Chemoresistance remains a major clinical obstacle to effective management of ovarian cancer. Cancer stem cells (or tumor-initiating cells, TICs) have been discovered recently, and have played a pivotal role in changing the view of cancer development; however, the molecular mechanisms by which these cells escape conventional therapies remain elusive. In this study, TICs were isolated from ovarian cancer cells as tumor spheres with specific stem properties under TIC-selective conditions. Unlike non-TICs, TICs strongly express stem cell factor (SCF) and c-Kit. Blocking SCF-c-Kit by SCF neutralizing antibodies, c-Kit small interfering RNA (siRNA) or imatinib (Gleevec), a clinical drug that inhibits c-Kit signaling, significantly inhibited TIC proliferation. Although cisplatin and paclitaxel killed the non-TICs, they did not eliminate TICs. Importantly, the combination of cisplatin/paclitaxel with c-Kit siRNA or imatinib inhibited the growth of both non-TICs and TICs. Similar results were obtained when patient-derived TICs were used. The findings also indicate that tumor-predisposing microenvironment, such as hypoxia, may promote ovarian TICs through upregulating c-Kit expression. Furthermore, I have showed that c-Kit expression induced activation of Phosphatidylinositol 3-kinases (PI3K)/Akt, -catenin, and ATP-binding cassette G2, which could be reversed by treatment with the PI3K/Akt inhibitor or -catenin siRNA. I further studied potential gene expression in TICs using cDNA and microRNA (miRNA) microarrays. The result from these microarrays provided a general profile in gene expression of TICs compared with the bulk tumor cells. In particular, let-7a, b, and c were shown to be downregulated in TICs compared to bulk tumor cells, suggesting that their loss may contribute to ovarian cancer development. Together, this study reveals a previously undescribed therapeutic effect of SCF-c-Kit signaling blockade to prevent ovarian cancer progression by eliminating TICs and the altered genes or miRNAs may represent possible molecular targets. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Ovarian cancer is the most lethal gynecological cancer. This is mainly due to widespread peritoneal dissemination and malignant ascites, in which angiogenesis, the formation of new blood vessels, is critical to both ascites development and its metastasis. Loss of E-cadherin is a well-established marker that characterizes the progression of metastatic tumors, including ovarian cancer. The release of a soluble form of E-cadherin (sE-cad) has been frequently associated with a rapid reduction of functional E-cadherin at the cell surface. Importantly, sE-cad is significantly present in ascites from women with stage III/IV ovarian cancer when compared to women with benign ovarian cysts. However, despite the clinical significance, most studies have focused on its role in weakening cell-cell adhesion, whether sE-cad itself has any biological function is not fully understood. Here it is shown for the first time a potent angiogenic role for sE-cad released from ovarian carcinoma. Soluble form of E-cadherin promoted the migration, permeability, and tubulogenesis of endothelial cells. These activities were also observed with a sE-cad/Fc chimera, and targeted inhibition using E-cadherin blocking antibodies completely prevented the sE-cad mediated effects. In addition, it was further revealed that sE-cad could be released from ovarian cancer cells in form of exosomes, a form of extracellular vesicles that play an important role in distant intercellular communication. sE-cad-positive exosomes were able to stimulate the angiogenic phenotype in vitro and functional neovascularization in a Matrigel implant model in vivo. The use of E-cadherin blocking antibodies resulted in diminished angiogenesis, confirming that the effect was sE-cad-positive exosomes specific. In search of the underlying mechanism by which sE-cad-positive exosomes promoted angiogenesis in endothelial cells which lacked E-cadherin, sE-cad was found to heterodimerize with VE-cadherin. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector β-catenin, but not p120 catenin. Similarly, the angiogenic phenotype could be reversed by inhibition of VE-cadherin, PI3K/Akt and β-catenin. A mass spectrometric proteomic analysis of the isolated exosomes revealed distinct membrane-bound proteases, especially disintegrin and metalloproteinase 10 (ADAM10) and matrix metalloproteinase 25 (MMP25) commonly associated with ovarian cancer progression, are implicated in sE-cad production. Small interfering RNA-mediated down-regulation of ADAM10 and MMP25 significantly inhibited sE-cad production. Moreover, hepatocyte growth factor, a multifaceted cytokine which is frequently elevated in ovarian cancer ascites, was shown to increase the expression of ADAM10 and MMP25 concomitant with an elevated level of sE-cad. Together, these results uncover a novel angiogenic role of sE-cad and a new mechanism of the action of sE-cad in tumor progression. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Epithelial ovarian cancer is a common female malignancy with a relatively high mortality rate worldwide. This may due to a lack of efficient diagnostic methods at early stage and worsen by complications caused by metastasis at advanced stage. For successful metastasis, cancer cells detached from the original growing sites have to survive in the body circulation before conquering a distant location within the body. Resistance to anoikis (apoptosis induced without appropriate extracellular matrix) is therefore utmost important during metastatic spreading. In addition, a pre-metastatic niche remodeled by cancer cells is also a pre-requisite for metastatic colonization. Emerging evidence has suggested the dysregulation of miRNAs is associated with different aspects of tumorigenesis. However, the specific roles of miRNAs in anoikis resistance and in remodeling of distant niche remain unknown thus far. This study attempted to investigate the functional roles of miR-141, in particularly anoikis resistance of ovarian cancer cells and the reprogramming of stromal cells. The miR-200 family is frequently upregulated and associated with human cancer metastasis. In this study, by cDNA array profiling together with biochemical and functional studies, miR-141, a member of miR-200 family, was identified as an oncomiR enhancing cell viability in low serum medium and anoikis resistance. Moreover, enforced expression of miR-141 led to bigger tumor sizes and promoted metastatic colonization in mouse models. Further studies demonstrated miR-141 directly targets tumor suppressive KLF12 in ovarian cancer cells, depletion of KLF12 could mimic function of miR-141. Clinical study revealed the upregulated miR-141 was significantly correlated with the downregulated KLF12, serous subtype, advanced and distant metastatic ovarian cancer. Furthermore, Genechip profiling, Human Apoptosis Array and Luciferase reporter assay revealed the upregulated miR-141 and downregulated KLF12 enhanced anoikis resistance via elevation of survivin which protect cells against intrinsic apoptotic activity. On the other aspect, miR-141 was found to be a secretary miRNA and commonly detected in the serum of ovarian cancer patients. The upregulated miR-141 expression was also correlated to levels of common cancer biomarker CA125. Importantly, the serum miR-141 level was significantly correlated with the tumor burden of patients during treatments, indicating it could be used as a non-invasive biomarker for ovarian cancers. Finally, based on miR-141 as tumor-secreted and circulated miRNA, a series of functional studies demonstrated that miR-141 could be transferred to hFF-1 fibroblast cells. Intriguingly, ovarian cancer cells cultured in miR-141-fibroblast culturing medium showed a remarkable increase of cell migration, suggesting that the remodeled-miR-141 fibroblast cells can secrete stimulating factors and promote ovarian cancer cells aggressiveness. This is the first study showing miR-141 could reprogram fibroblast cells to be a niche for ovarian cancer cell dissemination and metastatic progression. However, further investigations for verifying such functions are warranted. In conclusion, this study provides strong evidence that miR-141 is oncoMir enhancing ovarian cancer cell plasticity in metastasis e.g. anoikis resistance. Moreover, the finding of secretary form miR-141 not only gives the feasibility to be a potential biomarker for detecting ovarian cancer but also shows a possible mechanism of how miRNAs reprogram the distant niche for metastatic colonization. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

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Every year more than 10 million people develop cancers globally. Ovarian cancer, specifically, results in more than 22,000 new cases and 16,000 deaths from this disease yearly, more than any other cancer of the female reproductive system. In addition, because of non-specific symptoms and poor screening techniques, most ovarian cancer cases are discovered after the disease is in an advanced state. Consequently, aggressive and effective treatment options that incur minimal toxic effects to healthy tissue are in great need. In the present research, stealth biodegradable nanoparticles were developed as vehicles for the controlled and targeted delivery of chemotherapeutic agents for the treatment of ovarian cancer. The design of this delivery system consisted of nanoparticles of biodegradable polymers of the poly(lactic-co-glycolic acid) family loaded with