Yes / The synthesis and characterization of three aromatic oligoamides, constructed from the same pyridyl carboxamide core but incorporating distinct end groups of acetyl (Ac) 1, tert-butyloxycarbonyl (Boc) 2 and amine 3 is reported. Single crystal X-ray diffraction analysis of 1-3 and a dimethylsulfoxide (DMSO) solvate of 2 (2-DMSO), has identified the presence of a range of intra- and intermolecular interactions including N-H⋯N, N-H⋯O=C and N-H⋯O=S(CH3)2 hydrogen-bonding interactions, C-H⋯π interactions and off-set, face-to-face stacking π-π interactions that support the variety of slipped stack, herringbone and cofacial crystal packing arrangements observed in 1-3. Additionally, the cytotoxicity of this series of aromatic oligoamides was assessed against two human ovarian (A2780 and A2780cisR), two human breast (MCF-7 and MDA-MB-231) cancerous cell lines and one non-malignant human epithelial cell line (PNT-2), to investigate the influence of the terminal functionality of these aromatic oligoamides on their biological activity. The chemosensitivity results highlight that modification of the terminal group from Ac to Boc in 1 and 2 leads to a 3-fold increase in antiproliferative activity against the cisplatin-sensitive ovarian carcinoma cell line, A2780. The presence of the amine termini in 3 gave the only member of the series to display activity against the cisplatin-resistance ovarian carcinoma cell line, A2780cisR. Compound 2 is the lead candidate of this series, displaying high selectivity towards A2780 cancer cells when compared to non-malignant PNT-2 cells, with a selectivity index value >4.2. Importantly, this compound is more selective towards A2780 (cf. PNT-2) than the clinical platinum drugs oxaliplatin by > 2.6-fold and carboplatin by > 1.6-fold. / University of Bradford Development Fund; University of Birmingham - Birmingham Fellowship; UKRI Future Leaders Fellowship (MR/T041315/1); UKRI Future Leaders Fellowship (MR/S035486/2)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25486号 / 医博第5086号 / 新制||医||1073(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 伊藤 貴浩, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

BACKGROUND:Recent advances suggest fallopian tube as the main cellular source for women's pelvic serous carcinoma (PSC). In addition to TP53 mutations, many other genetic changes are involved in pelvic serous carcinogenesis. IMP3 is an oncofetal protein which has recently been observed to be overexpressed in benign-looking tubal epithelia. Such findings prompted us to examine the relationship between IMP3 over-expression, patient age and the likelihood of development of PSC.METHODS:Fallopian tubes from three groups (low-risk, high-risk, and PSC) of patients with matched ages were studied. Age was recorded in 10years intervals ranging from age 20 to older than 80. The number of IMP3 signatures (defined by 10 or more tubal secretory cells stained positively and continuously in benign appearing tubal mucosa) from both tubal fimbria and ampulla segments was measured. The data was analyzed by standard contingency table and Poisson distribution methods after age adjustment. IMP3 overexpression was also examined in serous tubal intraepithelial carcinoma and PSC.RESULTS:The positive IMP3-stained cells are mainly tubal secretory cells. The absolute number of tubal IMP3 signatures increased significantly within each age group. Age remained a significant risk factor for serous neoplasia after age adjustment. IMP3 signatures were more frequent in the patients of both high-risk and PSC groups. The presence of IMP3 signatures in tubal mucosa was significantly associated with tubal or pelvic serous carcinogenesis (p<0.001).CONCLUSIONS:The findings suggest that tubal secretory cells with IMP3 signatures showing growth advantage could potentially serve as a latent precancer biomarker for tubal or pelvic serous carcinomas in women.

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.

