Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I.DOI: http://dx.doi.org/10.7554/eLife.02242.001

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Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

Approximately 85% of lung cancers are non-small-cell lung cancers (NSCLCs), which are often diagnosed at an advanced stage and associated with poor prognosis. Currently, there are very few therapies available for NSCLCs due to the recalcitrant nature of this cancer. Mutations that activate the small GTPase KRAS are found in 20% to 30% of NSCLCs. Here, we report that inhibition of superoxide dismutase 1 (SOD1) by the small molecule ATN-224 induced cell death in various NSCLC cells, including those harboring KRAS mutations. ATN-224-dependent SOD1 inhibition increased superoxide, which diminished enzyme activity of the antioxidant glutathione peroxidase, leading to an increase in intracellular hydrogen peroxide (H 2 O 2 ) levels. We found that ATN-224-induced cell death was mediated through H 2 O 2 -dependent activation of P38 MAPK and that P38 activation led to a decrease in the antiapoptotic factor MCL1, which is often upregulated in NSCLC. Treatment with both ATN-224 and ABT-263, an inhibitor of the apoptosis regulators BCL2/BCLXL, augmented cell death. Furthermore, we demonstrate that ATN-224 reduced tumor burden in a mouse model of NSCLC. Our results indicate that antioxidant inhibition by ATN-224 has potential clinical applications as a single agent, or in combination with other drugs, for the treatment of patients with various forms of NSCLC, including KRAS-driven cancers. IntroductionLung cancer is the leading cause of cancer deaths in the United States and worldwide. Non-small-cell lung cancers (NSCLCs) represent 80% of all lung cancers and are often diagnosed at an advanced stage with poor prognosis. Adenocarcinoma, a subtype of NSCLC, is the most common form of lung cancer and is characterized by activating mutations in the KRAS proto-oncogene in 20% to 30% of cases and by inactivating mutations in the tumor suppressor TP53 in 50% of cases (1).With the goal of identifying new therapies for NSCLCs, a largescale chemical screen recently identified a small molecule that selectively induced cell death in oncogenic KRAS-driven (KRAS onc -driven) cancer cells compared with normal cells. The small molecule induced cell death by increasing intracellular ROS (2). However, the mechanism by which ROS was induced and caused cell death has not yet been established. Also, it is known that oncogenic-driven cancer cells generate increased ROS as byproducts of their augmented metabolism to promote and maintain tumorigenicity (3-5). Since high levels of ROS can induce cell death (2, 6), cancer cells adapt to ROS stress by upregulating intracellular antioxidant proteins (7-10) in order to maintain ROS levels that allow protumorigenic signaling without inducing cell death. This reliance on antioxidants potentially makes cancer cells selectively vulnerable to antioxidant inhibition, as nontransformed cells generate lower basal levels of ROS and are therefore less dependent on their detoxification. In fact, studies have shown that disabling antioxidant mechanisms triggers ROS-mediated cell death in a variety of cancer cell ty...