HIF-1 Target Genes Research Articles

Abstract 2998: MLN4924 inhibition of Cullin-Ring ligases activates a hypoxic response and secretion of tumor promoting factors in human MCF7 breast cancer cells

Abstract Hypoxia-inducible factors (HIFs) are the key transcription factors that mediate the cellular response to low oxygen tension. During normoxia, HIF activity is tightly controlled by its oxygen-dependent prolyl hydroxylation which allows polyubiquitination by von Hippel-Landau tumor suppressor (VHL), leading to HIF degradation by the ubiquitin-proteasome pathway. During hypoxia, low oxygen tension prevents HIF prolyl hydroxylation resulting in HIF accumulation and subsequent transcription of HIF target genes. VHL is a known Cullin-RING E3 ubiquitin ligase (CRL) that is dependent on neddylation of the Cul-2 subunit by NEDD8 Activating Enzyme (NAE) for its enzymatic activity. The small molecule inhibitor MLN4924 inhibits NAE neddylation of Cullins by covalently binding the nucleotide-binding site of NAE. HIF-1α is a well-known CRL substrate that increases in abundance upon MLN4924 treatment. We investigated the response of MCF-7 human breast cancer cells to MLN4924 treatment compared to hypoxia (1% O2, 48 hours) treatment. Cell lysates tested using the Human Oncology Antibody Array and Western blot showed that both treatments induced accumulation of HIF-1α protein, with protein levels in MLN4924 treated cells greater than levels in hypoxia treated cells. This correlated with the expected attenuation of HIF-1α ubiquitination as detected by the Human Ubiquitin Antibody Array. The specificity of MLN4924 targeting Cullin proteins was tested using Western blots. The neddylation of the VHL subunit Cul-2, as well as Cul-1, was not detectable in MLN4924 treated cells, but was readily detectable in hypoxia treated cells. Since MLN4924 was reported to inhibit mTORC1 activity, Western blots were used to evaluate signaling of the mTORC1 complex. Thr-389 phosphorylation of p70 S6K was not detectable upon treatment with MLN4924 and attenuated in hypoxia treated cells. Thr-389 phosphorylated p70 S6K was not detectable in Rapamycin-treated cells, a potent mTORC1 inhibitor. The relative protein levels of several known HIF-1α target genes were tested using the Human Angiogenesis Antibody Array. VEGF was detected in cell culture supernates from normoxia, hypoxia, and MLN4924 treated cells. ELISAs were used to measured VEGF and Carbonic Anhydrase IX and were found to be highest in MLN4924 treated cells. Interestingly, these same array profiles detected tumor promoting factors Angiogenin, Amphiregulin, Endothelin 1, and PDGF-AA in cell supernates from MLN4924 treated cells at higher levels than hypoxia treated MCF-7 cells. Recent reports found MLN4924 to inhibit tumor angiogenesis in mouse models, indicating that the effects of MLN4924 on secretion of tumor promoting factors by MCF-7 cells may be context dependent. Citation Format: James Rivard, Amy James, Erin Eleria, Neal Lee, Kathy Brumbaugh, Greta Wegner, Kevin Reagan. MLN4924 inhibition of Cullin-Ring ligases activates a hypoxic response and secretion of tumor promoting factors in human MCF7 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2015-2998

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis.

BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

The importin α/β-specific inhibitor Ivermectin affects HIF-dependent hypoxia response pathways.

Hypoxia-inducible transcription factors (HIFs) regulate hundreds of genes involved in cellular adaptation to reduced oxygen availability. HIFs consist of an O2-labile α-subunit (primarily HIF-1α and HIF-2α) and a constitutive HIF-1β subunit. In normoxia the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1-3, resulting in recognition by von Hippel-Lindau protein, ubiquitination and proteasomal degradation. In contrast, reduced oxygen availability inhibits PHD activity resulting in HIF-1α stabilisation and nuclear accumulation. Nuclear import of HIF-1α mainly depends on classical nuclear localisation signals (NLS) and involves importin α/β heterodimers. Recently, a specific inhibitor of nuclear import has been identified that inhibits importin α/β-dependent import with no effects on a range of other nuclear transport pathways involving members of the importin protein family. In this study we evaluated the physiological activity of this importin α/β-inhibitor (Ivermectin) in the hypoxia response pathway. Treatment with Ivermectin decreases binding activity of HIF-1α to the importin α/β-heterodimer. Moreover, HIF-1α nuclear localisation, nuclear HIF-1α protein levels, HIF-target gene expression, as well as HIF-transcriptional activity are reduced upon Ivermectin treatment. For the first time, we demonstrate the effect of specific importin α/β-inhibition on the hypoxic response on the molecular level.

