Circadian rhythms orchestrate physiological processes such as metabolism, immune function, and tissue regeneration, aligning them with the optimal time of day (TOD). This study identifies an interplay between the circadian clock within muscle stem cells (SCs) and their capacity to modulate the immune microenvironment during muscle regeneration. We reveal that the SC clock triggers TOD-dependent inflammatory gene transcription after injury, particularly genes related to neutrophil activity and chemotaxis. These responses are driven by cytosolic regeneration of the signaling metabolite nicotinamide adenine dinucleotide (oxidized form) (NAD+), as enhancing cytosolic NAD+ regeneration in SCs is sufficient to induce inflammatory responses that influence muscle regeneration. Mononuclear single-cell sequencing of the regenerating muscle niche further implicates the cytokine CCL2 in mediating SC-neutrophil cross-talk in a TOD-dependent manner. Our findings highlight the intersection between SC metabolic shifts and immune responses within the muscle microenvironment, dictated by circadian rhythms, and underscore the potential for targeting circadian and metabolic pathways to enhance tissue regeneration.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

Men taking antioxidant vitamin E supplements have increased prostate cancer (PC) risk. However, whether pro-oxidants protect from PC remained unclear. In this work, we show that a pro-oxidant vitamin K precursor [menadione sodium bisulfite (MSB)] suppresses PC progression in mice, killing cells through an oxidative cell death: MSB antagonizes the essential class III phosphatidylinositol (PI) 3-kinase VPS34—the regulator of endosome identity and sorting—through oxidation of key cysteines, pointing to a redox checkpoint in sorting. Testing MSB in a myotubular myopathy model that is driven by loss of MTM1 —the phosphatase antagonist of VPS34—we show that dietary MSB improved muscle histology and function and extended life span. These findings enhance our understanding of pro-oxidant selectivity and show how definition of the pathways they impinge on can give rise to unexpected therapeutic opportunities.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Metformin is among the most prescribed antidiabetic drugs, but the primary molecular mechanism by which metformin lowers blood glucose levels is unknown. Previous studies have proposed numerous mechanisms by which acute metformin lowers blood glucose, including the inhibition of mitochondrial complex I of the electron transport chain (ETC). Here, we used transgenic mice that globally express the Saccharomyces cerevisiae internal alternative NADH dehydrogenase (NDI1) protein to determine whether the glucose-lowering effect of acute oral administration of metformin requires inhibition of mitochondrial complex I of the ETC in vivo. NDI1 is a yeast NADH dehydrogenase enzyme that complements the loss of mammalian mitochondrial complex I electron transport function and is insensitive to pharmacologic mitochondrial complex I inhibitors including metformin. We demonstrate that NDI1 expression attenuates metformin’s ability to lower blood glucose levels under standard chow and high-fat diet conditions. Our results indicate that acute oral administration of metformin targets mitochondrial complex I to lower blood glucose.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia 1 – 6 . Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD + and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD + without proton pumping 7 , 8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor 9 , 10 or NAD + precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd , which maintains NAD + regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD + regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Background Volatile anesthetics induce hyperpolarizing potassium currents in spinal cord neurons that may contribute to their mechanism of action. They are induced at lower concentrations of isoflurane in noncholinergic neurons from mice carrying a loss-of-function mutation of the Ndufs4 gene, required for mitochondrial complex I function. The yeast NADH dehydrogenase enzyme, NDi1, can restore mitochondrial function in the absence of normal complex I activity, and gain-of-function Ndi1 transgenic mice are resistant to volatile anesthetics. The authors tested whether NDi1 would reduce the hyperpolarization caused by isoflurane in neurons from Ndufs4 and wild-type mice. Since volatile anesthetic behavioral hypersensitivity in Ndufs4 is transduced uniquely by glutamatergic neurons, it was also tested whether these currents were also unique to glutamatergic neurons in the Ndufs4 spinal cord. Methods Spinal cord neurons from wild-type, NDi1, and Ndufs4 mice were patch clamped to characterize isoflurane sensitive currents. Neuron types were marked using fluorescent markers for cholinergic, glutamatergic, and γ-aminobutyric acid–mediated (GABAergic) neurons. Norfluoxetine was used to identify potassium channel type. Neuron type–specific Ndufs4 knockout animals were generated using type-specific Cre-recombinase with floxed Ndufs4. Results Resting membrane potentials (RMPs) of neurons from NDi1;Ndufs4, unlike those from Ndufs4, were not hyperpolarized by 0.6% isoflurane (Ndufs4, ΔRMP –8.2 mV [–10 to –6.6]; P = 1.3e-07; Ndi1;Ndufs4, ΔRMP –2.1 mV [–7.6 to +1.4]; P = 1). Neurons from NDi1 animals in a wild-type background were not hyperpolarized by 1.8% isoflurane (wild-type, ΔRMP, –5.2 mV [–7.3 to –3.2]; P = 0.00057; Ndi1, ΔRMP, 0.6 mV [–1.7 to 3.2]; P = 0.68). In spinal cord slices from global Ndufs4 animals, holding currents (HC) were induced by 0.6% isoflurane in both GABAergic (ΔHC, 81.3 pA [61.7 to 101.4]; P = 2.6e-05) and glutamatergic (ΔHC, 101.2 pA [63.0 to 146.2]; P = 0.0076) neurons. In neuron type–specific Ndufs4 knockouts, HCs were increased in cholinergic (ΔHC, 119.5 pA [82.3 to 156.7]; P = 0.00019) and trended toward increase in glutamatergic (ΔHC, 85.5 pA [49 to 126.9]; P = 0.064) neurons but not in GABAergic neurons. Conclusions Bypassing complex I by overexpression of NDi1 eliminates increases in potassium currents induced by isoflurane in the spinal cord. The isoflurane-induced potassium currents in glutamatergic neurons represent a potential downstream mechanism of complex I inhibition in determining minimum alveolar concentration. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.

