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Cancer is a leading cause of death worldwide. All major tumor suppressors and oncogenes are now recognized to have fundamental connections with metabolic pathways. A hallmark feature of cancer cells is a reprogramming of their metabolism even when nutrients are available. Increasing evidence indicates that most cancer cells rely on mitochondrial metabolism to sustain their energetic and biosynthetic demands. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca

Cytosolic calcium (cCa2+) entry into mitochondria is facilitated by the mitochondrial membrane potential (ΔΨm), an electrochemical gradient generated by the electron transport chain (ETC). Is has been assumed that as long as mutations that affect the ETC do not affect the ΔΨm, the mitochondrial Ca2+ (mCa2+) homeostasis remains normal. We show that knockdown of NDUFAF3 and SDHB reduce ETC activity altering mCa2+ efflux and influx rates while ΔΨm remains intact. Shifting the equilibrium toward lower [Ca2+]m accumulation renders cells resistant to death. Our findings reveal an unexpected relationship between complex I and II with the mCa2+ homeostasis independent of ΔΨm.

In the last few years, metabolism has been shown to be controlled by cross-organelle communication. The relationship between the endoplasmic reticulum and mitochondria/lysosomes is the most studied; here, inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated calcium (Ca2+) release plays a central role. Recent evidence suggests that IP3R isoforms participate in synthesis and degradation pathways. This minireview will summarize the current findings in this area, emphasizing the critical role of Ca2+communication on organelle function as well as catabolism and anabolism, particularly in cancer.

Cancer is a leading cause of death worldwide. All major tumor suppressors and oncogenes are now recognized to have fundamental connections with metabolic pathways. A hallmark feature of cancer cells is a reprogramming of their metabolism even when nutrients are available. Increasing evidence indicates that most cancer cells rely on mitochondrial metabolism to sustain their energetic and biosynthetic demands. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca2+) storage organelle in mammalian cells, through special domains known as mitochondria-ER contact sites (MERCS). In this domain, the release of Ca2+ from the ER is mainly regulated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), a family of Ca2+ release channels activated by the ligand IP3. IP3R mediated Ca2+ release is transferred to mitochondria through the mitochondrial Ca2+ uniporter (MCU). Once in the mitochondrial matrix, Ca2+ activates several proteins that stimulate mitochondrial performance. The role of IP3R and MCU in cancer, as well as the other proteins that enable the Ca2+ communication between these two organelles is just beginning to be understood. Here, we describe the function of the main players of the ER mitochondrial Ca2+ communication and discuss how this particular signal may contribute to the rise and development of cancer traits.

International audience; T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.

Spontaneous Ca(2+) signaling from the InsP(3)R intracellular Ca(2+) release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca(2+) uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP(3)R activity or mitochondrial Ca(2+) uptake increased α-ketoglutarate (αKG) abundance and the NAD(+)/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)–to–mitochondria Ca(2+) transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca(2+) inhibited αKGDH activity and increased NAD(+), which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca(2+) from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca(2+) regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca(2+) transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