Epithelial Ovarian Cancer (EOC) kills more women annually in the United Kingdom than any other gynaecological cancer. Survival rates for women diagnosed with EOC have not improved over the past 30 years, due to the often advanced stage at presentation, where widespread intra-peritoneal dissemination has occurred. The natural history of the disease remains uncertain but the ovarian surface epithelium (OSE) is a strong candidate for the tissue of origin. The OSE undergoes cyclical damage and repair in women of reproductive age following ovulation, which can be considered an acute inflammatory event. Factors that prevent ovulation (pregnancy, breastfeeding and contraceptive pill use) also protect against the development of EOC. Previously published data show that the OSE is able to upregulate the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) in response to inflammation, the enzyme responsible for converting inactive cortisone to anti-inflammatory cortisol. This thesis hypothesises that 11βHSD isozymes are deregulated in ovarian cancer; that the peritoneal surface epithelium (PSE) is indistinguishable from the OSE in its response to inflammation and should be considered a potential source of some “ovarian cancers”; and finally that the expression of the tumour suppressor gene OPCML (OPioid binding Cell adhesion Molecule-Like) is altered by inflammation. These hypotheses were examined at three levels. Firstly, primary cultures of EOC were established, and glucocorticoid metabolism and the response to inflammation was compared to normal OSE. Results from these investigations reveal that the11βHSD1 response to IL-1α stimulation is impaired in EOC compared to normal OSE at the mRNA level but there is no significant difference when 11βHSD1 enzyme activity is measured in these tissues. When basal levels of 11βHSD1, 11βHSD2 and COX2 are compared amongst untreated samples of EOC and OSE, there was a significant correlation between 11βHSD1 and COX2 mRNA expression (P<0.001). 11βHSD2 mRNA expression was significantly higher in the EOC specimens compared to OSE (P<0.05). Secondly the response to inflammation was compared in primary cultures of human peritoneal surface epithelial (PSE) cells and OSE. The data suggest that the mRNA response to inflammation was similar in OSE and PSE, but that the 11βHSD1 enzyme activity was reduced in PSE (P<0.05), which may result in differences in tissue healing. Finally, the effect of inflammation on the expression of the ovarian cancer associated tumour suppressor gene (TSG), OPCML (OPioid binding Cell adhesion Molecule-Like) and the other members of the IgLON family, was examined in OSE. These results suggest that OPCML mRNA expression can be induced by IL-1α, an effect that is inhibited by cortisol.

Le cancer endométrioïde de l’ovaire (CEO) ainsi que l’implant endométriosique sont deux tumeurs caractérisées par la présence de cellules épithéliales et stromales ressemblant aux cellules endométriales. Des études antérieures ont mis en évidence des anomalies immunologiques communes à la cellule épithéliale endométriale (CEE), à la cellule endométriosique (CENDO) et à la cellule du CEO (CCEO) suggérant une origine commune endométriale de l’endométriose (ENDO) et du CEO. Selon la théorie de l'implantation, la migration des cellules endométriales vers d'autres sites ectopiques constitue un événement important dans l'étiopathogénie de l’endométriose (ENDO). Récemment, il a été démontré que CXCL12/CXCR4, un axe œstrogèno-dépendant, joue un rôle important dans l’homéostasie cellulaire et contrôle la migration des cellules dans les conditions normales et pathologiques. Ainsi, l'implication des œstrogènes dans la régulation de ce système nous amène à nous questionner sur l’effet du bisphénol A (BPA), un xéno-œstrogène utilisé dans une large gamme de produits, sur l’expression de CXCL12/CXCR4 et sur la migration de la CEE et son développement ectopique en tumeur. Dans ce projet, nous avons utilisé des CEE primaires provenant d’endomètres et d’implants endométriosiques, ainsi que deux lignées néoplasiques (endométriale et endométrioïde ovarienne). Nous avons étudié le rôle du BPA dans l'expression du couple CXCL12/CXCR4 par des méthodes classiques d’ELISA, RT-PCR en temps réel, Western blot et immunocytochimie. La prolifération a été évaluée par méthode colorimétrique et la migration par la technique des deux chambres de Boyden. Le dosage du BPA a été fait par chromatographie en phase gazeuse. Les principaux résultats montrent que l’expression basale du CXCR4 au niveau des différents types de cellules est augmentée par le traitement au BPA en comparaison aux contrôles. Ces résultats démontrent, une fois de plus, une possible origine commune de l’ENDO et CEO. D’autre part, nous avons démontré que le traitement au BPA entraine une augmentation de la migration et de la prolifération des CEE selon un axe CXCL12/CXCR4 dépendant. Ceci pourrait être un mécanisme impliqué dans la genèse de l’endométriose et suggère que le BPA pourrait être le facteur responsable par l’activation de l’axe CXCL12/CXCR4. Ce travail a aussi permis d’identifier une anomalie inhérente au tissu endométrial de patientes endométriosiques et qui consiste en une augmentation de l’expression du récepteur CXCR4, ce qui pourrait constituer un biomarqueur de diagnostic de l’ENDO. En dernier lieu, compte tenu de l'absence de lien entre les concentrations de BPA et celles du CXCL12, la réponse chimiotactique qui serait responsable de la migration de la CEE vers la cavité pelvienne pourrait être dépendante d'autres facteurs du microenvironnement péritonéal, ce qui appuie la nature multifactorielle de l’étiopathogénie de endométriose.

