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Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here we show that NEAT1 lncRNA is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to A-to-I editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1 – JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer.

Context Renal cancer is a common urologic malignancy, and therapeutic options for metastatic disease are limited. Most clear cell renal cell carcinomas (ccRCC) are associated with loss of von Hippel-Lindau tumor suppressor (pVHL) function and deregulation of hypoxia pathways. Objective This review summarizes recent evidence from genetic and biological studies showing that hypoxia and hypoxia-related pathways play critical roles in the development and progress of renal cancer. Evidence acquisition We used a systematic search for articles using the keywords hypoxia, HIF, renal cancer, and VHL. Evidence synthesis Identification of the tumor suppressor pVHL has allowed the characterization of important ccRCC-associated pathways. pVHL targets α-subunits of hypoxia-inducible transcription factors (HIF) for proteasomal degradation. The two main HIF-α isoforms have opposing effects on RCC biology, possibly through distinct interactions with additional oncogenes. Furthermore, HIF-1α activity is commonly diminished by chromosomal deletion in ccRCCs, and increased HIF-1 activity reduces tumor burden in xenograft tumor models. Conversely, polymorphisms at the HIF-2α gene locus predispose to the development of ccRCCs, and HIF-2α promotes tumor growth. Genetic studies have revealed a prominent role for chromatin-modifying enzyme genes in ccRCC, and these may further modulate specific aspects of the HIF response. This suggests that, rather than global activation of HIF, specific components of the response are important in promoting kidney cancer. Some of these processes are already targets for current therapeutic strategies, and further dissection of this pathway might yield novel methods of treating RCC. Conclusions In contrast to many tumor types, HIF-1α and HIF-2α have opposing effects in ccRCC biology, with HIF-1α acting as a tumor suppressor and HIF-2α acting as an oncogene. The overall effect of VHL inactivation will depend on fine-tuning of the HIF response. Patient summary High levels of hypoxia-inducible transcription factors (HIF) are particularly important in the clear cell type of kidney cancer, in which they are no longer properly regulated by the von Hippel-Lindau protein. The two HIF-α proteins have opposing effects on tumor evolution.

Hypoxia is central to both ischaemic and neoplastic diseases. However, the non-coding transcriptional response to hypoxia is largely uncharacterized. We undertook integrated genomic analyses of both non-coding and coding transcripts using massively parallel sequencing and interfaced this data with pan-genomic analyses of hypoxia-inducible factor (HIF) and RNApol2 binding in hypoxic cells. These analyses revealed that all classes of RNA are profoundly regulated by hypoxia and implicated HIF as a major direct regulator of both the non-coding and coding transcriptome, acting predominantly through release of pre-bound promoter-paused RNApol2. These findings indicate that the transcriptional response to hypoxia is substantially more extensive than previously considered.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.Constant availability of molecular oxygen is crucial for the structure and function of any mammalian cell. Therefore, cellular responses to reduced oxygen tensions (hypoxia) play an important role in development and many aspects of physiological homeostasis. Many important disease processes, including ischemic vascular diseases and cancer, involve reduced tissue oxygenation, and cellular adaptation to this is implicated in disease progression and clinical outcome. The hypoxia-inducible transcription factor (HIF) 2 is a central mechanism responding to low cellular oxygenation and mediates a variety of systemic and local adaptive responses, including the control of red cell production, regulation of angiogenesis, modulation of vascular tone, enhancement of glycolysis, and cellular glucose uptake (for a review, see Refs. 1-3). HIF consists of a heterodimer of ␣-and ␤-subunits, both being basic helix-loop-helix-Per Arnt Sim domain proteins. Whereas HIF␤ is constitutively expressed, HIF␣ subunits are unstable and inversely correlated to the availability of molecular oxygen. At least two oxygen-regulated isoforms of HIF␣ have been identified, HIF-1␣ and HIF-2␣, sharing a high degree of sequence homology and a similar domain structure (4). Regulation of HIF is primarily governed by oxygen-dependent hydroxylation of its HIF␣ subunits, which influences protein stability and transcriptional activity.Growth and behavior of tumor cells is strongly dependent on their microenvironment, where hypoxia is both a stress factor and an important signal (for a review, see Refs. 5 and 6). Dating back to 1927, Otto Warburg had already described that tumor cells have a much increased utilization of the glycolytic pathway (7). Since then, a number of studies have established that indeed HIF is necessary to activate glycolysis in tumor cells in order to maintain energy homeosta...

Hypoxia-inducible factors (HIFs) transduce transcriptional responses to hypoxia that involve hundreds to thousands of target genes. • The oxygen-sensitive signal regulating HIF activity is generated by 2-oxoglutarate-dependent dioxygenases that catalyze the hydroxylation of specific HIF prolyl and asparaginyl residues to inactivate HIF in the presence of oxygen. • Inhibition of the HIF prolyl hydroxylases by 2-oxoglutarate analogues mimics hypoxia and activates many, but not all, components of the HIF transcriptional response. • Erythropoietin production by cortical interstitial fibroblasts in the kidney is very sensitive to activation of the HIF pathway. • In diseased kidneys, erythropoietin production is reduced, but can be increased by HIF prolyl hydroxylase inhibitors. • Activation of HIF has the potential to generate many other renal and systemic effects that will require consideration when HIF prolyl hydroxylase inhibitors are used clinically.

Though genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma1, modulates the binding and function of hypoxia inducible factor (HIF) at a previously unrecognized, transcriptional enhancer of cyclin D1 specific for renal cancers characterized by pVHL inactivation. The protective haplotype impairs binding of HIF-2 resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control.

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1–3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.