Background: Historically, metaplasia of ovarian surface epithelial (OSE) cells in the cortical inclusion cysts is associated with ovarian cancer development. Recent studies have also suggested fallopian tube epithelium as another origin of ovarian cancer cells. MicroRNA-200 (miR200) family is related to epithelial-mesenchymal transition (EMT) and overexpressed in ovarian cancer. However, the potential involvement of miR200 in ovarian pathogenesis is largely unknown.
Objective: To study any morphological and signaling differences between immortalized normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells with changes in miR200 expression and control cells growing in three-dimensional (3D) cultures.
Methods: OSE cells with stable ectopic expression of miR200 and ovarian cancer cells with stable miR200 knockdown were established using lentiviral agents. The resulting cells and control cells were grown as 3D cultures first in Matrigel to form the cyst-like spheroids and then transferred to Collagen I matrix for cell migration study. Immunofluorescence and confocal microscopy was applied to study the morphologies of the 3D structures, and in conjunction with Western blot analysis, to study changes in signaling pathways in response to changes of miR200 expression and in the presence of a variety of pharmacologic inhibitors. Clinical samples were examined for miR200 expression to evaluate the results from 3D in vitro cultures.
Results: In comparison with the wild-type control counterparts, OSE cells with ectopic expression of miR200 had elevated E-cadherin expression, showed more compact and prolonged cyst stabilization in both Matrigel and Collagen I 3D cultures; whereas the ovarian cancer cells with miR200 knockdown showed increased TGFβ expression and multiple lumens with enhanced cell death in Matrigel culture, and change from collective migration mode to single cells in Collagen I culture. Both acetylated tubulin and Histone H3 staining of mitotic cells in Matrigel culture demonstrated ovarian cancer cells with miR200 knockdown had abnormal spindle orientation and defective cell cycle division. Application of TGFβ inhibitors reversed the single cell migration phenotype of miR200 knockdown cancer cells to collective movement. However, the downstream pathways responsible for the switching of migration phenotype were not the same between the serous and mucinous cell lines. For the mucinous cell line, miR200 controlled the switching through TGFβ-ROCK pathway, whereas the downstream pathway for the switching in the serous cell line remained unknown. qRT-PCR analysis of micro-dissected cells from clinical samples verified the elevated expression of miR200 in endosalpingiosis cysts and tumor tissues when compared with normal ovarian surface epithelia and fallopian tube epithelia.
Conclusion: The qRT-PCR validation study verifies the findings of our 3D in vitro culture study and demonstrates that miR200 is important for inclusion cyst formation and collective movement of ovarian cancer cells during tumor invasion. Perturbation of this pathway in cancer cells will accelerate cell death through abnormal cell division. Hence, miR200 may be an interesting target in ovarian cancer therapy.
Citation Format: Pui Wah Choi, Junzheng Yang, Wing Ping Fong, Ross S. Berkowitz, William R. Welch, Gregory J. Goodall, Shu Wing Ng. The dual roles of microRNA-200: From inclusion cyst formation to cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A23.