miRNAs are critical modulators in the development and progression of cancer. Emerging evidence suggests that they are drivers of ovarian cancer. A better understanding of the molecular underpinnings of the development, progression and chemoresistance of the disease is critical for the development of new, more effective therapies. Here we explore the expression patterns of miRNAs as they relate to gene expression, as they differ across molecular subtypes of the disease. We examine the correlation structure of miRNA expression with mRNA expression in two distinct genomic datasets and report on patterns in correlation structure in several subsets of the data. We find that the datasets show consistency in their correlation structure, and in the specific miRNA-mRNA pairs that are either highly positively or negatively correlated. The data include a larger number of strong positive and strong negative correlations than would be expected by chance, indicating that biological relationships between the types of data are detectable in these datasets. We further find an enrichment for positively-correlated miRNA-mRNA pairs in which the miRNA is encoded in close proximity to the mRNA. The correlation of miRNA and mRNA is apparently unaffected by miRNA and mRNA expression level; similarly the two molecular subtypes do not contain differences in their correlation. We find that the recently described poorer prognosis, or angiogenic, subtype has a generally lower miRNA activity than the second, non-angiogenic, subtype. The subtypes are characterized by a consistent pattern of differential miRNA expression. We also report on a switch-like relationship between the expression levels of certain miRNAs and the genes that are anticorrelated with them. We propose these miRNAs drive many of the differences in the subtypes both directly, by RISC-mediated repression of target messages and indirectly, by repressing transcription factors that regulate expression in the cell. We build models of patient survival and time-to-relapse based on these miRNA expression data and inferred miRNA activity scores, using several types of univariate and variable selection models. We find essentially no survival-predictive information provided by the RE score data. While the direct miRNA expression measurements may contain some predictive power, we find that a larger dataset and the segretation of that dataset into distinct molecular phenotypes is likely to be necessary to produce a useful model of survival in ovarian cancer.

The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.

Ovarian cancer is the most lethal gynecological malignancy among women. The current first-line treatments for ovarian cancer are cisplatin, carboplatin and paclitaxel. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Therefore, the resistance to cisplatin therapy has been a critical hurdle in the management of recurrent ovarian cancer. The mechanisms responsible for cisplatin resistance are not completely understood. In the search for new therapies to overcome/bypass cisplatin resistance, melanoma differentiation gene-7 (MDA-7) IL-24, which is a new cytokine, has anti-cancer efficacy by suppressing cell growth and inducing apoptosis in a broad range of tumor cells and does not induce any toxicity in normal cells, thus, making it a potentially effective therapeutic gene for ovarian cancer. The purpose of this study was to evaluate the potential therapeutic efficacy of MDA-7 to treat ovarian carcinoma. Since adenoviral-mediated MDA-7 gene therapy has been shown to be well tolerated and showed biological activity in clinical studies in the context of other carcinomas we assessed the anticancer effects of Ad.mda-7 and in combination with cis-platinum on ovarian cancer cells. Our results show that the purified recombinant MDA-7 protein, GST-MDA-7, and Ad.mda7 virus (5) induced growth arresst and apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7 virus (5). The use of a modified Ad.mda-7 virus type5, Ad.mda-7 virus type(5/3), inhanced infectivity and significantly enhanced ovarian cancer cell killing in human ovarian cancer cell lines in vitro compared to unmodified Ad.mda-7 virus, Ad.mda-7 virus type5. Also Ad-mda7 synergizes with cis-platinum in vitro and enhances ovarian cancer cell death. Taken together, these findings demonstrate that MDA-7 is capable of promoting growth suppression and inducing cell death in ovarian cancer cells, at least OVCAR cells and support the pharmacological interest of the combination of MDA-7 and cis-platinum.

