An essential role for Ran GTPase in epithelial ovarian cancer cell survival

Abstract

Background: We previously identified that Ran protein, a member of the Ras GTPase family, is highly expressed in high grade and high stage serous epithelial ovarian cancers, and that its overexpression is associated with a poor prognosis. Ran is known to contribute to both nucleocytoplasmic transport and cell cycle progression, but its role in ovarian cancer is not well defined.

Results: Using a lentivirus-based tetracycline-inducible shRNA approach, we show that downregulation of Ran expression in aggressive ovarian cancer cell lines affects cellular proliferation by inducing a caspase-3 associated apoptosis. Using a xenograft tumor assay, we demonstrate that depletion of Ran results in decreased tumorigenesis, and eventual tumor formation is associated with tumor cells that express Ran protein.

Conclusion: Our results suggest a role for Ran in ovarian cancer cell survival and tumorigenicity and suggest that this critical GTPase may be suitable as a therapeutic target.

Figure 1

Ran expression in parental cell lines, mixed populations and clones. Comparison of Ran mRNA levels by Q-PCR in TOV112D TetR (a) and TOV1946 TetR (b) parental cell lines, shRNA Ran or LacZ mixed populations and clones. Levels were normalized to parental cell line without induction. Values represent the mean ± SD of two separate experiments each performed in duplicate. Western blot analysis of TOV112D TetR (c) and TOV1946 TetR (d) parental cell lines, mixed populations and clones shRNA Ran or LacZ without and after three days of induction. ß-actin was used as loading control. P: Parental cell line, M.P.: Mixed population, #1, #2 and #3: independent clones. Mann-Whitney test indicates a significant difference with a p-value < 0.05 (*).

Figure 2

Effect of Ran depletion on proliferation. Growth curves for TOV112D TetR (a) and TOV1946 TetR (b) parental cell lines, mixed populations and clones expressing shRNA Ran. Cells were induced with tetracycline three days prior to day 0. Cells were trypsinized and counted every 24h for five days. Values represent mean ± SD of duplicate wells from three independent experiments. Cellular morphology of TOV112D TetR (c) and TOV1946 TetR (d) on day 5 of the growth curve. Loss of Ran GTPase expression results in cell number reduction and round shaped cells. All pictures were taken at 100× magnification. P: Parental cell line, M.P.: Mixed population, #1, #2 and #3: independent clones. Mann-Whitney test indicates a significant difference relative to the non-induced parental cell line with a p-value < 0.05 (*). There was no significant difference between the different non-induced cells.

Figure 3

Loss of Ran expression results in a caspase-3 associated apoptosis in ovarian cancer cell lines. TOV112D TetR (a) and TOV1946 TetR (b) parental cell lines, mixed populations and clones expressing shRNA LacZ and Ran were induced with tetracycline for the indicated times and then fixed in 70% ethanol. Cells were treated with propidium iodide and sub-G₁ peaks were quantified by flow cytometry. Values represent the mean ± SD of three independent experiments. Western blot analysis showing caspase-3 cleavage in cell depleted in Ran expression after 3 days of induction in TOV112D TetR (c) and TOV1946 TetR (d). Kinetic of caspase-3 cleavage by western blot analysis in TOV112D TetR (e) and TOV1946 TetR (f) parental cell lines, mixed populations and clones expressing shRNA LacZ and Ran. TOV112D TetR (g) and TOV1946 TetR (h) parental cell lines, mixed populations and clones expressing shRNA LacZ and Ran were induced with tetracycline for the indicated time and then treated with TMRE for 20 minutes. ΔΨm reduction was measured by flow cytometry. Values represent the mean ± SD of three independent experiments. P: Parental cell line, M.P.: Mixed population, #1, #2 and #3: independent clones. Mann-Whitney test indicates a significant difference relative to the non-induced sample with a p-value < 0.05 (*).

Figure 4

Loss of Ran expression results in tumor regression in vivo. A. TOV112D TetR mixed population and clone expressing shRNA Ran were subcutaneously injected in SCID mice and tumor growth was measured biweekly. All mice developed tumors albeit of varying size and tumor volume. Doxycycline-supplemented food inducing shRNA expression started at day 9. M.P: Mixed population, Ran #2: clone. Values represent mean ± SE of groups of 4 mice. Mann-Whitney test indicates a significant p-value < 0.05 (*). B. Immunohistochemistry analysis of the xenografts on day 6 of the doxycycline-supplemented food showing morphological hematoxylin eosin (HE) staining and Ran, cleaved caspase-3 and Ki67 expression. Slides were obtained from contiguous tissues.