Publications

THE SILENT CARRIER ALLELE: β-THALASSEMIA WITHOUT A MUTATION IN THE β-GLOBIN GENE REGION

Data from Decreased Expression of Cystathionine β-Synthase Promotes Glioma Tumorigenesis

<div>Abstract<p>Cystathionine β-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding shRNA targeting CBS or a nontargeting control shRNA, and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation <i>in vitro</i> but decreased the latency period before rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate–putamen. In soft-agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF2α/EPAS1). HIF2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and VEGF transcripts, which are HIF2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF2α protein levels and HIF2 target gene expression, which promotes glioma tumor formation.</p><p><b>Implications:</b> CBS loss-of-function promotes glioma growth. <i>Mol Cancer Res; 12(10); 1398–406. ©2014 AACR</i>.</p></div>

Methylation of hypoxia-inducible factor (HIF)-1α by G9a/GLP inhibits HIF-1 transcriptional activity and cell migration

Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator in response to hypoxia and its transcriptional activity is crucial for cancer cell mobility. Here we present evidence for a novel epigenetic mechanism that regulates HIF-1 transcriptional activity and HIF-1-dependent migration of glioblastoma cells. The lysine methyltransferases G9a and GLP directly bound to the α subunit of HIF-1 (HIF-1α) and catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. K674 methylation suppressed HIF-1 transcriptional activity and expression of its downstream target genes PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Inhibition of HIF-1 by K674 methylation is due to reduced HIF-1α transactivation domain function but not increased HIF-1α protein degradation or impaired binding of HIF-1 to hypoxia response elements. K674 methylation significantly decreased HIF-1-dependent migration of U251MG cells under hypoxia. Importantly, we found that G9a was downregulated by hypoxia in glioblastoma, which was inversely correlated with PTGS1 expression and survival of patients with glioblastoma. Therefore, our findings uncover a hypoxia-induced negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma.

MCM Proteins Are Negative Regulators of Hypoxia-Inducible Factor 1

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

Tie2-dependent VHL knockdown promotes airway microvascular regeneration and attenuates invasive growth of Aspergillus fumigatus

Microvascular ischemia and infections are associated with the development of chronic rejection following lung transplantation. The von Hippel–Lindau protein (VHL) controls protein levels of hypoxia-inducible factors (HIFs), regulates vascular repair, and improves tissue perfusion. Here, we studied the role of VHL in microvascular repair by orthotopically transplanting tracheas into mice with VHL haplodeficiency in Tie2 lineage cells. We showed that VHL haplodeficiency prolonged airway microvascular perfusion and promoted tissue blood flow through the production of the angiogenic factors, SDF-1 and angiopoietin 1. VHL-haplodeficient pulmonary endothelial cells exhibited increased angiogenic activity, resistance to serum deprivation-induced cell death, and enhanced microvascular repair. By contrast, in recipient mice with HIF-1α deficiency in Tie2 lineage cells, microvascular repair was significantly diminished and suggested that recipient-derived HIF-1α normally participates in the repair of alloimmune-mediated microvascular damage. To evaluate the translational impact of our findings, we compared VHL-haplodeficient mice with wild-type controls using a model of Aspergillus airway infection. In 83% of the VHL-haplodeficient recipients, Aspergillus fumigatus was noninvasive in contrast to 75% of wild-type mice in which the mold was deeply invasive. Our study demonstrated that stabilization of HIF-1α in angiogenic cells, through Tie2 cell VHL haplodeficiency, promoted airway microvascular regeneration and vascular normalization and thereby minimized tissue ischemia and hypoxia. By also mitigating the virulence of A. fumigatus, a common pathogen and itself a risk factor for the development of lung transplant rejection, the selective enhancement of HIF-1α expression has the prospect of offering several novel therapeutic effects to transplant recipients.

Mutations in Genes Encoding Transcription Factors: The Nuclear Receptor Superfamily

Abstract In Chapters 5 through 14, mutations that affect the structure and/or function of specific transcription factors and result in human disease will be described. As in the previous chapter, the intent is not to catalogue all known mutations but rather to provide examples that illustrate principles of molecular pathophysiology that will be applicable to the ever expanding list of such conditions that is being reported in the molecular biology and clinical literature. In contrast to the mutations in cis-acting elements, the mutations in trans-acting factors affect the structure of the protein product of a gene. In the text, gene symbols will be indicated by italicized letters (all capitalized for human genes, first letter only for mouse genes), whereas the protein product of a gene will be indicated by unitalicized capital letters (for both human and mouse proteins). Missense mutations result in substitution of one amino acid for another and will be designated in the text using the single-letter code for amino acids (see Table 5.1). For example, the mutation C23S (due to a TGT AGT transversion) would result in substitution of serine for cysteine at residue 23 of the protein. Nonsense mutations result in premature termination of translation, which is designated by an X. Thus, the mutation C23X (due to a TGT TGA transversion) would result in translation of a truncated polypeptide containing only 22 amino acid residues.