the chemotherapeutic agent doxorubicin or the imaging agents rhodamine 6G, indocyanine green or gadopentetic acid. Nanoparticles were modified by incorporation of functional poly(ethylene glycol) on their surface to improve the stability of the colloidal suspension, increase their circulation lifetime in vivo, and provide a site for conjugation of targeting agents specific to ovarian tissue. Various methods were evaluated for this surface modification, including the use of polymer blends, the chemical conjugation of the polymers, and the polymerization of lactide and glycolide monomers initiated by heterofunctional poly(ethylene glycol). Nanoparticles incorporating poly(ethylene glycol) presented improved characteristics compared to unmodified particles including smaller size, higher stability and slower release of the chemotherapeutic agent doxorubicin The actual drug or agent content was decreased in the case of doxorubicin and rhodamine, but increased for indocyanine green as a result of improved agent-polymer interactions. Poly(ethylene glycol)-containing nanoparticles were conjugated to monoclonal antibody mAb106-105, which is specific to the extracellular domain of human folliclestimulating hormone (FSH) receptors. These receptors are only expressed in ovarian cells in women, thus providing a system that is highly specific to ovarian tissue. The interaction and therapeutic potential of nanoparticles with or without targeting antibodies were tested on OVCAR-3, Caov-3, and MDA-MB-231 cancer cells.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Ovarian cancer is the fifth leading cause of cancer death among women in the U.S. and kills approximately 14,000 women each year (Nezhat et al., 2015). Survival increases with early diagnosis; the five-year survival rate in stage I is 90%. Symptoms are vague and common to many health diseases, which may well explain why upwards of 70% of women with ovarian cancer are diagnosed at stage III or IV (Slatnik & Duff, 2015). Preventative guidelines in the U.S. do not recommend screening for ovarian cancer in women of average risk (AAFP, 2016b; ACOG, 2011; Doubeni et al., 2016; Moyer, 2012; NCCN, 2015; Qaseem et al., 2014; Wilt et al., 2015). A lack of screening recommendations and a subtle presentation point to the need for greater healthcare professional recognition of symptoms and risk factors of ovarian cancer, which can then lead to a prompt diagnosis. While healthcare professionals have the opportunity to improve women’s health, gaps in knowledge exist related to ovarian cancer risk factors and symptom recognition (Gajjar et al., 2012). Continuing education improves healthcare professionals’ performance and patient health outcomes (Cervero & Gaines, 2015). Increasing healthcare professionals’ knowledge of ovarian cancer may help to detect ovarian cancer in earlier stages and enhance health outcomes of women. Based on the need for an increase in awareness and knowledge among healthcare professionals, a local ovarian cancer conference was developed and offered to healthcare professionals. The conference focused on presenting ovarian cancer risk factors and symptoms. Attendees were provided with an ovarian cancer resource for patient education. The conference was evaluated through pretests and posttests and a conference evaluation survey. Data was collected the evening of the conference with 29 attendees responding. After the conference, correct responses increased in the areas of risk factor and symptom recognition. The number of correct responses increased from 106 on the pretest to 122 on the posttest. In regards to ability to educate women about ovarian cancer, 62% of respondents indicated that they were “very confident” in their ability. / Pam Solseng Ovarian Cancer Endowment Fund

Epithelial ovarian cancer is a leading fatal malignancy in women. The high mortality rate of this cancer is due to the poor prognosis and that the majority of patients are diagnosed at late stage. Therefore, a combination of cytoreduction and adjuvant chemotherapy is the only choice for this disease at late stage. Platinum-based chemotherapy regimens are the first line treatment for ovarian cancer. However, the repetitive challenges of platinum-based agents and the frequent relapse cause acquired chemoresistance which is the major obstacle for clinical management of this disease. The underlying molecular mechanism for acquired chemoresistance remains largely unclear. Therefore, understanding the molecular mechanism in acquired chemoresistance of ovarian cancer is urgently needed. Emerging evidence has suggested that dysregulation of miRNAs is significantly involved in acquired chemoresistance in human cancers. In this study, the expression status and functional roles of miR-199b-5p were characterized in