Target Genes Vascular Endothelial Growth Research Articles

Clinical implications of activation of the LIMD1-VHL-HIF1α pathway during head-&-neck squamous cell carcinoma development

Background & objectives Given the importance of the role of hypoxia induced pathway in different cancers including head-and-neck squamous cell carcinoma (HNSCC), this study delved into elucidating the molecular mechanism of hypoxia-inducible factor-1α (HIF1α) activation in HNSCC. Additionally, it analyzes the alterations of its regulatory genes [von Hippel–Lindau (VHL) and LIM domain containing 1 (LIMD1)] and target gene vascular endothelial growth factor (VEGF) in head-and-neck lesions at different clinical stages in relation with human papillomavirus (HPV) infection. Methods Global mRNA expression profiles of HIF1α, VHL, LIMD1 and VEGF were evaluated from public datasets of HNSCC, followed by validation of their expression (mRNA/protein) in an independent set of HPV+ve/–ve HNSCC samples of different clinical stages. Results A diverse expression pattern of the HIF1α pathway genes was observed, irrespective of HPV infection, in the datasets. In validation in an independent set of HNSCC samples, high mRNA expressions of HIF1α/VEGF were observed particularly in HPV positive samples. However, VHL/LIMD1 mRNA expression was low in tumours regardless of HPV infection status. In immunohistochemical analysis, high/medium (H/M) expression of HIF1α/VEGF was observed in basal/parabasal layers of normal epithelium, with significantly higher expression in tumours, especially in HPV-positive samples. Conversely, high cytoplasmic VHL expression in these layers gradually decreased with the progression of HNSCC, regardless of HPV infection. A similar trend was noted in LIMD1 expression (nuclear/cytoplasmic) during the disease development. The methylation pattern of VHL and LIMD1 promoters in the basal/parabasal layers of normal epithelium correlated with their expression, exhibiting a gradual increase with the progression of HNSCC. The H/M expression of HIF1α/VEGF proteins and reduced VHL expression was associated with poor clinical outcomes. Interpretation & conclusions The results of this study showed differential regulation of the LIMD1-VHL-HIF1α pathway in HPV positive and negative HNSCC samples, illustrating the molecular distinctiveness of these two groups.

Promyelocytic leukemia protein regulates angiogenesis and epithelial-mesenchymal transition to limit metastasis in MDA-MB-231 breast cancer cells.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor-suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Long Non-coding RNA H19 Suppression Protects the Endothelium Against Hyperglycemic-Induced Inflammation via Inhibiting Expression of miR-29b Target Gene Vascular Endothelial Growth Factor a Through Activation of the Protein Kinase B/Endothelial Nitric Oxide Synthase Pathway.

It has been shown that non-coding RNAs (ncRNAs) play an important regulatory role in pathophysiological processes involving inflammation. The vascular endothelial growth factor A (VEGFA) gene also participates in the inflammatory process. However, the relationships between ncRNAs and VEGFA are currently unclear. Here, this study was designed to determine the relationship between long non-coding RNA (lncRNA) H19, mircoRNA29b (miR-29b), and VEGFA in the development of diabetes mellitus (DM). We demonstrate that H19 is upregulated and miR-29b downregulated in individuals with DM and directly binds miR-29b. VEGFA is the target of miR-29b in the vascular endothelium of individuals with DM. We found that positive modulation of miR29b and inhibition of H19 and VEGFA significantly attenuates high glucose-induced endothelial inflammation and oxidative stress. We also found that the protein kinase B/endothelial nitric oxide synthase (AKT/eNOS) signal pathway in endothelial cells is activated through regulation of miR29b and H19 endogenous RNAs. We conclude that H19 suppression protects the endothelium against high glucose-induced inflammation and oxidative stress in endothelial cells by upregulation of miR-29b and downregulation of VEGFA through AKT/eNOS signal pathway activation. These results suggest a novel link between dysregulated ncRNA expression, inflammation, and the signaling pathway in the vascular endothelium of individuals with DM, indicating a promising strategy for preventing cardiovascular disease in such individuals.

