La formation de métastases osseuses implique de nombreuses interactions entre les cellules de cancer du sein et le microenvironnement osseux. Les gradients d’hypoxie et l’activation de HIF (hypoxia inducible factor) 1alpha sont essentiels au maintien de l’homéostasie osseuse. Le rôle de la signalisation HIF dans les ostéoblastes lors du processus métastatique n’a pourtant jamais été exploré. Dans cette étude, nous montrons que les cellules ostéoprogénitrices (OPC), se situent dans des niches hypoxique, et que l’activation de la signalisation HIF dans ces cellules augmente la masse osseuse et favorise les métastases osseuses du cancer sein. L’effet de la signalisation HIF dans les OPC n’est pas limité au squelette, en effet celle-ci stimule aussi la croissance des tumeurs mammaires et la dissémination tumorale dans les poumons et d’autres organes distants. Nous avons mis en évidence que la signalisation HIF dans les OPC induit l’augmentation de la concentration plasmatique de la chimiokine C-X-C motif ligand 12 (CXCL12), qui entraine une augmentation systémique de la prolifération et de la dissémination tumorale, via l’activation de son récepteur CXCR4 sur les cellules cancéreuses. Ainsi, nos résultats mettent en évidence le rôle protumorigénique de l’hypoxie dans le lignage ostéoblastique, lors de la formation de métastases osseuse, mais également par une action systémique sur les tumeurs mammaires et les métastases dans les tissus mous. Nous démontrons également que des altérations de l’anabolisme osseux peuvent affecter la progression du cancer du sein, révélant un nouveau rôle du squelette au sein du macroenvironnement tumoral. / Bone metastasis involves dynamic interplay between tumor cells and thelocal stromal environment. In bones, local hypoxia and activation of the hypoxiainducible factor (HIF)-1alpha in osteoblasts are essential to maintain skeletalhomeostasis. However, the role of osteoblast-specific HIF signaling in cancermetastasis is unknown. Here we show that osteoprogenitor cells (OPC) are locatedin hypoxic niches in the bone marrow, and that activation of HIF signaling in thesecells increases bone mass and favors breast cancer metastasis to bone locally.Remarkably, HIF signaling in osteoblast lineage cells also promotes breast cancergrowth and dissemination remotely, in the lungs and in other tissues distant frombones. Mechanistically, we found that activation of HIF signaling in OPC increasesblood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to asystemic increase of breast cancer cell proliferation and dissemination, throughdirect activation of the CXCR4 receptor. Hence, our data reveal a previouslyunrecognized role of the hypoxic osteogenic niche in promoting tumorigenesisbeyond the local bone microenvironment. They also indicate that alterations inbone formation can affect breast cancer progression, and support the concept thatthe skeleton is an important regulator of the systemic tumor environment.

