The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1␣ exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1␣-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.Decreased environmental oxygen forces cells and tissues to adapt in multiple ways. In response to hypoxia, a significant number of changes in gene expression occur, resulting in elevated transcription of angiogenic factors, hematopoietic factors, and some metabolic enzymes (21). The switch between the two forms of respiration utilized by animal cells, aerobic versus anaerobic, was first noted by Pasteur in the late 19th century (12,22). As the oxygen level decreases, the generation of ATP shifts from the oxidative phosphorylation pathway in the mitochondria to the oxygen-independent pathway of glycolysis in the cytoplasm. Although glycolysis is less efficient than oxidative phosphorylation in the generation of ATP, in the presence of sufficient glucose glycolysis can sustain ATP production due to increases in the activity of the glycolytic enzymes (12,22). Perhaps nowhere has this forced adaptation been the focus of so much study as in transformed cells; this is because in solid tumors it is clear that a large percentage of the cell population is at least transiently hypoxic (1).Earlier in the 20th century, Otto Warburg demonstrated that tumors differed from normal tissues in their utilization of the glycolytic pathway (26). For a given amount of glucose, tumor fragments ex vivo produced far more lactate than sections of nontransformed tissues under normoxic conditions. In vivo the situation is likely to be more complex. Within individual tumors, there are some areas that may respond to hypoxia by exhibiting the normal physiological switch to glycolysis similar to that employed by all nontransformed cells in response to lowered oxygen levels. Concurrently, many other areas of transformed cells in solid tumors may adapt to h...

To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.

The Hexosamine Biosynthetic Pathway leads to elevated post-translation addition of O-linked-βN-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation in cancer cells inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via HIF-1α and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation and interaction with VHL resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of ER stress and apoptosis of cancer cells mediated through CHOP induction of BCL2-family proteins. HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects and apoptosis. Human basal-like breast cancers with high levels of HIF-1α contain elevated OGT, O-GlcNAcylation and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.

Adaptation to hypoxia is a critical step in tumor progression and is, in part, regulated by the transcription factor hypoxiainducible factor-1A (HIF-1A). Xenograft models have been extensively used to characterize the role of HIF-1A in experimental cancers. Although these models provide an understanding of tumor growth at terminal stages of malignancy, they do not address tumor initiation or metastatic progression. To elucidate these roles, HIF-1A was conditionally deleted in the mammary epithelium of a transgenic mouse model for metastatic breast cancer. Conditional deletion of HIF-1A in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth; this was correlated with decreased tumor cell proliferation. Tumors with conditional deletion of HIF-1A were also less vascular during early tumor progression. Perhaps most surprisingly, deletion of HIF-1A in the mammary epithelium resulted in decreased pulmonary metastasis. These results show that whereas HIF-1A is not required for the initiation of breast tumor growth or tumor cell metastasis, the transcriptional activity of HIF-1A is a significant positive regulator of tumor progression and metastatic potential. [Cancer Res 2007;67(2):563-72]