DETERMINING THE EFFECT OF DOUBLE-STRANDED RNA TREATMENT IN OVARIAN CANCER By Charlotte Faye Roberts, B.A. A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science in Biochemistry at Virginia Commonwealth University.Virginia Commonwealth University, 2011Major Director: Jessica K. Bell, Ph.D.Assistant Professor, Department of Biochemistry & Molecular Biology. Epithelial ovarian cancer is a lethal gynecological malignancy. Due to its asymptomatic nature it is typically detected in the latter metastatic stages. Standard treatment protocol involves surgical cytoreduction, followed by a combination of taxane and platinum-based chemotherapeutics. Initially this treatment is successful however, most patients face recurring tumors that over time become resistant to current drug regimens. Thus, novel chemotherapeutic development is necessary. Cancer cells express receptors of the innate immune system, pattern recognition receptors (PRRs) that function to alert the host of invading pathogens. PRRs such as toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and dsRNA-dependent protein kinase receptor (PKR) recognize double-stranded RNA (dsRNA), a viral replication intermediate, and trigger apoptosis. Numerous studies have been conducted on the four dsRNA receptors in cancer. The findings have shown that stimulation of individual or a group of these receptors have led to a multitude of responses such as activation of apoptosis, inhibition of tumorigenic growth, and inhibition of metastasis in several cancer types (prostate, breast, nasopharyngeal, and melanoma cancer). Previous work in the Bell lab has shown that within a panel of ovarian cancer cell lines, one subset upregulates dsRNA receptors upon stimulation with polyinosinic-polyuridylic acid (pI:pC) and leads to apoptosis. A second subset of ovarian cancer cells do not upregulate dsRNA receptors and their survival is not affected by dsRNA treatment. We hypothesize that all or a subset of dsRNA receptors are required to elicit a dsRNA-induced apoptotic response. To test this hypothesis we examined the dsRNA-induced apoptotic response the responding cell lines (CAOV3 and OVCAR3) via three methods: selective ligand assays, transient knockdowns with siRNA, and stable lentiviral knockdowns with shRNA. Then we examined the dsRNA-induced apoptotic response in the non-responders (DOV13 and SKOV3). The first objective was to determine if all or a subset of these four dsRNA receptors were required for the dsRNA-induced apoptotic response. The second objective of this thesis was to examine if dsRNA receptor expression in cell lines resistant to dsRNA-induced apoptosis could restore dsRNA responsiveness. To execute the first objective, we first examined receptor contribution to the dsRNA-induced apoptotic response via a selective antagonist (2- aminopurine) to PKR and a selective agonist (polyadenylic-polyuridylic acid, pA:pU) to TLR3. Inhibition of PKR did not blunt the apoptosis levels in the responders and was determined to be inessential for the dsRNA-induced apoptosis. Selective ligation of TLR3 with pA:pU showed an increase in apoptosis, but not to levels seen with pI:pC. Objective one was also carried out via transient knockdown using siRNA. Knockdowns via this method were less than 70% and the lipid vehicle of one of the transfection reagents was found to be sensitizing to the cells. Stable lentiviral knockdowns with shRNA were utilized to conduct the knockdown assays. By qPCR, lentiviral knockdown of TLR3 showed an 85% decrease and showed a great decrease in the dsRNA-induced apoptotic response in the cell death assay. The lentiviral knockdown of RIG-I showed a 54% decrease via qPCR and did not alter dsRNA-induced apoptotic responses. The lentiviral knockdown of MDA5 could only be assessed via the TLR3/MDA5 double knockdown, and it showed a 53% decrease via qPCR analysis. The cell death assay of the TLR3/MDA5 double knockdown showed a great decrease in the dsRNA-induced response. The work presented in this thesis is the first to address the contribution of all four dsRNA receptors to the dsRNA-induced apoptotic response in one study. In this work, we have found that PKR is not needed for the dsRNA-induced apoptosis. Loss of TLR3 in the responders reduces death, but not back to basal levels. This may be due to the delivery method of pI:pC such that it goes directly to the endosome. Forced expression of the dsRNA receptors (TLR3, MDA5, and RIG-I) can all induce apoptosis to similar levels indicating redundancy. The importance of this work reveals that any of the three dsRNA receptors, TLR3, MDA5, and RIG-I, could be possible targets for individualized chemotherapeutic regimens for women with ovarian cancer expressing these receptors.

