Hypoxia-inducible factor (HIF): fuel for cancer progression

Hypoxia is an integral part of the tumor microenvironment, caused primarily due to rapidly multiplying tumor cells and a lack of proper blood supply. Among the major hypoxic pathways, HIF-1 transcription factor activation is one of the widely investigated pathways in the hypoxic tumor microenvironment (TME). HIF-1 is known to activate several adaptive reactions in response to oxygen deficiency in tumor cells. HIF-1 has two subunits, HIF-1β (constitutive) and HIF-1α (inducible). The HIF-1α expression is largely regulated via various cytokines (through PI3K-ACT-mTOR signals), which involves the cascading of several growth factors and oncogenic cascades. These events lead to the loss of cellular tumor suppressant activity through changes in the level of oxygen via oxygen-dependent and oxygenindependent pathways. The significant and crucial role of HIF in cancer progression and its underlying mechanisms have gained much attention lately among the translational researchers in the fields of cancer and biological sciences, which have enabled them to correlate these mechanisms with various other disease modalities. In the present review, we have summarized the key findings related to the role of HIF in the progression of tumors

HIF-I alpha é um substrato da via da ubiquitina-proteossoma e da autofagia mediada por chaperones

A via da ubiquitina-proteossoma é considerada a principal via de degradação selectiva de proteínas solúveis nas células eucarióticas. No entanto, os lisossomas desempenham também um papel essencial na degradação de proteínas intracelulares, processo designado de autofagia. Existem três vias autofágicas distintas, macroautofagia, microautofagia e autofagia mediada por chaperones, que diferem entre si mecanisticamente. A autofagia mediada por chaperones é uma forma selectiva de autofagia. Nesta via, as proteínas citosólicas, que contêm uma sequência peptídica específica, bioquimicamente semelhante ao KFERQ, são primeiro reconhecidas pela chaperone Hsc70 e, posteriormente, dirigidas para a membrana do lisossoma, local onde interagem com a proteína associada à membrana do lisossoma 2A (LAMP-2A). Estes substratos são então desenrolados e translocados para o lúmen do lisossoma, onde são degradados. Este projecto explora a possibilidade da existência de um diálogo transversal entre a via da ubiquitina-proteossoma e autofagia mediada por chaperones, até há pouco tempo consideradas independentes. O HIF-1 (Hypoxia-Inducible Factor 1) é um heterodímero composto por uma subunidade estável, o HIF-1β, e uma subunidade lábil, o HIF-1α. Em condições de normoxia, dois resíduos de prolina presentes no HIF-1α são hidroxilados e a proteína é ubiquitinada e subsequentemente degradada pelo proteossoma. Assim, propõe-se que o HIF-1α, substrato típico da via da ubiquitina-proteossoma, possa em circunstâncias específicas, ser dirigido para a autofagia mediada por chaperones. De facto, os resultados obtidos neste trabalho mostram que uso de inibidores do lisossoma, mas não os que inibem a macroautofagia, promovem uma estabilização do HIF-1α. Também foi demonstrado que o HIF-1α interage com a proteína associada à membrana do lisossoma 2 2A (LAMP-2A) e que esta interage com a chaperone Hsc70 citosólica. Estas interacções aumentam após inibição do proteossoma. Uma vez que, todos os substratos conhecidos da autofagia mediada por chaperones interagem com a chaperone Hsc70 citosólica e com a proteína associada à membrana do lisossoma 2A (LAMP-2A) podemos inferir que o HIF-1α é degradado não só pela via da ubiquitina-proteossoma como também pela autofagia mediada por chaperones, mostrando uma possível comunicação entre estas duas vias. Deste modo, o HIF-1α poderá constituir um substrato modelo para o estudo dos mecanismos moleculares envolvidos na intercomunicação entre estas duas vias proteolíticas

