microRNAs (miRNAs) control a multitude of pathways in human cancers. Differential expression of miRNAs among different histological types of tumors within the same type of tissue offers insight into the mechanism of pathogenesis and may help to direct treatment to improve prognosis. We assessed expression of 667 miRNAs in endometrial endometrioid and serous adenocarcinomas using RNA extracted from benign endometrium as well as from primary endometrial tumors. Quantitative miRNA profiling of endometrial adenocarcinomas revealed four overlapping groups of significantly overexpressed and underexpressed miRNAs. The first group was composed of 20 miRNAs significantly dysregulated in both adenocarcinoma types compared with benign endometrium, two groups were composed of miRNAs significantly dysregulated in either endometrioid adenocarcinomas or in serous adenocarcinomas compared with benign endometrium, and the fourth group was composed of 17 miRNAs that significantly distinguished between endometrioid adenocarcinomas and serous adenocarcinomas themselves. Validation of the expression levels of the selected miRNAs was carried out in a second panel composed of ten endometrioid and five serous tumors. Experimentally validated mRNA targets of these dysregulated miRNAs were identified using published sources, whereas TargetScan was used to predict targets of miRNAs in the first and fourth profile groups. These validated and potential miRNA target lists were filtered using published lists of genes displaying significant overexpression or underexpression in endometrial cancers compared to benign endometrium. Our results revealed a number of dysregulated miRNAs that are commonly found in endometrial (and other) cancers as well as several dysregulated miRNAs not previously identified in endometrial cancers. Understanding these differences may permit the development of both prognostic and diagnostic biomarkers.

Progesterone, acting through its receptor, PR (progesterone receptor), is the natural inhibitor of uterine endometrial carcinogenesis by inducing differentiation. PR is downregulated in more advanced cases of endometrial cancer, thereby limiting the effectiveness of hormonal therapy. Our objective was to understand and reverse the mechanisms underlying loss of PR expression in order to improve therapeutic outcomes. Using endometrial cancer cell lines and data from The Cancer Genome Atlas, our findings demonstrate that PR expression is downregulated at four distinct levels. In well-differentiated cancers, ligand-induced receptor activation and downregulation are intact. miRNAs mediate fine tuning of PR levels. As differentiation is lost, PR silencing is primarily at the epigenetic level. Initially, recruitment of the polycomb repressor complex 2 to the PR promoter suppresses transcription. Subsequently, DNA methylation prevents PR expression. Appropriate epigenetic modulators reverse these mechanisms. These data provide a rationale for combining epigenetic modulators with progestins as a therapeutic strategy for endometrial cancer.Significance: Traditional hormonal therapy for women with endometrial cancer can be molecularly enhanced by combining progestins with epigenetic modulators, thereby increasing progesterone receptor expression and significantly improving treatment efficacy.

Cancer-testis (CT) antigens are a large family of genes that are selectively expressed in human testis germ cells, overexpressed in a variety of tumors and predominantly located on the X chromosome. To date, all known CT antigens are protein-coding genes. Here, we identify miR-888 as the first miRNA with features characteristic of a CT antigen. In a panel of 21 normal human tissues, miR-888 expression was high in testes and minimal or absent in all other examined tissues. In situ hybridization localized miR-888 expression specifically to the early stages of sperm development within the testes. Using The Cancer Genome Atlas database, we discovered that miR-888 was predominately expressed in endometrial tumors, with a significant association to high-grade tumors and increased percent invasion. In a separate panel of endometrial tumor specimens, we validated overexpression of miR-888 by real-time polymerase chain reaction. In addition, miR-888 expression was highest in endometrial carcinosarcoma, a rare and aggressive type of endometrial tumor. Moreover, we identified the progesterone receptor (PR), a potent endometrial tumor suppressor, as a direct target of miR-888. These data define miR-888 as the first miRNA CT antigen and a potential mediator of an aggressive endometrial tumor phenotype through down-regulation of PR.

Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer in women. With accumulating evidence, microRNAs have emerged as significant players in the development and progression of cancers. To investigate the role of miRNAs in endometrial cancer, we used quantitative real-time PCR arrays to identify miRNAs dysregulated in a panel of endometrial cancer tissue samples. Out of 667 miRNAs, miR-888 was identified as one of the most highly over-expressed miRNAs, specifically in mixed mϋllerian tumors, arguably the most aggressive form of endometrial cancer. Furthermore, realtime PCR validations revealed a significant inverse correlation between age onset of cancer and the level of miR-888 expression. Notably, a potential target of miR-888 is the progesterone receptor, whose role in endometrial cancer is well characterized. Together, these data point to miR-888 playing an important functional role in the development of aggressive endometrial tumors. Future research will focus on identifying and validating the targets of miR-888 to elucidate its mechanism of action and support this hypothesis.

Josh Lobb for maintaining a great program to complete my dissertation in during my graduate career. Many facilities and groups provided me with services and samples that made significant contributions to my work. Special thanks to Dr. Beverly Davidson for allowing me to attend her lab meetings and to her lab as a whole for helping me with experiments and providing me with useful suggestions. Megan Keiser in the Davidson lab helped me learn and troubleshoot the miRNA in situ hybridization technique along with advice from Hayley McLoughlin and Pavitra Ramachandran. Also from the Davidson Lab, Alex Mas Monteys helped with molecular cloning techniques, Patrick Staber with qPCR data analysis and Ryan Spengler with miRNA target identification. Overall the Davidson Lab provided me with tools and protocols to advance my research. Thank you to Mary Boes and Garry Hauser in the DNA Core Facility for their help in performing several RNA analyses and real-time PCR experiments. Thank you to James Schappet for his help in downloading data from the Cancer Genome Atlas via the University of Iowa Institute for Clinical and Translation Science Compass website. I used several instruments within the Central Microscopy Research Facility for my work and I would like to thank to Katherine Walters and Chantal Allamargot for their training and advice. Thank you to Joe Galbraith and Rita Sigmund in the Tissue Procurement Facility for providing me with tissues to optimize the miRNA in situ hybridization protocol and for their friendly advice. I would also like to thank Dr. Michael Goodheart for providing me with all the endometrial tumor specimens from the Obstetrics and Gynecology Tumor Bank, which were essential to my thesis project.