Bortezomib attenuates HIF-1- but not HIF-2-mediated transcriptional activation.

Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the α subunit of hypoxia-inducible factor (HIF)-1 is not degraded with bortezomib treatment, the heterodimeric HIF-1 fails to transactivate target genes. HIF-1 and HIF-2 are related hypoxia-inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIF-1, but not HIF-2. The present study investigated the effects of bortezomib on HIF-2 activity in cancer cells with different levels of HIF-1α and HIF-2α subunits. HIF-α subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription-polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIF-1-specific target gene; however, it had minimal effects on EPO and GLUT-1, which are target genes of both HIF-1 and HIF-2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF-1, and not HIF-2. This novel finding on the lack of an inhibitory effect of bortezomib on HIF-2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs.

Oxygen sensing in intestinal mucosal inflammation.

Hypoxia is a hallmark of chronically inflamed tissue. Hypoxia develops from vascular dysfunction and increased oxygen consumption by infiltrating leukocytes. With respect to inflammatory bowel disease (IBD), hypoxia is likely to be of particular importance: Impairment of the intestinal barrier during IBD allows anoxia from the lumen of the gut to spread to formerly normoxic tissue. In addition, disturbed perfusion of inflamed tissue and a higher oxygen demand of infiltrating immune cells lead to low oxygen levels in inflamed mucosal tissue. Here, cells become hypoxic and must now adapt to this condition. The hypoxia inducible factor (HIF)-1 complex is a key transcription factor for cellular adaption to low oxygen tension. HIF-1 is a heterodimer formed by two subunits: HIF-α (either HIF-1α or HIF-2α) and HIF-1β. Under normoxic conditions, hydroxylation of the HIF-α subunit by specific oxygen-dependent prolyl hydroxylases (PHDs) leads to ubiquitin proteasome-dependent degradation. Under hypoxic conditions, however, PHD activity is inhibited; thus, HIF-α can translocate into the nucleus, dimerize with HIF-1β, and bind to hypoxia-responsive elements of HIF-1 target genes. So far, most studies have addressed the function of HIF-1α in intestinal epithelial cells and the effect of HIF stabilization by PHD inhibitors in murine models of colitis. Furthermore, the role of HIF-1α in immune cells becomes more and more important as T cells or dendritic cells for which HIF-1 is of critical importance are highly involved in the pathogenesis of IBD. This review will summarize the function of HIF-1α and the therapeutic prospects for targeting the HIF pathway in intestinal mucosal inflammation.

9B.09

Objective: Pheochromocytomas and paragangliomas (PPGL) are relatively rare and mostly benign tumours. Approximately 10% of PPGL are malignant, as defined by the presence of metastases, i.e chromaffin tissue at a location that usually does not contain chromaffin cells. However, up to 35% of tumours in patients carrying an SDHB mutation appears to be malignant. Nowadays, no reliable marker allows to predict whether a PPGL is, or will become malignant. In addition, there are no curative treatments if metastases occur. The aim of the present study was to dentify genetic markers allowing to distinguish benign from malignant tumours. Design and method: An mRNA expression array was performed on benign and malignant PPGL. The genes showing a different expression between the benign and malignant tumours were selected to be confirmed and validated by qRT-PCR. Finally, the remaining genes were stained by immunohistochemistry on Tissue MicroArray including a large series of PPGL. Results: Forty benign and 12 malignant PPGL were investigated for differences in mRNA expression with Affymetrix arrays. Expression data were normalized according to Affymetrix recommendations. Then, using Pomelo II (http://pomelo2.bioinfo.cnio.es/), a Limma t-test was performed, to assess which genes were differentially expressed between benign and malignant PPGL. First, a non-clustered analysis was performed and 10 genes with a False Discovery Rate (FDR) below 0.05 and a relative overexpression ratio of at least 4 were found, including Interleukin 13 Receptor alpha 2 (IL13RA2) and Monooxygenase DBH-like 1 (MOXD1). Secondly, a supervised cluster analysis was performed (based on HIF target genes), resulting in 2 groups, which were both investigated for differences in mRNA expression between benign and malignant tumours. Five genes showed an FDR below 0.01 and were overexpressed in malignant tumours with a ratio higher than 4, including Contactin 4 (CNTN4), Iroquois Homeobox 3 (IRX3), and Sulfatase 2 (SULF2). These genes were further investigated using qRT-PCR, and immunohistochemistry on Tissue Micro Array including 91 benign and 12 malignant PPGL. Conclusions: Significant overexpression of Contactin 4 was shown in malignant compared to benign tumours, and may therefore contribute to distinguish malignant from benign PPGL.