Hypoxia Inducible Factors (HIF) functions are master regulators of oxygen homeostasis and have a key role in the physiological responses to hypoxia including angiogenesis and erythropoiesis. Under hypoxia, levels of HIF-α subunits increase, they hetereodimerise with HIF-1β sub unit and promote the initiation of transcription of target genes. Under normoxia, oxygen dependent HIF-α degradation is promoted by hydroxylation of either of two proline residues (Pro402 and Pro564). The interaction of prolylhydroxylated HIF-α with the Von Hippel-Lindau protein (pVHL) promotes hydrolytic degradation of HIFα through an E3 ubiquitin ligase proteasomal pathway. HIF prolyl hydroxylation is catalysed by three 2-oxoglutarate (2OG)-dependent oxygenases known as prolyl hydroxylase domain (PHD 1-3) proteins, through an Fe(II) mediated catalytic process using 2OG, and oxygen. The PHDs are part of the family of Fe(II) bound 2OG dependent oxygenases. There are approximately 70 human 2OG oxygenases many of which have biologically important roles. Small-molecule inhibitors have reached advanced clinical trials; however, many clinical candidates inhibit other structurally similar 2OG oxygenases (OGFOD1 and vCPH) potentially altering the therapeutic effect. This thesis describes the design and synthesis of potent and 2OG oxygenase selective inhibitors for the PHDs. The 1,3,8-triazaspiro[4.5]decane-2,4-dione and 4-hydroxy-2-(pyrazole)pyrimidine-5-amide series were chosen as initial 'hits' (reported in the patent literature). The main analogues of the series were characterised in vitro and in cells as potent and selective PHD inhibitors over structurally similar 2OG oxygenases (Chapter 2). Broad structure activity relationship (SAR) of both initial series demonstrated the sensitivity for PHD2 inhibition (Chapter 3). Combination of SAR work described in Chapters 2 and 3 lead to the development of the novel 4-hydroxy pyridine series. In-depth SAR resulted in optimised analogues including 1 (IC50 69 nM) and highly selective over structurally similar 2OG oxygenases including OGFOD1. The completed SAR work led to the development of two novel pharmacophores 2 and 3. Both pharmacophores displayed potent PHD inhibition and selectivity over OGFOD1. Analogues including 1 and 3 displayed on target cellular activity stabilising HIF-1α at 20 μM (Chapter 4). The 4-dimethylamine pyridine analogue displayed an increase in substrate hydroxylation on PHD2 in contrast to the DMSO control (Chapter 3). SAR and cellular characterisation indicated that the effect observed was not an assay artifact (Chapter 5). Fenofibrate was used as a starting point for the development of novel inhibitors of the oxygen consumption rate (OCR) via mitochondrial inhibition (Chapter 6). Analogues were synthesised in order to conduct broad SAR and on-target cellular activity was observed in a Seahorse XF assay (50% reduction in the OCR at 1 μM). A selection of amino and amide analogues warrant further investigation.

Le taux physiologique d’oxygène est finement régulé dans les tissus des mammifères, l’oxygénation diffère d’un tissu à l’autre, ainsi qu’au sein même de ces derniers, ce phénomène est en grande partie dû à l’architecture vasculaire. L’oxygène est un stimulus clef dans la destinée des cellules durant l’embryogenèse et la progression tumorale. Il est actuellement reconnu que les cellules souches se distribuent en suivant un gradient d’oxygène, où les faibles concentrations en oxygène (Hypoxie) favorisent la conservation d’un état indifférencié. Les cellules souches hématopoïétiques (CSH) résident dans des niches osseuses où la disponibilité en oxygène est limitée voir nulle. Le modèle de l’hématopoïèse a été largement décrit au niveau cellulaire et moléculaire et est un des principaux modèles utilisé dans l’étude des mécanismes gouvernant le caractère souche d’une cellule. Des études récentes révèlent dans des modèles murins que les facteurs de transcriptions induits par l’hypoxie (HIF) affectent le comportement des cellules souches hématopoïétiques. Ici, nous étudions le rôle potentiel des facteurs HIF-1α et HIF-2α dans les CSH Humaines. Le knockdown des deux facteurs a été obtenu en combinant une stratégie de short-hairpin RNA et de vecteurs de transferts lentiviraux. La transduction de cellules de sang de cordon CD34-positive révèle que les deux knockdowns affectent à court terme la croissance des progéniteurs et CSH et à long terme leurs capacités de reconstitution dans des souris immuno-déficientes NOD/LtSz-scid IL2rgnull. Cependant, nous observons un effet plus délétère de HIF-2α sur le maintien des cellules souches en comparaison à HIF-1α. / Physiological oxygen level is tightly regulated in mammalian tissues. Moreover, oxygenation differs from one tissue to the other, as well as within a single tissue, due to vasculature modeling. Oxygen levels have been shown to be a key stimulus of cell fate during embryogenesis and cancer progression. It is now widely admitted that stem cells fate followes oxygen gradients with low levels (hypoxia) promoting an undifferentiated state. Hematopoietic stem cells (HSC) reside in bone marrow niches in which oxygen availability is low, even absent. Hypoxia-inducible factors (HIF) are the main factors regulating the cell-response to oxygen variation. Studies on mouse models reveal that HIFs affect HSC activity. Here, we investigate the potential function of HIF-1α and HIF-2α within human HSCs. Knockdown of both factors was obtained by lentivirus-mediated short-hairpin RNA strategy. Transductions of CD34-positive cord blood cells reveal that HIF-2α knockdown affects myelo-erythroid differentiation. Both HIF-α are required for long-term reconstitution in immune-deficient NOD/LtSz-scid IL2rγnull mice, whereas a more pronounced deficiency was observed for HIF-2α. Overall, our data strongly suggest the existence of multiple but preponderant functions in human stem cell maintenance and differentiation.

