abstract: Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2014

O câncer de ovário é uma neoplasia ginecológica e alternâncias hormonais poderiam ter um papel na manifestação da doença. As ADAMTS´s são proteases secretadas dependentes de Zn2+/Ca2+. Nosso objetivo foi avaliar se há Influência de hormônios sexuais nos níveis de expressão de mRNA, proteínas e distribuição de ADAMTS 1 e 4 e alteração na migração e invasão em células tumorais humanas de ovário. As linhagens foram tratadas com progesterona, estrógeno ou testosterona e o controle não recebeu tratamento. O estrógeno e a testosterona induziram uma menor e a progesterona uma maior expressão gênica de ADAMTS 1 e 4 em relação ao controle para a linhagem ES-2. A progesterona foi capaz de induzir aumento nos níveis proteicos de ADAMTS 1 e 4 no lisado de ambas as linhagens. A progesterona diminuiu a capacidade migratória e de invasão das linhagens. Observamos na imunofluorescência ADAMTS 1 no núcleo das células. Concluímos que a progesterona regula a expressão das ADAMTS 1 e 4, e reduz a invasão e migração de células derivadas de câncer de ovário. / Ovarian carcinoma is the leading cause of gynecological neoplastic death, being associated primarily with deregulation of sex hormones. ADAMTS are secreted proteases. Our aim is to assess whether sex hormones would affect ADAMTS1 and 4 expressions in ovarian cancer cells. Estrogen and testosterone induced a decrease on gene expression of ADAMTS 1 and 4 compared to the control for the ES-2 cell line, while progesterone led to an increase in the mRNA levels of these same proteases. Progesterone increases ADAMTS\'s protein levels in the lysate and in conditioned medium from NIH-OVCAR-3 and in the lysate from ES-2 cells. Immunofluorescence showed ADAMTS 1 located at the cell nucleus. NIH-OVCAR-3 cells treated with progesterone exhibited decrease migratory activity compared to control and ES-2 exhibited decrease invasion activity. We conclude that progesterone modulates ADAMTS1 and 4 levels in ovarian cancer cell lines and decrease migratory and invasion behavior in ovarian cancer cells.

