Epithelial ovarian cancer is the most lethal gynecologic malignancy, with a 5-year survival rate of less than 40%. This is due in part to a lack of early detection markers and lack of specific symptoms during early disease. The laying hen is the only accessible animal model which develops epithelial ovarian cancer spontaneously, with features closely resembling the human disease. It has been estimated that approximately 30% of all cancers can be prevented with diet, exercise, and maintenance of an optimal weight, and the chronic low-grade inflammation that accompanies obesity is implicated as a causal factor in the development of cancer. Flaxseed, a rich plant source of anti-inflammatory omega-3 fatty acids and lignans which act as phytoestrogens and antioxidants, exhibits chemosuppressive effects against the development and progression of ovarian cancer. We have shown that a diet of 10% flaxseed reduces the incidence and severity of ovarian cancer when fed to laying hens over 4 years, due in part to the ability of flaxseed to suppress the production of proinflammatory PGE2 in the ovary by decreasing expression of COX enzymes. To investigate other potential specific mechanisms by which flaxseed acts to suppress ovarian cancer, we examined expression and activity of pathways known to be involved in the etiology and progression of human epithelial ovarian cancer in ovarian cancer in the laying hen, and determined whether flaxseed affected these pathways during cancer development. We investigated the effect of flaxseed and its individual components upon oxidative stress in the normal ovary and in ovarian cancer by analyzing expression of target genes of the NRF2 transcription factor. The NRF2 pathway is a "master switch" that regulates expression of ROS-responsive detoxification genes. Results revealed that expression of four genes was significantly downregulated in then ovaries of hens on the defatted flaxmeal (DFM) and whole flaxseed (WF) diets compared to hens on diets that are high in pro-inflammatory omega-6 fatty acids, suggesting that flaxseed decreases oxidative stress in the ovary. Conversely, one target gene was upregulated in ovarian cancer compared to normal ovaries, and this observation was not affected by flaxseed. Additionally, nuclear accumulation Nrf2 protein was not observed in tumor cells, suggesting that flaxseed does not exert chemosuppressive effects by modulating NRF2 signaling in ovarian cancer. To further investigate pathways potentially regulated by flaxseed, we performed a microarray with 44k features and found that a set of genes involved in branching morphogenesis was upregulated in ovarian cancer and significantly decreased by flaxseed, including E-cadherin and miR-200, suggesting that flaxseed impedes the activity of an aberrantly activated developmental program that controls gland formation during ovarian cancer progression. Lack of nuclear accumulation of ZEB1 protein in tumor cells suggests that this decrease in expression is likely not due to EMT. Finally, due to its known roles in controlling developmental programs such as EMT as well as regulating cell growth and proliferation, we performed a set of experiments to examine activity of the TGF-beta pathway. PCR array analysis revealed that SMAD target genes, ligands, receptors, and co-regulatory proteins were upregulated in ovarian tumors from hens on both diet groups, suggesting TGF-beta signaling is enhanced in ovarian cancer. However, expression of SMAD6 and SMAD7 was upregulated in tumors from hens on the flaxseed diet but not control diet, with SMAD7 protein being expressed in both epithelial tumor cells and intratumoral stromal cells. Additionally, immunohistochemical staining for pSMAD2/3 was decreased in epithelial tumor cells and absent from intratumoral stromal cells in tumors from hens on the flaxseed diet compared to tumors from hens on the control diet, and these data together suggest that flaxseed may inhibit pro-oncogenic TGF-beta signaling in ovarian cancer. Finally, flaxseed prevents the downregulation of expression of p15 and the upregulation of CCNA and CCNE in ovarian tumors, suggesting that flaxseed may slow cell cycle progression. Data from these studies provides preliminary evidence that flaxseed exerts pleiotropic effects upon gene expression to negatively regulate pathways driving the progression of ovarian cancer, including aberrant TGF-beta signaling and glandular development. These studies provide groundwork for in vitro studies to test the specific effects of flaxseed upon proteins involved in TGF-beta signaling and upon the expansion of tumor epithelia.

Colorectal cancer (CRC) has a 5-year survival rate of 68% yet it still has a mortality rate of 50,000 per year. While CRC has a host of causes, one that stands out is TGFβ deficient signaling, which is disrupted in a majority of high-microsatellite-instability or inflammation-associated CRCs. Since TGFβ is a multifunctional cytokine, it has been elusive to determine whether its effect on cancer development is operating through inflammation, differentiation or developmental pathways. Additionally, it is now becoming apparent that a great number of CRC cases can be associated with and possibly caused by gut bacteria dysbiosis. Here, I present a metagenomic and metatranscriptomic study of the interactions between TGFβ deficient signaling, inflammatory signaling, and the microbiome in a CRC mouse model. TGFβ deficient mice have reduced amounts of Firmicutes as well as mRNA counts of a key butyrate enzyme. Lack of butyrate, as shown by previous literature, could be inhibiting apoptosis and promoting growth. Also, TGFβ deficient mice have increased mRNA counts of polyamine producing genes, which could act synergistically with butyrate reduction. I find that H. hepaticus inoculation, as a source of inflammatory signaling, affects another species, M. schaedleri, to produce pro- inflammatory lipopolysaccharides. Additionally, H. hepaticus itself has increased oxidative phosphorylation; reactive oxygen species from this process could be adding to cancer-promoting DNA damage. Taken together, TGFβ deficient signaling and H. hepaticus inoculation, disrupt enough pathways to cross the threshold of carcinogenicity in 40% of the mice in our study. The results of this study emphasize the importance of microbiome function and represent possible new avenues of treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18894号 / 医博第4005号 / 新制||医||1009(附属図書館) / 31845 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 戸井 雅和, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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In patients with acute myeloid leukemia (AML), cytarabine-based chemotherapy usually achieves remission, but this is commonly followed by relapse and chemo-resistance. In this study, we aim to establish next-generation sequencing (NGS)-based microRNA expression profiling and pathway analysis to identify pathways regulated differentially between chemo-sensitive and -resistant AML as potential therapeutic targets. MicroRNA expression profiles differ significantly between chemo-sensitive and chemo-resistant AML cells and reflect differences in the activity of intracellular signaling cascades. Alterations in signaling pathway activities contribute to treatment resistance and thus represent potential drug targets. Our microRNA-led approach indicates a role for activin receptor type 2A in ARA-C resistance of AML cells and suggests activin receptor signaling to be a candidate pathway for targeted therapy.

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