Clinicians and researchers know that women experience distress related to the diagnosis of and treatment for ovarian cancer. A review of the literature revealed that while there is interest in the topic, distress is inconsistently defined and measured. Women have been reported to have a variety of distress experiences including the challenges of late diagnosis and the treatment regimen, communication difficulties with healthcare providers, and concern about the effect of their diagnosis on their loved ones. Without information directly from women, assumptions predominate about what the experience is like and what they would find helpful from support persons. Women’s perceptions about distress was identified as a gap in the knowledge leading to the present study which asked “What do women with ovarian cancer want their spouse/significant other, family, friends, and healthcare providers to know about their experience of distress during diagnosis and treatment?” A qualitative method, Grounded Theory as outlined by Glaser and Strauss in 1967 was chosen to guide this IRB approved study. Twelve women participated in audiotaped interviews contributing data for analysis using the constant comparative method. Six common themes or subcategories emerged across all the interviews and resulted in a conceptualization of the experience as an “existential assault.” Though individual experience differed, abstraction and conceptualization of the data revealed the common themes as (a) “out of the blue like lightning”; (b) “no stone left unturned”; (c)“knowing what I don’t want to know and not knowing what I want to know”; (d) “watching you, watching me- we are both afraid”; (e) “talking yet not talking, about death”; and (f) “now I have to take care of me.” Participants expressed the need for professional support people who contribute their efforts to cure, but who also listen to the participant’s need to manage and control their own experience and to live in ways that give their life meaning and purpose. The experience of distress for the participants was intensified by the needs of those in their social network (spouse/significant other, family, friends, and healthcare providers) who also experienced distress, at times requiring participants to provide support for those who would be expected to be providing support.

The present studies sought to determine whether the lethality of the drug combination [sorafenib + sildenafil] could be enhanced by the anti-inflammatory agent celecoxib, using ovarian cancer and other tumor cell lines as models. Also, in a dose dependent fashion celecoxib enhanced [sorafenib + sildenafil] lethality in multiple ovarian cancer cell lines. In a dose dependent fashion celecoxib enhanced the ability of [sorafenib + sildenafil] to reduce expression of multiple chaperone proteins in parallel with lower levels of the drug efflux pumps ABCB1 and ABCG2. Over-expression of GRP78 and HSP27 maintained pump expression in the presence of drugs. Cell killing by the 3 drug combination was mediated by mitochondrial / caspase 9 -dependent apoptotic signaling and by RIP-1 / caspases 2 and 4 / AIF -dependent necroptotic signaling. Pre-treatment of intrinsically resistant primary ovarian cancer cells with [celecoxib + sorafenib + sildenafil] significantly enhanced tumor cell killing by a subsequent cisplatin exposure. Similar data were obtained in some cancer cell lines, but not all, using the related platinum containing drugs, oxaliplatin and carboplatin. As our prior publications have also validated in vivo the combinations of [celecoxib + sildenafil] and [sorafenib + sildenafil] as cytotoxic to multiple tumor cell types, combined with the present findings, we would argue that the combination of celecoxib/sorafenib/sildenafil should be explored in a new phase I trial in ovarian cancer.

