The inherited bone marrow failure syndrome Fanconi Anaemia (FA) is caused by mutations in any one of the multiple FANC genes, which encode proteins that collaborate in the FA/BRCA DNA damage response pathway. FA is characterised by extreme predisposition to acute myeloid leukaemia (AML). AML in FA is associated with typical chromosomal aberrations involving gains of the long arm of chromosome 3 (3q gains). These are linked to overexpression of the oncogene ecotropic viral integration site 1 (EVI1). Based on this clinical observation, the hypothesis that EVI1 confers leukaemic transformation, in particular in the context of FA, was tested. Mouse embryonic stem cells with either functional or disrupted FA/BRCA-pathway were used to model normal and FA-associated embryonic haematopoiesis, and the effect of EVI1 overexpression was assessed in this model. EVI1 functions were also investigated with respect to protein interactions, focusing on the interaction with the co-repressor C-terminal binding protein 1 (CtBP1), in the context of genotoxic stress. To study this, in vitro haematopoietic differentiation assays, flow cytometry, mass spectrometry, immunoprecipitations and immunofluorescence were employed. In vitro haematopoietic differentiation using mouse embryonic stem cells with defective Fanca was successfully developed and applied. The analysis revealed that EVI1 overexpression in haemangioblast-like cells prevented the generation of haematopoietic precursors through endothelial to haematopoietic transition. Studies into EVI1 protein interaction dynamics showed that DNA damage-induced phosphorylation of EVI1 modifies interaction with the co-repressor CtBP1. This interaction was demonstrated to be partially required for the EVI1-induced block of the development of haematopoietic precursors using the mESC-based model. An EVI1-mediated modulation of the FA phenotype characteristic G2-arrest and of the FA-associated embryonic haematopoiesis was not demonstrated. This study contributes to the understanding of the function of the EVI1 oncogene in normal and FA-associated haematopoiesis and the DNA damage response. FA-associated haematopoiesis and leukaemogenesis can be further studied using the embryonic haematopoiesis model developed here, and further studies can build on the data generated with respect to EVI1.

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

Genomic aberrations are primary contributors to the pathophysiology of cancer [11]. Dysregulated expression of genes located within these aberrations are important predictors of chemoresistance, disease prognosis, and patient outcome [12]. This dissertation is focused on understanding the regulation and/or functions of specific genes located at dysregulated genomic regions such as 3q26 and 14q32 in the biology of ovarian and renal cancer, respectively. Serous epithelial ovarian cancer (EOC) manifest amplification at the 3q26.2 locus [2], an observation consistent with the cancer genome atlas (TCGA) [13]. The most amplified gene in this region is EVI1 which has been extensively studied in hematological malignancies [2]. However, its contribution to the pathophysiology of solid cancers remains unknown. We hypothesized that dysregulated EVI1 and SnoN/SkiL expression (located at the 3q26.2 amplicon) leads to the altered cellular functional response, thereby contributing to the pathophysiology of ovarian cancer. Our group has previously shown that EVI1 splice forms may exhibit altered subcellular localization and functional properties relative to the wild type form [14]. In Chapter 3 of this dissertation, we identified that EVI1 splice forms could modulate epithelial-mesenchymal transition. Our findings indicate that siRNA construct targeting the splice junction between exon 2 of MDS1 to exon 2 of EVI1, (reduces the expression of MDS1/EVI1 and EVI1Del190-515 splice forms) increases epithelial cell markers while decreasing mesenchymal markers and reducing migratory potential of ovarian and breast cancer cells. SnoN/SkiL, another gene overexpressed at the 3q26 is reported by our group to be induced upon As2O3 treatment in ovarian cancer cells via unknown mechanisms [15]. This induction of SnoN opposes the apoptotic cell death pathway induced by the drug treatment [15]. We have previously identified that the PI3K/AKT pathway (also dysregulated in ovarian cancer [16]) contributes to the up-regulation of SnoN upon treatment with As2O3 [17]. However, SnoN is regulated via multiple mechanisms including post-translational modifications [18]. Additionally, c-Ski (a homolog of SnoN) is regulated post-transcriptionally by numerous miRNAs in cancer cells [19-22]. In Chapter 4, we attempted to identify potential miRNAs that could regulate SnoN expression post-transcriptionally. We discovered that miR-494 reduces both SnoN mRNA and protein levels. Our experimental outcomes also demonstrate that miR-494 further sensitizes ovarian cancer cells to drug treatment. Interestingly, miR-494 is located at the 14q32 region which has been shown to be down-regulated in renal cancers [23]. Several reports indicate miR-494 to be involved in tumor suppressive responses including apoptosis and cell cycle arrest in various cancers [24-26]. However, its role in renal cancer biology remains unknown. We hypothesized that miR-494 elicits a tumor suppressive response in renal cancer cells. Through our studies in Chapter 5, we demonstrate that miR-494 reduces cell viability and increases apoptotic response in renal cancer cells. We also show that miR-494 increases LC3B mRNA and protein levels. A 3’UTR luciferase assay indicated that LC3B may be a potential target of miR-494. Intracellular lipid droplets (LDs) increased in miR-494 expressing in a LC3B-dependent manner. This was accompanied with reduced intracellular cholesterol content, increased mitochondrial structural disorganization, and altered Drp1 localization. The outcome of our findings have improved our understanding of the regulation and functional response of these genes/miRNAs (EVI1, SnoN, and miR-494) in ovarian and renal cancers. The studies reported in Chapter 5 identified a novel function of miR-494 in increasing LDs and reducing renal cell survival. However, additional studies are warranted to fully understand the underlying mechanism of increased LDs formation in miR-494 expressing cells and the implication of miR-494 and other miRNAs at the 14q32 region in renal cancer biology. In future, these studies will aid in the development of better treatment strategies which will contribute towards the management of cancer.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24522号 / 医博第4964号 / 新制||医||1065(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 小川 誠司, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19261号 / 理博第4116号 / 新制||理||1592(附属図書館) / 32263 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM