Il devient de plus en plus clair que pour traiter efficacement les tumeurs solides, nous devons également nous intéresser au microenvironnement tumoral. Physiologiquement, les zones intratumorales peuvent présenter une disponibilité anormale en nutriments, un pH altéré ou encore des niveaux d’oxygène bas (hypoxie). Il est connu que l’adaptation hypoxique engendre des cellules tumorales qui sont plus difficiles à traiter indépendamment de l’approche thérapeutique. De plus, l’adaptation hypoxique est nécessaire pour la progression tumorale puisque cette dernière favorise des processus tels que: la survie cellulaire, la motilité, l’angiogenèse, le métabolisme du glucose, l’immunomodulation ainsi que la résistance aux médicaments. Ces phénotypes passent par la régulation des ARN messager (ARNm) et des micro ARN (miARN). Pour ces raisons, des efforts importants ont été déployés pour comprendre l’adaptation hypoxique et les interventions thérapeutiques potentielles pouvant la contrer. À l’heure actuelle, il y a un manque de cohérence et de variété dans les protocoles de traitement hypoxique in vitro qui ne tient pas compte des aspects importants de l’hypoxie in vivo, comme la réduction de la disponibilité en éléments nutritifs, la durée de l’exposition hypoxique ainsi que le degré d’hypoxie. Pour mieux simuler le microenvironnement hypoxique in vivo, nous avons développé de nouveaux protocoles hypoxiques in vitro qui visent à simuler ces aspects. Tout d’abord, en utilisant une lignée cellulaire B16-HIF1a-eGFP, nous avons optimisé le stress métabolique à court terme en conjonction avec l’hypoxie pour augmenter la stabilisation de l’HIF1a. Pour déterminer comment le programme HIF1 adapte les cellules à ces différentes conditions, nous avons analysé les données de séquencage d’ARN qui démontrent que le stress métabolique induit un programme transcriptionnel HIF1 plus robuste et diversifié dans les cellules hypoxiques, et que ce dernier est représentatif du stress hypoxique in vivo. Nous avons également identifié de nouveaux miARN induits par l’hypoxie et démontré que notre protocole d’incubation régule davantage les miRNA associés au pronostic négatif du patient. Nous avons aussi étudié l’adaptation hypoxique à long terme et extrême in vitro. Nous avons observé que l’incubation hypoxique à long terme induit une transition épithéliale à mésenchymateuse (TME), indépendante de l’expression différentielle des facteurs de transcription du TME canonique. Ce changement se produit à des niveaux spécifiques d’oxygène, et nécessite une pré-incubation à des niveaux hypoxiques plus faible. Avec ce protocole, nous avons découvert une nouvelle isoforme de WT1 (tWT1), un moteur potentiel du TME. tWT1 commence la transcription dans l’intron 5 du gène WT1, une région avec plusieurs séquences d’ADN contenant des éléments de réponse à l’hypoxie. La protéine tWT1 a une fonctionnalité limitée : elle est localisée au niveau du noyau, et conserve la liaison de l’ADN aux régions précédemment connues. Nous avons aussi identifié l’expression de tWT1 dans les échantillons de patients atteints de leucémie ainsi qu’une isoforme tWT1 potentiellement plus fonctionnelle grâce à des analyses par kmer. Pour cibler ces phénotypes identifiés dans nos expériences d’adaptation hypoxiques, nous avons développé une nouvelle catégorie d’ARN intérférent (ARNi) thérapeutique : le microARN synthétique (synmiR). Les synmiR sont des molécules de RNAi avec des multiples cibles. En utilisant des expériences in vivo, nous avons établi de nouveaux principes de RNAi qui élargissent considérablement l’espace de conception pour les synmiR. Nous avons mis au point deux algorithmes de conception de synmiR distincts et avons testé leur efficacité dans le contrôle de l’activité transcriptionnelle du génome du VIH in vivo. En conclusion, nous avons montré que l’inclusion de facteurs physiologiques supplémentaires associés à l’hypoxie in vitro entraîne un engagement plus robuste de l’adaptation de l’hypoxie. À ce jour, aucun de nos protocoles d’hypoxie n’a été reproduit dans la littérature. Nous contribuons aux connaissances dans le domaine en décrivant les nouveaux ARNm/miARN induits par l’hypoxie, ainsi que la méthode d’induction fiable de l’EMT par l’hypoxie seulement. Nous faisons également état de l’existence de nouveaux isoformes de WT1 et de leurs liens avec le cancer et l’hypoxie. La connaissance de ces isoformes est importante pour l’avenir de la recherche sur WT1, car elle pourrait faire la lumière sur des résultats auparavant inexplicables. Notre travail dans les synmiR ouvre une nouvelle voie d’investigation pour le traitement de certaines maladies, et fournit un mécanisme d’action testable pour les miRNA endogènes. Une fois suffisamment développés, les synmiR offrent une occasion thérapeutique unique d’exploiter leur multi-ciblage pour avoir un impact spectaculaire sur une seule voie, ou affecter plusieurs voies par le ciblage simultané de gènes clés. / It is becoming increasingly clear that in order to effectively treat solid tumours, we must also address the tumour microenvironment. Physiologically, intratumoral areas may have abnormal nutrient availability, pH, or lower oxygen levels (hypoxia). It is known that hypoxic adaptation results in tumour cells which are harder to treat regardless of therapeutic approach, and hypoxic adaptation is necessary for disease progression due to the induction of tumour promoting phenotypes such as, but not limited to: cell survival, motility, angiogenesis, glucose metabolism, immunomodulation, and drug resistance. This is accomplished through the regulation of both mRNAs and miRNAs. For these reasons, significant effort has been applied to understanding hypoxic adaptation and potential therapeutic interventions. Currently, there is a lack of consistency and protocol variety in in vitro hypoxic treatments that leaves out important aspects of in vivo hypoxia, such as reduced nutrient availability, length of hypoxic exposure, and degree of hypoxia. To better simulate the in vivo hypoxic microenvironment, we have developed new in vitro hypoxic protocols which aim to simulate these aspects. First, using a B16-HIF1α-eGFP hypoxia reporter cell line, we optimized short-term metabolic stress in conjunction with hypoxia to enhance HIF1α stabilization. To ascertain how the HIF1 program adapts the cells to these different conditions, deep transcriptome profiling were performed and demonstrated metabolic stress induces a more robust and diversified HIF1 transcriptional program in cells under hypoxia, which was more representative of in vivo hypoxic stress. We identified novel hypoxia-induced miRNAs as well, and demonstrated our incubation protocol regulated more miRNAs associated with negative patient prognosis. We also investigated long-term and extreme hypoxic adaptation in vitro. Long term hypoxic incubation induced a epithelial to mesenchymal transition (EMT), independent of canonical EMT factor differential expression. This switch occurred at specific oxygen levels, and required pre-incubation at milder hypoxic levels, highlighting the relevance of simulating in vivo hypoxia development in vitro. Through this protocol, we discovered a novel isoform of WT1 (tWT1), a potential driver of our EMT. tWT1 begins transcription within intron 5 of the WT1 gene, a region with several Hypoxia Response Elements DNA sequences. tWT1 retains limited functionality: it is able to localize to the nucleus, and retains DNA binding to previously known gene promoter regions. We also identified the expression of tWT1 in leukemic patient samples as well as a potentially more functional tWT1 isoform through kmer-based analyses. To target these multiple phenotypes identified in our hypoxia adaptation experiments, we worked towards developing a new category of RNA-interference (RNAi) therapeutic, the synthetic microRNA (synmiR). SynmiRs are single-sequence, multi-targeted RNAi molecules. Using in vivo knock-down experiments, we established new RNAi principles which dramatically expand the design space for synmiRs. We developed two philosophically distinct synmiR design algorithms, and validated their efficacy in controlling HIV genome transcriptional activity in vivo. In conclusion, we have shown the inclusion of additional physiological factors associated with hypoxia in vitro results in a more robust engagement of hypoxia adaptation. To date, neither of our hypoxia protocols have been replicated in the literature. We contribute to the literature by describing novel hypoxia induced mRNAs/miRNAs, as well as methods for reliably inducing EMT through hypoxia alone. We also discovered the existence of novel WT1 isoforms and their links to cancer and hypoxia. Knowledge of these isoforms is important for WT1 research moving forward, as it may shed light on previously unexplainable results. Our work in synmiRs opens a new therapeutic avenue for multiple disease states, and provides a testable mechanism of action for endogenous miRNAs. Once sufficiently developed, synmiRs offer a unique therapeutic opportunity to harness their multi-targeting to dramatically impact a single pathway, or affect multiple pathways through simultaneous targeting of key genes.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24879号 / 医博第5013号 / 新制||医||1068(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 後藤 慎平, 教授 渡邊 直樹, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Transient Receptor Potential channels (TRP) are a superfamily of cationic specific ionchannels that are regulated by various stimuli such as temperature, pH, mechanical stress, ligandsand ion concentration. The role of TRP channels in disease states such as autosomal dominantpolycystic kidney disease, cancer metastasis, and developmental defects lend credence to thebelief that they play an important part in epithelial morphogenesis events. The development ofsomites, neural tube closure and migration of neural crest cells to form things such as the faceand heart is a good developmental model for the aforementioned cellular processes. We haveshown that TRP channels can be found in the developing ectoderm, hindbrain, and heart and thatthe inhibition of TRP channels in a developing embryo results in phenotypes suggestingperturbation of cellular remodeling processes. This leads to the question of the specific role ofTRP channels in the epithelial mesenchymal transition and remodeling in developing chickembryos.