Hypoxia-inducible factor-1α is involved in isoflurane-induced blood-brain barrier disruption in aged rats model of POCD

Prolonged exposure to inhaled anesthetics may lead to postoperative cognitive dysfunction (POCD). Nevertheless, the underlying mechanisms are not known. Hypoxia-inducible factor-1α (HIF-1α) and its target gene vascular endothelial growth factor (VEGF) were shown to be activated by inhaled anesthetics. The aim of the present study was to determine the role of HIF-1α in isoflurane-induced blood–brain barrier (BBB) disruption and resultant cognitive impairment. After a 4-h exposure to 1.5% isoflurane in 20-month-old rats, increases in vascular permeability, and disrupted BBB ultrastructure were accompanied by the degradation of tight junction proteins occludin and collagen type IV in brain blood vessels. Increases in HIF-1α and VEGF proteins and activation of MMP-2 in the hippocampus were also observed in the hippocamp of isoflurane-exposed rats compared with control rats. Pharmacological inhibition of HIF-1α activation by 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) markedly suppressed the expression of HIF-1α, VEGF and MMP-2, and mitigated the severity of BBB disruption.YC-1 pretreatment also significantly attenuated isoflurane-induced cognitive deficits in the Morris water maze task. Overall, our results demonstrate that hippocampal HIF-1α/VEGF signaling seems to be the upstream mechanism of isoflurane-induced cognitive impairment, and provides apotential preventive and therapeutic target for POCD.

Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model.

The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P < 0.01]. In addition, experimental groups revealed a down-regulation of E-selectin (-57%) and MCP1 (-33%) expression after ICA pretreatment compared with controls, going along with decreased T-cell [1.4% CD4+ T-cell infiltration vs 8.4% (control) and 4.9% CD8+ T-cell infiltration vs 10.7% (control)], dendritic cell (0.6% dendritic cells infiltration vs 1.9% infiltration(control)] and macrophage infiltration [4.8% macrophages (ICA) vs 10.9% (control)] within vascular grafts. These data of an animal transplant model show that the pharmaceutical activation of HIF with endogenous up-regulation of protective target genes leads to adaptation of the graft to low oxygen-saturation and hereby attenuates the development of transplant arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

The ETS Factor Myeloid Elf-1-Like Factor (MEF)/Elf4 Is Transcriptionally and Functionally Activated by Hypoxia.

Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HβD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl2 or low O2 incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (-200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HβD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

Activating Injury-Responsive Genes with Hypoxia Enhances Axon Regeneration through Neuronal HIF-1α

Injured peripheral neurons successfully activate a proregenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators coordinate the expression of such program remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the preconditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knock out in vivo impairs sensory axon regeneration. The HIF-1α target gene Vascular Endothelial Growth Factor A (VEGFA) is expressed in injured neurons and contributes to stimulate axon regeneration. Induction of HIF-1α using hypoxia enhances axon regeneration in vitro and in vivo in sensory neurons. Hypoxia also stimulates motor neuron regeneration and accelerates neuromuscular junction re-innervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration.

Gambogic Acid Suppresses Hypoxia-Induced HIF-1α/VEGF Expression Via Inhibiting PI3K/Akt/mTOR Pathway in Multiple Myeloma Cells

In multiple myeloma (MM), the hypoxic environment is an important factor causing tumor angiogenesis which is strongly correlated to disease progression and unfavorable outcome by activating the key transcription factor, hypoxia-inducible factor-1α (HIF-1α). Gambogic acid (GA) is the major active ingredient of gamboge, which has been shown to possess antitumor effect by in vitro and in vivo study. However, the underlying molecular mechanism that whether GA inhibits tumor angiogenesis remains not well understood. In this study, we investigated the effects of GA on expression of HIF-1α, and its downstream target gene vascular endothelial growth factor (VEGF) in human MM U266 cells. We found that hypoxia induced increase in the level of HIF-1α subunit protein and activated the PI3K/Akt/mTOR pathway. Moreover, the treatment with GA markedly decreased HIF-1α and VEGF expression under hypoxic condition. Mechanistic studies exhibited that GA inhibited the production of HIF-1α by reducing phosphorylation of Akt and mTOR in U266 cells. Furthermore, in vivo study revealed that intravenous injection of GA once every other day for two weeks could suppress tumor volumes by antiangiogenesis activity. Taken together, our results identify GA suppress hypoxia-activated pathways that linked to MM progression, at least partly, by the inhibition of PI3K/Akt/mTOR signaling pathway. Therefore, GA may be a new potent therapeutic agent against human MM cells. DisclosuresNo relevant conflicts of interest to declare.

Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing.

BackgroundHypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those.ResultsWith this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-145-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the factors involved in establishing the miRNA signature of GBM. Transcription factor binding sites for Hypoxia inducible factor 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia.ConclusionsOverall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-686) contains supplementary material, which is available to authorized users.

Enalapril and ASS inhibit tumor growth in a transgenic mouse model of islet cell tumors.

Accumulating evidence suggests a role for angiotensin-converting enzymes involving the angiotensin II-receptor 1 (AT1-R) and the cyclooxygenase pathway in carcinogenesis. The effects of ASS and enalapril were assessed in vitro and in a transgenic mouse model of pancreatic neuroendocrine neoplasms (pNENs). The effects of enalapril and ASS on proliferation and expression of the AGTR1A and its target gene vascular endothelial growth factor (Vegfa) were assessed in the neuroendocrine cell line BON1. Rip1-Tag2 mice were treated daily with either 0.6 mg/kg bodyweight of enalapril i.p., 20 mg/kg bodyweight of ASS i.p., or a vehicle in a prevention (weeks 5-12) and a survival group (week 5 till death). Tumor surface, weight of pancreatic glands, immunostaining for AT1-R and nuclear factor kappa beta (NFKB), and mice survival were analyzed. In addition, sections from human specimens of 20 insulinomas, ten gastrinomas, and 12 non-functional pNENs were evaluated for AT1-R and NFKB (NFKB1) expression and grouped according to the current WHO classification. Proliferation was significantly inhibited by enalapril and ASS in BON1 cells, with the combination being the most effective. Treatment with enalapril and ASS led to significant downregulation of known target genes Vegf and Rela at RNA level. Tumor growth was significantly inhibited by enalapril and ASS in the prevention group displayed by a reduction of tumor size (84%/67%) and number (30%/45%). Furthermore, daily treatment with enalapril and ASS prolonged the overall median survival compared with vehicle-treated Rip1-Tag2 (107 days) mice by 9 and 17 days (P=0.016 and P=0.013). The AT1-R and the inflammatory transcription factor NFKB were abolished completely upon enalapril and ASS treatment. AT1-R and NFKB expressions were observed in 80% of human pNENs. Enalapril and ASS may provide an approach for chemoprevention and treatment of pNENs.

Gambogic acid suppresses hypoxia-induced hypoxia-inducible factor-1α/vascular endothelial growth factor expression via inhibiting phosphatidylinositol 3-kinase/Akt/mammalian target protein of rapamycin pathway in multiple myeloma cells.

In multiple myeloma (MM), the hypoxic environment is an important factor causing tumor angiogenesis, which is strongly correlated to disease progression and unfavorable outcome by activating the key transcription factor, hypoxia-inducible factor-1α (HIF-1α). Gambogic acid (GA) is the major active ingredient of gamboge, which has been shown to possess antitumor effect by in vitro and in vivo study. However, the underlying molecular mechanism of whether GA inhibits tumor angiogenesis remains poorly understood. In this study, we investigated the effects of GA on expression of HIF-1α, and its downstream target gene vascular endothelial growth factor (VEGF) in human MM U266 cells. We found that hypoxia induced increase in the level of HIF-1α subunit protein and activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target protein of rapamycin (mTOR) pathway. Moreover, the treatment with GA markedly decreased HIF-1α and VEGF expression under hypoxic conditions. Mechanistic studies exhibited that GA inhibited the production of HIF-1α by reducing phosphorylation of Akt and mTOR in U266 cells. Furthermore, in vivo study revealed that intravenous injection of GA once every other day for 2 weeks could suppress tumor volumes by antiangiogenesis activity. Taken together, our results identify that GA suppresses hypoxia-activated pathways that are linked to MM progression, at least partly, by the inhibition of the PI3K/Akt/mTOR signaling pathway. Therefore, GA may be a new potent therapeutic agent against human MM cells.

