Mammalian cellular responses to hypoxia include adaptive metabolic changes and a G 1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G 1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G 1 /S checkpoint, we observed that the retinoblastoma protein is essential for the G 1 /S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G 1 checkpoint, suggesting that it is a key regulator of G 1 /S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G 1 /S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension.Cellular hypoxia is an environmental stress with important implications in developmental biology, normal physiology, and many pathological conditions, including cancer. Normal tissues display an oxygen gradient across a distance of 400 m from a blood supply; tumors often have disordered and diminished vascularization, and hypoxia occurs in tumor tissue that is Ͼ100 -200 m away from a functional blood supply (1-3). Cells may adapt to hypoxia in numerous ways, including a transition from oxidative phosphorylation to glycolysis and neovascularization. Many of these metabolic responses are mediated by the transcription factor HIF-1, 1 a heterodimer of a hypoxia-induced subunit HIF-1␣ and a constitutive subunit HIF-1␤ (ARNT), which transactivates genes encoding several glycolytic enzymes as well as the vascular endothelial growth factor gene (4 -7). Cells may also respond to hypoxia by diminishing their proliferative rates. Both invasive and noninvasive studies of a variety of normal tissues and tumors suggest that hypoxic cells may be viable but nonproliferating (8 -12). This low proliferative state may be related to the phenomenon of tumor dormancy, described in nonvascularized metastatic foci (13), and may also help explain why hypoxic tumors are relatively chemoresistant and radioresistant (14). Although some transformed cell lines undergo apoptosis in extreme hypoxia and an acidic environment, nontransformed hypoxic cells remain viable but arrest in G 1 (15)(16)(17).The best characterized molecular event necessary for the G 1 /S phase transition is phosphorylation of the retinoblastoma protein (RB) by specific cyclin-dependent kinase (CDK)-cyclin complexes (18). CDK activity can be inhibited by cyclin-dependent kinase inhibitors (CDKIs), such as p27 and p21, which then promote RB hypophosphorylation. Th...

Nonsense-mediated RNA decay (NMD) rapidly degrades both mutated mRNAs and nonmutated cellular mRNAs in what is thought to be a constitutive fashion. Here we demonstrate that NMD is inhibited in hypoxic cells and that this inhibition is dependent on phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣). eIF2␣ phosphorylation is known to promote translational and transcriptional up-regulation of genes important for the cellular response to stress. We show that the mRNAs of several of these stress-induced genes are NMD targets and that the repression of NMD stabilizes these mRNAs, thus demonstrating that the inhibition of NMD augments the cellular stress response. Furthermore, hypoxia-induced formation of cytoplasmic stress granules is also dependent on eIF2␣ phosphorylation, and components of the NMD pathway are relocalized to these granules in hypoxic cells, providing a potential mechanism for the hypoxic inhibition of NMD. Our demonstration that NMD is inhibited in hypoxic cells reveals that the regulation of NMD can dynamically alter gene expression and also establishes a novel mechanism for hypoxic gene regulation.Cellular hypoxia, a stress common in development, cancer, and numerous other pathological conditions, affects metabolism, proliferation, apoptosis, and differentiation in a cell-specific manner. Much of the work to determine the mechanism for hypoxia-induced phenotypes has emphasized the role of transcriptional activation mediated by the hypoxia-inducible transcription factor 1. However, multiple studies have demonstrated that hypoxic cells may also regulate gene expression through a variety of transcriptional activators and repressors as well as through translational and posttranslational mechanisms (reviewed in references 27 and 37). In addition, several mRNAs, including the mRNA for vascular endothelial growth factor, have been shown to be stabilized in hypoxic cells through AU-rich sequences in their 3Ј untranslated regions (UTR) (24). The exact motifs and trans-acting elements responsible for RNA stabilization in hypoxic cells are not wellcharacterized, however, and the extent, mechanism, and significance of hypoxic stabilization of mRNAs are not known.Nonsense-mediated RNA decay (NMD) is a multistep intricate pathway by which mRNAs with premature termination codons (PTCs) are targeted for rapid destruction. Recent studies have provided insight into the mechanism of NMD in mammalian cells (reviewed in references 22 and 10). After mRNA splicing, exon-exon junctions are marked by an exon junction complex. The most common characteristic of mRNAs degraded by NMD is a premature termination codon located upstream of an exon junction complex, a feature not typically found in "normal" cellular mRNAs and thus signifying a mutation. If the ribosome does not traverse the exon junction complex because of the presence of a premature termination codon, this complex is able to recruit a series of enzymes, including the Rent1/Upf1 helicase, which target the mRNA for degradation. Knockdown o...

