Gonadotropins Regulate Oncogenic Pathways in Ovarian Surface Epithelium in a Three-Dimensional Model

Ovarian cancer is the most lethal gynecological malignancy among American women, killing 70% of women diagnosed due to the lack of signs and symptoms leading to late diagnosis. Epithelial ovarian carcinomas are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation associated with ovulation, and the gonadotropins that regulate ovulation have been suggested to play a role in ovarian surface transformation and cancer progression. The elevated levels of gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), during ovulation, menopause, and premature ovarian failure have been hypothesized to play a role in the initiation and progression of ovarian cancer. Aspects of ovarian surface repair following ovulation include proliferation, migration, and surface regeneration. In order to study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 day of culture. Organ cultures were propagated in medium supplemented with bovine serum albumin (BSA) or fetal bovine serum (FBS) to determine optimal growth conditions. OSE from BSA cultured organs proliferated significantly more than controls until day 4 while organs cultured in FBS had significantly more surface area encapsulated by OSE. The role of the gonadotropins in normal OSE is not well characterized; however, previous in vivo studies demonstrated that the gonadotropins increased OSE proliferation but in vitro studies were inconclusive because the OSE cells studied were usually immortalized and underwent morphological changes. Using a three-dimensional (3D) organ culture system, the role of gonadotropins in normal OSE in the absence of ovulation was investigated to test the hypothesis that the gonadotropins increase OSE proliferation by activating oncogenic pathways that may lead to induction of ovarian cancer. The 3D ovarian organ culture confirmed previous in vivo studies that demonstrated FSH and LH promote proliferation of the OSE. Transcriptional array analyses demonstrated the gonadotropins activated pathways that control cell cycle, angiogenesis and metastasis. Akt and epidermal growth factor receptor (EGFR) signaling were integral to gonadotropin induced OSE proliferation as demonstrated by abrogation by specific chemical inhibitors of these pathways. Western blots confirmed that Akt expression is not only enhanced, but Akt is also phosphorylated from FSH and LH treatment. However, inhibition of Akt signaling did not completely suppress Akt expression in LH treated ovaries, likely due to upregulation of both Akt1 and Akt2. Soft agar transformation experiments indicated menopausal concentrations of LH initiated OSE transformation. When hydrogen peroxide was used to initiate transformation to mimic oxidative stress from ovulation, FSH progressed OSE colony formation at menopausal concentrations. Although investigating the initiation and progression of the disease is crucial, alternative options for drug therapy are just as important. The current drug therapy used to treat ovarian cancer is paclitaxel/carboplatin, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3β (GSK3β), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3β inhibitors (GSK3βi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, GSK3βi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4′6-diamidino-2-phenylindole-positive nuclear condensation (DAPI), poly (ADP-ribose) polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3βi upregulated phosphorylation of the inhibitory serine residue of GSK3β in OVCA432 and SKOV3 cell lines and also inhibited phosphorylation of the downstream target glycogen synthase. A xenograft study using SKOV3 cells demonstrated that tumor growth was hindered by 9ING41 in vivo. Therefore, GSK3β inhibitors alone or in combination with existing drugs may hinder the growth of serous ovarian cancers. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer. Also, the 3D ovarian organ culture identified key cancer pathways regulated by gonadotropins and revealed how these hormones might contribute to OSE transformation by increasing proliferation, initiating, and progressing colony formation stimulated by oxidative stress. By testing and validating drug targets downstream of FSH and LH signaling, such as GSK3, perhaps ovarian cancers can not only be stifled at an earlier stage but be treated with more targeted therapies.