ABC transporters: human disease and pharmacotherapeutic potential.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are a 48-member superfamily of membrane proteins that actively transport a variety of biological substrates across lipid membranes. Their functional diversity defines an expansive involvement in myriad aspects of human biology. At least 21 ABC transporters underlie rare monogenic disorders, with even more implicated in the predisposition to and symptomology of common and complex diseases. Such broad (patho)physiological relevance places this class of proteins at the intersection of disease causation and therapeutic potential, underlining them as promising targets for drug discovery, as exemplified by the transformative CFTR (ABCC7) modulator therapies for cystic fibrosis. This review will explore the growing relevance of ABC transporters to human disease and their potential as small-molecule drug targets.

Correction to "Selective PPARδ Modulators Improve Mitochondrial Function: Potential Treatment for Duchenne Muscular Dystrophy (DMD)".

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00287.].

PPARδ modulation rescues mitochondrial fatty acid oxidation defects in the mdx model of muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a recessive, fatal X-linked disease that is characterized by progressive skeletal muscle wasting due to the absence of dystrophin, which is an a essential protein that bridges the inner cytoskeleton and extra-cellular matrix. This study set out to characterize the mitochondria in primary muscle satellite cell derived myoblasts from mdx mice and wild type control mice. Compared to wild type derived cells the mdx derived cells have reduced mitochondrial bioenergetics and have fewer mitochondria. Here, we demonstrate that a novel PPARδ modulator improves mitochondrial function in the mdx mice, which supports that modulating PPARδ may be therapeutically beneficial in DMD patients.

Selective PPARδ Modulators Improve Mitochondrial Function: Potential Treatment for Duchenne Muscular Dystrophy (DMD).

The X-ray structure of the previously reported PPARδ modulator 1 bound to the ligand binding domain (LBD) revealed that the amide moiety in 1 exists in the thermodynamically disfavored cis-amide orientation. Isosteric replacement of the cis-amide with five-membered heterocycles led to the identification of imidazole 17 (MA-0204), a potent, selective PPARδ modulator with good pharmacokinetic properties. MA-0204 was tested in vivo in mice and in vitro in patient-derived muscle myoblasts (from Duchenne Muscular Dystrophy (DMD) patients); 17 altered the expression of PPARδ target genes and improved fatty acid oxidation, which supports the therapeutic hypothesis for the study of MA-0204 in DMD patients.

Sirtuin 1 Promotes Deacetylation of Oct4 and Maintenance of Naive Pluripotency.

The enhancer landscape is dramatically restructured as naive preimplantation epiblasts transition to the post-implantation state of primed pluripotency. A key factor in this process is Otx2, which is upregulated during the early stages of this transition and ultimately recruits Oct4 to a different set of enhancers. In this study, we discover that the acetylation status of Oct4 regulates the induction of the primed pluripotency gene network. Maintenance of the naive state requires the NAD-dependent deacetylase, SirT1, which deacetylates Oct4. The activity of SirT1 is reduced during the naive-to-primed transition; Oct4 becomes hyper-acetylated and binds to an Otx2 enhancer to induce Otx2 expression. Induction of Otx2 causes the reorganization of acetylated Oct4 and results in the induction of the primed pluripotency gene network. Regulation of Oct4 by SirT1 may link stem cell development to environmental conditions, and it may provide strategies to manipulate epiblast cell state.

SirT1 is required in the male germ cell for differentiation and fecundity in mice.

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

Global microRNA depletion suppresses tumor angiogenesis.

MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.

Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging.

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/ß-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/ß-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Reductive glutamine metabolism is a function of the α-ketoglutarate to citrate ratio in cells.

Reductively metabolized glutamine is a major cellular carbon source for fatty acid synthesis during hypoxia or when mitochondrial respiration is impaired. Yet, a mechanistic understanding of what determines reductive metabolism is missing. Here we identify several cellular conditions where the α-ketoglutarate/citrate ratio is changed due to an altered acetyl-CoA to citrate conversion, and demonstrate that reductive glutamine metabolism is initiated in response to perturbations that result in an increase in the α-ketoglutarate/citrate ratio. Thus, targeting reductive glutamine conversion for a therapeutic benefit might require distinct modulations of metabolite concentrations rather than targeting the upstream signalling, which only indirectly affects the process.

Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism.

Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer.

SIRT1 suppresses the epithelial-to-mesenchymal transition in cancer metastasis and organ fibrosis.

The epithelial-to-mesenchymal transition (EMT) is important for the development of cancer metastases and organ fibrosis, conditions prevalent in aging. Because sirtuins affect the pathology of aging, we tested the effect of SirT1 on EMT. Reduced SIRT1 levels in HMLER breast cancer cells led to increased metastases in nude mice, and the loss of SIRT1 in kidney tubular epithelial cells exacerbated injury-induced kidney fibrosis. SIRT1 reduces EMT in cancer and fibrosis by deacetylating Smad4 and repressing the effect of TGF-ß signaling on MMP7, a Smad4 target gene. Consequently, less E-cadherin is cleaved from the cell surface and ß-catenin remains bound to E-cadherin at the cell-cell junctions. Our findings suggest that the SIRT1/Smad4/ß-catenin axis may be a target for diseases driven by EMT.

Identification of CDCP1 as a hypoxia-inducible factor 2α (HIF-2α) target gene that is associated with survival in clear cell renal cell carcinoma patients.