The Cellular Oxygen Sensor PHD2 in Cancer Growth

Adequate supply of oxygen is essential for the survival of multicellular organisms. However, in several conditions the supply of oxygen can be disturbed and the tissue oxygenation is compromised. This condition is termed hypoxia. Oxygen homeostasis is maintained by the regulation of both the use and delivery of oxygen through complex, sensitive and cell-type specific transcriptional responses to hypoxia. This is mainly achieved by one master regulator, a transcription factor called hypoxiainducible factor 1 (HIF-1). The amount of HIF-1 is under tight oxygen-dependent control by a family of oxygen-dependent prolyl hydroxylase domain proteins (PHDs) that function as the cellular oxygen sensors. Three family members (PHD1-3) are known to regulate HIF of which the PHD2 isoform is thought to be the main regulator of HIF-1. The supply of oxygen can be disturbed in pathophysiological conditions, such as ischemic disorders and cancer. Cancer cells in the hypoxic parts of the tumors exploit the ability of HIF-1 to turn on the mechanisms for their survival, resistance to treatment, and escape from the oxygen- and nutrient-deprived environment. In this study, the expression and regulation of PHD2 were studied in normal and cancerous tissues, and its significance in tumor growth. The results show that the expression of PHD2 is induced in hypoxic cells. It is overexpressed in head and neck squamous cell carcinomas and colon adenocarcinomas. Although PHD2 normally resides in the cytoplasm, nuclear translocation of PHD2 was also seen in a subset of tumor cells. Together with the overexpression, the nuclear localization correlated with the aggressiveness of the tumors. The nuclear localization of PHD2 caused an increase in the anchorage-independent growth of cancer cells. This study provides information on the role of PHD2, the main regulator of HIF expression, in cancer progression. This knowledge may prove to be valuable in targeting the HIF pathway in cancer treatment.Siirretty Doriast

A multiplex comparative proteomic analysis of hypoxia influence in the presence and absence of p53 in HCT116 Cells

Master'sMASTER OF SCIENC

Étude de l'implication de la régulation hypoxique de la furine dans la progression tumorale

Le cancer est une pathologie complexe causée par plusieurs facteurs génétiques et environnementaux. Aux États-Unis seulement, près de 1 400 000 cas de cancer seront diagnostiqués en 2006 et plus de 500 000 personnes en mourront. Encore aujourd'hui, plusieurs types de cancer sont incurables et la plupart des traitements existants ne sont malheureusement pas curatifs. Cette maladie est caractérisée par une croissance cellulaire incontrôlée ainsi qu'une dissémination des cellules anormales. La prolifération accrue des cellules cancéreuses mène à l'apparition de régions hypoxiques chez plusieurs tumeurs solides. Un grand nombre de molécules participent aux différents stades de la progression tumorale et permettent à la tumeur de s'adapter à son microenvironnement. Plusieurs de ces dernières sont synthétisées sous forme de pro-peptides nécessitant un clivage enzymatique ciblé afin d'acquérir leur activité. Il a été démontré que la furine, le prototype de la famille des convertases de proprotéines, joue un rôle important dans la progression tumorale. La furine est surexprimée chez plusieurs types de tumeurs et elle est nécessaire à l'activation de substrats protéiques dont les rôles dans la croissance tumorale ainsi que dans la formation des métastases sont répertoriés. Dernièrement, l'étude des différents promoteurs de la furine a révélé qu'elle pouvait être induite en hypoxie, et. ce, via le facteur de transcription HIF-1. Notre étude avait pour but d'étudier l'implication de la régulation hypoxique de la furine dans le développement du cancer. Nous avons démontré, par immunohistochimie, la présence de régions hypoxiques dans les xénogreffes formées à partir de fibrosarcomes humains ainsi qu'une surexpression de la furine dans ces régions. Afin d'inhiber cette induction dans les régions hypoxiques tumorales, nous avons construit des vecteurs plasmidiques contenant un inhibiteur de la furine, soit l'[alpha]1-AT-PDX, sous le contrôle d'un promoteur inductible en hypoxie. Ces vecteurs possèdent 3 ou 6 répétitions d'une séquence du gène de l'érythropoïétine contenant un site de reconnaissance pour le HIF-1. Nous avons démontré que ces vecteurs augmentent les niveaux de PDX chez les cellules HT1080 cultivées en hypoxie. De plus, nous avons établi que l'augmentation de PDX en hypoxie est principalement due à la présence de HIF-1 dans la cellule. Nous avons aussi observé une diminution de la maturation de la MT1-MMP ainsi que de l'IGF-IR[bêta], deux substrats de la furine. Dans le but de vérifier la possibilité qu'une inhibition de la furine induite en hypoxie puisse affecter la croissance tumorale, nous avons par la suite transfecté les cellules HT1080 afin d'obtenir une population de cellules exprimant de façon stable les plasmides 3HRE-PDX et 6HRE-PDX. Nous avons démontré que, suite à l'implantation de ces cellules chez des souris immunodéficientes, la croissance des tumeurs exprimant du PDX en hypoxie est diminuée de façon significative. Nous avons également démontré, par immunobuvardage de type Western, la présence de PDX à l'intérieur de ces tumeurs. De plus, des expériences d'immunohistochimie ont révélé que l'expression de PDX était principalement située dans les régions hypoxiques des tumeurs. La furine est une enzyme ubiquiste, impliquée dans le maintien physiologique des fonctions cellulaires. Nos résultats démontrent pour la première fois la faisabilité d'inhiber de façon ciblée la furine, soit dans les zones hypoxiques de tumeurs. Cette inhibition diminue la maturation de substrats impliqués dans la modification du microenvironnemt des tumeurs et par conséquent, diminue la progression tumorale