the chemoresistance of ovarian cancer. By miRCURY LNA™ miRNA array, miR-199b-5p was one of the downregulated miRNAs identified in acquired chemoresistant ovarian cancer cells. Further Q-PCR analysis found that miR-199b-5p exhibited a pattern of progressive reduction from early to advanced stage and from low to high grade ovarian cancer tissue samples (N=79). Interestingly, the expression of miR-199b-5p could be restored upon 5-Aza-2’-deoxycytidine (5-Aza-dC) treatment in ovarian cancer cells. Methylation-specific PCR and bisulfite genomic sequencing revealed that the promoter region of miR-199b-5p (Chromosome9q34) was frequently hypermethylated in chemoresistant ovarian cancer cells. Functionally, enforced expression of miR-199b-5p remarkably diminished the cisplatin-resistance in miR-199b-5p low expressing ovarian cancer cells, whereas depletion of miR-199b-5p by siRNA approach augmented cisplatin-resistance in miR-199b-5p high expressing ovarian cancer cells. A tumor xenograft mouse model further confirmed that miR-199b-5p could sensitize ovarian cancer cells to cisplatin-mediated tumor growth inhibition in vivo. On the other aspect, computational analysis plus luciferase reporter assay and western blotting identified JAG1, a key ligand of Notch1 signaling frequently associated with chemoresistance of human cancers, as a direct target of miR-199b-5p in ovarian cancer cells. Intriguingly, an inverse relationship between the downregulation of miR-199b-5p and the upregulation of JAG1 was found by in situ hybridization (ISH) and immunohistochemical (IHC) analyses in a commercial ovarian cancer tissue array (OVC1021). Functionally, enforced expression of JAG1 increased cisplatin resistance of ovarian cancer cells. In contrast, depletion of JAG1 by siRNA approach abrogated the cisplatin-resistance of ovarian cancer cells. This finding was in consistent with the effect of enforced expression of miR199b-5p in cisplatin-resistant ovarian cancer cells. Mechanistically, the Notch1 luciferase reporter assay and western blotting analysis demonstrated that the Notch1 signaling activity could be activated by JAG1 or conversely be inhibited by enforced expression of miR-199b-5p. Further investigation using the Notch specific inhibitor γ-secretase showed that JAG1-Notch1 signaling is a crucial pathway associated with chemoresistance of ovarian cancer in vitro and in vivo. Taken together, this is the first study showing that the epigenetic silencing of miR-199b-5p leads to aberrant activation of JAG1-Notch1 signaling and such signaling cascade plays a critical role in tumor progression and chemoresistance of ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

[Truncated abstract] Ovarian cancer is currently the leading cause of death from gynaecological malignancies in women from developed countries. Serous ovarian cancer is the most prevalent type of all ovarian cancers, with the majority diagnosed in an advanced stage where treatment efficacy is reduced and patient survival is poor. Because of this fact, the development of improved detection and treatment strategies are necessary, with much research focussing on the complex molecular pathways involved in ovarian tumour growth as one potential avenue for intervention. Apoptosis, or programmed cell death, is one such area of investigation because currently successful cancer treatments induce apoptosis in tumour cells. Molecular analysis of apoptosis in both normal tissue and tumours has established a positive relationship between increased expression of secreted frizzled-related protein 4 (SFRP4) and apoptosis, however to date, very little research has focussed on the role of this gene in the ovary . . . An examination of SFRP4 and β-catenin expression in 163 primary serous ovarian carcinomas revealed high SFRP4 expression was associated with low β-catenin expression and conversely, low SFRP4 was associated with high β-catenin expression in the majority of the ovarian tumours analysed, reinforcing the inverse relationship observed in the ovarian cell lines. A positive trend was observed between cancer stage and the expression level of these proteins, with increased SFRP4 expression and reduced β-catenin expression as cancer stage increased. Additionally, patient survival revealed a trend towards increased survival among ovarian cancer patients who had tumours expressing low levels of SFRP4. Taken together, the novel findings of this study indicate that the increased expression of SFRP4 observed in a large proportion of serous ovarian cancers is a cellular response to down-regulate the level of β-catenin, and thus an attempt to maintain cellular homeostasis by counteracting the excessive proliferating signals present in these tumour cells.

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy