Ovarian cancer is the leading cause of gynecological cancer death in the United States. Even though no single genetic alteration can be attributed to all ovarian cancers, 90% of ovarian tumors express telomerase, a ribonucleoprotein that elongates telomeric (TTAGGG)n repeats de novo. In normal somatic cells, telomerase is absent. In cancer cells, the re-expression of telomerase allows senescence to be bypassed contributing to cellular immortalization, a key step for cellular transformation, making telomerase a potentially important target for therapeutic intervention. Ovarian cancer cells secrete vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) that feedback through their receptors present on ovarian cancer cells to promote cell growth. Since telomerase can be regulated by growth factors, I examined VEGF regulation of telomerase activity and the possible contribution of LPA as an upstream regulator of VEGF-mediated telomerase activity in ovarian cancer. My data reveal that both VEGF and LPA upregulate telomerase activity by ERK 1/2-dependent transcriptional activation within the -976 to the -378 bp hTERT promoter regions where Sp1 is one of the major mediators of VEGF- and LPA-induced transactivation of hTERT. It also identifies telomerase as a novel molecular target of LPA as well as a target of VEGF in non-endothelial cells. In addition I found that, vitamin E, a dietary supplement able to degrade and suppress LPA activity, consistently abrogrates LPA-mediated telomerase activity through transcriptional inhibition of the hTERT -976 to -578 bp promoter regions. Lastly, since epidermal growth factor (EGF) promotes ovarian surface epithelial (OSE) cell growth and EGF receptors are frequently constitutively activated in ovarian cancers, the potential contribution of EGF in the regulation of telomerase activity was also examined. While none of the ovarian cancer cell lines examined produced large amounts of EGF, EGF stimulation of telomerase activity was mediated by Sp1 and c-Myc transcription factors within the hTERT core promoter in an ERK 1/2 /Pyk2-dependent manner. In conclusion, my research shows differential regulation of telomerase activity by growth factor and/or anti-oxidant nutraceuticals. In the future, these factors may be exploited as adjuvant therapy for improved chemotherapeutic benefit to decrease the mortality associated with ovarian cancer.

Objectives: Adherence to National Comprehensive Cancer Network (NCCN) guidelines for ovarian cancer treatment improves patient outcomes. The aim of this study was to assess disparities associated with ovarian cancer treatment in the state of Kentucky and central Appalachia. Methods: Data on patients diagnosed as having ovarian cancer from 2007 through 2011 were extracted from administrative claims-linked Kentucky Cancer Registry data. NCCN compliance was defined by stage, grade, surgical procedure, and chemotherapy. Selection criteria were reviewed carefully to ensure data quality and accuracy. Descriptive analysis, logistic regression, and Cox regression analyses were performed to examine factors associated with guidelines compliance and survival. Results: Most women were age 65 years or older (62.5%), had high grade (65.9%) and advanced stage (61.0%) ovarian cancer. Two-thirds of cases (65.9%) received NCCN-recommended treatment for ovarian cancer. The hazard ratio (HR) of death for women who did not receive NCCN-compliant care was 62% higher compared to the women who did receive NCCN compliant treatment (HR 1.62, 95% CI 1.11-2.35). Results from the logistic regression showed that NCCN-compliant treatment was more likely for: women age 65-74 years compared to age 20-49 (OR=3.32, 95% CI=1.32- 8.32), late stage compared to early stage cancers (OR 0.32, 95% CI 0.20-0.53), receipt of care at tertiary hospitals (OR=1.92, 95% CI=1.10-3.34), and privately insured compared to Medicaid (OR=0.31, 95% CI=0.13-0.77) or Medicare (OR=0.31, 95% CI=0.15-0.66). Conclusions: When the treatment of ovarian cancer did not follow NCCN-recommendations, patients had a significantly higher risk of death. Women were less likely to receive NCCN-compliant care if they were of younger age (20-49 years), had early stage disease, were not privately insured, or had care provided at a non-tertiary hospital.