Introdução: O câncer de ovário é frequentemente diagnosticado em estágios avançados e é pouco responsivo aos tratamentos empregados atualmente. O tratamento de primeira linha consiste em esquemas incluindo cirurgia citorredutora seguido de quimioterapia adjuvante por derivados de platina e taxano. Os esquemas de segunda linha são baseados em gemcitabina e doxorrubicina lipossomal. Em seguida, a doença tende a progredir rapidamente e a quimioterapia não é indicada pela ausência de resposta e pela alta toxicidade desses medicamentos. Anteriormente, mostramos que nanopartículas lipídicas semelhantes à composição química da LDL, porém sem proteína, e associadas à agentes antineoplásicos são captadas por células neoplásicas. Objetivos: Testamos a hipótese se o paclitaxel associado à nanopartícula poderia beneficiar com segurança pacientes com câncer de ovário avançado e refratário ao tratamento, e portanto, não mais elegíveis para quimioterapia convencional. Métodos: Quatorze mulheres com câncer de ovário avançado de 61 ± 10 anos, com estadiamento clínico IV e TqNqM1 (FIGO e TNM Scale, respectivamente) que não eram responsivas à quimioterapia em terceira linha foram incluídas no estudo. O tratamento consistiu com o paclitaxel associado à nanopartícula na dose 175 mg/m2 de superfície corporal, a cada 3 semanas. As pacientes foram submetidas a exames clínicos antes de cada ciclo de quimioterapia. Determinações bioquímicas séricas e exames de imagem foram realizados para acompanhamento de novas lesões ou progressão da doença. Resultados: Foi realizado o total de 74 ciclos, e não foram observadas toxicidades laboratoriais e clínicas. Em quatro pacientes a doença permaneceu estável. Conclusões: A notável ausência de toxicidade, não registrada na literatura até hoje nos vários sistemas de veiculação descritos abre uma nova perspectiva de tratamento da doença. Evita-se o desconforto e os riscos da quimioterapia convencional, podem ser incluídos pacientes muito idosos ou com outras fragilidades que contraindiquem a quimioterapia e não há limite para a continuação do tratamento. / Introduction: Ovarian cancer is often diagnosed at advanced stages and is poorly responsive to standard treatment. First-line treatment consists in schemes including citorreductive surgery followed by adjuvant chemotherapy schemes with platinum and taxane derivatives. Second-line regimens are based in gemcitabine and liposomal doxorubicin. Thereafter, the disease progresses rapidly and chemotherapy is no longer indicated for lack of response rate together with high toxicity of antineoplastic agents. Previously, we showed that non-protein lipid nanoparticles resembling chemical structure of LDL, however without protein and associated with antineoplastic agents are uptaken by neoplastic cells. Aims: We tested the hypothesis whether paclitaxel associated to the nanoparticle could safely benefit patients with advanced ovarian cancer refractory to previous treatment, thus not eligible for further conventional chemotherapy. Methodology: Fourteen women with advanced ovarian cancer aged 61 ± 10 years were included, with clinical stage IV and TqNqM1 (FIGO and TNM Scale, respectively) that were unresponsive for third line chemotherapy treatments. Treatment consisted with paclitaxel associated to the nanoparticle at 175 mg/m2 body surface dose, every 3 weeks. Patients were submitted to clinical examinations before each chemotherapy cycle. Serum biochemistry determinations and imaging exams were performed to monitoring new lesions or disease progression. Results: Total of 74 cycles of chemotherapy was performed, clinical and laboratorial toxicities were not observed. Four patients stayed with stable disease. Conclusions: The notable absence of toxicity, not recorded in the oncology literature to date described in various drug delivery systems, opens a new perspective on the treatment of cancer. Avoiding the discomfort and risks of conventional chemotherapy, can be included very aged patients or with other weaknesses, that chemotherapy is contraindicated, and there is no limit for continued treatment.

Bakgrund: I Sverige drabbas 560 kvinnor av livmoderhalscancer och 700 kvinnor av äggstockscancer varje år. Detta påverkar kvinnornas livsvärld samt relationerna till sina närstående men även den intima relationen till partnern. Cancern har en negativ påverkan på kvinnornas välbefinnande och därför är det viktigt att de får ett bra stöd från vårdpersonal. Syfte: Syftet med uppsatsen var att beskriva kvinnors upplevelser av att leva med livmoderhalscancer eller äggstockscancer. Metod: En kvalitativ metod användes för att beskriva kvinnors upplevelser av att leva med livmoderhalscancer eller äggstockscancer. Datainsamlingen bestod av åtta utvalda bloggar, där en analys av narrativer användes. Resultat: I resultatet framkom fyra olika teman: Blandade känslor kring sjukdomen, förändrad existens, cancerns påverkan på kvinnornas relationer samt att hantera det nya livet. Utifrån dessa teman skapades underteman som beskrev mer djupgående kring kvinnornas upplevelser av cancern. Slutsats: Kvinnorna upplever att deras livsvärld påverkas i samband med cancern och att ovisshet och rädsla är ständigt närvarande. Ett behov av information och uppföljning från vårdpersonal anses viktigt för att öka kvinnornas välbefinnande. / Background: In Sweden, 560 women are diagnosed with cervical cancer and 700 women are diagnosed with ovarian cancer every year. This has an effect of the women’s lifeworld and their relationships toward their close relatives. It has also an impact of the intimate relationship with their partners. The cancer affects the women’s wellbeing in a negative way and therefore it is of great importance for them to get good support from the healthcare professionals. Aim: The aim of this study was to describe women’s experiences of living with cervical cancer or ovarian cancer. Results: In the result four sections where identified: Mixed feelings about the diseases, changed existence, the cancers impact on women’s relationships and to manage the new life. Out of these sections subsections emerged describing more deeply about the women’s experiences of the cancer. Conclusion: Women experienced that their lifeworld is affected by the cancer and that uncertainty and fear are constantly present. A need for information and follow-up from healthcare professionals is considered important to increase women’s well-being.

