京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第13353号 / 論理博第1571号 / 新制||理||1664(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 教授 沼田 英治, 教授 曽田 貞滋 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DFAM

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in both men and women, and one of the leading causes of cancer-related deaths worldwide. One frequently mutated pathway involved in oncogenesis in CRC is the RAS/RAF/MAP kinase pathway. Oncogenic activation of KRAS and BRAF occur in 30‒40% and 5‒15% of all CRCs, respectively, and the mutations are mutually exclusive. Even though KRAS and BRAF are known to act in the same pathway, KRAS- and BRAF-mutated CRCs have different clinical and histopathological features. For example, BRAF mutation in CRC is tightly linked to microsatellite instability (MSI) and a CpG island methylator phenotype (CIMP), which is not seen in KRAS-mutated tumours. BRAF-mutated CRCs are also more often found in right-sided tumours. However, the underlying molecular reasons for these differences have not yet been defined. The overall aim of this thesis was to investigate molecular differences between KRAS- and BRAF-mutated CRCs to understand how KRAS and BRAF mutations differentially affect tumour progression. We used an in vitro cell culture system to explore molecular differences between KRAS- and BRAF-mutated CRCs and verified our findings using CRC tissue specimens from the Colorectal Cancer in Umeå Study (CRUMS). We found that BRAF mutation, but not KRAS mutation, was associated with expression of the stem cell factor SOX2. Furthermore, SOX2 was found to be correlated to a poor patient prognosis, especially in BRAF-mutated cancers. We further investigated the role of BRAF in regulation of SOX2 expression and found that SOX2 is at least partly regulated by BRAF in vitro. We continued by investigating the functional role of SOX2 in CRC and found that SOX2-expressing cells shared several characteristics with cancer stem cells, and also had down-regulated expression of the intestinal epithelial marker CDX2. There was a strong correlation between loss of CDX2 expression and poor patient prognosis, and patients with SOX2 expression were found to have a particularly poor prognosis when CDX2 levels were down-regulated. In conclusion, in these studies we identified a subgroup of BRAF-mutated CRCs with a particularly poor prognosis, and having a cancer stem cell-like appearance with increased expression of SOX2 and decreased expression of CDX2. Tumour progression is regulated by interactions with cells of the immune system. We found that BRAF-mutated CRCs were more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours, while the opposite was true for KRAS-mutated CRCs. Interestingly, we found that part of this difference is probably caused by differences in secreted chemokines and cytokines between KRAS- and BRAF-mutated CRCs, stimulating different arms of the immune response. Altered levels of expression of miRNAs have been seen in several malignancies, including CRC. We found that BRAF- and KRAS-mutated CRCs showed miRNA signatures different from those of wild-type CRCs, but the expression of miRNAs did not distinguish KRAS-mutated tumours from BRAF-mutated tumours. In summary, our findings have revealed possible molecular differences between KRAS- and BRAF-mutated CRCs that may explain some of the differences in their clinical and histopathological behaviour.

Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.