Ovarian cancer is one of the most common causes of gynecological cancer related deaths in women. In 2014, the estimated number of deaths due to ovarian cancer is 14,270 with occurrence of over 22, 240 new cases (National Cancer Institute, http://seer.cancer.gov/statfacts/html/ovary.html). Despite improvement in treatment strategies, the 5-year survival rate is still below 50% mainly due to chemoresistance and relapse. Amplification of chromosomal region 3q26 is a common characteristic in various epithelial cancers including ovarian cancer. This region harbors various oncogenes including the TGFβ signaling mediators EVI1 and SnoN/SkiL, PKCι and PIK3CA amplified at 3q26.2 and 3q26.3, respectively, in ovarian cancers. Previous studies indicate that these genes can exhibit cooperative oncogenicity by cross-regulating one another and facilitating cancer development. Our earlier studies demonstrated that treatment of ovarian cancer cells with arsenic trioxide (As2O3) promotes cytoprotective autophagy regulated by induction of SnoN to antagonize the cytotoxic effects of As2O3. Since exact mechanisms underlying As2O3-induced SnoN expression and cytoprotective responses were unclear, we hypothesized that SnoN may be regulated by signaling pathways involving genes amplified at the 3q26 locus. Phospholipid scramblase 1 (PLSCR1) is located at 3q23 proximal to the amplified 3q26 region. It had been implicated in disruption of plasma membrane asymmetry by mediating phospholipid scrambling, a process critical for cellular events such as blood coagulation and apoptosis. However, recent findings have led to more investigations on the role and regulation of PLSCR1 in cancer development and immune responses. PLSCR1 expression is regulated by various stimuli including growth factors (EGF, G-CSF, and SCF), cytokines (IFN), and differentiation-inducing agents (ATRA). Despite these studies, transcriptional regulation of PLSCR1 remains incompletely understood. Numerous studies have suggested a critical role for PLSCR1 in the pathophysiology of various cancers including leukemia, ovarian cancer, colorectal cancer, and metastatic liver cancer. However, the precise contribution of PLSCR1 and its regulation in ovarian cancer development is unclear. Since PLSCR1 (at 3q23) is located in close proximity to SnoN/SkiL (at 3q26.2), we hypothesized that PLSCR1 expression in ovarian cancer cells could be regulated by SnoN. Herein, we present studies that primarily focus on understanding the role and regulation of SnoN/SkiL (a TGFβ pathway regulator) and PLSCR1 (an interferon-regulated gene), which are located at 3q26.2 and 3q23, respectively, in epithelial ovarian cancer. In Chapter 3, we determined that activation of the PI3K signaling pathway mediates SnoN expression and cytoprotective responses upon stimulation of ovarian cancer cells with As2O3. We first identified that As2O3 stimulation leads to activation of EGFR and its downstream signaling mediators as well as modulates its interaction with the adaptor proteins, ShcA and Grb2. Interestingly, while treatment with a general SFK inhibitor (PP2), reduced the As2O3-induced EGFR activation and SnoN induction, a more specific inhibitor SU6656 did not alter SnoN expression. Further, via studies utilizing specific inhibitors and siRNA targeting PI3K, we determined that inhibition of PI3K signaling pathway decreases SnoN induction and increases apoptosis in ovarian cancer cells in response to As2O3. This suggests that PI3K (PIK3CA) activity is required for the As2O3-mediated SnoN induction and the cell survival responses in ovarian cancer cells. Finally, we determined by siRNA-mediated knockdown that EGFR and MAPK1 alter As2O3-induced cell death response independently of SnoN induction. In Chapter 4, via bioinformatic analyses, we identified that PLSCR1 DNA copy number and mRNA expression is elevated in ovarian cancer patients and cell lines relative to immortalized (Tag/hTERT) normal ovarian surface epithelial (OSE) cells. Interestingly, altered PLSCR1 DNA and mRNA levels were correlated with SnoN in ovarian cancers. We next identified that SnoN knockdown leads to a significant (~35%, P2O3 transcriptionally downregulates PLSCR1 in a ROS-independent mechanism. Furthermore, PLSCR1 knockdown, similar to SnoN knockdown increases ovarian cancer cell sensitivity to As2O3. PLSCR1 knockdown increases cleaved PARP (marker of apoptosis) with a consequent reduction in LC3-II levels (marker of autophagosomes). Collectively, these studies implicate PLSCR1 in the pathophysiology of ovarian cancers and in altering the chemotherapeutic responses in ovarian cancer cells. PLSCR1 is an IFN-regulated gene and mediates antiviral/immune responses. More recent studies in plasmacytoid dendritic cells have implicated PLSCR1 in regulating TLR9 signaling upon stimulation with CpG ODN. However, whether PLSCR1 could mediate the innate immune responses upon stimulation with dsDNA remained unclear. In Chapter 5, we identified that stimulation of normal ovarian and mammary epithelial cells with dsDNA (empty plasmid) markedly induces PLSCR1 consequent with activation of IRF3, a downstream mediator of TLR signaling that transcriptionally regulates the expression of type 1 IFNs. Interestingly, IRF3 knockdown ablates the dsDNA-induced PLSCR1 expression suggesting that PLSCR1 induction in response to dsDNA could be mediated by IRF3. Additionally, we have determined that dsDNA stimulation induces nucleic acid sensing TLRs, TLR9 and TLR4 as well as IFN-α and IFN-β mRNAs. Interestingly, dsDNA stimulation did not induce PLSCR1 or IRF3 activation in ovarian cancer cells suggesting that the mechanisms of IRF3 activation and PLSCR1 induction in response to dsDNA might be dysregulated in ovarian cancers. Collectively, our studies demonstrate a possible synergistic role of SnoN and PLSCR1 in ovarian cancer pathophysiology and suggest a potentially dysregulated role of PLSCR1 in the dsDNA-induced immune responses of malignant epithelial cells relative to normal epithelial cells. These studies could potentially lead to development of a novel combinatorial therapeutic strategy that targets both these molecules for improving treatment of patients with ovarian carcinoma.