Molecular mechanisms of nutrient mediated regulation of hypoxia inducible factor -1a(HIF-1a) in endothelial cells.

Poorly-controlled diabetes mellitus is associated with the development of chronic vascular complications, which cause morbidity and premature mortality. Many studies have highlighted that maintenance of normal blood glucose levels in all people with diabetes is the most effective way in which chronic complications can be reduced and prevented. Presently, the underlying mechanisms associated with the manifestation and progression of vascular complications are poorly defined. Therefore, this study considered how the increased oxidative demand placed upon cells by high glucose concentration would result in a state of pseudo hypoxia, and potentially the expression of hypoxia-inducible factor-alpha (HIF-1alpha). In an animal model of diabetes, HIF-1alpha protein expression was seen to increase in the vasculature surrounding the sciatic nerve at ten- and twenty-four weeks of diabetes duration, which was seen to be reversed at twenty-four weeks in response to treatment with the antioxidant alpha-lipoic acid. In vitro, exposure of HUVEC to 20mM glucose for twenty-four hours induced perinuclear expression of HIF-1alpha protein, as opposed to nuclear expression evident under hypoxic conditions. Furthermore, at twenty-four hours in 20mM glucose, the detected HIF-1alpha mRNA level was significantly higher than that seen in all other conditions (p < 0.001). The increase in HIF-1alpha mRNA detected was seen to be dependent upon mRNA stability, and potentially its association with RNA-binding proteins and/or the natural antisense, aHIF. Pi3K - and, to a lesser extent, p42/44 MAPK signalling pathways - were also implicated, and alpha-lipoic acid treatment reversed the stability of HIF-1alpha mRNA. The effect of high glucose on HIF-1alpha mRNA level was not seen in response to partially/non-metabolisable glucose analogues, indicating glucose metabolism to be central to the stabilisation of HIF-1alpha mRNA. In conclusion, using the metabolism of high concentrations of glucose to regulate HIF-1alpha at the level of mRNA and protein may contribute to the generation of chronic vascular disease associated with diabetes, and should be further explored as a potential mechanism by which such complications may be prevented

Characterizing Hypoxia and Its Behavioral Effects In 3-Dimensional Cell Aggregates

Cell transplantation can be considered a regenerative therapy, an intervention which attempts to replace or restore the function of compromised tissue by harnessing innate properties of cells that cannot be replicated artificially. For such therapies to succeed, it will be necessary to understand and closely match the physiological conditions that govern cell behaviors in vivo. One important factor is low oxygen tension, termed hypoxia, which is often overlooked in vitro. Because oxygen insufficiency can lead to cell death, hypoxia has traditionally been viewed as a negative condition. However, hypoxia can also serve as a potent regulator of crucial cell behaviors involved in normal development, adaptation, and regeneration through transcriptional activity of hypoxia-inducible factors (HIFs). Characterizing HIF activity, especially in three-dimensional tissues which can experience oxygen heterogeneities, will be useful in investigating how culture parameters impact local microenvironmental conditions to affect cell behavior. In the present work, a novel fluorescent reporter system was used to detect cellular HIF activity in islet-like clusters of pancreatic &beta-cells and adipose-derived stem cell (ADSC) spheroids, two cell systems used in transplantation therapies. Fluorescent signaling was observed in cultures incubated in reduced oxygen conditions and in large cultures, indicating the development of tissue size-dependent oxygen gradients. HIF activity could be monitored in the same samples over time and could be correlated to other cell behaviors. In &beta-cells, signal was observed in all clusters incubated in 1% or 2% oxygen and in the center of large clusters (\u3e300ìm) incubated in 20% oxygen. In clusters that exhibited HIF activity, insulin secretion and cluster morphology were severely impaired. In ADSC spheroids, both reduction of external oxygen concentration and an increase in spheroid size increased HIF activity. Moderate HIF activity corresponded with increased vascular endothelial growth factor (VEGF) secretion from spheroids, while intense HIF activity corresponded with regional reduction of cell viability; therefore, an optimal size for VEGF secretion could be demonstrated. Altogether, the HIF reporter system represents a useful tool in monitoring cellular hypoxia so that culture parameters such as external oxygenation and culture size can be properly considered in order to guide desired cell behaviors in cell transplantation