Le carcinome spinocellulaire (SCC) est le 2ème cancer de la peau le plus fréquent avec plus d’un million de nouveaux patients diagnostiqués dans le monde chaque année. On retrouve également des SCCs associés à un pronostic plus sombre au niveau de la tête, du cou, de la cavité orale et de l’œsophage. Des travaux récents ont démontré l’existence de cellules souches cancéreuses (CSCs) dans les SCCs cutanés mais les mécanismes moléculaires contrôlant leurs fonctions restent indéterminés. Dans une première étude, nous avons montré que Sox2, un facteur de transcription (TF) associé aux cellules souches, est détecté de manière hétérogène dans une grande majorité des papillomes et des SCCs chez la souris et chez l’humain. La délétion conditionnelle de Sox2 dans l’épiderme réduit drastiquement l’apparition de tumeurs démontrant le rôle clé de Sox2 dans l’initiation tumorale. En utilisant une souris génétiquement modifiée Sox2-GFP knock-in, nous avons démontré que les cellules tumorales Sox2+ sont enrichies en cellules propagatrices de tumeurs dont la proportion augmente au fur et à mesure des transplantations sériées. L’ablation des cellules Sox2+ dans les papillomes et les SCCs conduit à une importante régression des tumeurs, indiquant que ces cellules ont un rôle crucial dans le maintien des tumeurs. La délétion conditionnelle de Sox2 dans des papillomes et SCCs préexistants provoque également une régression majeure des tumeurs, soulignant le rôle essentiel de Sox2 dans la régulation des fonctions des cellules tumorales. Une analyse transcriptionnelle et des expériences d’immunoprécipitation de chromatine nous ont permis de mettre en évidence un réseau de gènes associés à des fonctions essentielles des cellules tumorales et régulés par Sox2 dans les tumeurs primaires in vivo. Dans une 2ème étude, nous avons montré que les SCCs issus de l’épiderme inter-folliculaire (IFE) présentent en général un caractère différencié alors que ceux issus du follicule pileux (HF) présentent fréquemment des caractéristiques de transition épithélio-mésenchymateuse (EMT). En réalisant une analyse transcriptionnelle et épigénétique, nous avons démontré que les différentes cellules à l’origine expriment un réseau de gènes spécifiques et présentent une accessibilité différentielle à des sites de liaison d’importants TFs associés soit à un phénotype épithélial soit à l’EMT. Ces résultats démontrent que l’état transcriptionnel et épigénétique de la cellule à l’origine amorce spécifiquement les tumeurs vers le processus d’EMT. L’ensemble de ces résultats souligne des mécanismes cruciaux à l’établissement de l’hétérogénéité tumorale et seront essentiels pour parvenir à des pronostics affinés et au développement de nouvelles thérapies ciblées dans le traitement du cancer. / Skin squamous cell carcinoma (SCC) is the second most frequent skin cancer with more than a million new patients affected every year throughout the world. It is also the predominant cancer of the head, neck, oral cavity and esophagus, associated with a poor prognosis. Recent studies have identified cancer stem cells (CSCs) in skin SCC but the molecular mechanisms controlling their functions remain unclear. In a first study, we show that Sox2, a transcription factor (TF) associated with stemness, is expressed in a heterogeneous manner in the vast majority of benign and malignant skin tumors in mouse and human. Sox2 conditional deletion in the epidermis impairs tumor development showing that Sox2 plays a crucial role in tumor initiation. Using a Sox2-GFP knock-in mouse model, we show that Sox2-expressing tumor cells are greatly enriched in tumor-propagating cells, which further increase upon serial transplantations. Lineage ablation of Sox2-expressing cells in primary benign and malignant SCCs leads to tumor regression, consistent with the critical role of Sox2-expressing cells in tumor maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumor regression, supporting the essential role of Sox2 in regulating cancer cells functions. Using transcriptional profiling and chromatin immunoprecipitation, we uncovered a gene network controlling many cancer hallmarks regulated by Sox2 in primary tumour cells in vivo.In a second study, by targeting the same oncogenic mutations to distinct skin compartments, we show that interfollicular epidermis (IFE)-derived SCCs are generally well-differentiated, while hair follicle stem cells (HFSCs)-derived SCCs frequently exhibit features of epithelial-mesenchymal transition (EMT). Using transcriptional and epigenetic profiling, we show that IFE and HF tumor-initiating cells harbor distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT. These different chromatin landscapes correlate with the differential accessibility of key epithelial and EMT TFs binding sites in the cancer cell of origin. These findings demonstrate that cell type-specific chromatin and transcriptional states differentially prime tumours towards EMT.Altogether, these results highlight crucial mechanisms for the establishment of tumor heterogeneity which will be relevant for better prognostic assessment and the development of novel targeted therapies for cancer treatment. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

The main functional cells of the inner ear are neurons and sensory cells that are formed from a common embryonic epithelial neurosensory domain. Discovering genes important for specification and differentiation of sensory cells and neurons in the inner ear is a crucial basis for understanding the pathophysiology of hearing loss. Some of these factors are necessary not only for the inner ear but also for the development of other neurosensory systems such as the visual and olfactory system. The aim of this work was to reveal functions of transcription factor SOX2 in inner ear development by using mouse models with different conditional deletions of Sox2 gene. Sox2 gene was deleted by cre-loxP recombination. In Isl1-cre, Sox2 CKO mutant, reduced number of hair cells differentiated only in some inner ear organs (utricle, saccule and cochlear base) and not in others (cristae and cochlear apex). Early forming inner ear neurons in the vestibular ganglion and neurons innervating the cochlear base developed in these mutants but died by apoptosis due to the lack of neurotrophic support from sensory cells. Late forming neurons in the cochlear apex never formed. In Foxg1-cre, Sox2 CKO mutant, only rudimental ear with no sensory cells was formed. The initial formation of vestibular ganglion with peripheral and...

<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)

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Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.