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α-, but not HIF-1α-, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

SIRT1 activates MAO-A in the brain to mediate anxiety and exploratory drive.

SIRT1 is a NAD(+)-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability.

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

The SirT3 divining rod points to oxidative stress.

Sirtuins are NAD(+) dependent deacetylases that counter aging and diseases of aging. Sirtuin research has focused on SirT1, which deacetylates transcription factors and cofactors in the nucleus. More recent findings highlight SirT3 as a mitochondrial sirtuin that regulates metabolism and oxidative stress. This review focuses on new data linking SirT3 to management of reactive oxygen species from mitochondria, which may have profound implications for aging and late-onset diseases.

Mitochondrial complex III-generated oxidants activate ASK1 and JNK to induce alveolar epithelial cell death following exposure to particulate matter air pollution.

We have previously reported that airborne particulate matter air pollution (PM) activates the intrinsic apoptotic pathway in alveolar epithelial cells through a pathway that requires the mitochondrial generation of reactive oxygen species (ROS) and the activation of p53. We sought to examine the source of mitochondrial oxidant production and the molecular links between ROS generation and the activation of p53 in response to PM exposure. Using a mitochondrially targeted ratiometric sensor (Ro-GFP) in cells lacking mitochondrial DNA (rho0 cells) and cells stably expressing a small hairpin RNA directed against the Rieske iron-sulfur protein, we show that site III of the mitochondrial electron transport chain is primarily responsible for fine PM (PM2.5)-induced oxidant production. In alveolar epithelial cells, the overexpression of SOD1 prevented the PM2.5-induced ROS generation from the mitochondria and prevented cell death. Infection of mice with an adenovirus encoding SOD1 prevented the PM2.5-induced death of alveolar epithelial cells and the associated increase in alveolar-capillary permeability. Treatment with PM2.5 resulted in the ROS-mediated activation of the oxidant-sensitive kinase ASK1 and its downstream kinase JNK. Murine embryonic fibroblasts from ASK1 knock-out mice, alveolar epithelial cells transfected with dominant negative constructs against ASK1, and pharmacologic inhibition of JNK with SP600125 (25 microM) prevented the PM2.5-induced phosphorylation of p53 and cell death. We conclude that particulate matter air pollution induces the generation of ROS primarily from site III of the mitochondrial electron transport chain and that these ROS activate the intrinsic apoptotic pathway through ASK1, JNK, and p53.

Hyperoxia-induced premature senescence requires p53 and pRb, but not mitochondrial matrix ROS.

Senescence is a potential tumor-suppressing mechanism and a commonly used model of cellular aging. One current hypothesis to explain senescence, based in part on the correlation of oxygen with senescence, postulates that it is caused by oxidative damage from reactive oxygen species (ROS). Here, we further test this theory by determining the mechanisms of hyperoxia-induced senescence. Exposure to 70% O(2) led to stress-induced, telomere-independent senescence. Although hyperoxia elevated mitochondrial ROS production, overexpression of antioxidant proteins was not sufficient to prevent hyperoxia-induced senescence. Hyperoxia activated AMPK; however, overexpression of a kinase-dead mutant of LKB1, which prevented AMPK activation, did not prevent hyperoxia-induced senescence. Knocking down p21 via shRNA, or suppression of the p16/pRb pathway by either BMI1 or HPV16-E7 overexpression, was also insufficient to prevent hyperoxia-induced senescence. However, suppressing p53 function resulted in partial rescue from senescence, suggesting that hyperoxia-induced senescence involves p53. Suppressing both the p53 and pRb pathways resulted in almost complete protection, indicating that both pathways cooperate in hyperoxia-induced senescence. Collectively, these results indicate a ROS-independent but p53/pRb-dependent senescence mechanism during hyperoxia.

Targeting the mitochondria for cancer therapy: regulation of hypoxia-inducible factor by mitochondria.

As tumors develop, they outgrow the vascular network that supplies cells with oxygen and nutrients needed for survival. In response to decreased oxygen levels, the tumor cells initiate a program of adaptation by inducing the transcription of multiple genes via the activation of the transcription factor hypoxia-inducible factor (HIF). Proteins encoded by a subset of genes induced by HIF promote tumorigenesis by acting directly on both the tumor cells and the microenvironment in which the tumor cells reside. The mechanism(s) by which hypoxia activates HIF is a subject of intensive research. Understanding how hypoxia activates HIF will provide targets for the development of therapies that could specifically target growing tumors by not allowing adequate adaptation to hypoxia, which is necessary for cancer progression. Here we outline how mitochondria regulate the activity of HIF during hypoxia.

Genetics of mitochondrial electron transport chain in regulating oxygen sensing.

Oxygen is the terminal electron acceptor in the mitochondrial electron transport chain and therefore is required for the generation of energy through oxidative phosphorylation. In environments of decreased oxygen levels (hypoxia), organisms have developed an adaptive response through the activation of the hypoxia-inducible transcription factor (HIF) to maintain their energetic demand. In order to sense hypoxic environments, cells have developed oxygen-sensing machinery that allows for the activation of HIF. The mitochondrial electron transport chain is required for the oxygen-sensing pathway. This chapter outlines methods used to explore the role of the electron transport chain and a by-product of electron transport, reactive oxygen species, in oxygen sensing.