Dissertations / Theses on the topic 'Acidic tumor microenvironment'

L'adénocarcinome canalaire pancréatique (PDAC) est une maladie mortelle caractérisée par un micro-environnement tumoral (TME) extrêmement acide (˂pHe 6,5) qui joue un rôle important dans son début et sa progression. Dans ce contexte, les canaux ioniques perméables au Ca2+ représentent de bons candidats cibles en raison de leur capacité à intégrer des signaux provenant de la TME. Les canaux Ca2+ sont en effet des capteurs de pH capables d'intégrer les signaux de la TME pour activer les voies intracellulaires en aval liées à la progression du PDAC. Bien que les rôles de l'acidose tumorale et de la signalisation du Ca2+ dans la progression du cancer soient bien établis, l'hypothèse d'une TME acide utilisant la signalisation du Ca2+ comme voie préférentielle pour soutenir la progression tumorale n'a pas encore été suffisamment explorée.Mon travail de doctorat visait à étudier les changements phénotypiques et génétiques des cellules PDAC lors d'un stress acide au cours des différentes étapes de sélection et à évaluer comment l'acidose tumorale module les signaux Ca2+ et les phénotypes dans les lignées cellulaires PDAC, avec un accent particulier sur les oscillations Ca2+ et Store-Operated Ca2+ entry (SOCE). À cette fin, les cellules PANC-1 et Mia PaCa-2 ont été soumises à une pression acide à court et à long terme et à une récupération à pHe 7,4. Ce dernier traitement visait à imiter les bords du PDAC et l'évasion consécutif des cellules cancéreuses de la tumeur. L'impact de l'acidose a été évalué sur la morphologie cellulaire, la prolifération, l'adhésion, la migration, l'invasion, l'activité des invadopodes et la transition épithélio-mésenchymateuse (TEM) par des tests fonctionnels in vitro et le séquençage de l'ARN, ainsi que sur les signaux Ca2+ intracellulaires avec Fura-2. Nos résultats indiquent qu'un court traitement acide limite la croissance, l'adhésion, l'invasion et la viabilité des cellules PDAC. Au fur et à mesure que le traitement acide progresse, il sélectionne les cellules cancéreuses ayant des capacités de migration et d'invasion accrues induites par l'EMT, ce qui augmente encore leur potentiel métastatique lorsqu'elles sont réexposées à un pHe 7,4. L'analyse RNA-seq des cellules PANC-1 exposées à une acidose de courte durée et récupérées à pHe 7,4 a révélé un recâblage transcriptomique distinct. Il est intéressant de noter que les cellules PANC-1 sont caractérisées par des oscillations Ca2+ plus lentes pendant une exposition à l'acide à court terme par rapport aux cellules de contrôle et par une tendance à la régulation négative d'ORAI1 au niveau de l'ARNm, tandis que l'acidose à long terme et le rétablissement à un pH neutre déterminent le rétablissement d'oscillations Ca2+ rapides et la régulation positive d'ORAI1. Dans tous nos modèles cellulaires, les oscillations du Ca2+ sont dépendantes de SOCE, car le blocage d'ORAI1 par Synta66 et siORAI1 entraîne une altération de l'initiation et du maintien des oscillations du Ca2+. Ces données sont en corrélation avec le SOCE dans les cellules PANC-1, qui est diminuée pendant le traitement acide à court terme, et augmentée dans les cellules sélectionnées pour l'acide avec et sans récupération à pHe 7,4. Enfin, l'entrée de Ca2+ médiée par ORAI1 pourrait être impliquée dans l'activation des cascades de signalisation qui conduisent à l'augmentation de la migration et de l'invasion de tous les modèles cellulaires exposés à un pHe acide, car le traitement par Synta66 et siORAI1 n'ont pas affecté l'invasion et la migration des cellules de contrôle.En conclusion, nos résultats montrent que la sélection induite par l'acide contribue à l'acquisition d'un phénotype plus agressif dans les cellules de PDAC, caractérisé par une augmentation du SOCE, nécessaire à la génération d'oscillations rapides de Ca2+ qui peuvent activer des voies de signalisation Ca2+-dépendantes impliquées dans la progression du PDAC