The mechanisms of volatile anesthetic action remain among the most perplexing mysteries of medicine. Across phylogeny, volatile anesthetics selectively inhibit mitochondrial complex I, and they also depress presynaptic excitatory signaling. To explore how these effects are linked, we studied isoflurane effects on presynaptic vesicle cycling and ATP levels in hippocampal cultured neurons from wild-type and complex I mutant (Ndufs4(KO)) mice. To bypass complex I, we measured isoflurane effects on anesthetic sensitivity in mice expressing NADH dehydrogenase (NDi1). Endocytosis in physiologic concentrations of glucose was delayed by effective behavioral concentrations of isoflurane in both wild-type (tau [unexposed] 44.8 +/- 24.2 s; tau [exposed] 116.1 +/- 28.1 s; p < 0.01) and Ndufs4(KO) cultures (tau [unexposed] 67.6 +/- 16.0 s; tau [exposed] 128.4 +/- 42.9 s; p = 0.028). Increasing glucose, to enhance glycolysis and increase ATP production, led to maintenance of both ATP levels and endocytosis (tau [unexposed] 28.0 +/- 14.4; tau [exposed] 38.2 +/- 5.7; reducing glucose worsened ATP levels and depressed endocytosis (tau [unexposed] 85.4 +/- 69.3; tau [exposed] > 1,000; p < 0.001). The block in recycling occurred at the level of reuptake of synaptic vesicles into the presynaptic cell. Expression of NDi1 in wild-type mice caused behavioral resistance to isoflurane for tail clamp response (EC50 Ndi1(-) 1.27% +/- 0.14%; Ndi1(+) 1.55% +/- 0.13%) and halothane (EC50 Ndi1(-) 1.20% +/- 0.11%; Ndi1(+) 1.46%+/- 0.10%); expression of NDi1 in neurons improved hippocampal function, alleviated inhibition of presynaptic recycling, and increased ATP levels during isoflurane exposure. The clear alignment of cell culture data to in vivo phenotypes of both isoflurane-sensitive and -resistant mice indicates that inhibition of mitochondrial complex I is a primary mechanism of action of volatile anesthetics.

