Date of Award

5-1-2016

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

Hayashi, Kanako Hayashi

Abstract

Type II endometrial carcinomas (ECs) are estrogen independent, poorly differentiated tumors that behave in an aggressive manner. As TP53 (Tumor Protein p53) mutation and E-cadherin (CDH1) inactivation occur in 80% of human endometrial type II carcinomas, we hypothesized that mouse uteri lacking both Trp53 and Cdh1 would exhibit a phenotype indicative of neoplastic transformation. Mice with conditional ablation of Cdh1 and Trp53 (Cdh1 d/dTrp53d/d) clearly demonstrate architectural features characteristic of type II ECs, including focal areas of papillary differentiation, protruding cytoplasm into the lumen (hobnailing) and severe nuclear atypia at 6 months of age. Further, Cdh1 d/dTrp53d/d tumors in 12-month-old mice were highly aggressive, and metastasized to nearby and distant organs within the peritoneal cavity, such as abdominal lymph nodes, mesentery and peri-intestinal adipose tissues, demonstrating that tumorigenesis in this model proceeds through the universally recognized morphological intermediates associated with type II endometrial neoplasia. We also observed abundant cell proliferation and complex angiogenesis in the uteri of Cdh1d/dTrp53d/d mice. Our microarray analysis found that most of the genes differentially regulated in the uteri of Cdh1 d/dTrp53d/d mice were involved in inflammatory responses. CD163 and Arg1, markers for tumor-associated macrophages, were also detected and increased in the uteri of Cdh1d/dTrp53d/d mice, suggesting that an inflammatory tumor microenvironment with immune cell recruitment is augmenting tumor development in Cdh1d/dTrp53d/d uteri. Further, inflammatory mediators secreted from CDH1-negative, TP53 mutant endometrial cancer cells induced normal macrophages to express inflammatory-related genes through activation of nuclear factor-κB signaling (NFκB). These results indicate that absence of CDH1 and TP53 in endometrial cells initiates chronic inflammation, promotes tumor microenvironment development following the recruitment of macrophages, and promotes aggressive ECs. The second study described focused on targeting inflammation in endometriosis. Endometriosis affects 6-10% of women of reproductive age. Although endometriosis is a benign disorder, approximately 50% of affected women experience severe chronic pelvic pain and infertility. Because endometriosis is estrogen-dependent, the most widely used medical drugs are oral contraceptives, GnRH agonists and progestins, which suppress ovarian function and reduce pelvic disease and associated pain. However, these hormonal treatments are often of limited efficacy, elicit side-effects, temporarily inhibit fertility, and have high recurrence rates of symptoms. Therefore, it is necessary to identify therapeutic targets and efficient drugs that improve current treatment. Proinflammatory signaling has an essential role in the progression of endometriosis, as the peritoneal fluid has increased macrophage, cytokine and chemokine content. Although nonsteroidal anti-inflammatory drugs, such as ibuprofen, have also been used for the treatment of endometriosis, these drugs primarily relieve dysmenorrhea. We have recently identified a small molecule, niclosamide, which modulates the NFĸB and STAT3 (signal transducer and activator of transcription 3) signaling pathways. Therefore, we hypothesized that niclosamide inhibits the endometriotic microenvironment suppressing proinflammatory mechanisms via NFĸB and STAT3 signaling. To determine the inhibitory mechanisms of niclosamide in endometriosis, endometriotic lesions were induced in mice. Both injection and suture technique were used to implant the uteri of donor mice into the peritoneal cavity of wild-type recipient females. After three days recovery from the surgery, mice began daily oral treatment of with niclosamide (0, 100, or 200 mg/kg BW) for 3 weeks. We observed a significant difference in the pattern of progression of endometriotic lesions after 3 weeks in the suture model. Daily oral administration of 200 mg/kg BW niclosamide reduced the total endometriotic lesion weight and volume when compared with control treatment. Niclosamide treated animals had fewer proliferating Ki67 positive epithelial cells in the lesions. RNA sequencing was performed to determine which genes had altered expression following treatment of niclosamide and revealed genes encoding structural proteins, inflammatory factors, and mediators of Wnt signaling. Immunohistochemical analysis revealed a reduction in phospho-STAT3, phospho-IKK and iNOS following treatment of niclosamide suggesting repression of inflammatory signaling. Current treatments for endometriosis temporarily inhibit fertility by repressing ovulation. However, niclosamide treatment did not disturb normal reproductive function in mice, but still reduced lesion development. Thus, these results suggest that niclosamide could be a potential therapeutic drug for the treatment of endometriosis by targeting inflammatory mechanisms.

Available for download on Wednesday, August 08, 2018

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