Class of 2006 Abstract / Objectives: To determine if Polycystic Ovarian Syndrome (PCOS) patients treated with lifestyle changes and metformin resulted in ovulation after six months of treatment. Methods: A retrospective chart review of initial patient visits at an infertility clinic were obtained. Patients that were given a diagnosis of PCOS were further reviewed for age at initial diagnosis, weight, height, ovarian cysts, lifestyle recommendations (diet, exercise, and vitamin use), metformin recommendations and usage, and if ovulation occurred after six months of treatment. Results: A total of 1011 charts were reviewed. At the initial office visit, 206 (20.38%) of these patients were classified as having PCOS. Of PCOS patients, 113 (54.85%) patients ovulated after six months of treatment. In the average initial weight, ovulators averaged slightly less weight than did non-ovulators (171.77 pounds ± 44.26 vs. 188.65 pounds ± 51.37, p=0.0121). This also follows true for the initial BMI of ovulators vs. non-ovulators (29.53 kg/m2 ± 10.14 vs. 32.69 kg/m2 ± 13.03, p=0.0521). There was a significant difference in metformin use between ovulators and non-ovulators (90.27% vs. 73.12%, p=0.0024). More ovulators were found to continue metformin treatment as compared to non-ovulators. Conclusions: In this specific infertility clinic setting, 20.3% of patients were diagnosed with PCOS at the initial office visit. Of these PCOS patients, treatment with lifestyle changes and metformin use resulted in 55% of patients achieving ovulation at six months. This study shows that weight loss, through lifestyle modification and metformin treatment, increases this population’s chances of ovulation within six months of therapy.

Ovarian cancer is the deadliest gynecologic cancer for women. Diagnosis at the local stage leads to 91% 5-year survival rates, but only 15% of cases are detected early. Existing screening methods have proven ineffective in large clinical trials. Screening is complicated by the heterogeneity of the disease with multiple types of ovarian cancer originating both on the ovary and in the fallopian tube. Early stage cancer is too subtle for non-invasive imaging techniques such as ultrasound or magnetic resonance imaging. This study evaluates the feasibility and design of dual modality, multispectral fluorescence imaging (MFI) and optical coherence tomography (OCT) endoscopes for improved ovarian cancer screening. The study is broken up into three sections. In the first study MFI is validated in an ex vivo imaging study of human ovarian and fallopian tube tissue samples. Tissue autofluorescence excited by ultraviolet and blue wavelengths is shown to be a promising discriminator between normal and cancerous tissue. The second study combines OCT and MFI into a sub millimeter diameter endoscope designed to screen for ovarian cancer by screening inside the fallopian tube and at the ovary. The small size is required for screening the full length of the fallopian tube. MFI is implemented as a wide-field navigational imaging technique with high sensitivity complemented by high resolution structural depth imaging of OCT over a limited field of view. The final study presents a novel lens design for a scanning fiber endoscope with forward-viewing navigation and side-viewing OCT. A piezo tube is used to scan an optical fiber providing both the navigation channel’s illumination and OCT imaging. The design spatially separates the forward-viewing illumination from the OCT. As the piezo fiber circularly scans at its maximum deviation the OCT beam focus is rotationally scanned out the side of the endoscope tip by a rotationally symmetric double reflection in the cover plate.