Le cancer de l'ovaire est la huitième cause de décès par cancer chez la femme dans le monde. Le traitement proposant la combinaison carboplatine-paclitaxel donne un taux de réponse complet dans 40 à 60 % des cas. Cependant, plus de 90 % des patientes récidive après 2 ans. Il a été montré que le complexe de neurotensine (NTS) et son récepteur à haute affinité 1 (NTSR1) favorise la progression du cancer par prolifération, migration, invasion et néoangiogenèse in vitro et/ou in vivo dans de nombreux cancers. A ce jour, le rôle du complexe NTS/NTSR1 dans la réponse à la chimiothérapie n'a pas été pris en compte. Dans ce travail, j'ai étudié si les inhibiteurs ce complexe pouvait améliorer la réponse à la chimiothérapie, et les mécanismes associés à cet effet. Dans une série de 46 patients, NTS et NTSR1 ont été détectés respectivement dans 72% et 74% des cas. L'étude du transcriptome de cancer de haut grade a montré que l'expression du NTSR1 était liée à des stades plus élevés et au statut "résistance". J'ai pu mettre en évidence la contribution de la voie NTS/NTSR1 à réponse la chimiothérapie en utilisant deux lignées cellulaires de cancer de l'ovaire. En présence d'un antagoniste spécifique du NTSR1, le SR48692, des cellules cancéreuses de l'ovaire ou des tumeurs expérimentales ont montré une réponse amplifiée au carboplatine. En effet, la présence du SR48692 diminue l'efflux de carboplatine des cellules, et ainsi augmente les dommages à l'ADN induit par le platine. L'apoptose a également été renforcée en présence de cet antagoniste. Ces résultats renforcent notre hypothèse selon laquelle le blocage de la voie NTS/NTSR1 améliore la réponse à la chimiothérapie. / Ovarian cancer (OC) is the eighth most common cause of cancer death in female worldwide. Because OC has often no apparent symptoms at the early stages, the majority is diagnosed at the advanced stages. The combination of carboplatin plus paclitaxel, results in a complete response rate in 40-60 % of the cases. However, more than 90% patients relapse after 2 years, and in most cases, recurrent patients becomes incurable due to the chemoresistance. The complex of neurotensin (NTS) and its high affinity receptor 1 (NTSR1) has been shown to promote cancer progression in many type of cancer, via proliferation, survival, migration, invasion cellular effects, and neoangiogenesis. To date, the role of the complex NTS/NTSR1 in the platinum-based chemotherapy has not been considered. I studied whether NTS/NTSR1 inhibitors could enhance chemotherapy and the related mechanism. In a series of 46 patients, NTS and NTSR1 were detected in 72% and 74% of cases, respectively. Transcriptome analysis in a large series of high grade OC showed that NTSR1 expression was correlated with higher stages and with platinum resistance. We studied the contribution of NTS/NTSR1 pathway to chemotherapy by using two OC cell lines. In the presence of NTSR1 specific antagonist, SR 48692, OC cells or experimental tumors showed an enhanced response to carboplatin. SR 48692 decreased the efflux of carboplatin and increase the DNA damage induced by the platinum. Apoptosis was also enhanced in the presence of NTSR1 antagonist. These results strengthen our hypothesis that the blockade of NTS/NTSR1 pathway enhances the response to chemotherapy and potentially sensitizes tumor cells resistant to platinum.

