Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism.

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

The ER-mitochondria Ca2+ signaling in cancer progression: Fueling the monster.

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.

In the Right Place at the Right Time: Regulation of Cell Metabolism by IP3R-Mediated Inter-Organelle Ca2+ Fluxes.

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.

Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells.

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.

Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum-to-mitochondria Ca2+ transfer for survival.

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ 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 Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

Concerted Action of AMPK and Sirtuin-1 Induces Mitochondrial Fragmentation Upon Inhibition of Ca2+ Transfer to Mitochondria.

Mitochondria are highly dynamic organelles constantly undergoing fusion and fission. Ca2+ regulates many aspects of mitochondrial physiology by modulating the activity of several mitochondrial proteins. We previously showed that inhibition of constitutive IP3R-mediated Ca2+ transfer to the mitochondria leads to a metabolic cellular stress and eventually cell death. Here, we show that the decline of mitochondrial function generated by a lack of Ca2+ transfer induces a DRP-1 independent mitochondrial fragmentation that at an early time is mediated by an increase in the NAD+/NADH ratio and activation of SIRT1. Subsequently, AMPK predominates and drives the fragmentation. SIRT1 activation leads to the deacetylation of cortactin, favoring actin polymerization, and mitochondrial fragmentation. Knockdown of cortactin or inhibition of actin polymerization prevents fragmentation. These data reveal SIRT1 as a new player in the regulation of mitochondrial fragmentation induced by metabolic/bioenergetic stress through regulating the actin cytoskeleton.

Complex I and II are required for normal mitochondrial Ca2+ homeostasis.

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.

Publisher Correction: Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics.

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.

FR58P1a; a new uncoupler of OXPHOS that inhibits migration in triple-negative breast cancer cells via Sirt1/AMPK/ß1-integrin pathway.

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 ß1-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.

Endoplasmic Reticulum-Mitochondria Calcium Communication and the Regulation of Mitochondrial Metabolism in Cancer: A Novel Potential Target.

Cancer is characterized by an uncontrolled cell proliferation rate even under low nutrient availability, which is sustained by a metabolic reprograming now recognized as a hallmark of cancer. Warburg was the first to establish the relationship between cancer and mitochondria; however, he interpreted enhanced aerobic glycolysis as mitochondrial dysfunction. Today it is accepted that many cancer cell types need fully functional mitochondria to maintain their homeostasis. Calcium (Ca2+)-a key regulator of several cellular processes-has proven to be essential for mitochondrial metabolism. Inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ transfer from the endoplasmic reticulum to the mitochondria through the mitochondrial calcium uniporter (MCU) proves to be essential for the maintenance of mitochondrial function and cellular energy balance. Both IP3R and MCU are overexpressed in several cancer cell types, and the inhibition of the Ca2+ communication between these two organelles causes proliferation arrest, migration decrease, and cell death through mechanisms that are not fully understood. In this review, we summarize and analyze the current findings in this area, emphasizing the critical role of Ca2+ and mitochondrial metabolism in cancer and its potential as a novel therapeutic target.

Diabetic concentrations of metformin inhibit platelet-mediated ovarian cancer cell progression.

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.

Selective Vulnerability of Cancer Cells by Inhibition of Ca(2+) Transfer from Endoplasmic Reticulum to Mitochondria.

In the absence of low-level ER-to-mitochondrial Ca(2+) 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 that 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 Ca(2+) transfer to mitochondria for viability of tumorigenic cells and suggest that mitochondrial Ca(2+) addiction is a feature of cancer cells.