Introdução: O câncer de ovário é considerado a primeira causa de óbito entre os tumores ginecológicos. Entretanto, com os avanços nos tratamentos as pacientes com câncer de ovário apresentam uma sobrevida maior, sendo fundamental discutir sua qualidade de vida. Objetivos: Validar e analisar a confiabilidade de dois questionários existentes na literatura, a saber: EORTC-OV28 e FACT-O e avaliar a compreensão, preferência e aceitação dos mesmos. Metodologia: O estudo foi realizado nos Ambulatórios de Ginecologia Oncológica e Oncologia Clínica do Hospital do Câncer AC Camargo São Paulo. Foram analisadas 114 mulheres com diagnóstico de câncer de ovário, em qualquer estádio da doença. Para cada questionário foi analisada a consistência interna (alpha de Cronbach), a validade de constructo convergente (coeficiente de correlação de Spearman com os domínios do questionário SF-36 e HADS), a validade de constructo discriminante (comparação das médias dos escores, segundo estadiamento inicial (I e II) x avançado (III e IV), Karnosfky (80-100 por cento x 5070 por cento ), doença atual (sem evidência de doença X com evidência de doença) e tratamento atual (em tratamento e pós tratamento)) e analisado o grau de compreensão dos mesmos. A reprodutibilidade foi verificada após duas semanas e foi analisada por meio da comparação de médias (Wilcoxon), coeficiente de correlação intra-classe e gráficos de Bland-Altman. Resultados: As escalas de sintomas dos questionários EORTC-OV28 e bem estar familiar do FACT-O, respectivamente, apresentaram valor de alpha de Cronbach 0,85 e 0,79. A maioria das escalas apresentou correlação significativa com os domínios do SF-36 e HADS e foi capaz de discriminar entre os grupos de comparação. Ambos apresentaram boa compreensão. Quanto ao tempo de preenchimento o questionário EORTC-OV 28 apresentou menor média (5,10 min.). Todos os questionários apresentaram bons índices de reprodutibilidade. Conclusões: O questionário EORTC OV 28 apresentou melhores parâmetros de validade, mas as demais análises foram muito semelhantes entre os questionários. A escolha do questionário a ser aplicado pelo pesquisador dependerá dos aspectos considerados por ele relevantes na pesquisa / Introduction: Ovarian cancer is considered the main cause of death among gynecological tumors. Therefore, ovarian cancer patients have presented higher survival rates due to advances in treatments and it becomes necessary to discuss quality of life. Aim: Validate and analyze the reliability of two questionnaires in the literature, namely OV28-EORTC and FACT-O and assess comprehension, preference and acceptance. Methodology: This study was conducted in outpatient clinics of Gynecology Oncology and Medical Oncology at the Cancer Hospital AC Camargo - São Paulo. The total amount of subjects analyzed was 114 women diagnosed with ovarian cancer at any stage of the disease. Moreover, in each questionnaire, internal consistency (Cronbach\'s alpha), the convergent construct validity (Spearman correlation coefficient with domains of the SF-36 and HADS), the discriminant construct validity (comparison of mean scores of the second stage initial (I and II) x advanced (III and IV), Karnosfky (80-100 per cent vs. 50-70 per cent ), current disease (without evidence of disease X with evidence of disease), current treatment (treatment and post treatment) and degree of understanding were analysed. Reproducibility was checked after 2 weeks and analyzed by comparing means (Wilcoxon), coefficient of intraclass correlation and Bland-Altman. Results: The prognostic scales of the EORTC questionnaires OV28-and family welfare of the FACTO, had a Cronbach alpha value of 0.85 and 0.79, respectively. The majority of scales correlated significantly with the SF-36 and HADS; nonetheless, the ability of differentiation between comparison groups could be realised; therefore. both have presented good understanding. The questionnaire EORTC OV-28 had a lower average (5.10 min) in relation to the time spending to complete it. Furthermore, all questionnaires showed good levels of reproducibility. Conclusions: To sum up, the questionnaire EORTC OV 28 presented the best validity parameters, although the analysis were very similar between the questionnaires. Therefore, the investigator should firstly, consider which are the required aspects to evaluate to be able to make the correct choice