Lysophosphatidic acid (LPA) and transforming growth factor beta (TGFβ) are platelet-derived intercellular mediators of cell proliferation and motility. LPA is a general growth, survival and motility-stimulating factor in mammalian cells. TGFβ prevents proliferation of normal epithelial cells. However, the growth-inhibitory effect of TGFβ is lost or reduced in most malignant cells. Instead, TGFβ promotes migration and invasion of advanced cancer cells. Since LPA and TGFβ are both present in the blood and tumor microenvironments, we were interested in signal integration and functional outcomes in malignant epithelial cells in an LPA and TGFβ co-stimulatory context. In a subset of breast and ovarian cancer cell lines which remain sensitive to the cytostatic effect of TGFβ, we found that LPA up-regulated expression of the cyclin-dependent kinase inhibitor p21Waf1. But this up-regulation was not observed in TGFβ-resistant ones. We examined the possibility that LPA-induced p21 might contribute to the cytostatic response to TGFβ. Indeed, TGFβ alone induced p21 expression weakly in TGFβ-sensitive cells. Serum or serum-borne LPA cooperated with TGFβ to elicit the maximal p21 induction. LPA stimulated p21 via LPA1 and LPA2 receptors and Erk-dependent activation of the CCAAT/enhancer-binding protein beta (C/EBPβ) transcription factor independent of p53. Loss or gain of p21 expression led to a shift between TGFβ sensitive and resistant phenotypes in breast and ovarian cancer cells, indicating that LPA-induced p21 is a key determinant of the growth inhibitory activity of TGFβ. The p21-stimulatory action of LPA is absent from most breast and ovarian cancer cells, leading to their resistance to TGFβ. Therefore we reveal a novel crosstalk between LPA and TGFβ that underlies TGFβ sensitive and resistant phenotypes of breast and ovarian cancer cells. In the next part of our study, we examined the role of interactions between LPA and TGFβ in regulation of tumor cell motility. LPA and, to a much less extent, TGFβ stimulate chemotactic migration and invasion of breast and ovarian cancer cells. However, when combined together with LPA, TGFβ strongly attenuated LPA-driven migration and invasion of breast and ovarian cancer cells. This inhibitory effect was most likely mediated through TGFβ downregulation of expression of LPA1, the major receptor subtype responsible for LPA-regulated cell migration. Knockdown of Smad3 or Smad4 with small hairpin RNA (shRNA) eliminated the inhibitory effects of TGFβ on the LPA1 expression and LPA-dependent cell migration. There are two potential TGFβ inhibitory elements (TIE) (-40 bp and -401 bp) present in the human LPA1 gene promoter. Deletion or point mutation of the distal TIE at around -401 bp abolished the inhibitory effect of TGFβ on the LPA1 promoter activity as revealed by luciferase assays. A DNA pull-down assay showed that the -401-TIE-E2F4/5 sequence was capable of binding Samd3, Smad4, and E2F4/5 in TGFβ-treated cells. The binding of the Smad complex to the native TIE-E2F4/5 sequences of the LPA1 gene promoter was further verified by chromatin immunoprecipitation assay. Our results identify a novel role of TGFβ in the control of LPA1 expression and LPA1-coupled biological activities, adding LPA1 to the list of TGFβ-repressed target genes.