Studies on molecular understanding of endometrial diseases and development of graphene-based biotechnology

Abstract

Endometrial cancer and adenomyosis are well-known gynecological disorders of the female reproductive system. The endometrial cancer and adenomyosis are ovarian steroid hormone-dependent gynecological disorders and are related to unopposed E2 exposure. These disease are associated with several risk factors, such as infertility, pelvic pain, menorrhagia, and dysmenorrhea. Nevertheless, molecular mechanisms leading to endometrial cancer and adenomyosis remain unclear.Hormone therapy, radiation therapy, and chemotherapy are variously implemented to treat gynecological disorders such as endometrial cancer, endometriosis, and adenomyosis. In particular, there are reports of bone damage such as osteoporosis, osteolysis, and osteosarcoma after radiation therapy. Also, Research has been reported that osteoporotic disease occurs in postmenopausal women and endometrial cancer patients. The occurrence of these bone disease can lead to huge losses in health care costs and reduce the quality of life and life expectancy of patients. Recently, the field of regenerative medicine using stem cells and biomaterials has been actively studied to prevent bone damage.

The main purpose of the first study was to understand the role of epithelial Mig-6 in the uterus. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4-resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, I generated a mouse model in which I specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of signal transducers and activators of transcription 3 (STAT3) and proliferation of the endometrial epithelial cells. The results from my immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr-positive cells (Pgrcre+Mig-6f/f). However, Sprr2fcre+Mig-6f/f mice were P4-responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. I showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells, and the antitumor effects of P4 are mediated by the endometrial stroma. These data help to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer.

The second and third studies were to understand the pathophysiological function of β-catenin in adenomyosis development. Previous study showed that aberrant activation of β-catenin develops adenomyosis through EMT. To identify the molecular pathways regulated by aberrant activation of β-catenin, I performed DNA microarray and ChIP-seq analysis in the uteri of mutant mice which expressed a dominant stabilized β-catenin in the uterus. Pathway analysis of the microarray and ChIP-seq identified activation of TGF-β signaling in the mutant mice (Pgrcre/+ and Ctnnb1f(ex3)/+). Further ChIP analysis revealed Tgf-β2 as a direct transcriptional β-catenin target gene in the uterus. Immunohistochemistry analysis also showed aberrant overexpression of TGF-β2 in epithelial cells of mutant mice as well as women with adenomyosis. There is a strong positive correlation between β-catenin and TGF-β2 proteins in women with adenomyosis. TGF-β2 positive epithelial cells of eutopic endometrium and adenomyosis lesions showed lower expression of E-cadherin compared to control epithelial cells in both mouse and human adenomyosis. Ishikawa cells with nuclear β-catenin induced the expression of TGF-β2 and vimentin but decreased the expression of E-cadherin. Interestingly, a cell invasion assay showed that nuclear β-catenin expression significantly increased invasiveness compared to the control group. Furthermore, Pirfenidone, a TGF-β inhibitor, treatment increased E-cadherin expression and reduced cell invasiveness in Ishikawa cells with nuclear β-catenin.

Also pathway analysis of the microarray and ChIP-seq revealed dysregulation of Wnt/β-catenin signaling in the mutant mice compared to control mice. Lymphoid enhancer-binding factor 1 (LEF-1) major transcription factor of Wnt/β-catenin pathway, was up-regulated in mutant uterus compared to control. Immunohistochemistry analysis showed that LEF-1 levels are remarkably increased in the uterine of mutant mice compared to control during development of adenomyosis. Further the ChIP analysis revealed Lef-1 as a direct transcriptional β-catenin target gene in the murine uterus. In human adenomyosis, the levels of LEF-1 were higher in women with adenomyosis compared to women without adenomyosis. HEC1A cells with nuclear β-catenin induced the expression of LEF-1 and ZEB1 but decreased the expression of E-cadherin. These data show that TGF-β2 and LEF-1 can be novel targets and biomarkers for treating adenomyosis.

The fourth study conducted research on study of regenerative medicine using graphene biomaterials and human adipose-derived mesenchymal stromal cells (hASCs) to treat bone disease after radiation therapy or in menopausal women, endometrial cancer patients. Recently, biomaterial graphene and stem cells have been actively studied in the field of regenerative medicine for bone regeneration. So, this study was conducted to examine the effect of multilayer graphene consisting of various layers on the osteogenic differentiation of hASCs. Various layers (1 to 7) of graphene film, which contained wrinkles between layers with 30 to 50 nm in width, were attached with chemical vapor deposition on a cell culture glass. hASCs were cultured on the multilayer graphene films, which consisted of different number of the wrinkles. Three primary hASC lines subpassaged different times were provided and cell activity and osteogenic differentiation was subsequently monitored. The osteogenic differentiation of the hASCs, which was confirmed by Alizarin red staining, were significantly promoted by the culture of multilayer graphene films compared with the control (no layer). The graphene with three layers yielded optimal differentiation. When the hASCs were cultured on the three-layer graphene films for 24 hours, expression of cell adhesion molecule (F-actin) and FAK, ERK and RUNX2 were activated compared with the control. Real-time RT-PCR analysis showed significant upregulation of ALP and OPN, pRUNX2-targeted mRNA genes in the cells cultured on the multilayer graphene films for 24 hours. These results suggest that changes in the physical environment due to multilayer graphene films promote osteogenic differentiation of hASCs through activation of the FAK-ERK. These studies will greatly contribute to our understanding of the molecular mechanism and to the development of new biomarkers and therapeutic approaches in endometrial cancer and adenomyosis. In addition, it may help to understand the field of regenerative medicine to treat bone disease that can occur after traditional treatments such as radiation therapy, hormone therapy of gynecological disorders.