Hypoxia-associated factor, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leading to its oxygen-independent degradation

The hypoxia-inducible factor 1α (HIF-1α) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1α is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1α in normoxia. Here, we describe a new regulator of HIF-1α, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1α leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1α in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1α levels in normoxia and hypoxia in both pVHL-competent and -deficient cells, whereas HAF knockdown increases HIF-1α levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2α is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1α accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1α-specific degradation pathway that degrades HIF-1α through a new, oxygen-independent mechanism.     

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

Background

The endothelin B receptor (ET_BR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.

Principal Findings

Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET_BR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET_BR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET_BR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ET_BR expression is associated with low PHD2 levels. In melanoma xenografts, ET_BR blockade by ET_BR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.

Conclusions

In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET_BR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.     

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.     

MicroRNA 211 Functions as a Metabolic Switch in Human Melanoma Cells

MicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1α (HIF-1α) protein levels and decreased cell growth during hypoxia. HIF-1α protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1α protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations—a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.     

Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background

Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling.

Methods and Results

Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO₂ = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression.

Conclusion

The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression.     

Adenomatous Polyposis Coli and Hypoxia-inducible Factor-1α Have an Antagonistic Connection

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of β-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α–dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of β-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.     

HIF-1-Dependent Induction of Jumonji Domain-Containing Protein (JMJD) 3 under Hypoxic Conditions

Jumonji domain-containing proteins (JMJD) catalyze the oxidative demethylation of a methylated lysine residue of histones by using O₂, α-ketoglutarate, vitamin C, and Fe(II). Several JMJDs are induced by hypoxic stress to compensate their presumed reduction in catalytic activity under hypoxia. In this study, we showed that an H3K27me3 specific histone demethylase, JMJD3 was induced by hypoxia-inducible factor (HIF)-1α/β under hypoxia and that treatment with Clioquinol, a HIF-1α activator, increased JMJD3 expression even under normoxia. Chromatin immunoprecipitation (ChIP) analyses showed that both HIF-1α and its dimerization partner HIF-1β/Arnt occupied the first intron region of the mouse JMJD3 gene, whereas the HIF-1α/β heterodimer bound to the upstream region of the human JMJD3, indicating that human and mouse JMJD3 have hypoxia-responsive regulatory regions in different locations. This study shows that both mouse and human JMJD3 are induced by HIF-1.     

Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells

Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β₁ integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β₁ integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β₁ integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β₁ integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β₁ integrins.     

Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1α) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1α protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1α level in some normoxic tumor types.