Of oxygen, fuels, and living matter

Effects of Aging and Hypoxia-Inducible Factor-1 Activity on Angiogenic Cell Mobilization and Recovery of Perfusion After Limb Ischemia

Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1α, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1α gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

Abstract 378: Endothelial Cell Hypoxia-inducible Factor (HIF) Activity is Necessary for Right Ventricular Remodeling in a Murine Model of Chronic-hypoxic Pulmonary Hypertension

Introduction: Pulmonary hypertension induced myocardial remodeling is required to minimize RV wall stress as pulmonary arterial pressures increase. Myocardial angiogenesis is presumed to play an integral role in early RV remodeling, though molecular regulators remain undefined. Endothelial cell (EC) expression of HIF-1 is necessary for adaptive LV remodeling during chronic pressure overload, with loss of EC-specific HIF-1 activity associated with reduced LV capillary density. We hypothesized that EC HIF activity may also be necessary for adaptive RV remodeling and angiogenesis in a model of chronic RV pressure overload. Methods: We used a murine model of chronic hypoxic pulmonary hypertension (CH-PH) to induce RV remodeling. Conditional transgenic mice lacking expression of the obligate HIF-1α/HIF-2α co-factor ARNT (aryl hydrocarbon receptor nuclear translocator) in Tie2+ lineage cells ( ARNT fl/fl ; Tie2-Cre +/- ) and ARNT fl/fl ;Tie2-Cre -/- controls were exposed to normobaric hypoxia (10% FiO 2 ) for 1-5 weeks. We measured RV hypertrophy (RVH), RV systolic pressure (RVSP), and myocardial EC proliferation (flow cytometry) as a marker for sprouting angiogenesis. Results: After three weeks of CH-PH, RVH was observed in ARNT fl/fl ;Tie2-Cre -/- control mice, as anticipated (0.332±0.02 vs. 0.231±0.02; P < 0.05). CH-PH induced RVH in ARNT fl/fl ;Tie2-Cre -/- mice was associated with an early (1 week) increase in RV EC proliferation (vs. LV, 4.5±1.3% vs.1.8±0.5%; P < 0.05). Conversely, ARNT fl/fl ; Tie2 - Cre +/- mice did not develop RVH after 3 weeks of CH ( 0.222±0.01 vs. 0.241±0.02; P = NS), and early RV EC proliferation was attenuated (vs. LV, 2.9±1.2% vs. 1.5±0.3%; P = NS). Despite similar increases in CH-PH induced RVSP after 3 weeks, after 5 weeks RVSP normalized in ARNT fl/fl ; Tie2 - Cre +/- mice, but remained elevated in ARNT fl/fl ;Tie2-Cre -/- controls. Conclusions: These preliminary findings confirm that EC-specific HIF activity is necessary for CH-PH induced RVH, and suggest that reduced EC proliferation (and potentially angiogenesis) may contribute to abrogated RV remodeling in ARNT fl/fl ; Tie2 - Cre +/- mice. We hypothesize that late normalization of RVSP in these mice may be indicative of premature RV failure.

Hypoxia-inducible factor-dependent signaling between triple-negative breast cancer cells and mesenchymal stem cells promotes macrophage recruitment

Significance The recruitment of host stromal cells, such as macrophages and mesenchymal stem cells (MSCs), to the primary tumor is a critical step toward cancer malignancy. We have identified signals that are exchanged between breast cancer cells (BCCs) and MSCs. This signaling increases the recruitment of both MSCs and macrophages to primary tumors and increases metastasis of BCCs to lymph nodes and lungs. Reduced oxygen levels (hypoxia) in breast cancers are associated with increased risk of metastasis and decreased patient survival. We show that hypoxia stimulates signaling between BCCs and MSCs due to the activity of hypoxia-inducible factors (HIFs). Drugs that block HIF activity prevent signaling and macrophage recruitment, which suggests that they may be useful additions to breast cancer therapy.