A STUDY OF THE MOLECULAR GENETICS OF RENAL CELL CARCINOMA IN MAN

The aim was to measure the expression levels of hypoxia inducible factor 1-α (HJF-lα), glucose transporter one (GLUT-1), and vascular endothelial growth factor (VEGF) isoforms 165 and 189 mRNA in patients with renal cell carcinoma (RCC). Patients with RCC underwent radical nephrectomy at Derriford Hospital, Plymouth. Tumour as well as matched normal tissue from the kidney was harvested, snap frozen and stored in liquid nitrogen, and used to quantitate VEGF 165, VEGF 189, GLUT-1 and HJF-1α mRNA expression using ribonuclease protection assays, and quantified using a Phosphor-imager system. The VEGF 165 isoform was increased in the tumour tissue in comparison with the adjacent normal tissue (3.05 vs. 1.56 P=0.00002) as was the VEGF 189 isoform (2.41 vs. 1.43 P=0.0002). Forty four patients were analysed for the expression of HIF-lα and GLUT-1 with statically significant differences seen between the tumour tissues with respect to the normal tissue for both HIF-lα (1.34 vs. 1.10 P=0.01) and GLUT-1 (1.99 vs. 1.63 P=0.003). Hypoxia inducible factor I (HIF-1) is a key regulator of genes involved in the hypoxic response. HIF consists of alpha and beta subunits, with the alpha subunit being degraded under normoxic conditions and stabilised under hypoxia. Polymorph isms in exon 12 of the HIF gene have been recently been identified consisting of nucleotide changes (C+ 1772T and G+ 1790A) resulting in an amino acid substitution from Proline 582 to Serine, and Alanine 588 to Threonine respectively. These polymorphisms are found within the oxygen-dependent degradation domain (ODD) of the HIF-lα protein when transcribed which is important in the oxygen regulation of the protein via hydroxylation of the proline residue at position 564 (P564) by HIF-α- prolylhydroxylase (HIF-PH). The regulation of HIF-1α by this method is a novel way of regulating the levels of the protein, and polymorphisms in the ODD of HIF-lα may affect the ability of VHL to direct the alpha subunit for destruction. We have genotyped 146 patients and 288 controls for the G+ 1790A, and 160 patients and 162 controls for the C+ 1772T polymorphisms respectively. We found an increase in both the GA and CC (P<0.00001 and P= 0.00002) genotypes in our patients with renal cell carcinoma, and a decrease in GG and CT (P<0.00001 and P=0.00002) genotypes respectively. Haplotype analysis revealed there to be an increase in the T-A and C-A haplotypes (P=0.00008, and P=0.02) and a decrease in the T-G haplotype P=0.01. No statistical difference was found for the other haplotypes. We have shown that these HIF-lα polymorphisms are present in RCC with increased frequency and may play an important role in the disease process, leading to increased angiogenesis in the tumour. Vascular endothelial growth factor (VEGF) is a highly specific mitogen that is able to stimulate the proliferation of endothelial cells. There have been a number of findings linking the expression of VEGF with RCC, with it also being used to assess the prognosis of the disease. Polymorphisms in the VEGF gene have been recently identified. A possible link between promoter polymorphisms and expression of mRNA isoforms has been found in a variety of cytokines. Certain polymorphisms in renal cell carcinoma patients can lead to an up regulation of the expression of the mRNA, and may be a factor in the highly vascularized nature of the tumours studied. The aim was to investigate the frequency of polymorphisms within the VEGF gene (C-2578A) in 173 patients with RCC and 142 normal controls. No differences were seen between the patients and control populations, and the polymorphism did not correlate with Robson stage, Fuhrman grade or age and gender. Although a trend was seen between the C- 2578A polymorphism and expression of VEGF mRNA species in RCC patients