Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2alpha gene (EPAS1) in normal cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2alpha gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumours and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2alpha pathway in the hypoxic tumour microenvironment, which is necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumour growth in vivo. In addition, restoring HIF-2alpha expression in DNMT3a-reintroduced cancer cells restores full tumorigenic potential, including the capacity to traverse the hypoxic barrier. These data support a tumour-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2alpha oxygen-sensing pathway and the cellular response to hypoxia.

<p>Tumor hypoxia is an enduring problem for traditional cancer therapies such as radiation and chemotherapy. This obstacle has traditionally been attributed to the widespread presence of chronic, diffusion-limited hypoxia in solid tumors; recent data suggests that tumor hypoxia may also be spatially and temporally variable. In this work we characterize the presence of spatial and temporal fluctuations in hypoxia, as well as use mathematical modeling to predict the impact of fluctuations on the hypoxic cytotoxin, tirapazamine, and examine potential mechanisms of fluctuations in tumor oxygenation. Using phosphorescence lifetime imaging on preclinical tumors, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and that previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Then, using a one-dimensional theoretical model, we examine the effects of fluctuating hypoxia on metabolized tirapazamine concentration; we find that fluctuating oxygen reduces the concentration of metabolized tirapazamine at distances farther from the source, thereby limiting its ability to reach and kill the most hypoxic cells. Finally, we use a three-dimensional Green's function oxygen transport model to explore the effects of temporal fluctuations in hemoglobin saturation, blood flow, and overall perfusion on tumor tissue oxygenation. Results from the model predict that hemoglobin saturation has a dominant effect on tissue oxygenation. These studies collectively suggest that the pervasive temporal and spatial heterogeneity in tumor oxygenation are highly therapeutically relevant, and future clinical and preclinical studies are needed.</p> / Dissertation

abstract: Hypoxia is a pathophysiological condition which results from lack of oxygen supply in tumors. The assessment of tumor hypoxia and its response to therapies can provide guidelines for optimization and personalization of therapeutic protocols for better treatment. Previous research has shown the difficulty in measuring hypoxia anatomically due to its heterogenous nature. This makes the study of hypoxia through various imaging modalities and mapping techniques crucial. The potential of hypoxia targeting T1 contrast agent GdDO3NI in generating hypoxia maps has been studied earlier. In this work, the similarities between hypoxia maps generated by MRI using GdDO3NI and pimonidazole based immunohistochemistry (IHC) in non-small cell lung carcinoma bearing mice have been studied. Six NCI-H1975 tumor-bearing mice were studied. All animal studies were approved by Arizona State University’s Institute of Animal Care and Use Committee (IACUC). Post co-injection of GdDO3NI and pimonidazole, T1 weighted 3D gradient echo MR images were acquired. For ex-vivo analysis of hypoxia, 30 μm thick tumor sections were obtained for each harvested tumor and were stained for pimonidazole and counter-stained with DAPI for nuclear staining. Pimonidazole (PIMO) is clinically used as a “gold standard” hypoxia marker. The key process involved stacking and iterative registration based on quality metric SSIM (Structural Similarity) Index of DAPI stained images of 5 consecutive tumor sections to produce a 3D volume stack of 150 μm thickness. Information from the 3D volume is combined to produce one final slide by averaging. The same registration transform was applied to stack the pimonidazole images which were previously thresholded to highlight hypoxic regions. The registered IHC stack was then co-registered with a single thresholded T1 weighted gradient echo MRI slice of the same location (~156 μm thick) using an elastic B-splines transform. The same transform was applied to achieve the co-registration of pimonidazole and MR percentage enhancement image. Image similarity index after the co-registration was found to be greater than 0.5 for 5 of the animals suggesting good correlation. R2 values were calculated for both hypoxic regions as well as tumor boundaries. All the tumors showed a high boundary correlation value of R2 greater than 0.8. Half of the animals showed high R2 values greater than 0.5 for hypoxic fractions. The RMSE values for the co-registration of all the animals were found to be low further suggesting better correspondence and validating the MR based hypoxia imaging. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