HIF-1α and TAZ serve as reciprocal co-activators in human breast cancer cells.

Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality. Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization. Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α. Inhibition of TAZ expression impairs the hypoxic induction of HIF-1 target genes, such as PDK1, LDHA, BNIP3 and P4HA2 in response to hypoxia, whereas inhibition of HIF-1α expression impairs TAZ-mediated transactivation of the CTGF promoter. Taken together, these results complement our previous findings and establish bidirectional crosstalk between HIF-1α and TAZ that increases their transcriptional activities in hypoxic cells.

Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.

Muc1 is protective during kidney ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

Pharmacological HIF2α inhibition improves VHL disease-associated phenotypes in zebrafish model.

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

Endothelin-converting enzyme is a plausible target gene for hypoxia-inducible factor

Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.

HIF-prolyl hydroxylase is a potential molecular target for esculetin-mediated anti-colitic effects

We investigated a potential molecular target for anti-colitic effects of esculetin, 6,7-dihydroxycoumarin. Esculetin administered rectally effectively ameliorated TNBS-induced rat colitis and attenuated the expression of pro-inflammatory mediators in the inflamed colon. In human colon carcinoma HCT116 cells, esculetin induced hypoxia-inducible factor-1α (HIF-1α), leading to secretion of vascular endothelial growth factor, a HIF-1 target gene product involved in ulcer healing of the gastrointestinal mucosa. Esculetin directly inhibited HIF prolyl hydroxylase-2 (HPH-2), an enzyme playing a major role in negatively regulating HIF-1α protein stability. Esculetin inhibition of HPH and consequent induction of HIF-1α were attenuated by escalating dose of either ascorbate or 2-ketoglutarate, the required factors of the enzyme. Structurally, the catechol moiety in esculetin was required for HPH inhibition. Collectively, HPH may be a molecular target for esculetin-mediated anti-colitic effects and the catechol moiety in esculetin is the pharmacophore for HPH inhibition.

HIF1α Regulates mTOR Signaling and Viability of Prostate Cancer Stem Cells.

Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1α (HIF1A/HIF1α) in CSCs and a basal stem cell subpopulation (Lin(-)/Sca-1(+)/CD49f(+)) was identified, in primary prostate tumors of mice, with elevated HIF1α expression. To further analyze the consequences of hypoxic upregulation on stem cell proliferation and HIF1α signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1(+)/CD49f(+)) as well as from a human prostate cancer cell line (CD44(+)/CD49f(+)) were isolated and characterized. HIF1α levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Interestingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1α upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1α is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer.

Differential responses of blood-brain barrier associated cells to hypoxia and ischemia: a comparative study

BackgroundUndisturbed functioning of the blood–brain barrier (BBB) crucially depends on paracellular signaling between its associated cells; particularly endothelial cells, pericytes and astrocytes. Hypoxic and ischemic injuries are closely associated with disturbed BBB function and the contribution of perivascular cells to hypoxic/ischemic barrier regulation has gained increased attention. Regardless, detailed information on the basal hypoxic/ischemic responses of the barrier-associated cells is rare and the outcome of such cell-specific responses on BBB modulation is not well understood. This study investigated crucial parameters of hypoxic/ischemic adaptation in order to characterize individual perivascular cell responses to stress conditions.MethodsThe brain microvascular endothelial cell line RBE4 (EC cell line) as well as primary rat brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were exposed to 24 and 48 hours of oxygen deprivation at 1% and 0.2% O2. All primary cells were additionally subjected to combined oxygen and glucose deprivation mimicking ischemia. Central parameters of cellular adaptation and state, such as HIF-1α and HIF-1 target gene induction, actin cytoskeletal architecture, proliferation and cell viability, were compared between the cell types.ResultsWe show that endothelial cells exhibit greater responsiveness and sensitivity to oxygen deprivation than ACs and PCs. This higher sensitivity coincided with rapid and significant stabilization of HIF-1α and its downstream targets (VEGF, GLUT-1, MMP-9 and PHD2), early disruption of the actin cytoskeleton and metabolic impairment in conditions where the perivascular cells remain largely unaffected. Additional adaptation (suppression) of proliferation also likely contributes to astrocytic and pericytic tolerance during severe injury conditions. Moreover, unlike the perivascular cells, ECs were incapable of inducing autophagy (monitored via LC3-II and Beclin-1 expression) - a putative protective mechanism. Notably, both ACs and PCs were significantly more susceptible to glucose than oxygen deprivation with ACs proving to be most resistant overall.ConclusionIn summary this work highlights considerable differences in sensitivity to hypoxic/ischemic injury between microvascular endothelial cells and the perivascular cells. This can have marked impact on barrier stability. Such fundamental knowledge provides an important foundation to better understand the complex cellular interactions at the BBB both physiologically and in injury-related contexts in vivo.

