Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma

Abstract

Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

Keywords: fallopian tube; ovarian cancer.

Figure 1. The Dicer-Pten DKO mouse tumor cells express a mixture of epithelial and mesenchymal markers

A. Immunohistochemistry of the Dicer-Pten DKO mouse tumor tissues for different markers. Scale bars represent 50μm. B. Western blot analysis of marker expression in different cell lysates. The position of the full-length E-cadherin is marked by an arrowhead. β–actin was used as loading control.

Figure 2. The Pten disruption contributed to the rapid proliferation of the Dicer-Pten DKO mouse cancer cells

A. Cell growth assay to compare the growth rates of different mouse cancer cell lines. B. Confocal microscopic images of three-dimensional in vitro spheres formed by the Dicer-Pten DKO mouse cancer cell lines in Collagen I matrix and stained for β–catenin (red), F-actin (green) and nucleus (blue). C. Western blot analysis for the expression for growth and metabolic markers. β–actin was used as loading control.

Figure 3. The Dicer-Pten DKO mouse cancer cells expressed elevated levels of tRNA fragments

A. The result of the Nanostring® nCounter miRNA expression assay. The relative expression of mmu-miR-720, mmu-miR-1937a, 1937b, and 1937c in different cell lines is shown in the inset. B. Normalized counts of the miRNAs representing tRNA fragments that are elevated (red), as well as some other miRNAs that are significantly down-regulated (dark) in the Dicer-Pten DKO mouse cancer cells relative to normal fallopian tube (FT) and ovary-derived tumor cell lines. C. qRT-PCR of mmu-miR-720 in different RNA preparations.

Figure 4. Introduction of a Dicer1-expression construct reversed the epithelial and growth phenotypes of the Dicer-Pten DKO mouse cancer cells

A. Western blot to confirm the expression of FLAG-tagged Dicer1 in the lentivirus-infected cancer cells. β–actin was used as loading control. B. Cell growth assay to compare the growth rates of control and lentivirus-infected cancer cell lines. ^*, P < 0.005; ^#, P < 0.02. C. Flow cytometric density graphs to show the BrdU incorporation and cell cycles of the mouse cancer cell lines. The Y-axis represents the FITC fluorescence of BrdU labeled cells, whereas the X-axis represents the propidium iodide (PI) fluorescence. The boxed areas show the percentages of cell populations in G0/G1 phase (R4), G2/M phase (R5), and S phase (R6), respectively. D. Relative adhesion of different cell lines to collagen I extracellular matrix. The reading of FTdT172 cells was defined as 1. E. Fluorescence micrographs of ZO-1 staining (green) in both FTdT967 cells (top) and FTdT967 cells infected with Dicer1-expressing lentivirus (bottom). The DAPI-stained nuclei were shown in blue.