<p>In this thesis, a model of tissue oxygenation is presented, that takes into account the heterogeneous nature of tumor vasculature. Even though the model is rather simple, the resulting oxygen distributions agree very well with clinically observed oxygen distributions for most tumors and healthy normal tissues. The model shows that the vascular density may not describe the oxygenation of a tissue sufficiently well, unless the heterogeneity of the vascular system is taken into account. Based on the oxygen distributions from the tissue model, the associated radiation response at low and high doses can be determined. </p><p>The radiation response of heterogeneous tumors should preferably be described by two clonogen compartments, one resistant and one sensitive, dominating the response at high and low radiation doses, respectively. Furthermore, each compartment should be characterized by the effective radiation resistance and the effective clonogen number. The resistant-sensitive model of radiation response has been analyzed in great detail. It accurately describes the response of severely heterogeneous tumors, both at low and high doses and LET values. The effective response parameters are given as integrals, averaged over the whole spectrum of radiation resistance. The parameters can also be determined from clinically established dose-response relations. </p><p>The main properties of the dose-response relation for a generally heterogeneous tumor is described in some detail. The normalized dose-response gradient has been generalized to take heterogeneities in both dose delivery and radiation response into account. This quantity is important for accurate treatment plan optimization using intensity modulated radiation therapy for individual patients. </p>

In this thesis, a model of tissue oxygenation is presented, that takes into account the heterogeneous nature of tumor vasculature. Even though the model is rather simple, the resulting oxygen distributions agree very well with clinically observed oxygen distributions for most tumors and healthy normal tissues. The model shows that the vascular density may not describe the oxygenation of a tissue sufficiently well, unless the heterogeneity of the vascular system is taken into account. Based on the oxygen distributions from the tissue model, the associated radiation response at low and high doses can be determined. The radiation response of heterogeneous tumors should preferably be described by two clonogen compartments, one resistant and one sensitive, dominating the response at high and low radiation doses, respectively. Furthermore, each compartment should be characterized by the effective radiation resistance and the effective clonogen number. The resistant-sensitive model of radiation response has been analyzed in great detail. It accurately describes the response of severely heterogeneous tumors, both at low and high doses and LET values. The effective response parameters are given as integrals, averaged over the whole spectrum of radiation resistance. The parameters can also be determined from clinically established dose-response relations. The main properties of the dose-response relation for a generally heterogeneous tumor is described in some detail. The normalized dose-response gradient has been generalized to take heterogeneities in both dose delivery and radiation response into account. This quantity is important for accurate treatment plan optimization using intensity modulated radiation therapy for individual patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21257号 / 医博第4375号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 妹尾 浩, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O tumor sob hipóxia apresenta resistência a terapia antitumoral convencional por diferentes mecanismos. O uso de métodos diagnósticos moleculares não invasivos, como imagem por PET, permite a identificação de tumores sob hipóxia e auxilia no delineamento da estratégia terapêutica mais adequada. Atualmente, diversas pesquisas têm demonstrado alternativas ao tratamento de tumores sob hipóxia, explorando características como, potencial redutor do tumor e inibição de mecanismos de adaptação celular para a sobrevivência sob essa condição. Assim, neste trabalho foi realizada a síntese e avaliação in vivo de novo derivado 2-nitroimidazol, contendo o grupo hidrofílico zwiteriônico amôniometil-trifluoroborato (AMBF3), 18F-AmBF3-bu-2NI, com potencial para geração de imagens de tumores sob hipóxia. O composto AmBF3-bu-2NI foi facilmente preparado em 4 etapas sintéticas. A marcação com 18F foi realizada via reação de troca isotópica 18F-19F e 18F-AmBF3-bu-2NI foi obtido em 14,8 ± 0,4% de rendimento radioquímico (n = 3) com decaimento corrigido, 24,5 ± 5,2 GBq/?mol de atividade específica e >99% de pureza radioquímica. Estudos de imagem e biodistribuição ex vivo em camundongos, portando tumores HT-29, demonstraram que 18F-AmBF3-bu-2NI possui rápido clearance do sangue, com excreção pelas vias hepatobiliar e renal. No entanto, o tumor não foi visualizado em imagens de PET até 3 h pós-injeção devido à baixa captação tumoral (0,54 ± 0,13 e 0,19 ± 0,04% AI/g em 1 e 3 h pós-injeção, respectivamente), devido à não difusão de 18F-AmBF3-bu-2NI através da membrana celular. Adicionalmente, compostos quinazolinicos com potencial aplicação em diagnóstico foram também sintetizados contendo unidades biorredutives, nitro-benzil e nitro-imidazol, além de grupo fluoroetil, inicialmente contendo 19F (frio), como padrão analítico para a síntese do radiotraçador. Entretanto, devido a formação de produtos voláteis durante a radiossíntese da unidade 2-[18F]fluoroetil 4-metilbenzenosulfonato (34*), para incorporação no anel quinazolínico, a obtenção do radiotraçador e os correspondentes estudos de biodistribuição e imagem não foram realizados. Em paralelo ao trabalho anterior, foi realizada a síntese de um conjunto de 12 compostos aminotriazolil-quinazolínicos com potencial atividade antitumoral, via reação de cicloadição CuAAC. Inicialmente todos derivados quinazolínicos obtidos no trabalho para aplicação no diagnóstico foram testados em uma série de linhagens de células tumorais sob condições de normóxia e hipóxia (MDA-MB-231, SKBR3, BT474, PC3, MKN45, U251, U87, MIA PaCa-2, Skmel37, e A549, na concentração de 10 ?M), empregando cisplatina como referência. Neste estudo, apenas os derivados contendo grupo nitro-benzil-triazólico 61 e 63, apresentaram cerca de 50% de inibição de células MKN45 em normóxia e 40% em células SKBR3 sob hipóxia, respectivamente. Na sequência, os 12 derivados aminotriazolil-quinazolínicos foram submetidos a avaliação da citotoxicidade in vitro sob as linhagens de células tumorais de mama (MDA-MB-231, SKBR3, BT474, na concentração de 30 ?M), empregado os controles positivos Erlotinib e ii Lapatinib. Apenas o derivado contendo a função ftalimida 9, não substituído nas posições C-6 e C-7 do anel quinazolínico, apresentou cerca de 60% de inibição de células SKBR3 em hipóxia. Paralelamente, os derivados aminotriazolil-quinazolínicos foram submetidos à avaliação de triagem da atividade inibitória frente as quinases HER2, EGFR e PERK, na concentração de 10 ?M. Todavia, não houve inibição significativa nas enzimas avaliadas na concentração testada. Novos ensaios estão em andamento a fim de determinar a capacidade dos compostos atuarem como inibidores do crescimento de outras linhagens de células tumorais. / Tumor hypoxia is resistant to conventional antitumor therapy by different mechanisms. The use of non-invasive molecular diagnostic methods, such as PET imaging, allows the identification of tumors under hypoxia and assists in designing the most appropriate therapeutic strategy. Currently, several researches have provided alternative treatments for tumors under hypoxia, exploring some specific properties, such as tumor reducing potential and inhibition of adaptive mechanisms required for cell survival under hypoxia. Thus in this work, it was performed the synthesis and in vivo evaluation of new 2-nitroimidazole derivative, containing the zwitterionic hydrophilic group, ammonium methyl- trifluoroborate (AMBF3), 18F-AmBF3-bu-2NI, with potential for tumor imaging in hypoxia. The compound AmBF3-bu-2NI was easily prepared in four steps. 