Oxygen homeostasis

Abstract Metazoan life is dependent upon the utilization of O 2 for essential metabolic processes and oxygen homeostasis is an organizing principle for understanding metazoan evolution, ontology, physiology, and pathology. Hypoxia‐inducible factor 1 (HIF‐1) is a transcription factor that is expressed by all metazoan species and functions as a master regulator of oxygen homeostasis. Recent studies have elucidated complex mechanisms by which HIF‐1 activity is regulated and by which HIF‐1 regulates gene expression, with profound consequences for prenatal development, postnatal physiology, and disease pathogenesis. Copyright © 2009 John Wiley & Sons, Inc. This article is categorized under: Physiology > Organismal Responses to Environment

Regulation of the breast cancer stem cell phenotype by hypoxia-inducible factors

The small subpopulation of breast cancer cells that possess the capability for self-renewal and formation of secondary tumours that recapitulate the heterogeneity of the primary tumour are referred to as tumour-initiating cells or BCSCs (breast cancer stem cells). The hypoxic tumour microenvironment and chemotherapy actively induce the BCSC phenotype. HIFs (hypoxia-inducible factors) are required and molecular mechanisms by which they promote the BCSC phenotype have recently been delineated. HIF inhibitors block chemotherapy-induced enrichment of BCSCs, suggesting that their use may improve the response to chemotherapy and increase the survival of breast cancer patients.

The role of hypoxia‐inducible factors in carotid body (patho) physiology

Abstract Hypoxia‐inducible factors mediate adaptive responses to reduced O 2 availability. In patients with obstructive sleep apnoea, repeated episodes of hypoxaemia and reoxygenation (intermittent hypoxia) are sensed by the carotid body (CB). The ensuing CB chemosensory reflex activates the sympathetic nervous system and increased secretion of catecholamines by the adrenal medulla, resulting in hypertension and breathing abnormalities. In the CB, intermittent hypoxia induces the formation of reactive oxygen species (ROS) and increased intracellular Ca 2+ levels, which drive increased expression of hypoxia‐inducible factor (HIF) 1α and a decrease in the levels of HIF‐2α. Intermittent hypoxia increases HIF‐1α‐dependent expression of Nox2 , encoding the pro‐oxidant enzyme NADPH oxidase 2, and decreased HIF‐2α‐dependent expression of Sod2 , encoding the anti‐oxidant enzyme superoxide dismutase 2. These changes in gene expression drive persistently elevated ROS levels in the CB, brainstem, and adrenal medulla that are required for the development of hypertension and breathing abnormalities. The ROS generated by dysregulated HIF activity in the CB results in oxidation and inhibition of haem oxygenase 2, and the resulting reduction in the levels of carbon monoxide leads to increased hydrogen sulfide production, triggering glomus cell depolarization. Thus, the pathophysiology of obstructive sleep apnoea involves the dysregulation of O 2 ‐regulated transcription factors, gasotransmitters, and sympathetic outflow that affects blood pressure and breathing. image

HIF-1 promotes murine breast cancer brain metastasis by increasing production of integrin β3–containing extracellular vesicles

Brain metastasis is a major cause of breast cancer (BC) mortality, but the cellular and molecular mechanisms have not been fully elucidated. BC cells must breach the blood-brain barrier in order to colonize the brain. Here, we determined that integrin β3 (ITGB3) expression mediated by hypoxia-inducible factor 1 (HIF-1) plays a critical role in metastasis of BC cells to the brain. Hypoxia stimulated BC cell migration and invasion ex vivo and brain colonization in vivo. Knockdown of either HIF-1α or ITGB3 expression impaired brain colonization by human or mouse BC cells injected into the cardiac left ventricle. Exposure of BC cells to hypoxia increased expression of ITGB3 and its incorporation into small extracellular vesicles (EVs). EVs harvested from the conditioned medium of hypoxic BC cells showed increased retention in the brain after intracardiac injection that was HIF-1α and ITGB3 dependent. EVs from hypoxic BC cells showed binding to brain endothelial cells (ECs), leading to increased EC-BC cell interaction, increased vascular endothelial growth factor receptor 2 signaling, increased EC permeability, and increased transendothelial migration of BC cells. Taken together, our studies implicate HIF-1-stimulated production of ITGB3+ EVs as a key mechanism by which hypoxia promotes BC brain metastasis.

HIF-1 mediates the Warburg effect in clear cell renal carcinoma

General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.

Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.

Abstract 5427: Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α

Abstract Hypoxia-inducible factor 1 (HIF-1) is a main regulator of cellular response to hypoxia and its transcriptional activity is crucial for cancer cell migration and invasion. Post-translational modifications of the α subunit of HIF-1 (HIF-1α) play dominant roles in regulating its function. In this study, we demonstrated that the lysine methyltransferases G9a and GLP catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. Functionally, K674 methylation suppressed HIF-1 transcriptional activity as well as expression of the HIF-1 target genes including PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Moreover, mutation of K674 of HIF-1α significantly enhanced U251MG cell migration under hypoxia. Importantly, we found that G9a level was downregulated by hypoxia in glioblastoma, which was negatively correlated with PTGS1 expression and survival rate in glioblastoma patients. These findings reveal a novel naturally existing negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma. Citation Format: Lei Bao, Yan Chen, Hsien-Tsung Lai, Cheng-Ming Chiang, Gregg L. Semenza, Yingfei Wang, Weibo Luo. Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5427.