Introdução: A avaliação da reserva ovariana em reprodução assistida (RA) busca identificar mulheres em que a exaustão folicular determine as dificuldades reprodutivas. Além da idade, a presença de causas outras de subfertilidade, como a endometriose (EDT), implica interferências negativas potenciais sobre a resposta ovariana. Objetivo: Avaliar a reserva folicular ovariana de mulheres subférteis portadoras de endometriose e determinar o melhor preditor de má resposta em RA. Métodos: Foram analisados 87 ciclos de RA em mulheres com idade inferior a 40 anos, ciclos menstruais regulares, sem patologias endócrinas e com ambos os ovários, sendo 30 ciclos em portadoras de EDT (casos) e 57 ciclos em mulheres subférteis por fator masculino exclusivo (controles). A reserva ovariana foi inferida pelas dosagens basais dos hormônios anti-mülleriano (AMH) e folículo estimulante (FSH), a contagem de folículos antrais pequenos (CFA) e a medida do volume ovariano médio (VOM). Curvas Receiver Operating Characteristic (ROC AUC) foram traçadas para avaliação da capacidade discriminatória de cada teste em identificar má resposta. Resultados: Pacientes com EDT apresentaram FSH basal significativamente maior em relação aos controles (9,13 ± 5,09 mUI/mL vs. 6,28 ± 2,45 mUI/mL; p < 0,05), sem diferenças para AMH, CFA e VOM. O número total de oócitos aspirados foi menor na EDT em relação aos controles (5,33 ± 3,43 vs. 8,28 ± 5,8; p < 0,05) e correlacionou-se significativamente com o AMH em ambos os grupos (EDT: r = 0,61; Controles: r = 0,58; p<0,0001). O FSH basal apresentou correlação significativa com o total de oócitos aspirados apenas na EDT (r = -0,48; p<0,01); também na EDT, o AMH basal foi o marcador individual com melhor potencial discriminatório para má resposta (AUC = 0,875), seguido de FSH basal (AUC = 0,682) e VOM (AUC = 0,665), enquanto no grupo controle, o AMH foi o melhor marcador (AUC = 0,8372), seguido do VOM (AUC = 0,709) e da CFA (AUC = 0,686). Conclusões: O FSH basal está significativamente maior nas pacientes subférteis com EDT e correlaciona-se com a resposta em RA apenas nas portadoras da doença. Já o AMH basal, é o marcador com o melhor potencial discriminatório de má resposta, independentemente da presença da endometriose. CFA e VOM não se apresentaram como bons preditores de má resposta. Sendo assim, a associação entre endometriose e subfertilidade deve estar vinculada a alterações do crescimento/desenvolvimento do folículo ovariano e não a prejuízos sobre a reserva folicular gonadal propriamente dita. / Introduction: Ovarian reserve evaluation in assisted reproduction (AR) aims to identify women in whom follicular exhaustion determines reproductive difficulties. More than age, the presence of other subfertility factors, such as endometriosis (EDT), implies potential negative interference on ovarian response. Objective: To evaluate follicular ovarian reserve in subfertile patients with endometriosis and determine the best predictor of poor response in AR. Methods: We evaluated 87 AR cycles of women presenting with less than 40 years of age, regular menses, no endocrine diseases and with both ovaries, divided in 30 EDT cycles (cases) and 57 cycles in women with subfertility associated to male factor (controls). Ovarian reserve was determined based on basal levels of anti-müllerian hormone (AMH) and follicle- stimulating hormone (FSH), small antral follicle count (AFC) and medium ovarian volume (MOV). Receiver Operating Characteristic curves (ROC AUC) were obtained for evaluation of discriminatory capacity of each test in identifying poor response. Results: EDT patients presented significantly higher basal FSH levels when compared to controls (9,13 ± 5,09 mUI/mL vs. 6,28 ± 2,45 mUI/mL; p < 0,05) and there were no differences in AMH, AFC and MOV between groups. The total number of oocytes retrieved was lower in endometriosis when compared to controls (5,33 ± 3,43 vs. 8,28 ± 5,8; p < 0,05) and significantly correlated with AMH in both groups (EDT: r = 0,61; Control: r = 0,58; p<0,0001). Basal FSH presented significant correlation with the number of oocytes retrieved only among EDT patients (r = - 0,48; p<0,01); in EDT patients, either, basal AMH was the individual marker with the best discriminatory potential for poor responders identification (AUC = 0,875), followed by basal FSH (AUC = 0,682) and MOV (AUC = 0,665), whereas among controls, AMH was the best marker (AUC = 0,8372), followed by MOV (AUC = 0,709) and AFC (AUC = 0,686). Conclusions: Basal FSH is significantly higher in subfertile patients with endometriosis and correlates with ovarian response in AR among these patients. Basal AMH, by the way, is the individual marker with the best discriminatory potential in determining poor response, which is not dependent on endometriosis presence. AFC and MOV did not presented as good predictors of poor response. The association between endometriosis and subfertility, therefore, may be linked to prejudice to growth/development of the ovarian follicle, but not to damage to the real ovarian follicular reserve.

