京都大学 / 新制・課程博士 / 博士(医学) / 甲第23801号 / 医博第4847号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 竹内 理, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23759号 / 医博第4805号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 万代 昌紀, 教授 松村 由美, 教授 古川 壽亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ovarian cancer is the fifth leading cause of all cancer deaths in women and the most lethal gynecologic cancer in the United States. During metastasis, cancer cells exfoliate from the primary tumor and aggregate to form spheroids, enhancing their survival within the peritoneal cavity during dissemination to a secondary outgrowth site. The inability of removal of these aggregates by traditional surgical interventions may contribute to the high recurrence and mortality rate of ovarian cancer diagnosed at late stages. Obesity, particularly abdominal obesity, has been shown to increase ovarian cancer risk and decrease survival. The recruitment of stromal vascular fraction (SVF) present in adipose tissue represents a growth and proliferation advantage to ovarian tumors, and endogenous sphingolipids like sphingosine-1-phosphate are increased in ovarian cancer patients. These conditions, combined with the physiological conditions within malignant ascites (hypoxia and low glucose), represent a physiological environment that can impact the metabolic responses of ovarian cancer spheroids. Here, we investigated the metabolic adaptations of serous ovarian cancer cells across the metastatic cycle and in conditions that mimic those of the peritoneal cavity and malignant ascites. We first investigated the different in metabolic responses between adherent monolayers and 3D spheroids. We confirmed that spheroids have a reduced metabolic rate and drug response that is affected by the incorporation of obese SVF into aggregates. To investigate these changes in the next stages of the metastatic cycle, we used time trials to observe how adherence of spheroids to a secondary site changes metabolic response and substrate utilization in physiological conditions. Adhesion of spheroids showed changes in energy metabolism and substrate utilization, switching from mainly glutamine oxidation to glucose oxidation that could support successful outgrowth. Spheroids also were resilient to culture conditions, even non-permissive conditions such as those found in the peritoneal cavity. Finally, we utilized human malignant ascites from ovarian cancer patients as a further investigation into conditions that imitate in vivo characteristics that could affect spheroid metabolism. Exposure to malignant ascites reduced spheroid viability as well as basal respiration and ATP synthesis. However, spare respiratory capacity was increased, and human spheroids changed their substrate utilization in response to ascites. Taken together, these studies provide an identification of metabolic switches across different stages of ovarian cancer metastasis that contribute to their survival, which represents an emerging target for prevention and treatment for individuals with ovarian cancer. / Doctor of Philosophy / Ovarian cancer is the deadliest reproductive cancer in women, and most women who are diagnosed will die from the disease because of its high recurrence rate and because it is typically detected at late stages. When ovarian cancer metastasizes, cells or cell clusters from the original tumor aggregate together to form balls of cells called spheroids, which move through the abdominal cavity to other sites to grow additional tumors. These spheroids are thought to contribute to recurrence of this cancer, since they cannot be removed by surgery. As these spheroids move through the abdominal cavity, they are exposed to an environment that has a low amount of oxygen and glucose. These spheroids may also be exposed to bioactive lipids and cells from the adipose tissue called stromal vascular fraction, both of which are related to obesity and may help cancer spheroids survive. The survival of these spheroids is in part related to how their metabolism functions, which may help them make energy and the building blocks needed to continue growing and form successful secondary tumors. Identifying how these spheroids change their metabolism at different points during the disease may help identify points that can be targeted to prevent changes in metabolism that could support their growth. This dissertation identified metabolic changes that occur in spheroids, in conditions that are similar to those spheroids would be exposed to in an abdominal cavity. First, we compared single layers of cells to spheroids and found that spheroids had a lower metabolic rate and lower drug response, which may help them survive in the abdominal cavity. Next, we allowed the spheroids to lay down and grow out, like they would when they found a new location during metastasis, to see how their metabolism changed and what substances they used to make energy in conditions that mimicked the abdominal cavity. As spheroids adhered, they changed their energy metabolism and switched the substances they used to make energy, all while continuing to survive and grow out even in conditions that were not supportive. These switches could help them grow out and successfully metastasize. Lastly, we used ascites fluid from human ovarian cancer patients and treated spheroids with this to see how their metabolism changed in response. While some aspects of metabolism and survival was reduced, their ability to increase their metabolism when stressed increased and human spheroids used nutrients to make energy differently. Overall, we show that across the stages of metastasis, ovarian cancer spheroids can change their metabolism in response to their environment. Identifying these metabolic switches helps us understand how successful metastasis happens, and can inform future targets to slow or prevent metastasis, prolonging the life of women who have been diagnosed with ovarian cancer.

Academic: Ovarian cancer is the leading cause of death among female gynecologic cancers. Current treatments include surgical debulking, and chemotherapy. However, better interventions are needed to reduce the mortality rate of metastatic disease. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aimed to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease. Spheroids of both homogeneous and heterogeneous composition demonstrated overall lower metabolic capacity than their adherent counterparts. Spheroids had a lower basal energetic demand than adherent cells, paralleled by lower maximal respiration capacity, glycolytic capacity, and spare respiratory capacity. We conclude that the lower energetic demand of spheroids may be a mechanism to prolong death by reserving energy and metabolic cellular processes; this may render anti-metabolic drug treatment with AICAR or metformin ineffective against disseminating ovarian cancer aggregates. General: Ovarian cancer is currently the leading cause of death among female gynecologic cancers. While treatments exist, better interventions are needed to reduce the mortality rate in this form of cancer. Ovarian cancer cells have displayed the ability to aggregate and form 3D homogeneous and heterogeneous spheroids, which can function as micrometastases. Ovarian cancer spheroids survive independently prior to adhering to an endothelial tissue. Since aggregation has been shown to provide a survival advantage to the spheroids and increased their aggressive phenotype, this study aims to investigate how the metabolism of ovarian cancer cells change in 3-dimensional (3D) culture. Examining metabolic pathways and identifying markers of metabolic change could provide the scientific base for new, targeted interventions for this disease. / Master of Science

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the insufficient accurate screening programs for the early detection of EOC. To improve the accuracy of the early detection, there is a need to deeply understand the mechanism of EOC progression and the interaction between cancer cells with their unique microenvironment. Therefore, this work investigated the metabolic shift in the mouse model for progressive ovarian cancer, and evaluated the effects of hypoxic environment, spheroid formation as well as stromal vascular fractions (SVF) on the metabolic shift, proliferation rate, drug resistance and protein markers in functional categories. The results demonstrated an increasingly glycolytic nature of MOSE cells as they progress from a tumorigenic (MOSE-L) to a highly aggressive phenotype (MOSE-FFL), and also showed changes in metabolism during ovarian cancer spheroid formation with SVF under different oxygen levels. More specifically, the hypoxic environment enhanced glycolytic shift by upregulating the glucose uptake and lactate secretion, and the spheroid formation affected the cellular metabolism by increasing the lactate secretion to acidify local environments, modulating the expression of cell adhesion molecules to enhance cell motility and spheroids disaggregation, and up-regulating invasiveness markers and stemness makers to promote ovarian cancer aggressive potential. Hypoxia and spheroid formation decreased ovarian cancer cells growth but increased the chemoresistance, which leads to the promotion of aggressiveness and metastasis potential of ovarian cancer. SVF co-cultured spheroids further increased the glycolytic shift of the heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer. / Master of Science

Ovarian cancer is known as the "silent killer," due to its late diagnosis and frequent recurrence after initial treatment.  Finding a new way to diagnose and treat ovarian cancer in conjunction with current therapies is paramount.  By capitalizing on metabolic changes that occur during cancer progression, interventions can be developed.  The Nobel laureate Otto Warburg is credited with discovering an altered metabolic state within cancer cells known as the Warburg effect.  In the Warburg effect, cancer cells participate in an increased rate of aerobic glycolysis with an excess secretion of lactate, allowing for carbon flux into biosynthetic pathways.  Exactly which metabolic pathways are altered in ovarian cancer and at which stage in the progression of ovarian cancer they are occurring was unknown.  Therefore using the recently established mouse ovarian surface epithelial (MOSE) progression model, we were able to measure metabolic changes in varying states of disease and levels of aggressiveness.  As cells progressed from a benign early stage (MOSE-E), through a transitional intermediate stage (MOSE-I), to an aggressive late stage (MOSE-L), the MOSE cells became more glycolytic and lipogenic, establishing the MOSE model as a valuable model for studying ovarian cancer metabolism.  Treating the MOSE cells with the naturally occurring chemotherapeutic agent sphingosine decreased p-AKT  protein levels in the cell, decreased the glycolytic rate and decreased de novo cholesterol synthesis.  Cancer stem cells are known to be resistant to chemotherapy treatments and targeting their metabolism may be promising for combinatorial treatments.  Therefore, the metabolism of highly aggressive tumor-initiating cells (TIC), harvested from ascites of C57Bl/6 mice injected with MOSE-L cells were characterized.  Although the basal metabolism of the TICs was similar to the MOSE-L cells, TICs were more resistant to cell death as a consequence of external stresses and substrate depletion.  The TICs could also up-regulate oxygen consumption rate (OCR) when uncoupled and increase glycolysis when ATP Synthase was inhibited, highlighting their resiliency.  Taken together, we have identified targets for treatment strategies that could suppress the growth of primary tumors and may be effective against TICs, thereby suppressing tumor recurrence and possibly prolonging the life of women with ovarian cancer. / Ph. D.

