Skewed somatic X inactivation (XCI), X-linked gene overexpression and abnormal X content have been associated with breast and ovarian cancer. Partial or complete reactivation of the inactive X in females may be a step in breast and ovarian cancer progression, leading to overexpression of some tumour enhancing gene. Markers of an X reactivation event were examined: X gene dosage, expression, and methylation in 8 ovarian cancer cell lines. Another marker of an X reactivation event, skewed XCI, was assayed in peripheral blood DNA from 106 breast and/or ovarian cancer patients (52 BRCA1 mutation carriers, 24 BRCA2 mutation carriers, 30 non-mutation carriers), 147 age-matched population controls. Combined RNA/DNA FISH was used to quantify the number of inactive Xs compared to total number of Xs. Five cell lines had increased X content. Three cell lines localized XIST to the presumptive inactive X; however the numbers of inactive Xs were variable. Expression levels of 8 X-linked genes were assessed by real-time PCR. Expression was inconsistent between different genes and among cell lines, ranging from a 2 to 300-fold increase compared to a control. Overall, expression was greatly increased for genes subject to inactivation but not increased in genes that escape inactivation for most ovarian cancer cell lines. Methylation at AR and FMR1 was quantified by a real-time PCR based assay and SNuPE respectively. Methylation was lower than expected for 7 of 8 ovarian cancer cell lines at AR or FMR1, while three cell lines had low or no methylation for both genes. Skewed XCI was evaluated using a methylation-based PCR assay at AR. There was no significant increase in skewing above 90% for any cancer group assayed. In addition, two markers of X reactivation were assayed in two low passage cultures of normal ovarian surface epithelium from BRCA1 mutation positive breast cancer patients. One sample did not localize XIST to the inactive X and three of five genes subject to inactivation were overexpressed. In summary, there is evidence for loss of X silencing or gain of active X content in ovarian cancer cell lines and normal ovarian surface epithelium from BRCA1 mutation carriers. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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.

Little research has examined the effect of non-cancer life stressors on psychological well-being and recurrence in patients with cancer, and results have been mixed. Furthermore, no studies have examined specific types of stress, including loss, danger, and entrapment in patients with cancer, utilizing data obtained from the Life Events and Difficulties Schedule. Given that specifics stressors have been associated with certain psychological responses, this study sought to obtain a more nuanced understanding of the relationship between life stress and psychological well-being. This was examined in a sample of 135 women with ovarian cancer prior to surgery and during the year after diagnosis using latent growth curve analyses. Models of protective psychosocial resources examining social support, mastery, self-acceptance, and purpose in life as potential moderators and mediators of the relationship between life stress and psychosocial outcomes were also evaluated. Results indicated that cancer-related losses were most closely associated with psychological well-being across several analyses, and non-cancer losses had the greatest impact on psychological outcomes when cancer-related loss was low. Non-cancer losses were significantly related to greater fatigue prior to surgery. Additionally, major non-cancer danger stressors were associated with greater distress prior to surgery. In this sample, no stressors were significantly related to cancer recurrence. Social support was the most consistent moderator of life stress on psychological well-being, and its effects on distress and depression at baseline were mediated through self-acceptance. These findings highlight the importance of both cancer- and non-cancer-related stressors on psychological wellbeing among cancer patients in their first year following surgery and furthers our understanding of the role of protective psychosocial factors. This study has significant implications for distress screenings in patients with cancer, psychological interventions, and future research.

Ovarian cancer is a deadly gynecological disease that ranks fifth in all cancer-related deaths inwomen. The disease is often detected at an advanced stage and lack of sensitive predictive biomarkerscontribute to its poor prognosis, which is also responsible for the current five-year survival rate of only47%. Our laboratory has previously shown that a whole flaxseed supplemented diet decreases the onsetand severity of ovarian cancer in the laying hen, the only known animal model of spontaneous ovariancancer. Flaxseed is rich in omega-3 fatty acids (OM3FA), mostly α-Linoleic acid (ALA), which getsconverted to Docosahexaenoic acid (DHA) by the action of delta-6 desaturase enzyme. We have also shownthat the flaxseed diet in hens induces CYP1A1 expression in the liver while suppressing both CYP1B1 andCYP3A4 expressions. The upregulation of CYP1A1 parallels the increase in 2-hydroxyestradiol and the 2-methoxyestradiol (2MeOE2) level in the serum of the chickens. Flaxseed diet induces apoptosis to ovariantumors and not in the normal ovarian tissues. The current work explores the pro-apoptotic actions of oneof the biologically derived compounds of flaxseed diet, 2MeOE2, and describes one of its novel molecularactions. Our results indicate that 2MeOE2 -mediated pro-apoptotic actions are partly dependent on thecatalytic activation of protein kinase C delta (PKCd), which is responsible for key apoptotic histonemodifications and p38 MAPK (MAPK14) phosphorylation. Phosphorylated MAPK14, in turn, results inmore caspase-3 cleavage which activates more PKCd, therefore amplifying the apoptotic signal.The other part of this work explains one of the key anti-cancer actions of a flaxseed diet. Flaxseedreduces the number of endothelial cells and increases pericytes to endothelial cell ratio in ovarian tumorscompared to control diet-fed birds, indicating a reduction in tumor angiogenesis and an improvement inblood vessel maturation. Flaxseed decreases a-smooth muscle actin expression in ovarian tumors, whichmarks a decrease in cancer-associated fibroblasts (CAFs) in the tumor stroma. Flaxseed also reduces fibrosisin ovarian tumors. Flax -mediated reduction of ovarian fibrosis parallels an increased infiltration ofimmune cells in the preneoplastic ovaries and ovarian tumors. Therefore, this work describes how the flaxdiet differentially targets the ovarian tumor cells and the tumor stroma in preventing the incidence andseverity of ovarian cancer. Dietary supplementation with flaxseed can, therefore, be a healthy lifestylechoice as well as a potential adjuvant therapy for immunotherapy-based treatment modalities.

Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths in the United States. However, the mortality rate is relatively high, due to in part to the cancer being in an advance stage at diagnosis, since it is often asymptomatic at the early-stage with a ~94% of five-year survival rate if it is diagnosed at a localized stage (stage 1). Early detection of cancer would likely improve the survival rate. Scientists are searching for novel promising methods to detect ovarian cancer at an asymptomatic early stage; also, the method is cheap and user-friendly despite there are various techniques for ovarian cancer detection. Cancer antigen 125 (CA125), a type of serum biomarker that elevates ~50% of women with early-stage and ~80% of women with advanced-stage, is used mostly for screening epithelial ovarian cancer. However, the lack of sensitivity and specificity are known to be the main drawback of CA125. Finding new potential biomarkers that diagnose cancer at a localized stage will significantly reduce the mortality rate. Human epididymis protein 4 (HE4) is such a biomarker that has a higher sensitivity and specificity compared to all other known biomarkers, and recently it has been approved by food and drug administration (FDA) for clinical applications.In this project, we developed sandwich-type micro particles immunoassay for sensitive detection of HE4 biomarker in plasma. Here, we cross-link elemental particles to a specific functional group of the targeted biomolecules based on a covalent and non-covalent linking chemistry to improve the sensitivity and the selectivity of biomarker detection in which Fe3O4 and SiO2 microparticles were used to conjugate and purify the antibody-antigen in a media. The purified assay with the microparticles was analyzed with laser-induced breakdown spectroscopy (LIBS) for detection and quantization analysis of the HE4 biomarker. Furthermore, along with LIBS, Raman, Fourier transform infrared (FT-IR), and UV- Vis spectroscopic techniques were utilized to understand the conjugation dynamic and confirm the conjugation process.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / A subpopulation of tumor cells known as ovarian cancer initiating cells (OCICs) have been shown to be the cells that propagate the tumor phenotype in ovarian cancer. Studies have showed that a very small population (100) of these cells is sufficient to induce a tumor phenotype; while a large quantity of tumor cells (5 X 105) are required to induce such a phenotype. In this study we studied the functional changes in genes expressed in the OCIC phenotype which were important for such efficient propagation of cancers. To enable this analysis, we generated mRNA expression and DNA methylation profiles of OCICs and compared them with those of tumor and normal ovarian surface epithelial cells. We identified four pathways which regulated most of the observed changes and were predicted to be important factors in distinguishing the OCICs from tumors and normal cells. The gene signatures for these pathways were analyzed by unsupervised clustering in order to determine the similarities of OCICs with respect to tumor and normal samples. We further believed that the OCICs can be used as indicators towards the genesis and progression of early events in the ovarian cancers. In light of this, we considered two hypotheses which are currently addressing the genesis of ovarian cancer. The first hypothesis proposed ovarian surface epithelial cells to be cells of origin of the ovarian cancer while the other proposed the fallopian tube cells to be contributing the cell of origin for these cancers. It is also believed that these two cells can be reciprocal cells of origin for the cancer phenotype. In order to test these hypotheses, we integrated the in-house dataset with a public domain fallopian tube gene expression data. The integration of the results obtained from these analyses provided better understanding of the early events in ovarian carcinogenesis.

Background: When women from families with a known BRCA1 or BRCA2 mutation test negative for the family mutation, it is assumed that they will transition their personal cancer risk perception from high to average risk. However, there are scant data regarding the experience of mutation-negative women after genetic testing disclosure, particularly related to the shift of risk perception from assumed mutation-positive to actual mutation-negative. This study was designed to explore cancer risk perception and the experience of being a mutation-negative woman within a known BRCA1/2 mutation-positive family. Methods: We employed a qualitative descriptive design and convened a sample of 13 women who contributed in-depth, semi-structured telephone interviews (audio-recorded and transcribed verbatim) and performed qualitative content analysis with NVivo 2.0 software. Results: Six major content areas emerged from interview data: (i) rationale for initial involvement in the breast imaging study, (ii) rationale for continued participation, (iii) experience of living in a multiple-case family, (iv) risk perception: the personal meaning of mutation-negative status, (v) opinions regarding cancer aetiology and (vi) communication patterns between mutation-negative and mutation-positive family members. Conclusions: Living in a hereditary breast and ovarian cancer family is a complex experience that affects cognitive, emotional and social functioning. Our findings indicate that mutation-negative women may have unmet psychosocial needs that must be addressed by health-care professionals, particularly in the primary-care setting following genetic disclosure of a potentially reassuring result regarding their lack of the very high cancer risks associated with BRCA1/2 mutations.

Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

In this dissertation, we aimed to bring together a team of clinical experts, translational researchers, biostaticians and bioinformaticians to develop and implement innovative scientific methodologies in precision medicine applied to High Grade Serous Ovarian Cancer (HGS OvCa). We used a variety of translational and computational methods in order to generate impactful outcomes. These pipelines produced statistically robust results, with particular emphasis on drawing clinical and biological correlations. The results presented here contribute to the body of evidence necessary to substantiate these findings in a clinical setting. Bioassays, PDX models and ancillary specimen evaluation of previous clinical trials will help to validate our candidate biomarkers. Enhanced understanding of the molecular pathology of disease grounded in acquisition of genomic knowledge will facilitate the development of targeted treatment in cancer. Because clinical trials must be developed with correct metrics, patient selection and drug efficacy should incorporate adaptive designs. / Doctorat en Sciences médicales (Santé Publique) / info:eu-repo/semantics/nonPublished