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.

The mitochondrial electron transport chain (ETC) is necessary for tumour growth(1-6) and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies(7-9). Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria(10,11). However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)(12), which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)(13) targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

Mitochondrial complex I regenerates NAD+ and proton pumps for TCA cycle function and ATP production, respectively. Mitochondrial complex I dysfunction has been implicated in many brain pathologies including Leigh syndrome and Parkinson's disease. We sought to determine whether NAD+ regeneration or proton pumping, i.e., bioenergetics, is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NADH dehydrogenase (NDI1), a single enzyme that can replace the NAD+ regeneration capability of the 45-subunit mammalian mitochondrial complex I without proton pumping. NDI1 expression was sufficient to dramatically prolong lifespan without significantly improving motor function in a mouse model of Leigh syndrome driven by the loss of NDUFS4, a subunit of mitochondrial complex I. Therefore, mitochondrial complex I activity in the brain supports organismal survival through its NAD+ regeneration capacity, while optimal motor control requires the bioenergetic function of mitochondrial complex I.

A hallmark of advanced prostate cancer (PC) is the concomitant loss of PTEN and p53 function. To selectively eliminate such cells, we screened cytotoxic compounds on Pten(-/-); Trp53(-/-) fibroblasts and their Pten-WT reference. Highly selective killing of Pten-null cells can be achieved by deguelin, a natural insecticide. Deguelin eliminates Pten-deficient cells through inhibition of mitochondrial complex I (CI). Five hundred-fold higher drug doses are needed to obtain the same killing of Pten-WT cells, even though deguelin blocks their electron transport chain equally well. Selectivity arises because mitochondria of Pten-null cells consume ATP through complex V, instead of producing it. The resulting glucose dependency can be exploited to selectively kill Pten-null cells with clinically relevant CI inhibitors, especially if they are lipophilic. In vivo, deguelin suppressed disease in our genetically engineered mouse model for metastatic PC. Our data thus introduce a vulnerability for highly selective targeting of incurable PC with inhibitors of CI.

To avoid reactive oxygen species (ROS)-induced cell death, cancer cells increase their antioxidant defense system. In this issue of Cancer Cell, Takahashi et al. identify a novel, non-canonical oxidative stress defense mechanism involving TRPA1, a redox-sensitive Ca2+ channel, and the upregulation of anti-apoptotic pathways to promote cancer cell survival.

Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology.

Reactive oxygen species (ROS), now appreciated for their cellular signaling capabilities, have a dual role in cancer. On the one hand, ROS can promote protumorigenic signaling, facilitating cancer cell proliferation, survival, and adaptation to hypoxia. On the other hand, ROS can promote antitumorigenic signaling and trigger oxidative stress-induced cancer cell death. To hyperactivate the cell signaling pathways necessary for cellular transformation and tumorigenesis, cancer cells increase their rate of ROS production compared with normal cells. Concomitantly, in order to maintain ROS homeostasis and evade cell death, cancer cells increase their antioxidant capacity. Compared with normal cells, this altered redox environment of cancer cells may increase their susceptibility to ROS-manipulation therapies. In this review, we discuss the two faces of ROS in cancer, the potential mechanisms underlying ROS signaling, and the opposing cancer therapeutic approaches to targeting ROS.

Hypoxia-inducible factors (HIFs) are crucial for cellular and organismal adaptation to hypoxia. The mitochondrial respiratory chain is the largest consumer of oxygen in most mammalian cells; however, it is unknown whether the respiratory chain is necessary for in vivo activation of HIFs and organismal adaptation to hypoxia. HIF-1 activation in the epidermis has been shown to be a key regulator of the organismal response to hypoxic conditions, including renal production of erythropoietin (Epo). Therefore, we conditionally deleted expression of TFAM in mouse epidermal keratinocytes. TFAM is required for maintenance of the mitochondrial genome, and TFAM-null cells are respiratory deficient. TFAM loss in epidermal keratinocytes reduced epidermal levels of HIF-1α protein and diminished the hypoxic induction of HIF-dependent transcription in epidermis. Furthermore, epidermal TFAM deficiency impaired hypoxic induction of renal Epo expression. Our results demonstrate that the mitochondrial respiratory chain is essential for in vivo HIF activation and organismal adaptation to hypoxia.

