Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway

Adaptation to hypoxic stress provokes activation of the hypoxia-inducible-factor-1 (HIF-1) which mediates gene expression of, e.g., erythropoietin or vascular endothelial growth factor. Detailed information on signaling pathways that stabilize HIF-1 is missing, but reactive oxygen species degrade the HIF-1 alpha subunit, whereas phosphorylation causes its stabilization. It was believed that hypoxia resembles the only HIF-1 inducer but recent evidence characterized other activators of HIF-1 such as nitric oxide (NO). Herein, we concentrated on NO-evoked HIF-1 induction as a heretofore unappreciated inflammatory response in association with massive NO formation. We demonstrated that S-nitrosoglutathione induces HIF-1 alpha accumulation and concomitant DNA binding. The response was attenuated by the kinase inhibitor genistein and blockers of phosphatidylinositol 3-kinase such as Ly 294002 or wortmannin. Whereas mitogen-activated protein kinases were not involved, we noticed phosphorylation/activation of Akt in correlation with HIF-1 alpha stabilization. NO appears to regulate HIF-1 alpha via the PI 3K/Akt pathway under normoxic conditions.     

Regulation of the Hypoxia-Inducible Factor-1alpha (HIF-1alpha): a breath of fresh air in hypoxia research

Angiogenesis, a process that leads to the formation of new blood vessels, from a existing network of vessels is tightly regulated. The understanding of mechanisms that control its activity should lead to progress in the treatment of diseases such as cancer and ischemic disorders. In the case of cancer, the rapid growth of tumor cells results in a decrease in the concentration of oxygen, or hypoxia, in the center of the tumor. This stress is the signal that induces angiogenesis. Blood vessels bring nutrients and oxygen to the tumor, allowing it to grow and to metastase. The Hypoxia-Inducible Factor 1, HIF-1, plays a crucial role in this process. HIF-1 is a heterodimer composed of two subunits, alpha and beta. Under hypoxic conditions, HIF-1alpha is stabilized and enters the nucleus, to form a dimer with HIF-1beta, where it induces the expression of its target genes. Among these genes is vegf (vascular endothelial growth factor), a key player in blood vessel formation. The protein HIF-1alpha is subjected to post-translational modifications that are the molecular basis of the hypoxic response although the mechanisms are not completely understood. In this review, we will discuss in particular the multiple post-translational modifications regulating HIF-1alpha activity.     

1, 9-Pyrazoloanthrones downregulate HIF-1α and sensitize cancer cells to cetuximab-mediated anti-EGFR therapy

Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α.     

Antiangiogenic agents and HIF-1 inhibitors meet at the crossroads

Novel molecularly targeted therapies aim at exploiting oncogenic and non-oncogenic alterations that epitomize potential vulnerable aspects of tumorigenesis, with the hope to ultimately target cancer cells and spare normal tissues. Hypoxia, a decrease in tissue oxygen levels, is a feature of the tumor micro-environment that has attracted considerable interest for its potential contribution to increasing the tumorigenicity of cancer cells, by selecting more aggressive and metastatic clones and by activating pathways that contribute to cancer cells survival, all of which may have important therapeutic implications. In this article, we discuss how two therapeutic strategies, which have been developed over the last few years to target aspects dependent on or associated with intratumor hypoxia, may provide the rationale for a novel combination strategy aimed at blocking compensatory circuits that maintain cancer cells survival and propagate the cancer phenotype. We hypothesized that concurrent inhibition of HIF-1 and VEGF, which are mechanistically linked to intratumor hypoxia, represents a logical therapeutic combination that may find applications in a number of solid tumors, irrespective of their underlying genetic alterations. Indeed, intrinsic limitations of HIF-1 inhibitors and mechanisms of acquired resistance to anti-VEGF therapies may counterbalance each other in combination approaches that block vicious compensatory pathways exploited by cancer cells to overcome environmental stresses.

     

Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein.     

HIF-1 regulation: not so easy come, easy go

The hypoxia-inducible factor-1 (HIF-1) is the master regulator of the cellular response to hypoxia and its expression levels are tightly controlled through synthesis and degradation. It is widely accepted that HIF-1alpha protein accumulation during hypoxia results from inhibition of its oxygen-dependent degradation by the von Hippel Lindau protein (pVHL) pathway. However, recent data describe new pVHL- or oxygen-independent mechanisms for HIF-1alpha degradation. Furthermore, the hypoxia-induced increase in HIF-1alpha levels is facilitated by the continued translation of HIF-1alpha during hypoxia despite the global inhibition of protein translation. Recent work has contributed to an increased understanding of the mechanisms that control the translation and degradation of HIF-1alpha under both normoxic and hypoxic conditions.