Pancreatic ductal adenocarcinoma (PDAC) is the most common cancer affecting the pancreas, characterized by an unsatisfactory 5-year survival rate of around 10%, and to date, there are no effective therapeutic options for PDAC. This is in part due to a highly desmoplastic and immunosuppressive microenvironment that contributes to therapeutic failure. Moreover, the PDAC tumor microenvironment is featured by high acidosis (˂ pHe 6.5), a result of the metabolic reprogramming ("Warburg effect"), and hypoxic conditions, which offers important cues for its aggressiveness by selecting cancer cell phenotypes with competitive benefits for PDAC progression. In this context, Ca2+-permeable ion channels are known to regulate several hallmarks of cancer, including in PDAC. Therefore, they represent good target candidates due to their ability to integrate signals from the TME. Ca2+ channels are indeed pH and hypoxia sensors able to transduce TME signals to activate intracellular downstream pathways linked to PDAC progression. Although the roles of tumor acidosis and Ca2+ signaling in cancer progression are well established, the hypothesis of acidic TME employing Ca2+ signaling as a preferential route for sustaining tumor progression has not yet been sufficiently explored.My Ph.D. work aimed to study the phenotypic and genetic changes of PDAC cells upon acidic stress along the different stages of selection and to evaluate how tumor acidosis modulates Ca2+ signals and phenotypes in the PDAC cell lines, with a particular focus on Ca2+ oscillations and Store-Operated Ca2+ entry (SOCE). To this end, PANC-1 and Mia PaCa-2 cells were subjected to short- and long-term acidic pressure and recovery to pHe 7.4. The latter treatment was to mimic PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, invadopodia activity, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing, and for intracellular Ca2+ signals using Fura-2. Our results indicate that short acidic treatment limits the growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, thereby further enhancing their metastatic potential when re-exposed to pHe 7.4. RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pHe-selected recovered to pHe 7.4 revealed distinct transcriptome rewiring. We noted an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Interestingly, PANC-1 cells are characterized by slower Ca2+ oscillations during short-term acid exposure compared to control cells and a tendency of ORAI1 downregulation at mRNA levels, while long-term acidosis and recovery to neutral pHe determine the recovery of fast Ca2+ oscillations and upregulation of ORAI1. In all our cell models, Ca2+ oscillations are SOCE-dependent, as ORAI1 blockade with Synta66 and siORAI1 results in impaired Ca2+ oscillations' initiation and maintenance. These data correlate with SOCE in PANC-1 cells, which is decreased during the short-term acid treatment, and increased in acid-selected cells with and without recovery to pHe 7.4. Finally, ORAI1-mediated Ca2+ entry might be involved in the activation of signaling cascades that lead to the increased migration and invasion of all the cell models exposed to acidic pHe, as Synta66 treatment and siORAI1 didn't affect control cells' invasion and migration.In conclusion, our findings show that acid-induced selection contributes to the acquisition of a more aggressive phenotype in PDAC cells, characterized by upregulation of SOCE, required for the generation of fast Ca2+ oscillations which may trigger Ca2+-dependent signaling pathways involved in PDAC progression

Le canal TRPC1 (pour transient receptor potential canonical 1) est l'un des canaux cationiques non sélectifs les plus impliqués dans plusieurs maladies, y compris la progression du cancer. Les TRPC peuvent être activés par différents stimuli physico-chimiques dont le pH. Par ailleurs, l'une des caractéristiques du cancer représente les variations pH extracellulaire, notamment dans l'adénocarcinome canalaire pancréatique (ACP). Il existe de fortes indications quant à l'agressivité de l'ACP qui est causée par l'interaction entre le microenvironnement acide de la tumeur et la dérégulation des canaux ioniques. Cependant, cette interaction n'a jamais été étudiée. Lors de ce travail, nous avons étudié si TRPC1 ainsi que les fluctuations du pH acide du microenvironnement tumoral régulent la progression de l'ACP et plus particulièrement la prolifération et de migration cellulaires. Nous avons constaté que TRPC1 était surexprimé dans le tissu tumoral pancréatique par rapport au tissu normal, et dans la lignée cellulaire agressive PANC-1, par rapport à une lignée cellulaire de type canalaire, hTERT-HPNE. Pour déterminer si les fluctuations du microenvironnement acide de la tumeur affectent la dérégulation de TRPC1, les cellules PANC-1 ont été incubées dans un milieu présentant un pH de 7,4 ou 6,5 pendant 30 jours, après quoi les cellules ont été récupérées à pH 7,4 pendant 14 jours (7.4 R). L'adaptation acide (6.5) a réduit l'expression de la protéine TRPC1 mais a favorisé sa localisation membranaire par rapport au contrôle (7.4). Dans des conditions de pH 7,4R, les cellules cancéreuses présentaient une expression de TRPC1 exacerbée avec une localisation membranaire élevée, à la fois dans les modèles 2D et 3D. Nous avons constaté que les fluctuations de pH et l'invalidation moléculaire par siARN (KD) de TRPC1 affectaient respectivement la prolifération de PANC-1 dans un modèle 2D et sphéroïde. Dans notre modèle 2D, nous avons montré que TRPC1 KD affectait la progression à travers la phase G0/G1 dans toutes les conditions et la phase S lorsque les cellules sont cultivées dans un milieu pH 7,4, et la phase G2/M dans les conditions pH 6,5 et 7,4 R. En outre, pH 6,5 a amélioré tandis que le KD de TRPC1 a diminué la migration cellulaire. De plus, nous avons constaté que TRPC1 interagissait fortement avec PI3K dans des conditions acides, et CaM dans toutes les conditions. Le KD de TRPC1 diminuait à la fois cette interaction et l'activation de AKT et ERK1/2. Enfin, l'entrée basale de Ca2+ a été significativement réduite par le KD de TRPC1 dans les conditions de pH 6,5 et 7,4R. La réduction de la concentration de Ca2+ extracellulaire a entraîné une diminution additionnelle de la prolifération et de la migration des cellules transfectées avec siTRPC1 cultivées à pH 6,5 et 7,4R, mais pas dans des conditions normales de pH 7,4. Collectivement, nos résultats montrent que TRPC1 est régulé positivement dans les tissus et lignées d’ACP. Le microenvironnement acide de la tumeur favorise sa localisation membranaire et son interaction avec PI3K/CaM. Enfin, le Ca2+ transitant par TRPC1 participe à la prolifération et à la migration cellulaires. De plus, nous avons effectué un criblage du profil d'expression des canaux ORAI, de leur partenaire STIM1, d'un canal sodique activé par le voltage (Nav1.6) et d'un canal ionique détectant l'acidité (ASIC1) dans les tissus et lignées d'ACP. Nous avons examiné si le microenvironnement acide affecte la régulation épigénétique des canaux ioniques. Nous avons constaté qu'ORAI3 était régulé positivement dans le tissu ACP par rapport au tissu normal, tandis que STIM1 et NaV1.6 étaient régulés négativement. De plus, ORAI3 était préférentiellement localisé au niveau membranaire dans le tissu tumoral. De plus, nos résultats préliminaires montrent que le microenvironnement acide de la tumeur n'affecte pas les niveaux de méthylation de la région promotrice des gènes codant pour ASIC1 ou TRPC1, mais également du gène SCN8A
The transient receptor potential canonical 1 channel (TRPC1) is one of the most prominent nonselective cation channels involved in several diseases, including cancer progression. TRPCs can be activated by different physio-chemical stimuli of their surroundings, for instance, pH. Another hallmark of cancer is the variable extracellular pH landscape, notably in epithelial cancers such as pancreatic ductal adenocarcinoma (PDAC). PDAC progression and development are linked to the physiology and microenvironment of the exocrine pancreas. There are strong indications that PDAC aggressiveness is caused by the interplay between the tumor acidic microenvironment and ion channel dysregulation. However, this interaction has never been studied before. Here, we investigate if TRPC1 is involved in PDAC progression in the form of proliferation and migration and if the pH fluctuations of the acidic tumor microenvironment affect these processes. We found that TRPC1 was significantly upregulated in PDAC tumor tissue compared to adjacent normal tissue, and in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, hTERT-HPNE. To investigate if fluctuations of the acidic tumor microenvironment affect TRPC1 dysregulation, PANC-1 cells were incubated in a medium with a pH of 7.4 or 6.5 over 30 days, where after cells were recovered in pH 7.4 for 14 days (7.4R). Acid adaptation (6.5) reduced TRPC1 protein expression but favored its membrane localization compared to the control (7.4). pH recovery treatment (7.4R) resulted in an upregulation of TRPC1 expression with a high membrane localization, both in 2D and 3D models. We found that pH fluctuations and the siRNA-based knock-down (KD) of TRPC1 affected 2D and spheroid PANC-1 proliferation, respectively. In our 2D model, flow cytometry and cell cycle regulating protein immunoblotting showed that TRPC1 KD affected the progression through G0/G1 phase under all conditions and S-phase under control pH 7.4, which shifts to the G2/M phase in pH 6.5 and 7.4R. In addition, pH 6.5 enhanced, and the KD of TRPC1 decreased cell migration, respectively. Furthermore, we found that TRPC1 interacted strongly with PI3K under acidic conditions and CaM under all conditions, and a KD of TRPC1 decreased both this interaction and the activation of AKT and ERK1/2. Finally, basal Ca2+ entry was significantly reduced upon the KD of TRPC1 in pH 6.5 and 7.4R, where the entry was enhanced. The reduction of extracellular Ca2+ concentration resulted in an additional decrease in proliferation and migration of cells transfected with siTRPC1 growing in pH 6.5 and 7.4R, but not in normal pH 7.4 conditions.Collectively, our results show that TRPC1 is upregulated in PDAC tissue and cell lines. The acidic tumor microenvironment favors its plasma membrane localization, and its interaction with PI3K/CaM and Ca2+ entry leads to PDAC cells proliferation and migration. In addition, we performed an expression profile screening of ORAI channels, their partner STIM1, and a voltage-activated sodium channel (Nav1.6), and an acid-sensing ion channel (ASIC1) in PDAC tissues and cell lines, and investigated whether the acidic tumor microenvironment affects epigenetic regulation of ion channel expression. We found that ORAI3 was upregulated in PDAC tissue compared to normal tissue, where STIM1 and NaV1.6 were significantly downregulated. Moreover, ORAI3 was more localized in the plasma membrane in tumor tissue. Acid-adaptation had a differential effect on Ca2+ channel expression. Furthermore, our preliminary results show that the acidic tumor microenvironment does not affect the methylation levels of the ASIC1 or TRPC1 promoter region, but so some extend the SCN8A gene promoter

Defective apoptosis represents one of the major causative factors in the development and progression of cancer. The ability of tumour cells to evade engagement of apoptosis can play a significant role in their resistance to conventional therapeutic regimens. In the last few years, preclinical and clinical studies have indicated ceramide and the enzymes of its metabolism, in particular Acid Sphingomyelinase (A-SMase) which hydrolyzes sphingomyelin to ceramide and phosphocoline, as key players in tumour physiopathology. Different cancers have been shown to have reduced ceramide levels and, of interest, in our previous work we showed that A-SMase down-regulation was a key event in melanoma progression. This event is crucial for the tumours to become more aggressive, but the mechanisms responsible of it haven’t been investigated yet. Taking into account that there is a complex crosstalk between tumour cells and its immunological microenvironment, in this work we first investigated its possible role in A-SMase downregulation in a melanoma model. To this purpose we performed in vivo and in vitro experiments which led us to identify tumour associate macrophages (TAM) as possible responsible of A-SMase downregulation through the Ap2-α transcription factor. Moreover, we demonstrated that these molecular changes in tumour cells give, in turn, pro-tumoural and immunosuppressive features to the surrounding microenvironment, with the recruitment of Myeloid-derived suppressor (MDSCs) cells and Regulatory T lymphocytes (TREGS). From these and our previous data, we clearly showed that the ability to create this immunosuppression together with the acquisition of a more aggressive phenotype, both depend on the naturally occurring A-SMase decrease in melanoma cells during tumour progression. The broad role of A-SMase in tumour pathogenesis we identified, indicates that the enzyme is at the crossroad of key pathways in tumourigenesis. This aspects has clear potential in therapeutic perspective in which A-SMase overexpression or administration might be consider as an useful adjuvant for cancer therapy. Here we demonstrated for the first time that restoring A-SMase expression in melanoma cells not only reduces tumour growth and immunosuppression, but moreover accounts for a high, unexpected recruitment of immune cells with an anti-tumoural function in the tumour microenvironment, such as Dendritic cells (DCs) and CD4+ and CD8+ T lymphocytes. In conclusion our results reveal the central role of A-SMase expressed by melanoma cells in orchestrating the cross-talk with the surrounding microenvironment. These interactions are crucial for tumour fate, lying on its rejection or progression. Our observation that A-SMase overexpression “educate” tumour microenvironment against cancer cells, further encourage the use of this enzyme as an adjuvant for cancer therapy.

Extracellular acidity and high levels of lactate are commonly observed in solid tumours. Some tumours also exhibit a reversed cellular pH gradient with an intracellular pH that is higher than the extracellular. This has been shown to play a crucial part in not only the invasive and metastatic cascade of tumours, but also on their response to therapies. In this thesis, we present four different mathematical models that examine the possible causes of tumour acidity and its effect on cell metabolism and tumour invasion. In the second chapter, we derive an ordinary differential equation model that explicitly focus on the interplay between H+-ions and lactate. We subject the model to qualitative and quantitative analysis and, in particular, we study the effect in the variations of key parameter estimates on the emergence of a reversed transmembrane pH gradient within the tumour. The model predicts that a re- versed pH gradient is attainable under aerobic conditions when sourc es of H+-ions other than those from glycolysis are decreased and the lactate/H+ cell membrane transporter (MCT) activity is increased - but we find the intra- and extracellular pH values in this case to be too alkaline to be physiological. Under anaerobic conditions, we find that decreasing the sources of H+-ions other than those from glycolysis and also the glycolytic rate gives rise to a reversed cellular pH gradient, but again for intra- and extracellular pH values that are far from realistic biologically. In the third chapter, we present an extension to the first model by including the spatial diffusion of hydrogen ions and lactate. This spatial extension also predicts ii a reversed transmembrane pH gradient but this time for more realistic intra- and extracellular pH values. We find that low levels of blood lactate can give rise to a reversed pH gradient throughout the spatial domain independent of the levels of tissue lactate. Likewise, we have found the existence of a negative pH gradient to be strongly dependent on the combined activity of a lactate/H+ cell membrane transporter and other sources of H+-ion. In the fourth chapter, we study the role of oxygen and pH on early tumour growth using a hybrid cellular automaton model. We examine whether the levels of oxygen, intra- or extracellular pH are the dominating metabolites driving tumour growth and phenotypic transformations. This model predicts that when tumour cells are strongly sensitive to changes in the intracellular pH, a low activity of the Na+/H+ cell membrane transporter (NHE) or a high rate of anaerobic glycolysis can give rise to a "fingering" morphology. Furthermore, we show that as the activity of the MCT transporter increases, all the tumour cells within the spheroid can exhibit a reversed transmembrane pH gradient. In the fifth chapter, we examine the effect of extracellular acidity on tumour invasion focusing, in particular, on cellular adhesion, matrix-degrading enz yme activity and cellular proliferation. Our numerical simulations using a cellular Potts model show that, under acidic extracellular pH, cell-ECM adhesion strength has a comparable effect on tumour invasiveness as the rate at which the ECM is degraded by proteolytic enzymes. We also show that tumour cells cultured under physiological pH tend to be larger and develop a "diffuse" morphology compared to those cultured at acidic pH which display protruding "fingers" at the advancing tumour front.

A large proportion of human tumours exploit tryptophan catabolism as a means to suppress T cell activity in the tumour microenvironment. However, the mechanisms that allow tumour cells to resist the detrimental effects of local tryptophan depletion have so far remained obscure. Here we demonstrate that shortage of tryptophan induced by the expression of tryptophan-degrading enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) results in ATF4-dependent reprogramming of the amino acid transporter expression profile in tumour cells. Specifically, we show that tumour cells undergoing tryptophan starvation enhance the expression of several amino acid transporters including SLC1A5 and its truncated isoforms through activation of the ATF4 stress response pathway. While upregulation of SLC1A5 leads to improved glutamine and tryptophan uptake, the knockdown of this amino acid transporter inhibits tumour cell proliferation under low tryptophan conditions. In contrast to tumour cells, T cells fail to upregulate SLC1A5 during tryptophan starvation, but can enhance its expression upon cognate antigen T cell receptor engagement. SLC1A5 expression is also enriched in T regulatory cells and IDO-expressing dendritic cells, as well as in tumours growing under conditions of IDO-mediated tryptophan degradation in vivo. Finally, SLC1A5 expression correlates with IDO expression in several human tumours in silico, which is associated with poor prognosis in brain lower grade glioma. In conclusion, our results highlight key differences in the ability of tumour cells and T cells to adapt to tryptophan starvation and shed light on the previously unappreciated role of amino acid transport in IDO- and TDO-mediated immune escape.

Cholangiocarcinoma (CCA) is a fatal primary malignancy that is formed in the bile ducts. Cancer-associated myofibroblasts play a crucial role in CCA proliferation and invasion. Furthermore, there is a growing interest in the role of the exosome in the interaction between the cancer-associated myofibroblasts and cholangiocarcinoma which lead to CCA growth. However how cholangiocarcinoma-derived exosome affect the cancer-associated myofibroblasts in the tumor microenvironment remain unknown. In this study, we examined whether exosome produced by cholangiocarcinoma could involve in the prompt of CCA cells growth by regulation of myofibroblast. We found that cholangiocarcinoma-derived exosome could prompt elevated α-smooth muscle actin and stromal cell-derived factor one expression that induces myofibroblast proliferation. We then demonstrated that cholangiocarcinoma-derived exosome upregulated periostin expression that plays an important role in cancer metastasis. In 3D organotypic rat CCA coculture model, TCA and S1P considerably increase the growth of CCA cell. Conclusion: cholangiocarcinoma-derived exosome trigger cancer-associated myofibroblasts proliferation in the tumor microenvironment that leads to prompt CCA growth.

Les glioblastomes sont des tumeurs primaires du cerveau les plus malignes. L’identification des cellules souches cancéreuses de glioblastome (CSGs) a transformé notre vision globale des glioblastomes en révélant une hiérarchie cellulaire au sein de ces tumeurs. Les CSGs sont douées de propriétés d’auto-renouvellement, de différenciation et peuvent entrer en quiescence. Elles sont considérées comme les cellules entretenant les tumeurs, responsables de leur dissémination et des rechutes après traitement. La découverte des CSGs a conduit à un changement de paradigme dans le développement des thérapies anticancéreuses, avec la nécessité de cibler dans le traitement non seulement les cellules de la masse tumorale, mais aussi les CSGs. Un criblage différentiel de la chimiothèque Prestwick réalisé au laboratoire a permis d’identifier le bisacodyl comme une molécule présentant une cytotoxicité spécifique sur les CSGs en quiescence.Cette thèse présente un travail sur la caractérisation des CSGs, la compréhension du mode d’action du bisacodyl, ainsi que l’évaluation de son potentiel thérapeutique sur un modèle 3D in intro et des modèles in vivo
Glioblastomas are the most malignant primary brain tumors. The identification of glioblastoma stemcells (GSCs) has transformed our comprehension of those tumors by revealing a hierarchical organization. GSCs can self-renew, differentiate and enter into a quiescent state. They are considered as cells which fuel and as the main culprits of tumor relapse. The discovery of GSCs triggered a change in paradigm for cancer therapy. Indeed to gain in efficacy, therapies need to target, not only the cells forming the bulk of the tumor, but also GSCs particularly resistant and endowed with a high tumorigenic potential. Chemical screening of the Prestwick chemical library in our laboratory, unveiled bisacodyl with a specific activity on quiescent GSCs.This thesis presents work on the characterization of GSCs, study of the mode of action of bisacodyl on GSCs, as well as a preclinical evaluation of bisacodyl on a 3D model in vitro and animal models in vivo

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.

碩士
國立臺灣大學
口腔生物科學研究所
107
The microenvironment of cancer cells is considered to be an important indicator of cancer progression. Studies have shown that due to the specific metabolic mechanisms of cancer cells, the extracellular matrix of cell has a higher hydrogen ion concentration than the cytoplasm. Exposure of cancer cells to this environment has an effect on their function, including changing in the metabolic system, mediating the growth processes, and expression of autophagy proteins. Compared with cancer within other parts of the body, the oral cavity is the only access to the digestive system. The cancer cells that grow in are more frequently affected by the external environment, especially with acidic environment due to food digestion of secreting seliva. Therefore, Oral cancer cells are not only influenced by endogenous micro-acidification, but also by the exogenous oral digestive system, we presumed that oral cancer should have higher research value in related field about how the microenvironment acidosis changing cell performance. However, there are few related studies using oral cancer as a subject. In this thesis, the influence of the slightly acidic environment on the growth of cancer cells is taken as the main story. We collaboration in vivo and in vitro experiment. Observing the performance of stemness and extented to drug resistance, proofing that both in vivo and in vitro test can show consistent results. The results show that acidification has significant impact on different types of oral cancer cells line. For tongue cancer (SAS), long-term acid stimulation its ability to upregulate stemness and further have its influence on proliferation and chemoresistance, at the meanwhile acidosis cancer cell can also improve its vasculogenic mimicry ability. But the acidosis stimulation has totally different influence on oral squamous cell carcinoma (OECM1), stemness of the OEMC1 was down regulation after acid treated but proliferation and chemoresistance ability was same as SAS. Nevertheless, OECM1 in vivo tumor incident rate showed dramatic different from in vitro side population data that OECM1 had much more lower tumor incident rate than SAS. Leak of vasculogenic mimicry ability may be one of the reason that tumor couldn’t form enough vascular-like tube to gain nutrient and lead awful in vivo tumor incident rate.

碩士
國立成功大學
口腔醫學研究所
100
In clinical, tumor markers can be use to help cancer diagnosis , predict the patient’s response to particular therapies and follow-up disease. The squamous cell carcinoma antigen (SCCA) is a tumor marker and has a clinical value. In clinical diagnosis, patients with higher expression of SCCA show poor prognosis. However, SCCA in clinical use was more than 20 years, but the biological function and mechanism still remain unclear. Our previous studies, we found that overexpression of SCCA led to drug resistance, such as RSV (Resveratrol) and Cisplatin. Cancer cells usually grow faster than normal cells. In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed the “Warburg effect”. Many cancer cells consume glucose and produce lactic acid and cause acidification of the tumor extracellular environment. Acidification of the tumor extracellular environment may promote tumor formation and metastasis. In our previous studies showed the SCCA is a lysosomal proteases inhibitor and could inactivate cathepsin L and anti-cell death. This study we show the cancer cells cultured in low pH (Acidosis) media, increase SCCA expression. The cancer cells culture in low pH (Acidosis) media display drug resistance of RSV (Resveratrol) and Cisplatin. The acidosis enhances the formation of reactive oxygen species (ROS) and induces MAPK signaling pathway and transcription factor, CREB phosphorylation. The cancer cells treated with ROS scavenger reduce SCCA expression and CREB phosphorylation in acidosis condition. We found that, there is a CREB binding region in the promoter region of SCCA1 gene. These results suggested that acidosis increased phosphorylation of the transcription factor CREB via ROS, and them increased its transcriptional activity. Taken together, we suggest cancer resist drug-induced cell death in acidosis condition is through ROS-increased CREB transcriptional activity to promote SCCA1 gene expression .

Gli esosomi sono nanovescicole di origine endosomiale, che rappresentano un mezzo importante di comunicazione cellula-cellula. Essendo coinvolti in diversi tipi di processi, sia fisiologici che patologici, sono diventati oggetto di sperimentazione clinica, nonostante molti meccanismi alla base della loro biogenesi rimangono tuttora sconosciuti. Quindi nel nostro laboratorio è stata messa a punto una metodica atta a produrre esosomi fluorescenti, in modo da poterne seguire la biogenesi nei diversi compartimenti intracellulari, il rilascio nell’ambiente extracellulare e la fusione con la membrana delle cellule riceventi. È stato quindi studiato, in un modello cellulare di melanoma umano, come questo precursore lipidico fluorescente è assorbito dalla cellula, accumulato all’interno del reticolo endoplasmatico, per poi diventare parte integrante della membrana degli esosomi. Queste nanovescicole fluorescenti sono state caratterizzate per dimensione, densità e presenza di marcatori proteici e successivamente è stata studiata con esperimenti di cinetica (5min – 24h) la secrezione. In seguito tale metodica è stata applicata allo studio della progressione del melanoma in condizioni di acidità microambientale. Nel melanoma è già noto che il pH microambientale è in grado di promuovere invasione e migrazione cellulare, e in questa tesi abbiamo analizzato il ruolo degli esosomi in tali processi. Quindi abbiamo studiato come cellule di melanoma umano, corrispondente a uno stadio intermedio, coltivate in ambiente acido producano una maggior quantità di esosomi, con un’aumentata capacità di trasferimento in cellule bersaglio. Inoltre queste vescicole presentano un profilo proteico che permette alle cellule riceventi di acquisire capacità migratorie e invasive. Infine, attraverso studi di meta-analisi ed ex vivo su biopsie di pazienti, è stato confermato che le molecole arricchite negli esosomi secreti in condizioni di acidità possano rappresentare dei marcatori della progressione del melanoma, convalidando quindi il valore diagnostico e prognostico degli esosomi.
Exosomes, nanosized vesicles of endosomal origin, are worldwide recognized for their ability to transfer biological molecules, from cell to cell, crucial for both physiological and pathological processes. Hundred studies have been focused on exosome application also to clinics although biogenesis modalities are under investigation. Therefore, we set up a new effective fluorescent labelling strategy to trace exosome biogenesis and release with the aim to seek in human melanoma cell lines the impact that tumor progression may exert on their secretion and composition. Cells exposed to a fluorescent analogue of palmitic acid (Bodipy FL C16) were able to promptly synthetize fluorescent phospholipids, constituents of exosome membrane bilayer. Afterwards, we were able to follow exosome biogenesis from the intracellular sites of origin to cell secretion, chasing over time by direct cytofluorimetric analysis. To get insight into their function, we focused our studies on exosomes derived from melanoma cells maintained at low pH, which is a microenvironmental leverage for primary tumor to be transformed into widespread metastasis. When melanoma cells at specific intermediate stage were subjected to an acidic microenvironment, showed an increase in exosome release and transfer capability. Most importantly, when control melanoma cells were incubated with exosomes secreted in acidic medium acquired migratory and invasive capacities, demonstrating that exosomes carrying molecular payload can modify recipient cell program. Finally, meta-analysis and ex vivo studies confirmed the importance of acidic exosomes molecule content as marker of melanoma progression and so exosomes prognostic and diagnostic value.

ABSTRACT Breakthroughs in the understanding of the basic biology of melanoma have yielded continue progresses on a variety of fronts. In the past decade, several specific inhibitors have been utilized against melanoma. However, after a short period of remission, their clinical use inevitably evidenced drug resistance and disease exacerbation. Finding new arms against the advanced phase of melanoma is one of the major challenges in the struggle against this cancer. New insight highlighted the rewiring of cellular signaling and the reprogramming of metabolic pathways in cancer. In normal cells a precise balance between saturated and unsaturated Fatty Acid (FA) synthesis is required for maintaining cell homeostasis. Conversely the anabolic pathways responsible for constructing these molecules appear altered during tumorigenesis. It is now appreciated the importance of fatty acid regulation in malignant disease and the opportunities to target these pathways, required not only for cell growth and division, but also for tumor dissemination. The role of the stearoyl-CoA desaturase 5 (SCD5) was investigated in melanoma, being its pathophysiological function virtually unknown. This enzyme, converting saturated into monounsaturated FAs, is downregulated during progression of melanoma by epigenetic and miR221/-222-dependent mechanisms. SCD5 restored expression significantly reduced melanoma malignancy, in human A375M melanoma and in murine 4T1 mammary carcinoma cell lines, mainly by inducing oleic acid. In both cell systems we also evidenced SCD5 effects on tumor microenvironment, through reduced secretion of protumoral protein such as the Secreted Protein Acidic and Rich in Cysteine (SPARC), Collagen IV and Cathepsin B. The net effect of SCD5 action was evidenced by the intracellular acidification (pHe > pHi) and, in turn, by the inhibition of the vesicular trafficking across plasma membranes. This acidification also depends on SCD5-induced reduction of the C2 subunit of the proton pump vacuolar H+-ATPase. Supplementation of oleic acid was per se able to mimic SCD5 enforced expression by reducing the protumoral matrix protein secretion. Our data support a role for SCD5 and its enzymatic product, oleic acid, in protection against malignancy, offering an explanation for the beneficial Mediterranean diet. Furthermore, SCD5 appears to functionally connect tumor cells and the surrounding stroma, with consequences on tumor spread and eventually resistance to treatment.

碩士
高雄醫學大學
藥學研究所
102
Gastric cancer is a high prevalent carcinoma and the leading cause of cancer-related mortality in Taiwan. Betulinic acid (BA), a triterpene isolated from the white birch tree, has been reported to exhibit anti-inflammatory and anti-tumor properties. However, the mechanisms of BA responsible for the induction of gastric cancer death remain unclear. The endoplasmic reticulum (ER) is a membranous network within cells, and it is important for several cellular functions, including translation, folding of secretory proteins and sequestration of Ca2+. ER homeostasis is very sensitive to perturbations in cellular homeostasis and activates an adaptive response when it senses stressful conditions. This can occur in response to conditions such as, disturbed Ca2+ homeostasis, accumulation of misfolded proteins and oxidative stress. Numerous studies have also shown that ER stress-related chaperone proteins are overexpressed in many cancers, and are important for tumor development. Therefore, elimination of pro-survival chaperone proteins and induction of excessive ER stress may promote cancer cell apoptosis. Recently, cancer-associated fibroblasts (CAFs) in cancer microenvironments have been implicated in tumor growth and metastasis of various cancers. TGF-?? signaling may link with promoting fibroblast activation by up-regulation of ??-SMA expression during tumor development. In this regard, the present study assessed the anticancer effects of BA on human gastric cancer cells. A mechanistic analysis demonstrated that BA-induced human gastric cancer cell death was down-regulation of ER stress-related pro-survival chaperones and activation of mitochondrial-dependent apoptosis pathway. BA not only induced gastric cancer cell death, attenuated TGF-?? expression partially through HOXA9-mediated pathway, but also inhibited TGF-??-induced human fibroblasts (HS68) activation. In conclusions, these results provide a molecular basis for the ability of BA to mediate gastric cancer death, suppress fibroblasts activation. BA may be regarded as a promising agent in anti-gastric tumor therapy.