While the etiology of ovarian cancer (OC) is not completely understood, evidence suggests that chronic inflammation may promote malignant transformation. However, familial history remains the strongest risk factor for developing OC and is associated with germline BRCA1 mutations, such as the 185delAG mutation. Normal human ovarian surface epithelial cells expressing the 185delAG mutant, BRAT, exhibit molecular and pathological changes that may contribute to OC oncogenesis. In the current study, I sought to determine whether BRAT could promote an inflammatory phenotype by investigating BRAT's impact on the expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Using a culture model system of normal human ovarian surface epithelial (OSE) cells with and without the BRCA1 185delAG frameshift mutation, BRAT, I investigated BRAT's role in IL-1β expression. OSE cells stably expressing the 185delAG mutation and ovarian surface epithelial cells with endogenous 185delAG were analyzed for differential target gene expression by real time PCR, western blot, ELISA, luciferase reporter and siRNA assays. Normal and malignant breast epithelial cell lines transiently expressing BRAT were also evaluated by real time PCR to determine whether BRAT-induced IL-1β expression is tissue specific. BRAT-expressing OSE cells exhibited enhanced IL-1β mRNA and protein expression. However, expression of BRAT in all breast cell lines failed to significantly alter IL-1β expression levels so that BRAT-mediated IL-1β expression promoting a chronic inflammatory phenotype conducive to malignant transformation may be limited to the ovary. Secondly, since OSE cells expressing the BRCA1 185delAG mutation have increased levels of IL-1β that may contribute to malignant transformation, in a pilot study, I sought to assess whether elevated urinary levels of IL-1β are associated with OC as well as compare urinary IL-1β levels with clinical parameters. Urinary and serum levels of IL-1β were analyzed by ELISA and biostatistical analysis from a patient cohort consisting of healthy women (N=10), women with ovarian benign disease (N=23), women with OC (N=32), women with other benign gynecological conditions (N=22), and women with other gynecological cancers (N=6). Urinary IL-1β levels were elevated in patients with ovarian benign disease and a first degree family history of ovarian and/or breast cancer. Urinary IL-1β levels were also correlated with increased body mass index. Urinary and serum IL-1β levels were increased in ovarian benign and OC patient samples supporting the theory of elevated urinary IL-1β being associated with cancer progression. Lastly, I sought to begin early molecular characterization of BRCA1 185delAG to better understand its role in ovarian transformation. I isolated 185delAG protein expressed in E. coli and utilized web tools to analyze the amino acid sequence to determine the molecular and structural characteristics. The study results showed the predicted BRCA1 185delAG protein product is an ordered, self-aggregating, alpha helical protein structurally and molecularly distinct from wild-type BRCA1. The BRCA1 185delAG amino acid sequence contained domains with resemblance to the Peptidase M20 family. Isolation of the BRCA1 185delAG protein product will allow for further protein analysis to better understand its' oncogeneic functions; as well as, elucidate the mechanism of tissue-specific BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

Ovarian cancer has the highest mortality rate of all gynecological malignancies, due to inadequate treatment strategies and poor early diagnosis. Intraperitoneal (IP) chemotherapy administered on an intermittent schedule has been pursued for ovarian cancer treatment. However, local toxicities and complications associated with indwelling IP catheters required to deliver the chemotherapeutics have been documented. Furthermore, shortening or completely removing treatment-free periods between each chemotherapy cycle has shown improved efficacy compared to intermittent chemotherapy. The focus of this thesis was to develop and characterize a biocompatible and biodegradable IP injectable depot sustained drug delivery system as a new treatment strategy for ovarian cancer. A polymer-lipid injectable formulation (PoLigel) was developed and used for sustained docetaxel (DTX) delivery. The PoLigel resulted in homogeneous DTX peritoneal distribution and sustained plasma levels in healthy mice, which was in contrast to Taxotere®, the clinically used formulation of DTX. Sustained plasma, tissue, tumor and ascites DTX concentrations were observed in mice bearing IP SKOV3 tumors or ID8 ascites over a 3 week period following IP administration of the PoLigel. The intratumoral distribution and tumor penetration of DTX in subcutaneous (SC) and IP SKOV3 tumors were characterized. DTX distributed more towards the tumor core and diffused 1.5 fold further from blood vessels of the IP tumors compared to the SC tumors. The high efficacy observed in the IP SKOV3 and ID8 models and the SC SKOV3 model was attributed to favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure. Sustained chemotherapy with DTX alone and in combination with a drug efflux transporter inhibitor was investigated in multidrug resistant (MDR) ovarian cancer. In vitro, combination delivery via the PoLigel resulted in more apoptosis, greater intracellular accumulation of DTX, and lower DTX efflux in MDR ovarian cancer cells. Sustained combination chemotherapy was more than twice as efficacious as intermittent Taxotere® treatment in MDR ovarian cancer. Significant anti-tumor efficacy was also observed in the MDR model following sustained DTX chemotherapy compared to intermittent Taxotere®. Overall, results presented here encourage the clinical investigation of IP sustained chemotherapy for ovarian cancer treatment.

Prophylactic salpingo-oophorectomy, the preventive removal of the ovaries and fallopian tubes, is recommended to women who carry a BRCA1 or BRCA2 mutation in order to reduce the risk of breast, ovarian and fallopian tube cancer. The short and long term health and quality of life effects of this procedure are not well understood. We examined the actual and perceived reduction in cancer risk associated with this surgery. The impact of prophylactic salpingo-oophorectomy on health-related quality of life, psychological distress, cancer worry, menopausal symptoms, and sexual function during the year following surgery was also evaluated. In our prospective study, prophylactic salpingo-oophorectomy was associated with an 80% reduction in ovarian and fallopian tube cancer risk. The residual risk for primary peritoneal cancer was 0.2% per year or 4.3% at 20 years after salpingo-oophorectomy. Most women accurately perceived their risk of breast cancer. However, the risk for ovarian cancer was overestimated, particularly by women who carry a BRCA2 mutation. Physical and mental health-related quality of life did not decrease in the year following surgery; and psychological distress was similar to levels experienced by the general population. Most women were significantly less worried about cancer after the surgery, however, a subset of women continued to experience significant cancer specific distress after prophylactic salpingo-oophorectomy. Women who underwent prophylactic salpingo-oophorectomy when premenopausal experienced a significant worsening of vasomotor symptoms and a decline in sexual functioning. Hormone replacement therapy mitigated these symptoms, but not to pre-surgical levels. Dyspareunia was somewhat alleviated by hormone replacement therapy, however, the decrease in sexual pleasure was not. Satisfaction with the decision to undergo prophylactic salpingo-oophorectomy was high regardless of these symptoms. These studies will provide women who are considering prophylactic salpingo-oophorectomy with information about the reduction in cancer risk associated with the surgery and the possible effects experienced during the year following surgery.

Ovarian cancer has the highest mortality rate of all gynecological malignancies, due to inadequate treatment strategies and poor early diagnosis. Intraperitoneal (IP) chemotherapy administered on an intermittent schedule has been pursued for ovarian cancer treatment. However, local toxicities and complications associated with indwelling IP catheters required to deliver the chemotherapeutics have been documented. Furthermore, shortening or completely removing treatment-free periods between each chemotherapy cycle has shown improved efficacy compared to intermittent chemotherapy. The focus of this thesis was to develop and characterize a biocompatible and biodegradable IP injectable depot sustained drug delivery system as a new treatment strategy for ovarian cancer. A polymer-lipid injectable formulation (PoLigel) was developed and used for sustained docetaxel (DTX) delivery. The PoLigel resulted in homogeneous DTX peritoneal distribution and sustained plasma levels in healthy mice, which was in contrast to Taxotere®, the clinically used formulation of DTX. Sustained plasma, tissue, tumor and ascites DTX concentrations were observed in mice bearing IP SKOV3 tumors or ID8 ascites over a 3 week period following IP administration of the PoLigel. The intratumoral distribution and tumor penetration of DTX in subcutaneous (SC) and IP SKOV3 tumors were characterized. DTX distributed more towards the tumor core and diffused 1.5 fold further from blood vessels of the IP tumors compared to the SC tumors. The high efficacy observed in the IP SKOV3 and ID8 models and the SC SKOV3 model was attributed to favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure. Sustained chemotherapy with DTX alone and in combination with a drug efflux transporter inhibitor was investigated in multidrug resistant (MDR) ovarian cancer. In vitro, combination delivery via the PoLigel resulted in more apoptosis, greater intracellular accumulation of DTX, and lower DTX efflux in MDR ovarian cancer cells. Sustained combination chemotherapy was more than twice as efficacious as intermittent Taxotere® treatment in MDR ovarian cancer. Significant anti-tumor efficacy was also observed in the MDR model following sustained DTX chemotherapy compared to intermittent Taxotere®. Overall, results presented here encourage the clinical investigation of IP sustained chemotherapy for ovarian cancer treatment.

Prophylactic salpingo-oophorectomy, the preventive removal of the ovaries and fallopian tubes, is recommended to women who carry a BRCA1 or BRCA2 mutation in order to reduce the risk of breast, ovarian and fallopian tube cancer. The short and long term health and quality of life effects of this procedure are not well understood. We examined the actual and perceived reduction in cancer risk associated with this surgery. The impact of prophylactic salpingo-oophorectomy on health-related quality of life, psychological distress, cancer worry, menopausal symptoms, and sexual function during the year following surgery was also evaluated. In our prospective study, prophylactic salpingo-oophorectomy was associated with an 80% reduction in ovarian and fallopian tube cancer risk. The residual risk for primary peritoneal cancer was 0.2% per year or 4.3% at 20 years after salpingo-oophorectomy. Most women accurately perceived their risk of breast cancer. However, the risk for ovarian cancer was overestimated, particularly by women who carry a BRCA2 mutation. Physical and mental health-related quality of life did not decrease in the year following surgery; and psychological distress was similar to levels experienced by the general population. Most women were significantly less worried about cancer after the surgery, however, a subset of women continued to experience significant cancer specific distress after prophylactic salpingo-oophorectomy. Women who underwent prophylactic salpingo-oophorectomy when premenopausal experienced a significant worsening of vasomotor symptoms and a decline in sexual functioning. Hormone replacement therapy mitigated these symptoms, but not to pre-surgical levels. Dyspareunia was somewhat alleviated by hormone replacement therapy, however, the decrease in sexual pleasure was not. Satisfaction with the decision to undergo prophylactic salpingo-oophorectomy was high regardless of these symptoms. These studies will provide women who are considering prophylactic salpingo-oophorectomy with information about the reduction in cancer risk associated with the surgery and the possible effects experienced during the year following surgery.

<p>MAL (myelin and lymphocyte protein), has been implicated in several malignancies including esophageal, gastric, and cervical cancers. We have demonstrated that the MAL protein is expressed in the normal breast epithelium, and aberrantly expressed in breast cancer. Bisulfite sequencing of the MAL promoter CpG island revealed hypermethylation in breast cancer cell lines and 69% of primary tumors analyzed compared with normal breast epithelial cells. Differential methylation between normal and cancer DNA was confined to the proximal promoter region. In a subset of breast cancer cell lines, promoter methylation correlated with transcriptional silencing that was reversible with the methylation inhibitor decitabine. Furthermore, exogenous expression of MAL in breast cancer cell lines resulted in decreased cell proliferation, motility, reduced cell invasion through Matrigel and suppressed anchorage-independent growth in soft agar. In a cohort of 122 primary breast tumors, immunohistochemical analysis revealed that the MAL protein was an independent predictor of benefit from adjuvant chemotherapy. Moreover, overexpression of MAL in triple-negative MDA-MB-468 and BT20 breast cancer cell lines was sufficient to confer sensitivity to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibition and was associated with reduced phosphatidylinositol-3 kinase (PI3K)/Akt signaling. Immunohistochemistry studies conducted on 144 late-stage serous ovarian cancers showed that MAL expression was a significant predictor of survival. Knockdown of MAL expression in the SKOV8 ovarian cancer cell line reduced cell proliferation and resulted in increased sensitivity to the chemotherapeutic drug carboplatin. Thus, we have identified the MAL gene as a novel epigenetically regulated gene in breast cancer with implications for response to chemotherapy in both breast and ovarian cancer. Furthermore, we have shown that the MAL protein has predictive and prognostic value in breast and ovarian cancers, respectively.</p> / Dissertation