Peptide recognition domains (PRD) bind to short linear motifs on their biological partners and are found in several cellular pathways including those found to be critical in tumorigenesis. In this study, I aimed to generate peptide probes against PRDs present on proteins involved in ovarian cancer. Using bioinformatics, I identified 66 potential PRDs present on these proteins. I then used peptide phage display to successfully generate peptides against 27 of the 66 domains. To validate my results, I performed an extensive literature review and structural analysis. For several cases, the phage-display derived binding preferences are similar to previously reported studies. However, for a subset of domains, I identified non-canonical binding preferences that have not been reported previously in literature. The binding preferences obtained in this study can be used to design intracellular probes for studying the role of these PRDs in biological pathways important in ovarian cancer.

Proteins secreted or shed by tumors can be found in serum. Detecting these proteins by mass spectrometry (MS) is difficult, due to the wide dynamic range of protein concentrations in serum. To circumvent this issue, we mined the conditioned media of epithelial ovarian cancer (EOC) cell lines which is a less complex fluid to work with. We hypothesize that some of the proteins shed or secreted by EOC cell lines are similar to those secreted or shed by EOC tumors and that some of these proteins can be used as biomarkers. We mined the conditioned medium of four ovarian cancer cell lines (HTB75, TOV-112D, TOV-21G and RMUG-S) by two-dimensional liquid chromatography-mass spectrometry. Our study identified 1208, 1252, 885, and 463 proteins from the HTB-75, TOV-112D, TOV-21G, and RMUG-S cell lines respectively. In all, we identified 2039 proteins from which we focused on 420 extracellular and plasma membrane proteins. High abundance proteins such as albumin and immunoglobulins, which are problematic for serum proteomics, did not interfere with our study. Several known markers of EOC including CA-125, HE4, Mesothelin, and KLK6, were identified in this study. The list of 420 extracellular and membrane proteins was cross-referenced with the proteome of ascites fluid to generate a final list of 51 potential candidates. According to Ingenuity Pathway Analysis, two of the top 10 diseases associated with our list of 51 proteins were cancer and reproductive diseases. Of the 51 candidates, 10 proteins were selected for verification in sera from ovarian cancer patients and healthy individuals. Clusterin showed a significant difference between cancer patients and normal, with sera from cancer patients showing higher levels. Another protein, NPC2, did not show a difference in sera between cancer and normals. Protein expression studies using immunohistochemistry showed that NPC2 is highly expressed in ovarian cancer tissue and absent in normal ovarian surface epithelium. In summary, clusterin and NPC2 appear to play a role in ovarian cancer pathobiology and their role in EOC need to be studied further.

Ovarian high-grade serous cancer (HGSC) is the most lethal gynecologic malignancy and well-characterized models may improve patient outcomes. Patient-derived xenografts (PDXs) recapitulate disease heterogeneity; however, to be useful in predicting response to novel chemotherapeutics, they must reflect the response of the donor tissue to standard chemotherapy. The objectives of this study were: first, to evaluate the response of PDXs’ to platinum therapy and compare this response to that of the donor; and second, to determine whether treatment with chemotherapy enriches for tumourigenic cells. Eighteen samples formed tumours in the mammary fat pads of NOD-Scid-IL2Rγnull mice and were treated with Carboplatin. There was a 100% concordance between sample status and PDXs response to chemotherapy. HGS histology was confirmed for all cases. A conclusion regarding post-chemotherapy tumourigenicity could not be made due to inadequate statistical power. PDXs represent useful tools for evaluation of novel therapies and identification of patients who are platinum-resistant/sensitive.

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Class of 2009 / OBJECTIVES: To determine and characterize the relative impact of patient demographics on hospital inpatient charges, length of stay, and inpatient mortality in patients with ovarian cancer from 2002-2005. METHODS: A retrospective database analysis of AHRQ’s Health Care Cost and Utilization Project (HCUP) Nationwide Inpatient Sample databases was conducted spanning from January 1, 2002, to December 31, 2005.Data were collected regarding age, race, payer status, median household income, location of hospital (urban/rural), comorbidities, procedures, total charges, length of stay, and inpatient mortality. Multivariate and gamma regression methods were utilized to examine incremental risks associated with length of stay, total charges, and inpatient mortality, after controlling for all other variables. RESULTS: Overall, data from 246,012 hospital admissions were obtained. The average length of stay of patients was 6.58 days (SD = 7.22), the average number of diagnoses was 7.18 (SD = 3.36), the average number of procedures performed was 2.71 (SD = 2.66). A total of 14,485 (5.9%) patients died during hospitalization. The average total charge was $29,698 (SD = $42,951). The IRR was 0.886 (95%CI, -0.105 to -0.04) for patients who were Hispanic, and 1.089 (95%CI, 0.017–0.153) for patients who were Black compared to patients who were white. When compared to patients who lived in large, metropolitan areas, the IRR was 0.88 (95%CI, -0.146 to - 0.109) for patients located in smaller, metropolitan areas, and the IRR was 0.74 (95%CI, -0.335 to -0.268) for patients located in non- urban areas. CONCLUSIONS: Patient demographics were found to have associations, both directly and indirectly, with length o

Chemotherapy resistance in human epithelial ovarian cancer (EOC) is a significant reason for the high rate of death among patients. We hypothesized that chemotherapy- resistant EOC cells will be killed by novel drug treatments in non-adherent culture conditions. The objective of this study was to test the efficacy of novel drugs to affect platinum resistant EOC cell viability. To achieve this, the cell killing efficacy of several drugs were tested on drug-resistant EOCs growing in non-adherent cultures. Both EOC cell lines and primary EOC cells isolated from patient ascites were used for these studies. Two different classes of drugs were tested including multikinase inhibitors (dorsomorphin and LDN-193189), and an understudied class of novel chemotherapeutic agents called glycosylated antitumor ether lipids (GAELs). EOC cells were treated with the drugs at different doses alone or in combination with cisplatin. Because GAELs exhibited promising results in resistant EOC cells, the mechanism of GAEL-induced cell-death was evaluated. / October 2016

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.

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