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The extracellular matrix protein Fibronectin (FN) plays an important role in cell contractility, differentiation, growth, adhesion, and migration. The 12th -14th Type III repeats of FN (FNIII 12-14), also referred to as the Heparin-II domain, comprise a highly promiscuous growth factor (GF) binding region. This binding domain aids in cellular signaling initiated from the ECM. Additionally, FN has the ability to assemble into fibrils under certain conditions, mostly observed during cell contractile processes such as those that initiate due to upregulation of Transforming Growth Factor Beta 1 (TGF-β1) [1], [2]. Previous work from our lab has shown that self-assembly of FN into insoluble fibrils is crucial for Epithelial-Mesenchymal Transition (EMT) [3]. The transition from epithelial to mesenchymal cell type has been implicated as an early event in tumor formation and breast cancer. We were previously able to find that upregulation of FN fibrils drive EMT through contractility due to the increase of the GF latent TGF-β complex concentration at the cell membrane [3]. The challenge in the current work is to exploit the role of Heparin-II binding domain and to concentrate growth factors of interest, such as those that are pro-EMT or anti-EMT at the signaling sites of the cell membrane. Initially, we investigated the localization of the fragments FNIII 12-14 delivered to cell membrane using FITC conjugated protein. We then investigated the effects of exogenous FNIII 12-14 on EMT using breast epithelial cells (MCF10A) in the presence or absence of TGF-β1 to determine whether FNIII 12-14 alters EMT signaling. Quantification of mRNA expression, for EMT markers such as Slug, Snail, Twist, and ZEB1 were analyzed. Results showed that dosage increase of FNIII 12-14 appears to inhibit EMT transcription factors. This study will develop a new understanding of disease and gene control using ECM proteins. The exploitation of ECM natural protein interactions could become a new method in turning on/off genes of interest. While we are currently investigating this as a mechanism of blocking EMT, it could also have implications in wound healing, fibrosis, and tissue engineering, where EMT is an important aspect of the physiologic progression.

<p>The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.</p><p>The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, <i>Id2</i> and <i>Id3</i>. Interestingly, we found that TGF-β-induced repression of <i>Ids</i> is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of <i>Ids</i> and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.</p><p>Furthermore, we identified a serine/threonine kinase, <i>SNF1LK</i>, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling. </p>

The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread. The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, Id2 and Id3. Interestingly, we found that TGF-β-induced repression of Ids is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of Ids and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways. Furthermore, we identified a serine/threonine kinase, SNF1LK, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling.

La transició Epiteli-Mesènquima es dóna durant el desenvolupament embrionari i en els estadis tardans de la progressió tumoral permetent que es produeixi la metàstasi. Aquestes transicions necessiten una repressió de l'E-Cadherina i es pot reproduir en cèl·lules en cultiu amb l'expressió ectòpica de Snail1, un repressor de l'E-Cadherina. Durant la transició produïda per Snail es produeix la ràpida activació de gens mesenquimals com Fibronectina i LEF1. L'expressió forçada d'E-Cadherina fa disminuir els nivells de RNA de Fibronectina i LEF1, indicant que en l'activació d'aquests dos gens està implicat un cofactor sensible a l'E-Cadherina. En concordança, la transcripció de Fibronectina i LEF1 és depenent de &#61538;-Catenina i NF&#61547;B. La sobreexpressió d'E-Cadherina inhibeix l'activitat transcripcional d'aquests dos factors i disminueix la seva interacció amb el promotor de Fibronectina. De manera similar a la &#61538;-Catenina, NF&#61547;B es detecta associat a l'E-Cadherina i altres components dels contactes intercel·lulars. Quan es trenquen les unions adherents, com quan es sobreexpressa Snail, la interacció E-Cadherina-NF&#61547;B disminueix i augmenta l'activitat transcripcional de NF&#61547;B i&#61472;&#61472;&#61538;-Catenina. / Epithelial to mesenchymal transitions takes place during embryo development and in the late stages of tumorigenesis allowing metastasis formation. These transitions require E-Cadherin downregulation and can be reproduced in cell culture by ectopic expression of Snail1, an E-Cadherin gene repressor. During Snail-induced transition a rapid upregulation of mesenchymal genes such as Fibronectin and LEF1 has been characterized. Forced expression of E-Cadherin strongly down-regulates Fibronectin and LEF1 RNA levels, indicating that an E-Cadherin sensitive cofactor is involved in the activation of these genes. Accordingly, transcription of Fibronectin and LEF1 was dependent on &#61538;-Catenin and NF&#61547;B. E-Cadherin over-expression downregulated the transcriptional activity of both factors and decreased their interaction to Fibronectin promoter. Similarly to &#61538;-Catenin, NF&#61547;B was detected associated to E-Cadherin and other cell adhesion components. Association of NF&#61547;B to E-Cadherin required the integrity of this complex; conditions that disrupts adherens junctions, such as Snail over-expression, decreased E-Cadherin-NF&#61547;B interaction and up-regulates NF&#61547;B and &#61538;-Catenin transcriptional activity. Therefore, &#61538;-Catenin and NF&#61547;B transcriptional activities are required for expression of the studied mesenchymal genes and these activities are inactivated by immobilizing &#61538;-Catenin and NF&#61547;B to functional E-Cadherin structures.