Ovarian cancer (OC) has the highest mortality among gynecological cancers. The high mortality is associated with the lack of an accurate screening tool to detect disease in early stage. As a result the majority of OCs are diagnosed in late stage. Further, the molecular events responsible for malignant transformation in the ovary remain poorly understood. Consequently, delineating key molecular players driving OC could help elucidate potential diagnostic, prognostic and therapeutic targets. Receptor for hyaluronan-mediated motility (RHAMM) belongs to a group of hyaladherins, which share a common ability to bind to hyaluronan (HA). Intracellularly, RHAMM is involved in microtubule spindle assembly contributing to cell cycle progression. On the cell surface, loosely tethered RHAMM forms a complex with cluster differentiation 44 and HA to activate cell signaling pathways that promote cellular migration, invasion and proliferation. Since RHAMM is overexpressed in a number of cancer types and it is often associated with an aggressive cancer phenotype, I sought to determine if RHAMM similarly contributes to OC. I found that RHAMM is overexpressed in clinical specimens of OC by immuno-histochemistry and although both primary and metastatic OCs stain equally for RHAMM, RHAMM staining was most intense among clinically aggressive OC histologic subtypes. Further, using an in vitro model system, I was able to show that OC cells express and secrete RHAMM. Abrogation of RHAMM using silencing RNA technology inhibited OC cell migration and invasion suggesting that RHAMM may contribute, at least in part, to the metastatic propensity of OC. Since RHAMM lacks an export signal peptide sequence and has not been reported to employ alternate mechanisms for extracellular secretion, I utilized computational analyses to predict post-translational glycosylation events as a novel mode for RHAMM secretion. N- glycosylation inhibitors abrogated RHAMM secretion by OC cells in vitro validating my prediction and identify a novel and potentially unconventional mode for RHAMM secretion. Lastly, since RHAMM is secreted by OC cells, I sought to determine whether RHAMM could be detected in bodily fluids. In a pilot study, I found that urinary levels of RHAMM are elevated in OC patients as measured by enzyme-linked immunosorbant assays. Decreased urinary RHAMM levels noted following cytoreductive surgery support OC as the source of elevated urinary RHAMM levels. Finally, while obesity was associated with high urinary RHAMM levels in OC patients, combined measurements of urinary RHAMM and serum CA125 improved prediction of OC. Taken together, the studies described herein suggest that RHAMM contributes to OC and that further studies are warranted to further elucidate the clinical role of RHAMM in OC.

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery.

The overall objective of the research presented in this dissertation was to assess exposure to endocrine disrupting chemicals (EDCs), polychlorinated biphenyls (PCBs), phthalates, and bisphenol A (BPA) in the general population and evaluate their associations with adverse reproductive health effects, including cancers, in women. Given the proven contribution of unopposed estrogens to the risk for endometrial neoplasia or breast cancer, renewed health concerns have aroused about estrogen mimicking EDCs found in food, personal care products or as environmental contaminants. Our meta-analysis showed that exposure to estrogen mimicking PCBs increased summary risk of breast cancer and endometriosis. We further evaluated the relationship between endometriosis and breast cancer, and EDCs using a bioinformatics method. Our bioinformatics approach was able to identify genes with the potential to be involved in interaction with PCB, phthalates and BPA that may be important to the development of breast cancer and endometriosis. Therefore, we hypothesized that exposure to EDCs such as PCBs, phthalates, and BPA, results in adverse reproductive health effects in women. Using subject data and biomarkers available from the Center for Disease Controls National Health and Nutrition Examination Survey database we conducted a cross-sectional study of EDCs in relation to self-reported history of endometriosis, uterine leiomyomas, breast cancer, cervical cancer, ovarian cancer, and uterine cancer. Significantly higher body burdens of PCBs were found in women diagnosed with breast cancer, ovarian cancer, and uterine cancer compared to women without cancer. PCB 138 was significantly associated with breast cancer, cervical cancer, and uterine cancer, while PCBs 74 and 118 were significantly associated with ovarian cancer. The sum of dioxin-like PCBs were significantly associated with ovarian cancer (OR of 2.02, 95% CI: 1.06-3.85) and the sum of non-dioxin-like PCBs were significantly associated with uterine cancer (OR of 1.12, 95%CI: 1.03-1.23). Significantly higher body burdens of PCBs were also found in women diagnosed with endometriosis and uterine leiomyomas. Documenting the exposure to EDCs among the general U.S. population, and identifying agents associated with reproductive toxicity have the potential to fill research gaps and facilitate our understanding of the complex role environmental chemicals play in producing toxicity in reproductive organs.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.