Cancer is one of the leading causes of death. There have been many investigations into therapeutic ways to prevent and reverse cancerous growth. We report a new approach in this thesis, which is to investigate the functions of Vitamin B1 (thiamine) in cancerous cells and their regulation. A number of thiamine analogs were synthesized to carry out the structure-activity relationship (SAR) studies with two transporters THTR1 and THTR2. Initial results show that the modifications of thiazole reduced the uptake of thiamine.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most insidious forms of cancer whose incidence nearly equals its death rate. Despite extensive research studies, no effective therapeutic approaches for diminishing the morbidity associated with this disease are available. PDAC is characterized by activating Kras mutations and inactivation of Ink4a and the p53-Arf pathway in virtually all cases, while SMAD4¡Xa central regulator of Transforming growth factor-beta (TGF-£]) signaling¡Xis inactivated in 55% of PDAC. Our overall goal is to understand how perturbations in the inactivation of SMAD4 pathway contribute to the late stages of PDAC pathogenesis, and to elucidate the role of SMAD4 inactivation on the conversion of a benign form of the cancer to a more aggressive metastatic form. To address this important topic in cancer biology, we have devised a strategy to develop model cell lines to dissect the role of SMAD4 defect in PDAC cell lines and the potential synergistic effects of hypoxia and/or TGF-£]1 upon SMAD4 inactivation in their metastatic properties. Experiment results showed SMAD4 restored in PDAC model cell lines were down regulate HIF-1£\, VEGF, FGF10 and FGFR2 genes expression level, and also inhibited migration, chemoresistance and angiogenesis of cancer cells. We hypothesize that these effects are due to SMAD4 suppresses some cancer genes in PDAC. Further detailed investigations are also needed to fully elucidate the detail mechanisms for our findings here therefore, the future works of this study will go step on looking for those important downstream effect genes regulated by Smad4 protein in PDAC cells and try to find out the connection of all the dependence proteins.

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2009-08-19 21:09:13.707

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17781号 / 医博第3807号 / 新制||医||999(附属図書館) / 30588 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 上本 伸二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction: Since the era of Marshall, H. pylori has been to be implicated in many upper digestive tract diseases, such as gastritis, peptic ulcer disease, mucosa- associated lymphoid tissue lymphoma, and even gastric adenocarcinoma. In 1994, WHO recognized H. pylori as a definite carcinogen for gastric cancer. Many study had shown that microbial pathogens may induce oxidative stress in infected host cell. And this may also represent an important mechanism leading to epithelial injury in H. pyloric infection. Oxidative stress plays a role in altering epithelial cell turnover, accelerating apoptosis and increasing oxidative DNA damage. One of the evidences for this phenomenon is increasing level of reactive oxygen species (ROS) measured in the mucosa of infected stomach. ROS may activate HIF-11£\ transcription. HIF-1£\ overexpression had been detected in several human cancers. Furthermore its overexpression correlates significantly with highly aggressive disease, lymph node metastasis, clinicopathological status and poor prognosis in some cancer types. It may up regulate hypoxia-induced gene, such as the vascular endothelial growth factor (VEGF) transcription and angiogenesis. Therefore we propose Sp1-pVHL-HIF-1£\ pathway may play a role the carcinogenesis in Hp -associated gastric cancer. Material and methods: We took Paraffin-embedded specimens from 89patients, who had undergone UGI endoscopy and gastric mucosa biopsy. We assessed the Sp1, pVHL, HIF-1£\ in all cases by immunohistochemistry and then evaluated their correlation with the H pylori infection. Chi-square and Fisher¡¦s exact test was performed to determine the significance of the difference between Sp1, pVHL, HIF-1£\. Results: There are not significant difference in nuclear Sp1 expression and H. pylori different (p=0.59). Sp1 expression was not significant, (p=0.91, 0.93, 0.36, 0.42, 0.51) with sex, age, location, TNM stage and cell differentiation. pVHL protein was mainly expressed in the cytoplasm. There are no significant difference with H. pylori infection (p=0.14). The relationship pVHL protein expression between with sex, age, lesion site, TNM stage and cell differentiation were not significant (p=0.39, 0.70, 0.69, 0.83, 0.70). HIF-1£\ protein was mainly expressed in the nuclei. There are not significant association with H. pylori infection (p=0.49). There were no significant differences between HIF-1£\ protein expression with sex, age, location, TNM stage and cell differentiation (p=0.94, 0.32, 0.75, 0.35, 0.60). Furthermore, In normal tissue the expression of HIF-1£\ had significant association with pVHL(p=0.0002), and the expression also had no a mariginally significant association with Sp1(p=0.096). Expression of Sp1 had significant association with pVHL(p=0.0016)in tumor tissue, Therewas a significant association between normal and tumor tissue expression of the pVHL and Sp1(p=0.038, 0.019), but the expression of HIF-1£\ had no significant(p=0.23). Conclusion: In this study, we attempt to determine the association between Sp1-pVHL -HIF-1£\ pathway and a role in the carcinogenesis in H. pylori infection. Although we didn¡¦t confirm the hypothesis Sp1-pVHL -HIF-1£\ pathway playing an critical role in the mechanism of gastric cancer. We concluded that there is no significance between the expression of Sp1, pVHL and HIF-1£\ and gastric cancer, but the role of this pathway in the Hp infection associated carcinogenesis is still to be clarified.

Hypoxia Inducible Factors (HIFs) are very important transcription factors that can respond to low oxygen concentrations in the cellular environment. Inhibition of HIF’s transcriptional activity represents a promising approach to new anticancer compounds. Herein, we describe the design and synthesis of a series of HIF-1 inhibitors. Evaluation of these inhibitors using a cell-based luciferase assay led to the discovery compounds with sub-micromolar potency.

Le facteur de transcription HIF-1 ±hypoxia-inducible factor-1¿ est responsable de la réponse cellulaire à l'hypoxie. Cependant, il y a également des activateurs non-hypoxiques de HIF-1 incluant des hormones et des facteurs de croissance. L'angiotensine II (Ang II), l'hormone effectrice dans le système rénine-angiotensine, est un activateur non-hypoxique de HIF-1 puissant dans les cellules musculaires lisses vasculaires (CMLVs). Cette activation par Ang II implique des mécanismes de transcription, de traduction et de stabilisation protéiques. De plus, les espèces réactives d'oxygène (ERO) sont impliquées dans l'augmentation de la stabilisation de HIF-1 pendant un traitement à l'Ang II. Ce travail a pour but d'élucider le rôle des générateurs d'ERO dans l'induction de HIF-1 par l'Ang II dans les CMLVs. Ni un ARN interférence contre la sous-unité p22 de la NADPH oxydase ni le traitement avec un inhibiteur spécifique de la NADPH oxydase n'ont permi de diminuer l'accumulation de HIF-1 par l'Ang II. Néanmoins, l'inhibition pharmacologique du complexe III de la mitochondrie, l'épuisement cellulaire de la protéine mitochondriale Rieske Fe-S et le traitement des cellules avec un antioxydant mitochondrial diminuent de façon importante l'accumulation de HEF-1. En outre, l'inhibition des ERO mitochondriales (mtERO) supprime la stabilisation de HIF-1 et la transcription de gènes dépendante de HIF-1 par l'Ang II. De nombreuses études impliquent les ERO générées par la NADPH oxydase dans les voies de signalisation suivant la stimulation des CMLVs par l'Ang IL Toutefois, nos travaux identifient les mtERO et non les ERO dérivées de la NADPH oxydase comme étant des intermédiaires essentiels dans l'accumulation et la stabilisation de HIF-1.