An important mechanism of action of metformin as an antidiabetic drug involves inhibition of hepatic gluconeogenesis, but the molecular basis for this is controversial. A recent perspective (He and Wondisford, 2015He L. Wondisford F.E. Cell Metab. 2015; 21: 159-162Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) argued that direct action of the drug on AMPK is of key importance and occurs at clinically relevant concentrations of ∼70 μM. However, that review cited conflicting studies, which concluded either that AMPK-independent mechanisms are important in metformin action, or that AMPK activation plays a role but is secondary to energetic stress resulting from inhibition of oxidative phosphorylation by the drug. As work to define the molecular mechanisms of metformin in diabetes has proceeded, the hypothesis that biguanides may be "repurposed" for additional applications has received increased attention. This followed retrospective pharmacoepidemiologic studies that showed reduced cancer risk and/or improved cancer prognosis among diabetics treated with metformin relative to those treated with other drugs. Although some of these studies have been criticized on methodologic grounds, they nevertheless led to laboratory studies that revealed clear antineoplastic activity and provided a rationale for clinical trials (Buzzai et al., 2007Buzzai M. Jones R.G. Amaravadi R.K. Lum J.J. DeBerardinis R.J. Zhao F. Viollet B. Thompson C.B. Cancer Res. 2007; 67: 6745-6752Crossref PubMed Scopus (765) Google Scholar). More than 100 trials designed to detect clinical utility of metformin in oncology are now ongoing. There is also experimental evidence that metformin extends lifespan in model organisms, sparking interest in the possibility that these studies have clinical relevance. Not surprisingly in view of controversies regarding the molecular mechanisms of metformin action in diabetes, there is uncertainty regarding the mechanisms relevant to its putative antineoplastic and antiaging effects, and it cannot be assumed that these are identical to those involved in suppression of gluconeogenesis in diabetes (Foretz et al., 2014Foretz M. Guigas B. Bertrand L. Pollak M. Viollet B. Cell Metab. 2014; 20: 953-966Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). One possibility is that the drug acts indirectly by altering the host endocrine milieu in a manner that inhibits the growth of a subset of cancers, for example, by lowering insulin levels. While this is possible, the magnitude of such changes is small and may or may not be sufficient to alter neoplastic behavior. Another possibility relates to direct actions of biguanides on cancer cells or cells at risk for transformation, for example, by inhibiting oxidative phosphorylation (Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. Chandel N.S. eLife. 2014; 3: e02242Crossref Scopus (683) Google Scholar). However, it is unclear if clinically practical metformin doses are sufficient for such mechanisms to operate. Most in vitro assays reveal that millimolar (mM) levels of metformin are required to have antiproliferative effects. At first glance, this implies that direct clinical antineoplastic activity is unlikely, as diabetic patients receiving typical doses of 1.5–2.5 g of metformin per day (∼30 mg/kg) have plasma levels in the 10 μM range (Graham et al., 2011Graham G.G. Punt J. Arora M. Day R.O. Doogue M.P. Duong J.K. Furlong T.J. Greenfield J.R. Greenup L.C. Kirkpatrick C.M. et al.Clin. Pharmacokinet. 2011; 50: 81-98Crossref PubMed Scopus (802) Google Scholar). However, sensitivity to metformin is known to vary greatly with nutrient availability, and in vitro culture conditions are unlikely to mimic the in vivo microenvironment in this respect, as glucose, serine, and glutamine, each of which reduces metformin sensitivity, are likely present in media at higher concentration than in poorly vascularized tumors (Gravel et al., 2014Gravel S.P. Hulea L. Toban N. Birman E. Blouin M.J. Zakikhani M. Zhao Y. Topisirovic I. St-Pierre J. Pollak M. Cancer Res. 2014; 74: 7521-7533Crossref PubMed Scopus (92) Google Scholar). Thus, we suggest that the requirement for millimolar metformin concentrations in vitro may not be useful for predicting the drug concentration required for activity in vivo or clinically. Nevertheless, it is important to determine the in vivo concentration of metformin in mouse models that demonstrates antineoplastic activity in order to determine if this level is achievable in patients. We administered metformin to C57BL/6J mice at a dose of 250 mg/kg in drinking water for 2 weeks and used mass spectrometry to measure metformin concentrations at steady state. As shown in Figure S1 (available online), these mice had plasma concentrations in the 5 μM range and liver concentrations of ∼40 μM. Accumulation in liver is plausible as hepatocytes highly express the organic cation transporters (OCTs) that import metformin. We also studied J:Nu mice bearing human HCT116 p53−/− colon cancer, an OCT-expressing tumor previously shown to be growth inhibited in vivo when mice were provided with drinking water as a 1.25 mg/mL solution of metformin, which provided a daily oral dose of ∼250 mg/kg (Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. Chandel N.S. eLife. 