Functional regulation of hypoxia inducible factor-1α by SET9 lysine methyltransferase

HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.

Depletion of PHD3 protects heart from ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis

PHD3, a member of a family of Prolyl-4 Hydroxylase Domain (PHD) proteins, has long been considered a pro-apoptotic protein. Although the pro-apoptotic effect of PHD3 requires its prolyl hydroxylase activity, it may be independent of HIF-1α, the common substrate of PHDs. PHD3 is highly expressed in the heart, however, its role in cardiomyocyte apoptosis remains unclear. This study was undertaken to determine whether inhibition or depletion of PHD3 inhibits cardiomyocyte apoptosis and attenuates myocardial injury induced by ischemia–reperfusion (I/R). PHD3 knockout mice and littermate controls were subjected to left anterior descending (LAD) coronary artery ligation for 40min followed by reperfusion. Histochemical analysis using Evan's Blue, triphenyl-tetrazolium chloride and TUNEL staining, demonstrated that myocardial injury and cardiomyocyte apoptosis induced I/R injury were significantly attenuated in PHD3 knockout mice. PHD3 knockout mice exhibited no changes in HIF-1α protein level, the expression of some HIF target genes or the myocardium capillary density at physiological condition. However, depletion of PHD3 further enhanced the induction of HIF-1α protein at hypoxic condition and increased expression of HIF-1α inhibited cardiomyocyte apoptosis induced by hypoxia. In addition, it has been demonstrated that PHD3 plays an important role in ATR/Chk1/p53 pathway. Consistently, a prolyl hydroxylase inhibitor or depletion of PHD3 significantly inhibits the activation of Chk1 and p53 in cardiomyocytes and the subsequent apoptosis induced by doxorubicin, hydrogen peroxide or hypoxia/reoxygenation. Taken together, these data suggest that depletion of PHD3 leads to increased stabilization of HIF-1α and inhibition of DNA damage response, both of which may contribute to the cardioprotective effect seen with depletion of PHD3.

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.

Cardiomyocyte-Specific Transgenic Expression of Prolyl-4-Hydroxylase Domain 3 Impairs the Myocardial Response to Ischemia

Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. Methods and Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice. Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.

A HIF-1 network reveals characteristics of epithelial-mesenchymal transition in acute promyelocytic leukemia.

BackgroundAcute promyelocytic leukemia (APL) is a sub-type of acute myeloid leukemia (AML) characterized by a block of myeloid differentiation at the promyelocytic stage and the predominant t(15:17) chromosomal translocation. We have previously determined that cells from APL patients show increased expression of genes regulated by hypoxia-inducible transcription factors (HIFs) compared to normal promyelocytes. HIFs regulate crucial aspects of solid tumor progression and are currently being implicated in leukemogenesis.MethodsTo investigate the contribution of hypoxia-related signaling in APL compared to other AML sub-types, we reverse engineered a transcriptional network from gene expression profiles of AML patients’ samples, starting from a list of direct target genes of HIF-1. A HIF-1-dependent subnetwork of genes specifically dysregulated in APL was derived from the comparison between APL and other AMLs.ResultsInterestingly, this subnetwork shows a unique involvement of genes related to extracellular matrix interaction and cell migration, with decreased expression of genes involved in cell adhesion and increased expression of genes implicated in motility and invasion, thus unveiling the presence of characteristics of epithelial-mesenchymal transition (EMT). We observed that the genes of this subnetwork, whose dysregulation shows a peculiar pattern across different AML sub-types, distinguish malignant from normal promyelocytes, thus ruling out dependence on a myeloid developmental stage. Also, expression of these genes is reversed upon treatment of APL-derived NB4 cells with all-trans retinoic acid and cell differentiation.ConclusionsOur data suggest that pathways related to EMT-like processes can be implicated also in hematological malignancies besides solid tumors, and can identify specific AML sub-types.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0084-4) contains supplementary material, which is available to authorized users.

Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid.

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1α (HIF-1α) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1α was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1α protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1α and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1α and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1α and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.