While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2␣ phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumorpromoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis.During tumorigenesis, a disorganized vasculature leads to amino acid and glucose deprivation, cellular hypoxia, the accumulation of reactive oxygen species (ROS), and various other stresses (5, 12). Cellular adaptation to the hostile tumor microenvironment requires the regulation of stress-induced genes (reviewed in reference 16). For example, the transcription factor ATF-4, upregulated in human tumors due to the stress-induced phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF2␣), transactivates genes involved in amino acid metabolism, angiogenesis, and ROS attenuation (2,3,33). Cells that cannot phosphorylate eIF2␣ or that are deficient in ATF-4 and other stress-induced transcription factors do not form tumors in vivo (2, 13, 40), and therefore, a major goal in cancer biology has been to better understand and potentially target these adaptive mechanisms. However, while the translational and transcriptional responses that promote adaptation to the tumor microenvironment are well established, the role of mRNA stabilization in the cellular stress response has not been as thoroughly studied.Nonsense-mediated RNA decay (NMD) degrades up to 30% of all mutated protein-coding mRNAs, including those responsible for many genetic disorders, such as thalassemia, cystic fibrosis, and muscular dystrophy (11). During the processing of mammalian pre-mRNA, introns are excised and marked by an exon junction complex, which contains core NMD components. Newly synthesized mRNAs are thought to undergo a pioneering round of translation by a complex that includes eIF2␣ (6). When this translation complex pauses at a premature termination codon (PTC) upstream of an exon junction complex, the RNA helicase UPF1/Rent1, an essential component of the NMD process, is recruited and the...

The progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intra-tumoral immune tolerance are uncertain. Dectin-1 is an innate immune receptor critical in anti-fungal immunity, but its role in sterile inflammation and oncogenesis is not well-defined. Further, non-pathogen-derived ligands for Dectin-1 have not been characterized. We found that Dectin-1 is highly expressed on macrophages in pancreatic ductal adenocarcinoma (PDA). Dectin-1 ligation accelerated PDA, whereas Dectin-1 deletion or blockade of its downstream signaling was protective. We found that Dectin-1 ligates the lectin Galectin-9 in the PDA tumor microenvironment resulting in tolerogenic macrophage programming and adaptive immune suppression. Upon interruption of the Dectin-1–Galectin-9 axis, CD4+ and CD8+ T cells – which are dispensable to PDA progression in hosts with an intact signaling axis – become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that targeting Dectin-1 signaling is an attractive strategy for the immunotherapy of PDA.

Rationale Human atherosclerotic plaques contain large numbers of cells deprived of O2. In murine atherosclerosis, because the plaques are small, it is controversial whether hypoxia can occur. Objective To examine if murine plaques contain hypoxic cells, and whether hypoxia regulates changes in cellular lipid metabolism and gene expression in macrophages. Methods and Results Aortic plaques from apolipoprotein-E-deficient mice were immunopositive for hypoxia-inducible transcription factor (HIF-1α) and some of its downstream targets. Murine J774 macrophages rendered hypoxic demonstrated significant increases in cellular sterol and triglycerides. The increase in sterol content in hypoxic macrophages correlated with elevated 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity and mRNA levels. In addition, when macrophages were incubated with cholesterol complexes, hypoxic cells accumulated 120% more cholesterol, predominately in the free form. Cholesterol-efflux assays showed that hypoxia significantly decreased efflux mediated by ATP binding cassette sub-family A member 1 (ABCA1), whose sub cellular localization was altered in both J774 and primary macrophages. Furthermore, in vivo expression patterns of selected genes from cells in hypoxic regions of murine plaques were similar to those from J774 and primary macrophages incubated in hypoxia. The hypoxia-induced accumulation of sterol and decreased cholesterol efflux was substantially reversed in vitro by reducing the expression of the hypoxia-inducible transcription factor, HIF-1α. Conclusion Hypoxic regions are present in murine plaques. Hypoxic macrophages have increased sterol content due to the induction of sterol synthesis and the suppression of cholesterol efflux, effects that are in part mediated by HIF-1α.

Nonsense-mediated mRNA decay (NMD) is an mRNA quality control mechanism that destabilizes aberrant mRNAs harboring premature termination (nonsense) codons (PTCs). Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes. This raises the possibility that NMD itself is under regulatory control. Indeed, several recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including microRNA circuits and NMD itself. Cellular stress also modulates the magnitude of NMD by mechanisms that are beginning to be understood. Here, we review the evidence that NMD is regulated and discuss the physiological role for this regulation. We propose that the efficiency of NMD is altered in some cellular contexts to regulate normal biological events. In disease states—such as in cancer—NMD is disturbed by intrinsic and extrinsic factors, resulting in altered levels of crucial NMD-targeted mRNAs that lead to downstream pathological consequences.

Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTCs) and the rapid degradation of their mRNAs by nonsense mediated RNA decay (NMD). We used virtual library screening (VLS) targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD targeted mRNAs at nanomolar concentrations with minimal toxicity in cell based assays. As expected, pharmacological NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC mutated p53, pharmacological NMD inhibition combined with a PTC “read-through” drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacological NMD inhibitors can restore mRNA integrity in the presence of PTC and be used as part of a strategy to restore full length protein in a variety of genetic diseases.

Hypoxia-inducible factor-1A (HIF-1A) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1A is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1A can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1A is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1A in nonhypoxiamediated cancer cell proliferation remains speculative. We have disrupted HIF-1a by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1A significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1A expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1A promoted cell proliferation and survival under hypoxia in vitro.