación de la actividad de IF1 es la modulación de su expresión. En este trabajo, hemos demostrado que la proteína de unión a mRNA LRPPRC regula la traducción de IF1 en células HEK293T y cardiomiocitos. En cáncer, IF1 es una proteína altamente expresada en algunos de los carcinomas humanos más prevalentes. En este trabajo mostramos que la sobreexpresión de IF1, aunque inhibe la producción de ATP mitocondrial, correlaciona con una mejor prognosis en pacientes con cáncer de mama, especialmente en aquellas con cáncer de mama triple negativo. En este contexto, hemos estudiado el efecto de 1018 fármacos aprobados por la FDA sobre la respiración mitocondrial mediante el ensayo de Seahorse en busca de inhibidores de la OXPHOS que puedan ser reposicionados en la terapia del cáncer. Describimos cómo el nebivolol, un antihipertensivo β1-bloqueante, es capaz de inhibir la OXPHOS promoviendo la sobreexpresión de IF1 y la defosforilación del complejo I mitocondrial (CI). Así mismo, describimos en modelos xenograft de cáncer de colon y cáncer de mama que el tratamiento con nebivolol genera una crisis metabólica restringiendo la producción de ATP celular que inhibe la proliferación celular y activa la apoptosis en los tumores. Con ello, demostramos que el tratamiento con nebivolol aumenta la supervivencia in vivo de ratones portadores de carcinomas humanos de colon y mama. Por lo tanto, este fármaco conocido en terapia cardiovascular podría ser reposicionado en la terapia antitumoral.Mitochondria are essential organelles playing key roles in bioenergetics by controlling the production of ATP in the process known as oxidative phosphorylation (OXPHOS). The ATP synthase, the bottleneck of the OXPHOS, is a primary hub in the ATP production. The ATPase inhibitory factor 1 (IF1) is a mitochondrial protein that inhibits both the synthetic and hydrolytic activities of the ATP synthase. Studying the regulation of IF1 activity, we have demonstrated that phosphorylation of S39 of the protein impedes IF1 binding and inhibition of the enzyme, concluding that dephosphorylated IF1 is the active molecule that inhibits ATP synthase activities. A mitochondrial PKA activity seems to regulate mitochondrial respiration by improving the efficiency of the ETC when it is activated. A screening of protein kinase inhibitors identified SGK-1 as a promising candidate to phosphorylate IF1. SGK-1 partially colocalized within mitochondria and its chemical inhibition or silencing cause the dephosphorylation of IF1 inhibiting mitochondrial ATP production. On the other hand, the regulation of IF1 expression can also control the activity of the protein. In this work, we have demonstrated that the mRNA binding protein LRPPRC inhibits the translation of IF1 in HEK293T cells and in cardiomyocytes. In cancer, IF1 is highly overexpressed in some of the most prevalent human carcinomas. Herein, we show that IF1 overexpression, despite inhibiting mitochondrial ATP production, directly correlates with a better prognosis and survival of breast cancer patients bearing triple negative breast carcinomas. In this context, we have screened a library of 1,018 FDA-approved drugs searching for drugs that could be repurposed in cancer by inhibiting OXPHOS. We have found nebivolol, a β1-adrenergic blocker, as inhibitor of mitochondrial OXPHOS in colon and breast cancer cells. Nebivolol promotes an upregulation of IF1 amount and the inhibition of complex I activity by the dephosphorylation of the complex, both contributing to the inhibition of the production of ATP by the ATP synthase. Altogether, we show the anti-cancer effect of nebivolol in vivo by assessing the tumor growth arrest experienced in mice bearing colon or breast human carcinomas by triggering a metabolic crisis because of the limitations of cellular ATP. We conclude that nebivolol, a classic antihypertensive drug, is a valid candidate to be repurposed in cancer therapy

Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis

SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function

Critical requirement of SOS1 RAS-GEF function for mitochondrial dynamics, metabolism, and redox homeostasis

© The Author(s) 2021.SOS1 ablation causes specific defective phenotypes in MEFs including increased levels of intracellular ROS. We showed that the mitochondria-targeted antioxidant MitoTEMPO restores normal endogenous ROS levels, suggesting predominant involvement of mitochondria in generation of this defective SOS1-dependent phenotype. The absence of SOS1 caused specific alterations of mitochondrial shape, mass, and dynamics accompanied by higher percentage of dysfunctional mitochondria and lower rates of electron transport in comparison to WT or SOS2-KO counterparts. SOS1-deficient MEFs also exhibited specific alterations of respiratory complexes and their assembly into mitochondrial supercomplexes and consistently reduced rates of respiration, glycolysis, and ATP production, together with distinctive patterns of substrate preference for oxidative energy metabolism and dependence on glucose for survival. RASless cells showed defective respiratory/metabolic phenotypes reminiscent of those of SOS1-deficient MEFs, suggesting that the mitochondrial defects of these cells are mechanistically linked to the absence of SOS1-GEF activity on cellular RAS targets. Our observations provide a direct mechanistic link between SOS1 and control of cellular oxidative stress and suggest that SOS1-mediated RAS activation is required for correct mitochondrial dynamics and function.This work was supported by grants from ISCIII-MCUI (FIS PI19/00934 and CIBERONCCB16/12/00352), Junta de Castilla y Leon (SA264P18-UIC 076), and Ramon Areces Foundation (CIVP19A5942). The research was co-financed by FEDER funds

Metformin as an Adjuvant to Photodynamic Therapy in Resistant Basal Cell Carcinoma Cells

© 2020 by the authors.Photodynamic Therapy (PDT) with methyl-aminolevulinate (MAL-PDT) is being used for the treatment of Basal Cell Carcinoma (BCC), although resistant cells may appear. Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate energy, but cancer cells switch this metabolism to aerobic glycolysis (Warburg effect), influencing the response to therapies. We have analyzed the expression of metabolic markers (β-F1-ATPase/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio, pyruvate kinase M2 (PKM2), oxygen consume ratio, and lactate extracellular production) in the resistance to PDT of mouse BCC cell lines (named ASZ and CSZ, heterozygous for ptch1). We have also evaluated the ability of metformin (Metf), an antidiabetic type II compound that acts through inhibition of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway to sensitize resistant cells to PDT. The results obtained indicated that resistant cells showed an aerobic glycolysis metabolism. The treatment with Metf induced arrest in the G0/G1 phase and a reduction in the lactate extracellular production in all cell lines. The addition of Metf to MAL-PDT improved the cytotoxic effect on parental and resistant cells, which was not dependent on the PS protoporphyrin IX (PpIX) production. After Metf + MAL-PDT treatment, activation of pAMPK was detected, suppressing the mTOR pathway in most of the cells. Enhanced PDT-response with Metf was also observed in ASZ tumors. In conclusion, Metf increased the response to MAL-PDT in murine BCC cells resistant to PDT with aerobic glycolysis.This research was funded by Spanish grants from Instituto de Salud Carlos III MINECO and Feder Funds (FIS PI15/00974 and PI18/00708) and Ministerio de Ciencia, Innovación y Universidades (SAF2016-75916-R).Peer reviewe