18F labeling was conducted via 18F-19F isotope exchange reaction, and 18F-AmBF3-bu-2NI was obtained in 14.8 ± 0.4% (n = 3) decay-corrected radiochemical yield with 24.5 ± 5.2 GBq/?mol specific activity and > 99% radiochemical purity. Imaging and biodistribution ex vivo studies in HT-29 tumor-bearing mice showed that 18F-AmBF3-bu-2NI cleared quickly from blood, and was excreted via the hepatobiliary and renal pathways. However, tumor PET images were not visualized until 3 h post-injection due to low tumor uptake (0.54 ± 0.13 and 0.19 ± 0.04%ID/g at 1 h and 3 h post-injection, respectively) due to non-diffusion of 18F-AmBF3-bu-2NI through the cell membrane. Additionally, quinazolinic compounds with potential diagnostic application were also synthesized containing biorreductive units, nitrobenzyl and nitroimidazole, as well as a fluoroethyl group, initially containing 19F (cold), as an analytical standard for the synthesis of the radiotracer. However, due to the formation of volatile products during the radiosynthesis of the 2-[18F] fluoroethyl 4-methylbenzenesulfonate (34*) unit, for incorporation into the quinazoline ring, the radiotracer preparation and its corresponding biodistribution and imaging studies were not performed. Concomitantly to the previous work, the synthesis of a set of 12 aminotriazolyl-quinazoline compounds with potential antitumor activity was performed, via the CuAAC cycloaddition reaction. Initially, all quinazolinic derivatives obtained in the work for application in the diagnosis were tested in a range of tumor cell lines under normoxia and hypoxia conditions (MDA-MB-231, SKBR3, BT474, PC3, MKN45, U251, U87, MIA PaCa-2, Skmel37, and A549, at 10 ?M), using cisplatin as a reference. In this study, only the derivatives bearing the nitrobenzyltriazole group 61 and 63 showed about 50% inhibition of MKN45 cells in normoxia and 40% in SKBR3 cells under hypoxia, respectively. In the sequence, the 12 aminotriazolyl-quinazoline derivatives were submitted to in vitro cytotoxicity evaluation using breast tumor cell lines (MDA-MB-231, SKBR3, BT474, at 30 ?M), in the presence of the reference drugs Erlotinib and Lapatinib. Only the derivative containing the phthalimide function 9, unsubstituted at C-6 and C-7 positions of the quinazoline ring, displayed about 60% inhibition on SKBR3 cells under hypoxia. Concomitantly, the inhibitory iv activity of these aminotriazolyl-quinazoline derivatives were also subjected to a screening evaluation against the HER2, EGFR and PERK kinases, 10 ?M. However, there was no significant inhibition of these enzymes at the tested concentration. New assays are ongoing to determine the inhibitory activity under other tumor cell lines.

No description available.

Les gliomes malins de l’enfant (GME), de pronostic sombre, se distinguent des gliomes malins de l’adulte (GMA) sur le plan biologique mais aussi clinique, avec des taux de réponse au témozolomide (chimiothérapie alkylante de référence chez l’adulte) moindres. L’efficacité du temozolomide est réduite par l’action de l’enzyme de réparation de l’ADN, l’O6-methylguanine-DNA-methyltransferase (MGMT), dont l’expression est fréquemment inhibée par méthylation du promoteur de son gène dans les GMA. En première partie, la mise au point d’une nouvelle technique de PCR spécifique de méthylation a montré une fréquence plus faible dans les GME (15%) vs les GMA (45%, p<0,001). En deuxième partie, l’hypoxie intra-tumorale et la dérégulation en amont de l’axe mTOR-HIF-1α, connus pour être impliqués dans la chimio-résistance, ont été étudiés dans les GME et ciblés par l’association rapamycin-irinotecan dans une étude in vitro, pour laquelle des lignées dérivées de GME ont été développées. / Pediatric malignant glioma (PMGs), are associated with a very dismal prognosis. They are distinct from their adult counterparts (AMGs), biologically but also clinically, with a lower response to temozolomide (the current reference alkylating chemotherapy) compared to AMGs. Temozolomide efficacy is reduced by the activity of the DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), whose expression is frequently silenced by promoter methylation. First, the development of a new methylation-specific PCR showed a lower frequency of MGMT methylation in PMGs (15%) vs AMGs (45%, p<0,001). Secondly, intra-tumor hypoxia and the upstream deregulation of mTOR-HIF-1α axis, well-known to be involved in chemo-resistance and the up-regulation of MGMT expression, were studied in a PMG cohort. The targeting of this axis was then studied in vitro using a therapy combining rapamycin and irinotecan. For this, pediatric patient-derived malignant glioma cell lines were developed.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24801号 / 医博第4993号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 実, 教授 溝脇 尚志, 教授 江木 盛時 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM