京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20614号 / 医博第4263号 / 新制||医||1023(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 高田 穣, 教授 平井 豊博, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ovarian cancer (OVCA) is the most lethal gynecologic cancer. Chemoresistance remains a major hurdle to successful therapy and patient survival. The secreted isoform of the actin-associated protein, gelsolin (plasma gelsolin; pGSN), is highly expressed in chemoresistant than chemosensitive OVCA cells, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. DNA methylation plays a key role in the regulation of genes expression and contributing to cancer development and chemoresistance with the help of DNA methyltransferases (DNMTs) or Ten eleven translocation (TETs) enzymes. TET1 is the most studied isoform of TETs family and primarily responsible for 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) oxidation to initiate demethylation and increase in the expression of methylated genes. Whether pGSN expression in OVCA cells is regulated by DNA methylation and TET1 regulates the differential pGSN expression between chemosensitive and resistant OVCA cells is not known. In this study, we hypothesized pGSN overexpression in chemoresistant OVCA cells is due to the hypomethylation at its promoter region by TET1. Our objective was to investigate whether DNA methylation and specifically TET1 plays a role in the regulation of differential pGSN expression and chemosensitivity in OVCA. Chemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance and protein contents by qPCR and Western blotting respectively. Cisplatin-induced chemoresponsiveness was morphologically assessed by Hoechst staining (apoptosis). Infinium HumanMethylation450 BeadChip assay was used for global methylation analysis of twelve (12) different OVCA cells and to investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance. DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cell using specific inhibitors. Gain-and-loss-of-function assays were carried to identify the relationship between TET1 and pGSN in OVCA chemoresponsiveness. Differential protein and mRNA expressions of pGSN and TET1 were observed between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. Global methylation analysis revealed hypomethylation in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreases their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN and positively related to chemoresponsiveness of OVCA cells. This project does not only broaden our knowledge about the mechanistic insights into the epigenetic regulation of pGSN in OVCA chemoresistance, but it also reveals a new potential target to re-sensitize chemotherapy resistant OVCA cells. This may provide a future strategy to improve overall OVCA patient survival.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23763号 / 医博第4809号 / 新制||医||1056(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 中島 貴子, 教授 戸井 雅和, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells. / Master of Science in Life Sciences / Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium’s ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells

Cancer is one of the major causes of morbidity in the world. Although the overall survival of cancer has been significantly improved by chemotherapy in the last three decades, the success of cancer chemotherapy is still severely limited by the lack of selectivity of anti-cancer drugs to malignant cells leading to dose-limiting toxicity and the resistance of cancer cells to the conventional anti-cancer drugs. Gene-directed enzyme prodrug therapy (GDEPT) was designed to direct the anti-cancer drugs to specifically target the cancer cells by using cancer specific promoter to drive the expression of enzyme which can convert prodrug into anti-cancer drug specifically in cancer cells. However, this strategy is hindered by the lack of strong cancer specific promoters to specifically express drug-converting enzymes in cancer cells. In consequence, there is not enough anti-cancer drug activated inside the cancer cells. The first part of this study was to employ NF-κB binding sites as a novel enhancer system to improve the promoter activity of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) for GDEPT. In this system, the basal CEA promoter sequences were placed downstream of the 4 or 8 NF-κB DNA binding sites linked in tandem (κB4 or κB8). The system was designed to serve two particular purposes: to exploit the high levels of intratumoural NF-kB expression and keep the relative tumour specificity of the CEA and hTERT promoters. The results demonstrated that κB enhancer systems increased the transcriptional activity of CEA and hTERT promoter without compromising its cancer specificity. The fidelity of the κB4-CEA enhancer-promoter system was therefore improved by the increased transcriptional contrast between the cancer and normal cells. Moreover, in comparison with CEA promoter alone, κB-CEA enhancer-promoter system expressed human thymidine phosphorylase (TP) protein at significantly higher levels which were comparable to those expressed by CMV promoter. The κBCEA- TP system transfected cells demonstrated significantly higher sensitivity to 5'-Deoxy-5-Fluorouridine (5'-DFUR), a prodrug of 5-fluorouracil (5-FU). The second part of this study was involved in using NF-κB inhibitor as a chemosensitizer to sentizise the anti-cancer drug-induced chemoresistance cells to anti-cancer drugs. The results derived from this study manifested that the anti-alcoholism drug, Disulfiram (DS), and anti-inflammatory drug, triptolide (PG490), markedly enhanced the cytotoxicity of several conventional anti-cancer drugs in colon, lung and breast cancer cell lines. PG490 induced caspase-dependent cell death accompanied by a significant decrease in Bcl-2 levels. PG490 induced the expression of p53 and down-regulated p21 expression. This study indicated that some clinically used non-cancerchemotherapeutic drugs may be developed as chemosensitizers for cancer chemotherapy

No description available.

Le neuroblastome (NB) est une tumeur fréquente et agressive du jeune enfant. Les tumeurs de haut grade de forme métastatique, généralement développées chez l'enfant de plus de 1 an, sont associées à une importante mortalité malgré un traitement lourd incluant une chimiothérapie à haute dose. La chimiorésistance est donc un problème majeur qui caractérise ces NB de haut grade. Une des hypothèses pour expliquer cette chimiorésistance est celle de l’activation de l’autophagie, un mécanisme auquel recourent les cellules afin de faire face aux situations de stress. D’ailleurs, plusieurs études ont démontré que l'autophagie était activée à la suite des thérapies anticancéreuses. Son inhibition pourrait donc représenter une stratégie thérapeutique très intéressante pour le traitement de cancers. Le but de ce projet de recherche a été de mettre en évidence l'importance de l'autophagie dans les cellules du NB ainsi que l'effet de son inhibition sur la sensibilité des cellules du NB à la chimiothérapie conventionnelle. La présence d'autophagie dans les cellules de NB et sa valeur pronostic ont été évaluées par une étude immunohistochimique et par western blot sur 184 tumeurs patient. Ensuite, dans le but de déterminer l'effet de la chimiothérapie conventionnelle sur le niveau d'autophagie, des études in vitro sur 6 lignées cellulaires ont été effectuées utilisant des tests de mesure d'autophagie (MDC, monodanylcadaverine), de viabilité cellulaire (MTT) et de western blot. Celles ci ont été suivies par des tests d'inhibition de l'autophagie par deux méthodes: l’inactivation du gène ATG5 par un lentivirus contenant un shRNA ciblant ATG5 ou de l'hydroxychloroquine (HCQ), un inhibiteur pharmacologique de l’autophagie. Cette inhibition a été testée seule ou en combinaison avec une chimiothérapie conventionnelle dans le but de déterminer le rôle de l'autophagie dans la sensibilisation des cellules de NB à la chimiothérapie. Ensuite, l’intérêt de l’inhibition de l’autophagie a été évalué sur des modèles murins. Enfin, le niveau d'autophagie a été testé dans des cellules souches de NB. Notre étude a démonté que l'autophagie est présente à un niveau basal dans une majorité des NB mais qu'elle ne représentait pas un facteur pronostic dans ce type de tumeur. Les différentes chimiothérapies testées induisent une augmentation de l'autophagie dans les cellules du NB. Les deux tests d'inhibition ont démontré in vitro que l'autophagie participe à la résistance des cellules aux traitements chimiothérapeutiques classiques du NB. Le blocage de l’autophagie in vivo augmente l’efficacité de la chimiothérapie, cependant certaines données associées au traitement par HCQ devront être complétées. Cette étude démontre que l'inhibition de l'autophagie en combinaison avec la chimiothérapie classique représente une approche thérapeutique prometteuse dans le traitement du NB. / Neuroblastoma (NB) is a common tumor in childhood. Despite major advances in treatments, NB still have a poor prognosis with 40% of mortality. Chemoresistance is a major issue that characterizes aggressive NB. This is a consequence of an autophagic mechanism that tumor cells use to overcome stressful situations encountered during treatments. Autophagy has been the subject of several studies showing its activation in response to anticancer therapies. Its inhibition may therefore represent a very interesting therapeutic strategy for cancer treatment. The purpose of this research was to determine the presence of autophagy in NB cells and the effect of autophagy inhibition in sensitizing NB cells to conventional chemotherapy. The presence of autophagy was verified in 184 NB tumors. To determine the effect of conventional chemotherapy on autophagy, the MTT cell viability test and the autophagy measurement test (MDC, monodansylcadaverine) have been used to study 6 NB cell lines in vitro. An immunohistochemical study also allowed the verification of autophagy activation in tumors grown in mouse models. A lentivirus containing a shRNA against ATG5 was used to generate autophagy deficient cells. Using the MTT and MDC tests, we assessed their sensitivity to chemotherapy. In order to determine the effect of HCQ in sensitizing NB cells to chemotherapy, we used HCQ alone or in combination with conventional chemotherapy. This study demonstrated that autophagy is present at a basal level in NB cells. Unlike for LC3, the results showed that Beclin 1 is a factor of poor prognosis. The MTT and MDC tests have shown that vincristine, doxorubicin, cisplatin, temozolomide, rapamycin, and LY294002 induce autophagy in NB cells. Also, immunohistochemical studies showed that cisplatin treatment increased autophagy in vivo in xenograft model of human NB in mice. ATG5 deficient cells showed greater sensitivity to chemotherapy. Furthermore, the use of these cells in mouse models showed an important role of autophagy in tumor progression as well as an increased sensitivity to vincristine. Finally, combination of HCQ with conventional chemotherapy showed an increased sensitivity of NB cells to chemotherapy compared to cells receiving chemotherapy only. This study demonstrates that inhibition of autophagy in combination with conventional chemotherapy is an attractive therapeutic approach for the treatment of NB.

Pas de résumé français / Pas de résumé anglais

La surexpression de pompes d'efflux par les cellules cancéreuses permet l'élimination d'agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : P-gp/ABCB1, MRP1-ABCC1 et BCRP/ABCG2. La surexpression des ces transporteurs peut également être le "talon d'Achille" des cellules cancéreuses résistantes en les sensibilisant à certains composés. Ce phénomène, appelé sensibilité collatérale, pourrait constituer un nouvel outil thérapeutique conter les les cancers intrinséquement ou rendus résistants en éliminant sélectivement les cellules cancéreuse résistances. Ainsi, le S-vérapamil provoque la mort sélective par apoptose des cellules surexprimant suite à l'extrusion rapide et massive du glutathion 5GSH) intracellulaire par MRP1. Nous avons démontré que le vérapamil est capable de dépléter s"lectivement de leur contenu en GSH les tumeurs de cancer du poumon H69AR, MRP1 positives et résistantes, dès 3 heures d'exposition aiguë. Le vérapamil étant fortemnt carditoxique, nous avons développé de nouveaux agents de sensibilité collatérale, plus sélectif que le vérapamil, comme le xanthone 9, le flavonoïde 36 et le dimère de flavonoïde 4e. Enfin, grâce à l'étude de chimères MRP1/MRP2, nous avons démontré que la région comprenant les boucles L0 et L1-TM12 pourrait constituter les sites modualteurs et substrat du GSH sur MRP1. / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane, which prevent the accumulation of anticancer drugs in tumor cells. Three human ABC transporters are particulary involved in this chemoresistance : P-gp/ABCB1, MRP1-ABCC1 and BCRP/ABCG2. The overexpression of these trnasporters can also be an "Achille heel" for resistant cancer cells by sensitizing them to various drugs. This phenomenom, called collateral sensitivity, could constitute a new chemotherapy to eradicate cancers becoming resistant or cancer which ara resistant prioir to any treatment. Thus, S-verapamil triggers selective apoptosis of MRP1 overexpressing correlated to the massive and rapide extrusion of cellular glutathione by MRP1. We showed that verapamil is able to selectivity deprive H69AR MRP1 positive and resistant lung tumors, as soon as 3 hours of acute exposure. Verapamil being highly cardiotoxic, we have developed new collateral sensitivity drugs, more selective than verapamil, such as xanthone 9, flavonoïdd 36 and flavonoïd dimer 4e. Finally, thanks to the characterization of MRP1/MRP2 chimera, we showed that the MRP1 region including the intracellular loop L0 L1-TM12 might constitute the substrate and the modulator binding sites for GSH.