Oncolytic vaccinia virus has great potential in the treatment of cancer and two engineered strains have entered clinical trials. As the advent for oncolytic vaccinia virus as an approved therapy beckons, it is critical to consider some of the barriers that may hinder this progress. These include suboptimal delivery of the virus to tumour sites, incomplete destruction of the tumour mass, and a lack of full understanding of the way in which oncolytic vaccinia kills its target cells. This thesis attempts to address these issues, with a particular focus on ovarian cancer. As ovarian cancer is generally restricted to the peritoneal cavity, intraperitoneal delivery may be preferable over intravenous delivery. Here, it is shown that Lister-dTK, an engineered vaccinia strain, is able to selectively replicate in ovarian tumours, including metastases to the liver following intraperitoneal delivery. To determine whether Lister-dTK could potentially be used in combination with current therapies for ovarian cancer, the effect of cisplatin and Lister-dTK together was assessed in vitro but showed no improvement in overall cell death. In an attempt to further improve the anti-tumour efficacy of Lister-dTK, the extracellular matrix protein (ECM) decorin was expressed from the virus. Decorin interacts with various signalling pathways and is proposed to enhance virus spread. However, abrogation of EGFR and TGFβ signalling could not be demonstrated in vitro, nor could improved virus spread. In an intraperitoneal model of ovarian cancer, Lister-mDCN did not demonstrate enhanced efficacy over a control virus. To determine the mechanisms of ovarian cancer cell death induced by Lister-dTK, the roles of apoptosis, autophagy and necrosis were investigated. Whilst some features of both apoptosis and autophagy were observed, inhibition of these pathways did not attenuate Lister-dTK. It is proposed that necrosis is the primary cause of cell death but that this process may occur in a regulated manner.

Elevated levels of the inflammatory cytokine interleukin-6, IL-6, have been linked with poor prognosis in ovarian cancer patients by influencing tumour growth, invasion, angiogenesis and chemo-resistance. A clinical trial conducted in parallel with pre-clinical studies showed an anti-IL-6 antibody to have some activity in ovarian cancer patients and in xenograft models, via reduction in pro-inflammatory and angiogenic factors such as TNF-α, IL-8 and VEGF. Anti-IL-6 treatment also showed significant reductions in vascular area with decreased expression of an angiogenic factor Jagged1. The aim of my study was to investigate the effects of IL-6 on normal and tumour angiogenesis. I found that recombinant IL-6 stimulates angiogenesis in mouse and rat aortic ring assays and that it can also stimulate growth and migration of endothelial cells in vitro. IL-6 has similar potency as VEGF in inducing vessel sprouting. IL-6 itself does not induce VEGF in the endothelial cells I tested. Investigation of the effects of IL-6 on vessel maturation revealed a significant reduction in pericyte coverage of vessels treated with IL-6 compared with VEGF. Collectively, these data led to my hypothesis that ‘IL-6 drives aberrant angiogenesis, independent of VEGF signalling’. Investigating the mechanism by which IL-6 drives angiogenesis and leads to defective pericyte formation, I showed a link between IL-6 and the Notch ligands, Jagged1 and DLL4. My data suggested that IL-6 could stimulate Jagged1 in endothelial cells, whereas VEGF induces DLL4, the Notch ligand known to be involved in inducing stalk phenotype. Exploring previous findings to get a better understanding of the interaction of Notch ligands and pericyte recruitment also suggested a role of Angiopoeitin-2 in relation to IL-6 signalling. These observations were extended in IGROV-1 ovarian cancer xenografts treated with an anti-IL-6 antibody and by analysis of gene expression datasets from ovarian cancer biopsies. My results suggest therapeutic potential of combining inhibitors of IL-6 and VEGF in ovarian cancer.

Choi, Pui Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 202-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.