Inducers of Hypoxic Response: Marine Sesquiterpene Quinones Activate HIF-1

The hypoxia-inducible factor-1 (HIF-1) transcription factor regulates cellular oxygen homeostasis. Agents that activate HIF-1 and downstream HIF targets represent potential drug leads for the prevention and/or treatment of ischemic disorders. In a search for small-molecule HIF-1 activators, 1936 marine invertebrate and algal extract samples (U.S. National Cancer Institute's Open Repository) were evaluated for HIF-1 activation activity in a cell-based reporter assay. Bioassay-guided fractionation of two active extracts of the sponge Dactylospongia elegans afforded four new sesquiterpene quinones (2-5), one new sesquiterpene phenol (6), the known Golgi disruptor ilimaquinone (1), and three previously reported ilimaquinone analogues (7-9). While antiproliferative activity was observed at higher concentrations, the sesquiterpene quinones (1-3) possessing a 2-hydroxy-5-methoxy-1,4-benzoquinone moiety activated HIF-1 and increased the expression of HIF-1 target gene vascular endothelial growth factor (VEGF) in T47D cells.

Preconditioning and post-treatment with cobalt chloride in rat model of perinatal hypoxic–ischemic encephalopathy

Background: Hypoxia–ischemia (HI)-induced perinatal encephalopathy is a major cause of acute mortality and chronic neurologic morbidities such as cerebral palsy, mental retardation, and epilepsy. As the essential transcription factor for the activation of hypoxia-inducible genes, hypoxia-inducible factor 1 alpha (HIF-1α) plays an important role in the pathophysiological response to the stress of HI brain damage. Whether HIF-1α activation promotes neuroprotection in HI tissues is controversial. Methods: The left common carotid artery of rats aged 7days was ligated under anesthesia. The pups were then exposed to hypoxia in a normobaric chamber filled with 8% oxygen and 92% nitrogen for 2.5h. In the sham control group, the left common carotid artery was exposed but was not ligated or exposed to hypoxia. To assess the time window for effective treatment, the HIF-1α inducer cobalt chloride (CoCl2) was injected subcutaneously 1day before surgery, immediately or 1day after surgery. The brain tissues were harvested from the pups of each groups at 1, 2 and 7days after insult for HIF-1α protein ant its target genes expression and for investigating the injury. Morris water maze tests were performed at postnatal 7weeks. Results: HIF-1α protein levels and its target genes vascular endothelial growth factor, heme oxygenase-1, and insulin-like growth factor 1 were markedly increased after intraperitoneal injection of CoCl2 (60mg/kg). The target gene inducible nitric oxide synthase exhibited a biphasic time course. HI caused apoptosis and reduced capillary density, which were ameliorated by CoCl2. Both preconditioning with CoCl2 24h before HI and administration of CoCl2 24h after HI improved long-term reference memory compared with that in vehicle-injected littermate controls. Administration of CoCl2 immediately after HI did not improve spatial working memory. Conclusions: CoCl2 activates HIF-1α and protects against brain damage in vivo. The time of administration could be used to manipulate the activity of HIF-1α pathways and promote recovery.

Regulation of tumor growth and angiogenesis in colorectal cancer

Hypoxia in malignant tissues is associated with poor prognosis and treatment options. It is obvious that the grade of malignancy is associated with dedifferentiation of a given tissue. Our aim was to assess the expression of proteins that act as cellular oxygen sensors, which directly regulate the hypoxia-inducible factor 1 (HIF-1) pathway, i.e., prolyl hydroxylase domain proteins (PHD) PHD-1, PHD-2, PHD-3, and its target gene vascular endothelial growth factor (VEGF) in colorectal cancer (CRC). In this study, we cultured a human colon cancer cell line (SW480) and its correspondent lymph-node metastasis cell line (SW620) under normoxic or hypoxic conditions for different time periods. We then measured the HIF-1a expression using Western-Blot, the messenger RNA (mRNA)-expression of the PHDs with real-time polymerase chain reaction (PCR) and the amount of one of the target-genes using an VEGF-ELISA. SW620 shows a clearly elevated HIF-1a expression under normoxic and hypoxic states compared with SW480. Under hypoxic conditions, the amount of VEGF is elevated in both primary and secondary tumor-cell line, but the metastasis-cell line has a significant higher increase. PHD-1 shows no higher expression under hypoxic condition, PHD-2 expression is elevated but only in SW480. PHD-3 levels raised in both of the cell lines, with significant higher rates in SW480. HIF-regulating proteins are highly expressed in CRCs and their metastases. These molecules play an important role in the control of hypoxia-induced genes and may also have a function in the regulation of cellular proliferation and differentiation during tumorigenesis and dedifferentiation of CRCs. Our results emphasize the important role of PHDs not only as an oxygen-sensor, but also as a control-unit for tumor growth, apoptosis, and angiogenesis through HIF-1 pathway.

Expression of Hypoxia-Inducible Factors, Regulators, and Target Genes in Congenital Diaphragmatic Hernia Patients

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel-Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A ( VEGF-A ) and vascular endothelial growth factor receptor 2 ( VEGFR-2 ). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1α, HIF-2α, HIF-3α, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2α might indicate a role for these factors in the pathophysiology of CDH.

Knock-Down of Argonaute 2 (AGO2) Induces Apoptosis in Myeloid Leukaemia Cells and Inhibits siRNA-Mediated Silencing of Transfected Oncogenes in HEK-293 Cells

Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer.

Abstract 3083: The RNA stability factor HuR promotes an angiogenic switch in colon cancer

Abstract The initiation and progression of colorectal tumorigenesis is characterized by alterations in gene expression that allow for the overexpression of oncogenic and tumor-promoting factors. In normal cells, the mRNA transcripts of these cancer-associated genes are targeted for rapid decay through AU-rich elements (ARE) present in the mRNA 3’ untranslated region (3′UTR). However, growing evidence reveals that the loss of ARE-mediated mRNA decay is a characteristic feature contributing to pathogenic gene expression in colorectal cancer. Our prior work identified enhanced expression of the RNA-binding protein HuR to occur during colon tumorigenesis and HuR overexpression is correlated with poor clinical prognosis. In this capacity, HuR promotes overexpression of many cancer-associated factors by binding to their ARE and interfering with subsequent rapid mRNA decay. To determine the functional significance of HuR overexpression in vivo, a tissue-specific HuR-transgenic mouse (HuR-Tg) was created that overexpresses HuR in the epithelium of the small intestine and colon. HuR-Tg mice displayed normal growth and did not reveal any morphological changes in the gastrointestinal tract. To test if HuR overexpression can impact tumorigenesis downstream a tumor initiating event, crosses between HuR-Tg mice and the APCMin/+ mouse, a genetic model of gastrointestinal tumorigenesis, were performed. As early as 12 weeks of age, HuR-Tg/APCMin/+ lines display a 2- to 4-fold increase in tumor burden and size in both the colon and small intestine compared to APCMin/+control mice. Based on HuR's ability to bind various ARE-containing mRNAs associated with angiogenesis, vascular density was assayed in tissue derived from HuR-Tg/APCMin/+ mice. Micro-vessel density was 2-fold greater in the tumors and surrounding tissue in APCMin/+ mice overexpressing HuR. Angiogenic mRNA array analysis of tumors derived from HuR-Tg/APCMin/+ mice showed enhanced expression of multiple ARE-containing angiogenic genes including the HuR target genes vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). Furthermore, dietary treatment of HuR-Tg/APCMin/+ mice with celecoxib attenuated HuR-dependent tumor formation and suppressed micro-vessel density and VEGF expression. These findings indicate that HuR functions during colon tumorigenesis downstream of a tumor-initiating event to stabilize ARE-containing mRNAs associated with tumor vascularization and identify this mRNA stability factor as a new molecular target for therapeutic intervention in controlling angiogenic gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2011-3083

Constitutive expression of hypoxia-inducible factor-1 α in keratinocytes during the repair of skin wounds in horses

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia-inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin-dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia-independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.

Neural Stem Cells Adopt Tumorigenic Properties by Constitutively Activated NF-κB and Subsequent VEGF Up-Regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Myocyte Enhancer Factor 2 and Class II Histone Deacetylases Control a Gender-Specific Pathway of Cardioprotection Mediated by the Estrogen Receptor

Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied. We aimed to examine the effect of class II HDACs on post-myocardial infarction remodeling in male and female mice. Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, during which there is an upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER)alpha gene, which we show to be a direct MEF2 target gene. ERalpha also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a. Our results reveal a novel gender-specific pathway of cardioprotection mediated by ERalpha and its regulation by MEF2 and class II HDACs.