2014; 3: e02242Crossref Scopus (683) Google Scholar). This represents a dose 10-fold higher than the human dose on a mg/kg basis. In this model, tumors became resistant to metformin-induced growth inhibition when transfected with metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase, bypassing the need for respiratory complex I, suggesting that the drug acts by inhibiting oxidative phosphorylation in cancer cells (Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. Chandel N.S. eLife. 2014; 3: e02242Crossref Scopus (683) Google Scholar). The plasma (n = 4) and tumor (n = 9) metformin concentrations were both in the range of 3.2–12.4 μM (Figures S1A and S1B), a level achieved in routine diabetes treatment (Graham et al., 2011Graham G.G. Punt J. Arora M. Day R.O. Doogue M.P. Duong J.K. Furlong T.J. Greenfield J.R. Greenup L.C. Kirkpatrick C.M. et al.Clin. Pharmacokinet. 2011; 50: 81-98Crossref PubMed Scopus (802) Google Scholar). In an additional study, we utilized the metformin-sensitive human A549 lung adenocarcinoma. NMRI nu/nu mice were either intraperitoneally injected or given metformin (350 mg/kg) by mouth (P.O.) once daily for 2 or 3 weeks, respectively. After the last dose, we measured the metformin concentration over a 24 hr period in the plasma, liver, and tumors of these mice (Figures S1C and S1D). Intraperitoneal (i.p.) dosing of metformin resulted in a peak liver and tumor concentration of approximately 100 μM while P.O. dosing resulted in a significantly higher liver peak metformin concentration (∼1500 μM) than tumor metformin concentration (∼200 μM). Time-averaged plasma concentrations were ∼7.5 and ∼47 μM following i.p. and P.O. dosing, respectively. The 350 mg/kg dose of metformin activates AMPK in vivo and diminishes tumorigenesis in mouse models of cancer (Lévy et al., 2015Lévy J. Cacheux W. Bara M.A. L'Hermitte A. Lepage P. Fraudeau M. Trentesaux C. Lemarchand J. Durand A. Crain A.M. et al.Nat. Cell Biol. 2015; 17: 1062-1073Crossref PubMed Scopus (124) Google Scholar). Together, our findings suggest that the metformin plasma concentration in a murine model where the drug has antineoplastic activity attributable to inhibition of oxidative phosphorylation (Wheaton et al., 2014Wheaton W.W. Weinberg S.E. Hamanaka R.B. Soberanes S. Sullivan L.B. Anso E. Glasauer A. Dufour E. Mutlu G.M. Budigner G.S. Chandel N.S. eLife. 2014; 3: e02242Crossref Scopus (683) Google Scholar) is in the micromolar range, comparable to that of diabetic patients receiving a standard dose of metformin, despite the fact that the mg/kg dose in mice is considerably higher than the mg/kg dose in humans. This is in keeping with interspecies scaling of pharmacokinetics and suggests that ongoing clinical trials are reasonable from a pharmacokinetic perspective. It is also important to note that metformin is a cation and is predicted to accumulate 100- to 500-fold in the mitochondria due to the membrane potential. Thus, micromolar concentrations of metformin in the plasma may result in considerably higher concentrations in the mitochondria of cells that are capable of importing the drug. However, the first formal blinded clinical trial of metformin with a survival endpoint showed no benefit, even though plasma drug levels in the micromolar range were observed (Kordes et al., 2015Kordes S. Pollak M.N. Zwinderman A.H. Mathôt R.A. Weterman M.J. Beeker A. Punt C.J. Richel D.J. Wilmink J.W. Lancet Oncol. 2015; 16: 839-847Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). This underlines the need to take into account both pharmacokinetic and biologic factors in evaluating antineoplastic activity of biguanides. Even in the presence of a potentially adequate plasma metformin concentration, antineoplastic activity likely varies with tumor-related factors, including expression of cell-surface transport molecules important in drug influx and efflux, mutational status of LKB1, and genes encoding mitochondrial complex I components (Algire et al., 2011Algire C. Amrein L. Bazile M. David S. Zakikhani M. Pollak M. Oncogene. 2011; 30: 1174-1182Crossref PubMed Scopus (154) Google Scholar, Birsoy et al., 2014Birsoy K. Possemato R. Lorbeer F.K. Bayraktar E.C. Thiru P. Yucel B. Wang T. Chen W.W. Clish C.B. Sabatini D.M. Nature. 2014; 508: 108-112Crossref PubMed Scopus (484) Google Scholar). Therefore, further studies of pharmacokinetics and mechanism are needed to address the tantalizing possibility that metformin or novel biguanides may have a therapeutic role to play in treatment or prevention of diseases other than diabetes. N.S.C., M.P., C.A., S.C., and A.H. designed the study. C.R.R. and S.E.W. performed the experiments in Figures S1A and S1B. D.A. did the metformin quantitation in Figures S1A and S1B. N.S.C. and M.P. wrote the paper. C.A. edited the paper. S.M. and R.N. performed the experiments in Figures S1C and S1D. This work was supported by awards from the Terry Fox Cancer Research Institute to McGill University and NIH (RO1 CA12306708, PO1AG049665, and RO1 HL122062) to N.S.C., NIH (T32 T32HL076139) to S.E.W., and NIH (T32 HL076139-11) to C.R.R. Download .pdf (.18 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figure S1

