Yes / HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the con-text of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overex-pressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum–resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials. / This research was supported by GRACE, a gynaecological charity based in Surrey, UK.

Yes / Background: Epithelial ovarian cancer is a common malignancy, with no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We evaluated the expression of EN2 in Epithelial ovarian cancer, and reviewed its role as a biomarker. Methods: We evaluated 8 Epithelial ovarian cancer cell lines, along with > 100 surgical specimens from the Royal Surrey County Hospital (2009–2014). In total, 108 tumours and 5 normal tissue specimens were collected. En2 mRNA was evaluated by semi-quantitative RT-PCR. Histological sub-type, and platinum-sensitive/−resistant status were compared. Protein expression was assessed in cell lines (immunofluorescence), and in > 150 tumours (immunohistochemistry). Results: En2 mRNA expression was elevated in serous ovarian tumours compared with normal ovary (p < 0.001), particularly in high-grade serous ovarian cancer (p < 0.0001) and in platinum-resistant tumours (p = 0.0232). Median Overall Survival and Progression-free Survival were reduced with high En2 expression (OS = 28 vs 42 months, p = 0.0329; PFS = 8 vs 27 months; p = 0.0004). Positive cytoplasmic EN2 staining was demonstrated in 78% of Epithelial ovarian cancers, with absence in normal ovary. EN2 positive high-grade serous ovarian cancer patients had a shorter PFS (10 vs 17.5 months; p = 0.0103). Conclusion: The EN2 transcription factor is a novel ovarian cancer biomarker. It demonstrates prognostic value, correlating with worse Overall Survival and Progression-free Survival. It is hoped that further work will validate its use as a biomarker, and provide insight into the role of EN2 in the development, progression and spread of ovarian cancer. / Oncology Research and Development Departments at the Royal Surrey County Hospital and the University of Surrey

Ovarian cancer is one of the most lethal gynecological conditions among women today. Having around a 50% survival rate, it has been the 5th leading cause for cancer-related deaths for women. Delayed manifestation of symptoms with late stage diagnosis has been a major factor for relatively high mortality. The 5-year survival rate for the early stage is over 90%; therefore, early detection of cancer is essential to improve the survival rate. Even though technology has improved today, early detection has not improved, and still it has been posing challenges. In addition to the clinical practices in diagnosis, scientists are looking for other novel promising methods to detect it at the early stage that would be inexpensive and user-friendly. Currently, cancer antigen 125 (CA125), a type of biomarker that can become elevated in a patient’s blood serum, is recommended mostly for clinical tests in the screening of ovarian cancer. However, because of the lack of sensitivity and specificity associated with CA125, the search for new potential biomarkers is a research priority to diagnose cancer at a localized stage.In this work, I report a nano/micro particle labeled immunoassay method for the detection of ovarian cancer biomarker CA125 in a Phosphate-Buffered Saline (PBS) medium. Here, a sandwich type immunoassay method is presented. For this goal, CA125 biomarkers are immobilized on a solid surface (magnetic beads) using a bioconjugation technique. In order to specifically target CA125, antibody and aptamer molecules are used. Here, the elemental nano/micro particles are used to label the antibody and aptamer. This labeled immunoassay is subjected to surface enhanced Raman spectroscopy (SERS) and laser induced breakdown spectroscopy (LIBS) for the detection of CA125. I establish a calibration curve by acquiring the spectroscopic signal for the known concentration of CA125. In addition to the detection part, other spectroscopic techniques such as attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR), UV-Vis spectroscopy, dynamic light scattering (DLS) and scanning electron microscopy (SEM) are employed to study the bioconjugation steps. In this regard, chapter 1 gives a general overview about ovarian cancer with necessary statistics. In chapter 2, I have given necessary background information on bioconjugation techniques for immunoassay methods, particularly in the perspective of my experiments. Chapter 3 covers the antibody-based immunoassay using Raman labeled gold nanoparticles. It describes how to build a nano/micro particle based sandwich type immunoassay for CA125 detection and the corresponding results. Chapter 4 describes a similar immunoassay method to chapter 3, using aptamers instead of antibodies for specifically targeting CA125. In both chapters, SERS is employed for detection. In chapter 5, I use LIBS for the detection of an aptamer based assay. Following a similar technique in the previous chapter, I use silica microparticles to label the aptamer instead gold nanoparticles. Chapter 6 focuses on the computational aspect of our experimental work, detailing the molecular docking process and presenting preliminary results regarding the interactions of the antibody and aptamer with the CA125 antigen. Chapter 7 offers a summary of my findings along with the relevant background information.

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.