The simplest phospholipid, lysophosphatidic acid (LPA), is a heat stable component of serum known for its proliferative and migratory activities in cancer cells. Strong evidence suggests that LPA production and expression of its receptors are dysregulated in multiple human malignancies. The mechanism behind LPA-mediated tumor cell growth and oncogenesis remains poorly understood. In this thesis project I used ovarian and other cancer cells as a model system to examine the hypothesis that LPA present in the tumor microenvironment is a pathophysiological determinant of hyperactive de novo lipogenesis and aerobic glycolysis, two hallmarks of cancer cells. We demonstrated that LPA induced proteolytic activation of sterol regulatory element binding proteins (SREBPs) in a cancer specific manner, leading to activation of the SREBP-FAS (fatty acid synthase) lipogenic pathway. Treatment of cancer cell lines with LPA also led to dephosphorylation and inhibition of AMP-activated kinase (AMPK), thereby activating acetyl CoA carboxylase (ACC). Moreover, these effects of LPA were mediated by LPA2, a receptor subtype overexpressed in multiple cancers, providing an explanation for the cancer specific regulation of FAS and ACC by LPA. Downstream of the LPA2 receptor, we identified the Gα12-Rho-Rock pathway to activate SREBPs and the Gαq-PLC (phospholipase C) pathway to inactivate AMPK. Consistent with LPA mediated activation of the key lipogenic enzymes FAS and ACC, LPA stimulated de novo lipid synthesis via LPA2, leading to accumulation of intracellular triacylglycerol and phospholipids. Pharmacological and molecular inhibition of LPA2, FAS or ACC attenuated LPA-dependent cell proliferation, indicating that upregulation of lipid synthesis is an integral component of the proliferative response to LPA. In further support of this, downregulation of LPA2 expression led to dramatic inhibition of anchorage-dependent and –independent growth of ovarian cancer cells. To support increased biomass generation, rapidly proliferating cancer cells enhance carbon influx by activating glycolysis. In the next part of the study, we investigated if LPA signaling was also involved in activating aerobic glycolysis in cancer cells. LPA indeed activated glycolysis in ovarian and other cancer cells but failed to elicit this response in non-transformed cells, suggesting a cancer specific role of LPA in regulation of glucose metabolism. While LPA had no effect on glucose uptake, we found that LPA altered expression of multiple genes involved in glucose metabolism. The most significant observation was that LPA treatment dramatically upregulated expression of HK-2, one of the rate-limiting glycolytic enzymes. We explored the underlying mechanism and found that LPA activates HK-2 transcription through LPA2-mediated activation of SREBP-1. Two sterol regulator elements (SREs) on the human HK-2 promoter were identified to be responsible for LPA activation of the promoter. DNA pulldown and chromatin immunoprecipitation assays confirmed that SREBP-1 bound to these SREs in LPA-treated cells. Although in ovarian cancer cells, LPA treatment also stabilized Hif-1α protein, an established activator of HK-2 and glycolysis, LPA-regulated HK-2 expression and glycolysis was largely independent of Hif-1α. These results established that LPA stimulates glycolysis via the LPA2-SREBP-HK-2 cascade in neoplastic cells. Taken together, this dissertation provides the first evidence for regulation of cancer cell metabolism by LPA. The results indicate that LPA signaling is causally linked to lipogenic and glycolytic phenotypes of cancer cells. Therefore, targeting the key LPA2 receptor could offer a novel and innovative approach to blocking tumor-specific metabolism.

Lysophosphatidic acid (LPA) is a potent bioactive phospholipid mediator that functions through multiple G protein couple receptors (GPCRs). LPA is elevated in ascites of ovarian cancer patients and is involved in growth, survival and metastasis of ovarian cancer cells. Gene promoter analyses revealed that some LPA-target genes share similar sets of binding sites for prominent transcription factors posing the possibility of a general mechanism for activation of their expression by LPA. Detailed investigation of the mechanisms of regulation of cyclooxygenase 2 (Cox-2), a paradigm of LPA-regulated genes, showed that LPA robustly upregulated the expression of Cox-2 in ovarian cancer cells through multiple receptors. LPA induced rapid increase in Cox-2 mRNA and significantly enhanced the stability of Cox-2 transcript with the support of mRNA binding protein HuR. The effects of LPA on Cox-2 transcriptional activation include essential involvement of transcription factor, C/EBP-b. Further studies on mechanisms of activation of C/EBP-b demonstrated that LPA increased phosphorylation, binding and transcriptional activities of C/EBP-b. In addition, activation of C/EBP-b and LPA-target genes required contribution from EGFR. This novel crosstalk between LPA GPCRs and EGFR in mediating transcription factors activation was further explored by investigating the mechanisms of activation of AP-1 and NF-kB by LPA. Activation of AP-1 family of proteins by LPA relied heavily on basal inputs from EGFR as inhibition of EGFR kinase activity with AG1478 caused significant loss of LPA-induced AP-1 expression, binding and transcription activities. Although HGF and other agonists of RTK only weakly stimulate LPA-target genes and transcription factors in ovarian cancer cells, costimulation with HGF in the presence of AG1478 restored LPA signals to both C/EBP-b and AP-1. This suggests an obligatory role for a RTK in LPA-induced transcriptional activation, not necessarily inputs from EGFR. Interestingly, inhibition of EGFR with AG1478 did not interfere with LPA-induced NF-kB activation. Pharmacological inhibition and molecular targeting revealed that only a subset of G proteins participate in the crosstalk between LPA receptors and EGFR. Collectively, these results demonstrate the presence of at least two signals downstream of LPA receptors: one dependent on basal RTK activity and another mediated directly by LPA GPCRs.

Le cancer de l'ovaire représente la première cause de décès par cancer gynécologique. La survie globale des patientes à 5 ans est inférieure à 30%. Ce sombre pronostic s'explique à la fois par la découverte tardive de la maladie et par le développement d'une chimiorésistance. L'ascite est un fluide exsudatif qui est fréquemment accumulé dans la cavité péritonéale au cours de la progression des cancers de l'ovaire. Ce « microenvironnement tumoral » particulier contribue à la dissémination des cellules cancéreuses et à leurs implantations péritonéales.L'objectif global du travail de thèse a été, d'une part d'évaluer l'influence de l'ascite sur le comportement des cellules cancéreuses ovariennes et d'autre part, d'étudier les mécanismes de résistance à la perte d'ancrage des cellules cancéreuses ovariennes.Nous avons ainsi démontré que l'ascite induit une transition épithélio-mésenchymateuse partielle et que les modifications des comportements cellulaires observées sont dépendantes des intégrines alpha-v.Deux ligands de ces intégrines, la vitronectine et la fibronectine, ont été purifiés selon un protocole original permettant la caractérisation des deux protéines à partir d'une même ascite. Ces protéines ascitiques ont des propriétés différentes selon leur origine, donc selon les patientes dont elles sont issues, et influencent le comportement adhésif des cellules avec un degré variable. L'importance de la signalisation dépendante des intégrines alpha-v et des voies MAP Kinases a également été démontrée dans l'établissement d'une résistance des sphéroïdes tumoraux à l'anoïkis.En perspective, approfondir les connaissances des processus cellulaires et moléculaires conduisant à la dissémination intrapéritonéale et à l'émergence de chimiorésistance ainsi que déterminer le rôle potentiel de protéines ascitiques dans ces processus pourraient permettre la découverte de nouvelles cibles thérapeutiques. / Ovarian cancer is the most lethal gynaecological malignant disease, mainly due to late diagnosis and to acquired chemoresistance. An exudative fluid named ascites is frequently accumulates within the abdominal cavity during ovarian cancer progression. This unique tumor microenvironment contributes to cancer cell dissemination and peritoneal metastasis.The aim of the study was to evaluate the influence of ascites on cancer cell behaviors and to better understand the mechanisms of ovarian cancer cell resistance to the loss of anchorage.We demonstrate that ascites induces a partial epithelial–mesenchymal transition and that the modifications of cell behaviors observed are dependent of alpha-v integrins. A combined purification protocol has been established in order to purify vitronectin and Fibronectin, both ligands of these integrins, from a single pathological sample. These purified ascitic proteins have different molecular features according to the patients and impact the adhesive cell behavior with various degrees.Moreover we showed the importance of alpha-v integrins and MAP Kinases signalling pathways in the anoikis resistance of ovarian cancer spheroids.Our prospects are i) to increase the knowledge of the cellular and molecular processes leading to the intraperitoneal dissemination and to the emergence of chemoresistance and also ii) to determine the potential role of ascitics proteins in these processes. We will expect that these investigations couldlead to the discovery of new therapeutic targets.

Evolution of a Smart Girl is a collection of short stories that chronicles the evolution of the modern American female. The stories are arranged in three parts: "Dirty Barbie & Breakable Boys" focuses on adolescent relationships between boys and girls; "Some Things Can't Be Unbroken" centers on Maggie and Charlie Copper's marriage after Maggie is diagnosed with ovarian cancer at the age of twenty-five; and "Of Apples and Broken Scabs" presents four stories about four very different women who experience heartbreak in love, friendship, and lust. This work explores the ways in which a girl's interactions with others shape her into the young woman she becomes. Through same-sex friendships, romantic relationships, and sibling rivalries, the women in these stories experience intellectual and sexual awakenings.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg 2015. / Ovarian cancer is the most lethal gynaecological cancer. About 90% of ovarian cancers are epithelial (ovarian carcinomas), thought to arise from the ovarian surface epithelium. Diagnosed usually at clinically advanced stages, many patients show poor response to chemotherapy, with resistance and recurrent disease being prevalent. siRNA technology is currently being explored in clinical trials as a form of targeted therapeutic strategy in the disease. RBBP6 is a 250kD protein that enhances MDM2-mediated ubiquitination of p53 and also plays a role in cell cycle regulation and cell differentiation. It is upregulated in numerous cancers such as lung, oesophageal, colorectal and cervical cancer. RBBP6 suppresses p53 binding to DNA thereby inhibiting p53-dependent gene transcription. RBBP6 was knocked down using 30 nM siRNA in RMG-1 cells for 48 hours, after which the cells were treated with 50 nM paclitaxel and 0.5μM camptothecin for 24 hours. xCELLigence real time cell analysis was used to evaluate cell proliferation. qPCR and western blot were used to evaluate both gene expression and protein expressions respectively, of Bax, Bcl-2, MDM2, p53 and p21. Flow cytometry was used to determine the mode of cell death elicited apoptosis and also analyse changes in cell cycle progression. qPCR and Western blot analyses showed that RBBP6 expression reduced by approximately 57%. There was a significant upregulation of p53 and a significant downregulation of Bcl-2 in siRBBP6 transfected cells (p<0.05). Knockdown of RBBP6 resulted in a 37±5.8% cell death. There was a significant increase in cell death in paclitaxel and siRBBP6 co-treated cells (81.6±0.79%) as compared to cells treated with paclitaxel only (76.±1.14%). siRNA-mediated knock down of RBBP6 induces cell death in RMG-1 ovarian carcinoma cells. In addition, paclitaxel-induced cell death in RMG-1 cells is potentiated by RBBP6 siRNA transfection. A combination of chemotherapy with paclitaxel or camptothecin and RBBP6 siRNA could be a possible therapeutic strategy in combatting ovarian carcinomas.

No description available.

Dois peptídeos, designados Let#1 e Let#2, foram delineados do segundo laço extracelular da proteína NaPi2b. Após conjugação, os peptídeos induziram produção de anticorpos específicos em coelhos e camundongos. Os anticorpos com maiores títulos foram selecionados para ensaios complementares. Estes anticorpos reconheceram os peptídeos conjugados aos carreadores pelos métodos de ELISA e WB, não havendo reação cruzada quando testados com a proteína carreadora livre e com antígeno sintético inespecífico. Após a completa validação, foi possível observar que os anticorpos anti-peptídeos reconhecem NaPi2b nativa, presente na superfície de células OV-CAR, bem como mostraram reconhecimento de um alvo comum em todos os soros analisados, mesmo quando o soro era considerado negativo para COV pelo teste comercial. Este reconhecimento não foi observado quando as amostras foram analisadas pelo soro pré-imune dos animais teste, sugerindo um reconhecimento específico dos anticorpos produzidos. / Two peptides, designated Let#1 and Let#2, were outlined from the second extracellular loop of the NaPi2b protein. After conjugation, the peptides induced the production of specific antibodies in rabbits and mice. Antibodies with higher titers were selected for complementary tests. These antibodies recognized peptides conjugated to the carrier by ELISA and WB, with no cross-reaction when tested with the free carrier protein and nonspecific synthetic antigen. After complete validation, it was observed that the anti-peptide antibodies recognize native NaPi2b protein present on the OV-CAR cell surface and showed recognizing a common target in all sera tested, even when the serum was considered negative for ovarian carcinonoma by commercial test. This recognition was not observed when the samples were analyzed by pre-immune serum of test animals, suggesting specific recognition of antibodies produced.

O câncer de ovário (OvCa) se destaca dentre as neoplasias ginecológicas por ser um dos mais letais e de difícil diagnóstico. O OvCa ocorre devido ao acúmulo de alterações celulares progressivas promovidas por mutações no genoma de uma célula que, consequentemente, alteram as complexas vias de regulação celular que respondem a fatores internos, como reprogramação genética, ou externos, como a resposta a fatores de crescimento, que juntamente com outras alterações moleculares favorecem a progressão e a metástase. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem um caráter mais invasivo e migratório, além de se tornarem mais resistentes às drogas. A desregulação de fatores de transcrição como ZEB1, TWIST e SNAI1, vias de sinalização, microRNAs e fatores de crescimento incluindo EGF, TGF? e HGF podem desencadear a EMT. Após a eficiente indução da EMT com EGF na linhagem epitelial de adenocarcinoma de ovário humano Caov-3, foi realizada a análise proteômica quantitativa detalhada, baseada na análise de frações subcelulares enriquecidas em proteínas de membrana, citosol e núcleo, obtidas por centrifugação diferencial e subsequente fracionamento de proteínas por SDS-PAGE, a fim de compreender mais profundamente os mecanismos moleculares modulados pela EMT no OvCa. A partir da análise dos dados coletados em um sistema de espectrometria de massas de alta resolução acoplados a cromatografia líquida (LCMS/MS) e com o auxílio da bioinformática foram identificadas redes de interação proteína-proteína diferencialmente expressas, relacionadas principalmente com a regulação do ciclo celular e do metabolismo. A indução da EMT por EGF resultou na ativação de importantes vias de sinalização, tais como PI3K/Akt/mTOR e Ras/MAPK Erk, além da parada do ciclo celular na fase G1 regulada pelo aumento dos níveis de p21Waf1/Cip1, independentemente de p53, e diminuição de proteínas checkpoint. Através da proteômica dirigida, o monitoramento de reações múltiplas (MRM) revelou que, após a indução da EMT por EGF, o metabolismo das células Caov-3 foi alterado de uma maneira bastante peculiar. O estudo proteômico descrito permitiu a correlação entre processo da EMT induzido por EGF com o controle translacional, a regulação do ciclo celular e a alteração do metabolismo energético. / Ovarian cancer (OvCa) stands out among gynecological malignancies for being one of the most lethal and difficult to diagnose. OvCa occurs due to the accumulation of progressive cell changes promoted by mutations in the cell genome which, consequently, alter the complex cellular regulation pathways that respond to internal factors, such as genetic reprogramming, or external, such as response to growth factors, which together with other molecular changes favor the progression and metastasis. An important step of the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of epithelial phenotype and acquisition of mesenchymal phenotype by tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. Deregulation of transcription factors such as ZEB1, TWIST and SNAI1, signaling pathways, microRNAs and growth factors including EGF, TGF? and HGF can trigger EMT. After an efficient EMT induction by EGF in the epithelial cell line of human adenocarcinoma ovarian Caov-3, detailed quantitative proteomic analysis was performed based on analysis of subcellular fractions enriched in proteins from membrane, cytosol and nucleus, obtained by differential centrifugation and subsequent fractionation of proteins by SDS-PAGE, in order to understand deeply the molecular mechanisms modulated by EMT in OvCa. From the analysis of data collected in a highresolution mass spectrometry system coupled to liquid chromatography (LC-MS/MS) and with the aid of bioinformatics were identified protein-protein interaction networks differentially expressed, mainly related to regulation cell cycle and metabolism. EGF induced-EMT resulted in the activation of major signaling pathways such as PI3K/Akt/mTOR and Ras/MAPK Erk, in addition to G1 phase cell cycle arrest regulated by increased levels of p21Waf1/Cip1, regardless of p53, and reduction of checkpoint proteins. Through the targeted proteomics, multiple reaction monitoring (MRM) showed that after EGF induced-EMT, Caov-3 cells metabolism was changed in a very particular way. The proteomic study described allowed the correlation between EMT process induced by EGF with translational control, regulation of cell cycle and the change in the energy metabolism.

O câncer de ovário (OvCa) se destaca dentre as neoplasias ginecológicas por ser um dos mais letais e de difícil diagnóstico. O OvCa ocorre devido ao acúmulo de alterações celulares progressivas promovidas por mutações no genoma de uma célula que, consequentemente, alteram as complexas vias de regulação celular que respondem a fatores internos, como reprogramação genética, ou externos, como a resposta a fatores de crescimento, que juntamente com outras alterações moleculares favorecem a progressão e a metástase. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem um caráter mais invasivo e migratório, além de se tornarem mais resistentes às drogas. A desregulação de fatores de transcrição como ZEB1, TWIST e SNAI1, vias de sinalização, microRNAs e fatores de crescimento incluindo EGF, TGF? e HGF podem desencadear a EMT. Após a eficiente indução da EMT com EGF na linhagem epitelial de adenocarcinoma de ovário humano Caov-3, foi realizada a análise proteômica quantitativa detalhada, baseada na análise de frações subcelulares enriquecidas em proteínas de membrana, citosol e núcleo, obtidas por centrifugação diferencial e subsequente fracionamento de proteínas por SDS-PAGE, a fim de compreender mais profundamente os mecanismos moleculares modulados pela EMT no OvCa. A partir da análise dos dados coletados em um sistema de espectrometria de massas de alta resolução acoplados a cromatografia líquida (LCMS/MS) e com o auxílio da bioinformática foram identificadas redes de interação proteína-proteína diferencialmente expressas, relacionadas principalmente com a regulação do ciclo celular e do metabolismo. A indução da EMT por EGF resultou na ativação de importantes vias de sinalização, tais como PI3K/Akt/mTOR e Ras/MAPK Erk, além da parada do ciclo celular na fase G1 regulada pelo aumento dos níveis de p21Waf1/Cip1, independentemente de p53, e diminuição de proteínas checkpoint. Através da proteômica dirigida, o monitoramento de reações múltiplas (MRM) revelou que, após a indução da EMT por EGF, o metabolismo das células Caov-3 foi alterado de uma maneira bastante peculiar. O estudo proteômico descrito permitiu a correlação entre processo da EMT induzido por EGF com o controle translacional, a regulação do ciclo celular e a alteração do metabolismo energético. / Ovarian cancer (OvCa) stands out among gynecological malignancies for being one of the most lethal and difficult to diagnose. OvCa occurs due to the accumulation of progressive cell changes promoted by mutations in the cell genome which, consequently, alter the complex cellular regulation pathways that respond to internal factors, such as genetic reprogramming, or external, such as response to growth factors, which together with other molecular changes favor the progression and metastasis. An important step of the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of epithelial phenotype and acquisition of mesenchymal phenotype by tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. Deregulation of transcription factors such as ZEB1, TWIST and SNAI1, signaling pathways, microRNAs and growth factors including EGF, TGF? and HGF can trigger EMT. After an efficient EMT induction by EGF in the epithelial cell line of human adenocarcinoma ovarian Caov-3, detailed quantitative proteomic analysis was performed based on analysis of subcellular fractions enriched in proteins from membrane, cytosol and nucleus, obtained by differential centrifugation and subsequent fractionation of proteins by SDS-PAGE, in order to understand deeply the molecular mechanisms modulated by EMT in OvCa. From the analysis of data collected in a highresolution mass spectrometry system coupled to liquid chromatography (LC-MS/MS) and with the aid of bioinformatics were identified protein-protein interaction networks differentially expressed, mainly related to regulation cell cycle and metabolism. EGF induced-EMT resulted in the activation of major signaling pathways such as PI3K/Akt/mTOR and Ras/MAPK Erk, in addition to G1 phase cell cycle arrest regulated by increased levels of p21Waf1/Cip1, regardless of p53, and reduction of checkpoint proteins. Through the targeted proteomics, multiple reaction monitoring (MRM) showed that after EGF induced-EMT, Caov-3 cells metabolism was changed in a very particular way. The proteomic study described allowed the correlation between EMT process induced by EGF with translational control, regulation of cell cycle and the change in the energy metabolism.

The aim of this thesis is to investigate the role of inflammatory infiltrates and chemokines in metastasis of high-grade serous ovarian cancer, HGSC, to the omentum using human tissue biopsies and a 3- dimensional (3D) cell culture model. In ten patients with metastatic HGSC, omental tumour deposits contained a prominent leukocyte infiltrate of CD3+ T cells (9% of total cells) and CD68+ macrophages (11% of total cells). The presence of CD68+ macrophages showed a significant positive correlation with tumour cell proliferation analysed by Ki67 expression. Four ovarian cancer cell lines were co-cultured on a 3D model mimicking the microenvironment of the omentum for two weeks. The model was composed of collagen embedded human fibroblasts covered in a confluent layer of human primary mesothelial cells. The mesothelial cells in the 3D model significantly increased the growth (p = 0.002) and invasion (p = 0.0004) of the ovarian cancer cells. CXCL12 is the macrophage chemoattractant and ligand for the major chemokine receptor expressed on ovarian cancer cells. An association between CXCL12 and extracellular matrix remodelling was identified in two independent gene expression microarrays of ovarian cancer biopsies. The expression of CXCL12 in the HGSC omental metastases measured by quantitative Real Time-PCR positively correlated with decorin expression. Antibody mediated neutralisation of CXCL12 reduced growth (p = 0.012) and invasion (p = 0.029) in the 3D model. Mimicking an infiltrate of CD68+ macrophages in this multicellular 3D in vitro system also produced measurable changes in inflammatory cytokine and chemokine expression. There is currently a demand for more accurate models of HGSC and a necessity to study its metastasis that presents itself as the major clinical problem in patients. Therefore the development of this 3D model to mimic tumour-promoting inflammation in HGSC metastasis will provide researchers with an essential tool for testing novel therapeutic strategies.