Class of 2009 Abstract / OBJECTIVES: To develop a novel characterization system utilizing immunofluorescent FLAG tagged EP2 and EP4 receptors to assist in the explanation of their unique cell signaling properties for exploitation in future drug development design. METHODS: Plasmids were obtained and isolated that contained cDNAs encoding FLAG-tagged EP2 and EP4 receptors for transient expression in HEK-293 cells. The sequences of these plasmids were confirmed by restriction enzyme analysis and DNA sequencing. Transfected cells were treated with vehicle, PGE2 or forskolin to assess appropriate receptor functionality based on cAMP induction. RESULTS: The two PGE2 receptor subtypes, EP2 and EP4, are similar in their activation of adenylyl cyclase (AC) and subsequent up regulation of cAMP production. These receptors differ, however, in that EP2 more efficiently stimulates cAMP production and EP4 signaling involves the activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal related kinases (ERKs). The PGE2- treated cells responded as predicted with intracellular production of cAMP, with the EP2 receptor responding more efficiently than the EP4 receptor. CONCLUSIONS: The intent is for these cells to be used as a novel assay system for the development of future selective EP2 and EP4 agonists. This research could potentially benefit in selectively targeting EP2 or EP4 pathways linked to prevalent ailments such as pain, fever, inflammation, possibly cancer or bone growth.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18212号 / 薬博第802号 / 新制||薬||237(附属図書館) / 31070 / 京都大学大学院薬学研究科生命薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Inflammation and aberrant microRNAs expressions promote colon cancer growth and progression. However, the molecular mechanisms that link these pathways remain to be determined. In this dissertation, the causal relationship between inflammation and aberrant microRNAs expressions were explored. Elevated expression of prostaglandin E₂ (PGE₂) receptor EP4 has been seen in human colon cancer. However, the mechanism by which EP4 receptor protein is deregulated is not known. Experiments in this dissertation demonstrate, for the first time, that the EP4 receptor is negatively regulated by miR-101.In previous work, we show that S100P is induced by stimulation of the PGE₂/EP4 receptor signaling pathway. S100P is a ligand for Receptor for Advance Glycation End-products (RAGE). However, little is known about the downstream targets of S100P/RAGE signaling. Here, we demonstrated that S100P/RAGE receptor signaling induces expression of miR-155 via the transcription factor AP-1. In addition, we investigated the genes that are downstream of S100P/RAGE/miR-155 pathway. Our microarrays and bioinformatics analyses identified two novel miR-155 targets, WNK1 and ZNF493 that are down-regulated upon activation of the S100P/RAGE/miR-155 pathway. Lastly, we investigated whether inhibition of S100P/RAGE signaling pathway would be beneficial as a cancer therapy using methyl-2-acetamidoacrylate (M2AA). M2AA treatments decreased colon cancer cells viability and also suppressed colon tumor growth and metastasis in vitro and also in the CAM assay in vivo. Taken together, our results suggest that modulation of S100P/RAGE signaling by M2AA offers therapeutic potential as anti-metastatic agents. In summary, this dissertation provides new insights on the molecular events that link inflammation pathways and microRNAs to colon cancer as well as show that therapeutic strategies targeting these pathways could be effective in treatment of neoplasia.

Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E(2) in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE(2) and generation of PGE(3). Alone, PGE(3) bound and activated the PGE(2) EP4 receptor but with reduced affinity and efficacy compared with its "natural" ligand PGE(2). However, in the presence of PGE(2), PGE(3) acted as an antagonist of EP4 receptor-dependent 3',5' cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent "PGE(2)-to-PGE(3) switch" in human CRC cells and that PGE(3) acts as a partial agonist at the PGE(2) EP4 receptor.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23113号 / 医科博第124号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 篠原 隆司, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21403号 / 生博第404号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 HEJNA,James, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Postmenopausal osteoporosis is a disease characterized by bone loss and increased risk of fracture, and represents a significant burden on the Canadian health care system. Current treatments lack the ability to simultaneously address the therapeutic needs for promoting bone formation and inhibiting resorption. Our approach employs a novel conjugate drug in which an anabolic agent (EP4 receptor agonist) is reversibly joined with an anti-resorptive agent (alendronate) through a linker. This allows the bone-targeting ability of alendronate to deliver the EP4 agonist to bone sites, thereby mitigating the side effects associated with systemic administration of the EP4 agonist. This study investigated the in vivo efficacy of this drug in a curative experiment to treat postmenopausal osteoporosis using an ovariectomized rat model. Results showed that conjugate treatment dose-dependently stimulated bone formation and restored ovariectomy-induced bone loss, and conjugation between alendronate and the EP4 agonist was crucial to the drug’s anabolic effect.

Postmenopausal osteoporosis is a disease characterized by bone loss and increased risk of fracture, and represents a significant burden on the Canadian health care system. Current treatments lack the ability to simultaneously address the therapeutic needs for promoting bone formation and inhibiting resorption. Our approach employs a novel conjugate drug in which an anabolic agent (EP4 receptor agonist) is reversibly joined with an anti-resorptive agent (alendronate) through a linker. This allows the bone-targeting ability of alendronate to deliver the EP4 agonist to bone sites, thereby mitigating the side effects associated with systemic administration of the EP4 agonist. This study investigated the in vivo efficacy of this drug in a curative experiment to treat postmenopausal osteoporosis using an ovariectomized rat model. Results showed that conjugate treatment dose-dependently stimulated bone formation and restored ovariectomy-induced bone loss, and conjugation between alendronate and the EP4 agonist was crucial to the drug’s anabolic effect.