Cancer research is evolving. Historically concerned with the mechanisms by which malignant cells circumvent cell death signaling and maintain unchecked proliferation, focus has shifted to the complex interactions between the tumor cell and the surrounding microenvironment. Ovarian cancer has one of the highest incidence-to-death ratios of all cancers, and is typically asymptomatic until the later stages, often resulting in metastasis prior to discovery. Naturally occurring phenotypes like lactation and child-bearing (parity) reduce ovarian cancer incidence, but the mechanisms are not understood. As the primary site for ovarian cancer metastasis, and a secondary lymphoid organ capable of mounting potent innate and adaptive immune responses, we believe the omental fat band (OFB) provides a unique opportunity to study complex interactions within the tumor microenvironment. Additionally, we hypothesize that once understood, leukocyte populations within the OFB could be modulated to disrupt the pro-tumorigenic cascade. Using fluorescence-activated cell sorting (FACS) and quantitative realtime PCR (qRT-PCR), we comparatively evaluated the changes in the compositional immune profile of the OFB as a result of parity and cancer. Parous mice were associated with a reduction in macrophages and neutrophils in the OFB, resulting in an inherent "protective state" that was refractory to metastatic cancer cell growth after intraperitoneal implantation. This indicates that the leukocyte populations within the   OFB play an important role in tumor development. Therefore we utilized the potent TH1-type immunomodulatory cytokine IL-12 in a membrane-bound form to circumvent reported side effects, such as hepatic and renal damage, cardiotoxicity and death. Targeted IL-12 delivery to the OFB resulted in delayed disease development, although not protection from subsequent challenge. This was also associated with a reduction tumor-associated macrophages (TAMs) and neutrophils (TANs) within the OFB. Kinetic studies demonstrated that these changes were induced by a significant reduction in neutrophil and macrophage chemoattractants early on in the pro-tumorigenic cascade (7 days post-implantation). This work demonstrates that the OFB is a functionally plastic tissue that can be harnessed and re-mobilized to display an anti-tumorigenic microenvironment. / Ph. D.

Yes / HOX genes are vital for all aspects of mammalian growth and differentiation, and their dysregulated expression is related to ovarian carcinogenesis. The aim of the current study was to establish the prognostic value of HOX dysregulation as well as its role in platinum resistance. The potential to target HOX proteins through the HOX/PBX interaction was also explored in the con-text of platinum resistance. HOX gene expression was determined in ovarian cancer cell lines and primary EOCs by QPCR, and compared to expression in normal ovarian epithelium and fallopian tube tissue samples. Statistical analysis included one-way ANOVA and t-tests, using statistical software R and GraphPad. The analysis identified 36 of the 39 HOX genes as being overex-pressed in high grade serous EOC compared to normal tissue. We detected a molecular HOX gene-signature that predicted poor outcome. Overexpression of HOXB4 and HOXB9 was identified in high grade serous cell lines after platinum resistance developed. Targeting the HOX/PBX dimer with the HXR9 peptide enhanced the cytotoxicity of cisplatin in platinum-resistant ovarian cancer. In conclusion, this study has shown the HOX genes are highly dysregulated in ovarian cancer with high expression of HOXA13, B6, C13, D1 and D13 being predictive of poor clinical outcome. Targeting the HOX/PBX dimer in platinum–resistant cancer represents a potentially new therapeutic option that should be further developed and tested in clinical trials. / This research was supported by GRACE, a gynaecological charity based in Surrey, UK.

Yes / Background: Epithelial ovarian cancer is a common malignancy, with no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We evaluated the expression of EN2 in Epithelial ovarian cancer, and reviewed its role as a biomarker. Methods: We evaluated 8 Epithelial ovarian cancer cell lines, along with > 100 surgical specimens from the Royal Surrey County Hospital (2009–2014). In total, 108 tumours and 5 normal tissue specimens were collected. En2 mRNA was evaluated by semi-quantitative RT-PCR. Histological sub-type, and platinum-sensitive/−resistant status were compared. Protein expression was assessed in cell lines (immunofluorescence), and in > 150 tumours (immunohistochemistry). Results: En2 mRNA expression was elevated in serous ovarian tumours compared with normal ovary (p < 0.001), particularly in high-grade serous ovarian cancer (p < 0.0001) and in platinum-resistant tumours (p = 0.0232). Median Overall Survival and Progression-free Survival were reduced with high En2 expression (OS = 28 vs 42 months, p = 0.0329; PFS = 8 vs 27 months; p = 0.0004). Positive cytoplasmic EN2 staining was demonstrated in 78% of Epithelial ovarian cancers, with absence in normal ovary. EN2 positive high-grade serous ovarian cancer patients had a shorter PFS (10 vs 17.5 months; p = 0.0103). Conclusion: The EN2 transcription factor is a novel ovarian cancer biomarker. It demonstrates prognostic value, correlating with worse Overall Survival and Progression-free Survival. It is hoped that further work will validate its use as a biomarker, and provide insight into the role of EN2 in the development, progression and spread of ovarian cancer. / Oncology Research and Development Departments at the Royal Surrey County Hospital and the University of Surrey