Plus de 80% des cancers colorectaux sont initiés par la perte de fonction du gène Apc. Afin d’identifier de nouvelles cibles thérapeutiques, nous avons utilisé des modèles murins présentant des mutations du gène Apc et recherché par des analyses de puces à ADN de nouveaux événements moléculaires impliqués au cours de la carcinogenèse intestinale.Cette approche nous a permis d’identifier une activation de la signalisation Notch tout au long du processus tumoral. Toutefois, cette activation n’est pas un élément clé de la progression tumorale puisque son inhibition n’empêche pas le phénotype tumoral induit par la perte du gène Apc. En parallèle, nos travaux ont permis d’identifier une induction de l’autophagie tout au long de la carcinogenèse intestinale. L’activation de ce processus biologique ouvre, quant à lui, de nouvelles perspectives thérapeutiques dans le traitement du CCR. / Over 80% of colorectal cancers are linked to an Apc mutation. To identify new therapeutic targets, we used mouse models with Apc mutations and performed microarray experiments to identify key molecular events involved in intestinal carcinogenesis. This approach allowed usto identify an activation of the Notch signaling all along tumor progression. However, this induction is dispensable for tumor development since its inhibition did not prevent the Apc phenotype. In addition, we have identified an induction of autophagy throughout intestinal carcinogenesis which appears to be an attractive therapeutic target in the treatment of CRC patients.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21224号 / 生博第393号 / 新制||生||52(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 米原 伸, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)

Les lésions tumorales cortico-surrénaliennes sont majoritairement des adénomes bénins et très rarement des carcinomes. Les altérations génétiques impliquées dans le développement des tumeurs cortico-surrénaliennes sporadiques, plus particulièrement au stade malin, demeurent à ce jour très peu connues. Lors de travaux récents menant à l’identification d’altérations génétiques de β-CATÉNINE nous avons constaté que plusieurs tumeurs présentaient une accumulation nucléo/cytoplasmique de la protéine β-CATÉNINE sans toutefois contenir de mutations pour ce gène. Nous avons donc émis l’hypothèse que, comme pour d’autres types de cancers, d’autres composants de la voie de signalisation Wnt/β-CATÉNINE, tel qu’AXIN2, pourrait être impliqués dans le développement des tumeurs du cortex surrénalien. De plus, plusieurs aberrations dans l’expression d’AXIN2 et de β-CATÉNINE sont associées à des tumeurs présentant de l’instabilité microsatellite dans d’autres types de cancer, notamment le cancer gastrique et colorectal. Nous avons donc étudié une cohorte de 30 adénomes, 6 carcinomes, 5 AIMAH, 3 hyperplasies ACTH-dépendante et 5 PPNAD ainsi que les lignées cellulaires de carcinomes cortico-surrénaliens humains H295R et SW13. Une étude préliminaire du statut MSI a également été réalisée sur 10 tumeurs contenant une mutation pour AXIN2 et/ou β-CATÉNINE. Nous avons trouvé des mutations d’AXIN2 dans 7% des adénomes (2/30) et 17% des carcinomes (1/6) cortico-surrénaliens. L’analyse fonctionnelle des mutations par immunohistochimie, analyse western blot et analyse de RT-PCR en temps réel a révélé une diminution de l’expression d’AXIN2 associée à cette mutation. L’analyse préliminaire MSI a démontré 1 échantillon AIMAH MSI-H, c’est-à-dire instable pour le locus BAT-25 et BAT-26 et 3 autres adénomes sécrétant de l’aldostérone instables seulement pour le locus BAT-26. Ainsi, ces travaux permirent d’identifier une nouvelle altération génétique associée au développement des tumeurs du cortex surrénalien en plus de rapporter pour la première fois la présence de MSI-H dans ce type de tumeurs. / Adrenocortical lesions are mostly benign tumors and rarely carcinomas. From now on, genetic alterations implicated in sporadic adrecocortical tumour development remains largely unknown. In our previous work leading to identification of genetic alterations in β-catenin, we observed that many tumors presented a nucleo/cytoplasmic accumulation of β-catenin protein without β-catenin mutations. Thus, we hypothesised that, as for many others cancers, others components of the Wnt/ β-catenin signalling pathway, as AXIN2, are implicated in development of adrenocortical tumors. Also, many aberrations in AXIN2 and β-catenin expression have been reported in association with microsatellite instability in other types of cancers like gastroinstestinal and colorectal cancer. We have studied 30 adenomas, 6 carcinomas, 5 AIMAH, 3 ACTH-dependant hyperplasias and 5 PPNAD as well as the human carcinoma cancer cells lines H295R and SW13. Preliminary study for MSI was also realised on 10 tumors harbouring AXIN2 and/or Β-CATENIN mutations. We have found AXIN2 mutations in 7% of adrenocortical adenomas (2/30) and 17% of adrenocortiocal carcinomas. Functional analysis of this mutation by immunohistochemical, western blot and real-time RT-PCR analysis revealed a down-regulation of AXIN2 expression associated with this mutation. Preliminary analysis of MSI results in 1 AIMAH sample MSI-H, which means instable for BAT-25 and BAT-26 locus, and 2 aldosterone adenomas were unstable for BAT-26 locus. This work identified a new genetic alteration involved in adrenocortical tumour development and report for the first time MSI-H in this type of tumor.

Les lésions tumorales cortico-surrénaliennes sont majoritairement des adénomes bénins et très rarement des carcinomes. Les altérations génétiques impliquées dans le développement des tumeurs cortico-surrénaliennes sporadiques, plus particulièrement au stade malin, demeurent à ce jour très peu connues. Lors de travaux récents menant à l’identification d’altérations génétiques de β-CATÉNINE nous avons constaté que plusieurs tumeurs présentaient une accumulation nucléo/cytoplasmique de la protéine β-CATÉNINE sans toutefois contenir de mutations pour ce gène. Nous avons donc émis l’hypothèse que, comme pour d’autres types de cancers, d’autres composants de la voie de signalisation Wnt/β-CATÉNINE, tel qu’AXIN2, pourrait être impliqués dans le développement des tumeurs du cortex surrénalien. De plus, plusieurs aberrations dans l’expression d’AXIN2 et de β-CATÉNINE sont associées à des tumeurs présentant de l’instabilité microsatellite dans d’autres types de cancer, notamment le cancer gastrique et colorectal. Nous avons donc étudié une cohorte de 30 adénomes, 6 carcinomes, 5 AIMAH, 3 hyperplasies ACTH-dépendante et 5 PPNAD ainsi que les lignées cellulaires de carcinomes cortico-surrénaliens humains H295R et SW13. Une étude préliminaire du statut MSI a également été réalisée sur 10 tumeurs contenant une mutation pour AXIN2 et/ou β-CATÉNINE. Nous avons trouvé des mutations d’AXIN2 dans 7% des adénomes (2/30) et 17% des carcinomes (1/6) cortico-surrénaliens. L’analyse fonctionnelle des mutations par immunohistochimie, analyse western blot et analyse de RT-PCR en temps réel a révélé une diminution de l’expression d’AXIN2 associée à cette mutation. L’analyse préliminaire MSI a démontré 1 échantillon AIMAH MSI-H, c’est-à-dire instable pour le locus BAT-25 et BAT-26 et 3 autres adénomes sécrétant de l’aldostérone instables seulement pour le locus BAT-26. Ainsi, ces travaux permirent d’identifier une nouvelle altération génétique associée au développement des tumeurs du cortex surrénalien en plus de rapporter pour la première fois la présence de MSI-H dans ce type de tumeurs. / Adrenocortical lesions are mostly benign tumors and rarely carcinomas. From now on, genetic alterations implicated in sporadic adrecocortical tumour development remains largely unknown. In our previous work leading to identification of genetic alterations in β-catenin, we observed that many tumors presented a nucleo/cytoplasmic accumulation of β-catenin protein without β-catenin mutations. Thus, we hypothesised that, as for many others cancers, others components of the Wnt/ β-catenin signalling pathway, as AXIN2, are implicated in development of adrenocortical tumors. Also, many aberrations in AXIN2 and β-catenin expression have been reported in association with microsatellite instability in other types of cancers like gastroinstestinal and colorectal cancer. We have studied 30 adenomas, 6 carcinomas, 5 AIMAH, 3 ACTH-dependant hyperplasias and 5 PPNAD as well as the human carcinoma cancer cells lines H295R and SW13. Preliminary study for MSI was also realised on 10 tumors harbouring AXIN2 and/or Β-CATENIN mutations. We have found AXIN2 mutations in 7% of adrenocortical adenomas (2/30) and 17% of adrenocortiocal carcinomas. Functional analysis of this mutation by immunohistochemical, western blot and real-time RT-PCR analysis revealed a down-regulation of AXIN2 expression associated with this mutation. Preliminary analysis of MSI results in 1 AIMAH sample MSI-H, which means instable for BAT-25 and BAT-26 locus, and 2 aldosterone adenomas were unstable for BAT-26 locus. This work identified a new genetic alteration involved in adrenocortical tumour development and report for the first time MSI-H in this type of tumor.

[Truncated abstract] In recent years a better understanding of the biology of PPAR , a nuclear transcription factor, has emerged, leading to a resurgence in targeting PPAR for chemotherapy. The family of synthetic PPAR agonists, the thiazolidinediones (TZDs), and non-steroidal anti-inflammatory drugs (NSAIDs) have been implicated in the inhibition of cell proliferation, apoptosis and cell cycle arrest of androgen-sensitive (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cells generating much interest in their use for potential curative cancer therapies. In light of the potential use of TZDs and NSAIDs in prostate cancer prevention and their ability to induce inhibitory effects in vitro and in vivo, the first aim of this project was to undertake a comprehensive study of the effects of ciglitazone (TZD) and indomethacin (NSAID) on the androgen-independent prostate cancer cell line DU145, using standardised concentrations and time-points to compare the effects of TZDs and NSAIDs on cell proliferation, cell cycle and apoptosis. Treating the cells with either 10 µM ciglitazone or 10 µM indomethacin resulted in a time-dependent decrease in DU145 cell proliferation. The anti-proliferative effects were found to be in-part attributed to the slowing of cell progression through the G1/S-phase checkpoint of the cell cycle, and in the case of ciglitazone, apoptosis also played a role in its anti-proliferative effects in this cell line. Interestingly, although indomethacin failed to induce apoptosis, its antiproliferative effects were more potent than ciglitazone. The second aim of this project was to further investigate the underlying mechanisms responsible for the anti-proliferative effects of ciglitazone and indomethacin by evaluating their ability to modulate PPAR mRNA and protein expression, and to induce PPAR transcriptional activity. ... In addition, ligandinduced regulation of secreted frizzled related protein 4 (sFRP4) expression, a Wnt/ - catenin antagonists, was investigated. It was demonstrated that both ciglitazone and indomethacin attenuated Wnt/ -catenin signalling via the down-regulation of total - catenin levels within the cells, inhibition or slowing of the translocation of cytoplasmic -catenin into the nucleus and inhibition of cyclin–D1 expression An inverse relationship between PPAR and -catenin protein levels was also detected, suggesting that PPAR may directly bind to -catenin itself. sFRP4 expression was transiently upregulated by ciglitazone and indomethacin-treatment, suggesting that the antiproliferative effects of the ligands may be mediated in part through regulation of sFRP4 mRNA and protein levels. In summary, the anti-proliferative effects of ciglitazone and indomethacin on the androgen-independent prostate cancer cell line, DU145, described in this thesis are progressive steps in characterising the role of PPAR in prostate cancer cell proliferation. The identification of indomethacin as a more potent PPAR agonist than ciglitazone represents a novel target for the development of preventative strategies for advanced disease, and the relationship between PPAR and the Wnt/ -catenin signalling pathway provide an insight into the mechanisms involved in the anti-proliferative effects of ciglitazone and indomethacin. Further studies into this relationship would advance help identify novel preventative and curative therapeutic strategies for advanced prostate cancer.

Neurogenesis continues postnatally in two major neural stem cell (NSC) niches: The subventricular zone (SVZ) and dentate gurus of the hippocampus. SVZ NSCs self-renew and produce transit amplifying progenitor cells that, in turn, divide and give rise to neuroblasts. These neuroblasts migrate to the olfactory bulbs, via the rostral migratory stream (RMS), where they terminally differentiate into mature neurons. The postnatal SVZ (pSVZ) is more gliogenic than its adult counterpart (aSVZ), contributing to robust postnatal astrocytogenesis and oligodendrogenesis in the surrounding brain parenchyma. Studies examining Galectin-3 (Gal-3) in the aSVZ showed it has functions in regulating neuroblast migration, microglial activation, oligodendrocytic differentiation, and angiogenesis. However, the role of Gal-3 in pSVZ lineage progression is unknown. This thesis aims to unravel the roles of Gal-3 in regulating pSVZ lineage progression, fate choices, and NSC activation. In doing so, the thesis tackles the molecular pathways possibly involved in mediating the effects of Gal-3. I found through co-immunoprecipitation that Gal-3 was bound to β-catenin and both proteins were co-expressed in the aSVZ. In addition, expression of Gal-3 and Wnt/β-catenin signalling were downregulated as SVZ cells progressed through the lineage and became migratory. I hypothesised that Gal-3 may regulate lineage progression through regulation of Wnt/β-catenin signalling. To explore this hypothesis, Gal-3 overexpression, knockdown or control plasmids were co-electroporated with a Wnt/β-catenin reporter into the SVZ of postnatal day two mice. I found lineage progression was not altered by Gal-3 overexpression. Surprisingly, contrary to evidence described in the cancer literature, Gal-3 overexpression reduced Wnt/β-catenin signalling. This was accompanied by an acute reduction in proliferation. Also, more cells expressed p27/Kip1 in the SVZ, and more cells migrated into the RMS, suggesting increased cell cycle exit. However, NSC proliferation and clonal neurosphere forming capacity were not altered by Gal-3 overexpression, indicating that NSC activation was not influenced by Gal-3. While olfactory neuronogenesis was not altered by Gal-3 overexpression, striatal astrocytogenesis was increased while oligodendrogenesis was dampened. Further experiments revealed phosphorylation of Smad proteins 1/5/8 was increased in vivo and in vitro after Gal-3 overexpression. These findings indicate that Gal-3 positively regulated BMP signalling in the SVZ, possibly contributing to Gal-3's pro-gliogenic effects. Taken together, this thesis supports a model whereby a subpopulation of Gal-3-responsive pSVZ cells reacted to Gal-3 overexpression by acutely exiting the cell cycle, and possibly through the same mechanisms, switched from oligodendrocytic to astrocytic fate. These cellular responses might have been brought about, at least partially, by acute suppression of Wnt/β-catenin and activation of BMP signalling. These novel findings emphasise the regulatory actions of Gal-3 on pSVZ lineage progression through Wnt/β- catenin and BMP signalling.