// Rafaela Erices 1, 2 , Sofia Cubillos 2 , Raul Aravena 2, 3 , Felice Santoro 2 , Monica Marquez 2 , Renan Orellana 1, 15 , Carolina Ramirez 2 , Pamela Gonzalez 2, 4 , Patricia Fuenzalida 2 , Maria Loreto Bravo 2, 4, 5 , Barbara Oliva 2, 4 , Sumie Kato 1 , Carolina Ibanez 5, 6, 7 , Jorge Branes 1 , Erasmo Bravo 8 , Catalina Alonso 8 , Karen Garcia 7, 9 , Clemente Arab 10 , Vicente A. Torres 11, 16 , Alejandro S. Godoy 2, 12 , Jaime Pereira 6 , Galdo Bustos 13 , Julio Cesar Cardenas 13, 14 , Mauricio A. Cuello 1 , Gareth I. Owen 2, 4, 5, 7, 16 1 Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 2 Department of Physiological Sciences, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile 3 Universidad Santo Tomas, Santiago, Chile 4 Biomedical Research Consortium of Chile, Santiago, Chile 5 Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Catolica de Chile, Santiago, Chile 6 Hematology and Oncology Department, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 7 Center UC Investigation in Oncology, Pontificia Universidad Catolica de Chile, Santiago, Chile 8 Hospital Gustavo Fricke, Vina de Mar, Santiago, Chile 9 Hospital Sotero del Rio, Santiago, Chile 10 Hospital Luis Tisne, Santiago, Chile 11 Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile 12 Department of Urology, Roswell Park Cancer Institute, Buffalo, NY, USA 13 Anatomy and Developmental Biology, Institute of Biomedical Science, Geroscience Center for Brain Health and Metabolism, University of Chile, Santiago, Chile 14 Buck Institute for Research on Aging, Novato, CA, USA 15 Universidad Bernardo OHiggins, Facultad de Salud, Departamento de Ciencias Quimicas y Biologicas, General Gana, Santiago, Chile 16 Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago, Chile Correspondence to: Gareth I. Owen, email: gowen@bio.puc.cl Keywords: thrombocytosis, hemostasis, EA.hy926, SKOV3, UCI101 Received: July 27, 2016 Accepted: January 27, 2017 Published: February 15, 2017 ABSTRACT Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics. This work was funded by FONDECYT 1140549, FONDAP program 15150012, the Millennium Institute P09-015-F and the European Commission R&D MSCA-RISE 734749 (to C.H.); the Michael J. Fox Foundation for Parkinson’s Research target validation grant number 9277, FONDEF ID16I10223, FONDEF D11E1007, US Office of Naval Research-Global N62909-16-1-2003, US Air Force Office of Scientific Research FA9550-16-1-0384, ALSRP Therapeutic Idea Award AL150111, Muscular Dystrophy Association 382453, Seed grant Leading House for the Latin American Region, Switzerland and CONICYT-Brazil 441921/2016-7 (to C.H.), FONDECYT 1160332 and FONDAP15150012 (to J.C.C.); the Spanish Ministry of Economy and Competitiveness SAF2014-52228-R, Unidad de Excelencia María de Maeztu, funded by the MINECO (ref: MDM-2014-0370) and Fundació la Marató de TV3 20134030 (to R.V.); NIH NS095892 (to R.L.W.); FONDECYT 11180825 (to H.U.); FONDECYT 3150113 and EMBO ASTF 385-2016 (to A.C.-S.); FONDECYT 3140355 (to E.R.-F.); FONDECYT 3140458 and 11170291 (to F.J.); FONDECYT 3180427 (to Y.H.); FONDECYT 3190738 (to A.S.-C.); FONDECYT 11170546 and CONICYT PAI 77170091 (to C.T.-R.); R01DK113171, R01CA198103 and R01DK103185 (to R.J.K.); FONDECYT 1150766 (to F.A.C.); the Research Council KU Leuven grant OT14/101 (to G.B.); the Research Foundation – Flanders (FWO) G.0C91.14N, G.0A34.16N and the FWO Scientific Research Community “Ca2+ signaling in health, disease and therapy” W0.019.17 (to G.B.); FWO (G049817N, G076617N) and KU Leuven (C16/15/073) (to P.A.); a FWO doctorate fellowship (to M.K.); the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant 675448 (to M.L.S.); CONICYT fellowship PCHA/Doctorado Nacional/2016-21160232 (to M.G.-Q.); the George E. Hewitt Foundation for a postdoctoral fellowship (to D.E.M.); Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche – Projets blancs; under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer; Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale; a donation from Elior; the European Research Area Network on Cardiovascular Diseases (MINOTAUR), Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085); Institut National du Cancer; Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination; and the SIRIC Cancer Research and Personalized Medicine (to G.K.).

Highly malignant triple-negative breast cancer (TNBC) cells rely mostly on glycolysis to maintain cellular homeostasis; however, mitochondria are still required for migration and metastasis. Taking advantage of the metabolic flexibility of TNBC MDA-MB-231 cells to generate subpopulations with glycolytic or oxidative phenotypes, we screened phenolic compounds containing an ortho-carbonyl group with mitochondrial activity and identified a bromoalkyl-ester of hydroquinone named FR58P1a, as a mitochondrial metabolism-affecting compound that uncouples OXPHOS through a protonophoric mechanism. In contrast to well-known protonophore uncoupler FCCP, FR58P1a does not depolarize the plasma membrane and its effect on the mitochondrial membrane potential and bioenergetics is moderate suggesting a mild uncoupling of OXPHOS. FR58P1a activates AMPK in a Sirt1-dependent fashion. Although the activation of Sirt1/AMPK axis by FR58P1a has a cyto-protective role, selectively inhibits fibronectin-dependent adhesion and migration in TNBC cells but not in non-tumoral MCF10A cells by decreasing β1-integrin at the cell surface. Prolonged exposure to FR58P1a triggers a metabolic reprograming in TNBC cells characterized by down-regulation of OXPHOS-related genes that promote cell survival but comprise their ability to migrate. Taken together, our results show that TNBC cell migration is susceptible to mitochondrial alterations induced by small molecules as FR58P1a, which may have therapeutic implications.

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.

In the absence of low-level endoplasmic reticulum-to-mitochondrial Ca2+ transfer, ATP levels fall and AMPK-dependent, mTOR-independent autophagy is induced as an essential survival mechanism in many cell types. Here we demonstrate that tumorigenic cancer cell lines, transformed primary human fibroblasts and tumors in vivo respond similarly, but autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared. Cancer cell death is due to compromised bioenergetics that can be rescued with metabolic substrates or nucleotides, and caused by necrosis associated with mitotic catastrophe during their proliferation. Our findings reveal an unexpected dependency on constitutive Ca2+ transfer to mitochondria for viability of tumorigenic cells and suggest that mitochondrial Ca2+ addiction is a feature of cancer cells.