Many DNA repair factors act to suppress tumor formation by preserving genomic stability. Similarly, the CtIP protein, which interacts with the BRCA1 tumor suppressor, is also thought to have tumor suppression activity. Through its role in DNA end resection, CtIP facilitates DNA double-strand break (DSB) repair by homologous recombination (DSBR-HR) and microhomology-mediated end joining (MMEJ). In addition, however, CtIP has also been implicated in the formation of aberrant chromosomal rearrangements in an MMEJ-dependent manner, an activity that could potentially promote tumor development by increasing genome instability. To clarify whether CtIP acts in vivo to suppress or promote tumorigenesis, we have examined its oncogenic potential in mouse models of human breast cancer. Surprisingly, mice heterozygous for a null Ctip allele did not display an increased susceptibility to tumor formation. Moreover, mammary-specific biallelic CtIP ablation did not elicit breast tumors in a manner reminiscent of BRCA1 loss. Instead, CtIP inactivation dramatically reduced the kinetics of mammary tumorigenesis in mice bearing mammary-specific lesions of the p53 gene. Thus, unlike other repair factors, CtIP is not a tumor suppressor, but has oncogenic properties that can promote tumorigenesis, consistent with its ability to facilitate MMEJ-dependent chromosomal instability. Consequently, inhibition of CtIP-mediated MMEJ may prove effective against tumor types, such as human breast cancer, that display MMEJ-dependent chromosomal rearrangements.