Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.The survival of metazoan organisms is dependent upon their ability to efficiently generate energy through the process of mitochondrial oxidative phosphorylation in which reducing equivalents, derived from the oxidation of acetyl CoA in the tricarboxylic acid cycle, are transferred from NADH and FADH 2 to the electron transport chain and ultimately to O 2 , a process which produces an electrochemical gradient that is used to synthesize ATP (1). Although oxidative phosphorylation is more efficient than glycolysis in generating ATP, it carries the inherent risk of generating reactive oxygen species (ROS) 2 as a result of electrons prematurely reacting with O 2 at respiratory complex I or complex III. Transient, low level ROS production is utilized for signal transduction in metazoan cells, but prolonged elevations of ROS result in the oxidation of protein, lipid, and nucleic acid leading to cell dysfunction or death.O 2 delivery and utilization must, therefore, be precisely regulated to maintain energy and redox homeostasis.Hypoxia-inducible factor 1 (HIF-1) plays a key role in the regulation of oxygen homeostasis (2, 3). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1␤ subunit and an O 2 -regulated HIF-1␣ subunit (4). Under aerobic conditions, HIF-1␣ is hydroxylated on proline residue 402 and/or 564 by prolyl hydroxylase 2 a dioxygenase that utilizes O 2 and ␣-ketoglutarate as co-substrates with ascorbate as co-factor in a reaction that generates succinate and CO 2 as side products (5-8). Under hypoxic conditions the rate of hydroxylation declines, either as a result of inadequate substrate (O 2 ) or as a result of hypoxia-induced mitochondrial ROS production, which may oxidize Fe(II) in the catalytic center of the hydroxylase (9, 10). Hydroxylated HIF-1␣ is bound by the von HippelLindau protein, which recruits a ubiquitin protein ligase complex that targets HIF-1␣ for proteasomal degradation (11)(12)(13)(14).HIF-1 regulates the transcription of hundreds of genes in response to hypoxia (15, 16), including the EPO (17) and VEGF (18) genes that encode proteins required for erythropoiesis and angiogenesis, respectively, which serve to increase O 2 delivery. In addition, HIF-1 controls a series of molecular mechanisms designed to maintain energy and redox homeostasis. First, HIF-1 coordinates a switch in the composition of cytochrome c oxidase (mitochondrial electron-transp...

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O(2) is the ultimate electron acceptor for mitochondrial respiration, a process catalyzed by cytochrome c oxidase (COX). In yeast, COX subunit composition is regulated by COX5a and COX5b gene transcription in response to high and low O(2), respectively. Here we demonstrate that in mammalian cells, expression of the COX4-1 and COX4-2 isoforms is O(2) regulated. Under conditions of reduced O(2) availability, hypoxia-inducible factor 1 (HIF-1) reciprocally regulates COX4 subunit expression by activating transcription of the genes encoding COX4-2 and LON, a mitochondrial protease that is required for COX4-1 degradation. The effects of manipulating COX4 subunit expression on COX activity, ATP production, O(2) consumption, and reactive oxygen species generation indicate that the COX4 subunit switch is a homeostatic response that optimizes the efficiency of respiration at different O(2) concentrations. Thus, mammalian cells respond to hypoxia by altering COX subunit composition, as previously observed in yeast, but by a completely different molecular mechanism.

Many cancer cells are characterized by increased glycolysis and decreased respiration, even under aerobic conditions. The molecular mechanisms underlying this metabolic reprogramming are unclear. Here we show that hypoxia-inducible factor 1 (HIF-1) negatively regulates mitochondrial biogenesis and O(2) consumption in renal carcinoma cells lacking the von Hippel-Lindau tumor suppressor (VHL). HIF-1 mediates these effects by inhibiting C-MYC activity via two mechanisms. First, HIF-1 binds to and activates transcription of the MXI1 gene, which encodes a repressor of C-MYC transcriptional activity. Second, HIF-1 promotes MXI-1-independent, proteasome-dependent degradation of C-MYC. We demonstrate that transcription of the gene encoding the coactivator PGC-1beta is C-MYC dependent and that loss of PGC-1beta expression is a major factor contributing to reduced respiration in VHL-deficient renal carcinoma cells.

A library of drugs that are in clinical trials or use was screened for inhibitors of hypoxia-inducible factor 1 (HIF-1). Twenty drugs inhibited HIF-1-dependent gene transcription by >88% at a concentration of 0.4 M. Eleven of these drugs were cardiac glycosides, including digoxin, ouabain, and proscillaridin A, which inhibited HIF-1␣ protein synthesis and expression of HIF-1 target genes in cancer cells. Digoxin administration increased latency and decreased growth of tumor xenografts, whereas treatment of established tumors resulted in growth arrest within one week. Enforced expression of HIF-1␣ by transfection was not inhibited by digoxin, and xenografts derived from these cells were resistant to the anti-tumor effects of digoxin, demonstrating that HIF-1 is a critical target of digoxin for cancer therapy.cancer therapy ͉ hypoxia ͉ tumor xenograft

The identification of stem-cell-like cancer cells through conventional methods that depend on stem-cell markers is often unreliable. We developed a mechanical method of selecting tumourigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem-cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe-combined-immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as 10 such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Hypoxia has long been linked to the Warburg effect, yet the underlying mechanism remains largely unclear. It is also not known if lncRNAs are involved in the contribution of hypoxia to the Warburg effect. Here we show that lincRNA-p21 is a hypoxia-responsive lncRNA and is essential for hypoxia-enhanced glycolysis. Hypoxia/HIF-1α-induced lincRNA-p21 is able to bind HIF-1α and VHL and thus disrupts the VHL-HIF-1α interaction. This disassociation attenuates VHL-mediated HIF-1α ubiquitination and causes HIF-1α accumulation. These data indicate the existence of a positive feedback loop between HIF-1α and lincRNA-p21 that promotes glycolysis under hypoxia. The ability of lincRNA-p21 to promote tumor growth is validated in mouse xenograft models. Together, these findings suggest that lincRNA-p21 is an important player in the regulation of the Warburg effect and also implicate lincRNA-p21 as a valuable therapeutic target for cancer.

HIF-1 is a heterodimeric transcription factor that mediates adaptive responses to hypoxia and plays critical roles in cancer progression. Using a cell-based screening assay we have identified acriflavine as a drug that binds directly to HIF-1␣ and HIF-2␣ and inhibits HIF-1 dimerization and transcriptional activity. Pretreatment of mice bearing prostate cancer xenografts with acriflavine prevented tumor growth and treatment of mice bearing established tumors resulted in growth arrest. Acriflavine treatment inhibited intratumoral expression of angiogenic cytokines, mobilization of angiogenic cells into peripheral blood, and tumor vascularization. These results provide proof of principle that small molecules can inhibit dimerization of HIF-1 and have potent inhibitory effects on tumor growth and vascularization.cancer ͉ chemotherapy ͉ hypoxia ͉ xenograft