The Small GTPase Ran Increases Sensitivity of Ovarian Cancer Cells to Oncolytic Vesicular Stomatitis Virus

Abstract

Background/Objectives: Ovarian cancer is the deadliest gynecologic cancer, and with the majority of patients dying within the first five years of diagnosis, new therapeutic options are required. The small guanosine triphosphatase (GTPase) Ras-related nuclear protein (Ran) has been reported to be highly expressed in high-grade serous ovarian cancers (HGSOCs) and associated with poor outcomes. Blocking Ran function or preventing its expression were shown to be promising treatment strategies, however, there are currently no small molecule inhibitors available to specifically inhibit Ran function. Interestingly, a previous study suggested that the Vesicular stomatitis virus (VSV) could inhibit Ran activity. Given that VSV is an oncolytic virus (OV) and, therefore, has anti-cancer activity, we reasoned that oncolytic VSV (oVSV) might be particularly effective against ovarian cancer via Ran inhibition. Methods: We evaluated the sensitivity of patient-derived ovarian cancer cell lines to oVSV, as well as the impact of oVSV on Ran and vice versa, using overexpression systems, small interfering RNAs (siRNAs), and drug inhibition. Results: In this study, we evaluated the interplay between oVSV and Ran and found that, although oVSV does not consistently block Ran, increased Ran activation allows for better oVSV replication and tumor cell killing. Conclusions: Our study reveals a positive impact of Ran on oVSV sensitivity. Given the high expression of Ran in HGSOCs, which are particularly aggressive ovarian cancers, our data suggest that oVSV could be effective against the deadliest form of the disease.

Keywords: RanGTP; VSV; oncolytic viruses; ovarian cancer.

Figure 1

oVSV infects and kills human ovarian cancer cells. (A) Representative fluorescence pictures of TOV112D, TOV21G, TOV1946, OV1946, TOV3133G, OV2085, TOV2835EP, TOV3041G, TOV3392D, OV3331, and TOV2414 cells infected with oVSV-YFP at an MOI of 0.1 for 24 h. (B) Relative cell viabilities 24, 48, and 72 h post-infection with oVSV at an MOI of 0.1 (n = 6). Dotted lines highlight 100% and 50% viabilities. (C) Ran expression of ovarian cancer cells as measured by Western blot. GAPDH and vinculin are protein-loading controls.

Figure 2

oVSV infection does not consistently inhibit Ran. Ran activation assay measuring RanGTP (activated Ran) and total Ran (used here as a loading control) by Western blot upon Ran pull-down. A polyclonal antibody against VSV, recognizing multiple viral proteins, was also used to detect infection. Cells were either left untreated or infected with oVSV-YFP at the indicated MOIs for 8 h. MOIs used for each cell line were selected based on the viral sensitivities determined in Figure 1. (A–C) show different effects of infection on RanGTP expression. NV = no virus.

Figure 3

Ran KD decreases VSV replication. (A) Fluorescence intensities of TOV112D, TOV1946, TOV3041G, and TOV2835EP cells transfected with non-targeting (NT) or Ran-targeting siRNAs and infected 16–24 h later with oVSV-YFP at the indicated MOIs (n = 3). (B) Virus outputs (plaque forming units (PFUs)) were measured 24 h post-infection (n = 3). The dotted lines represent viral inputs. Unpaired multiple t-test: *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 4

Ran inhibition impairs VSV replication. (A) Fluorescent intensities of TOV112D, TOV1946, and OV3331 cells treated or not with M36 (40 µM) for 16 h prior to infection with oVSV-YFP at the indicated MOIs (n ≥ 3). (B) Virus outputs were quantified 24 h post-infection (n ≥ 3). The dotted lines represent viral inputs. Statistical analyses by unpaired multiple t-test: ns: p > 0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 5

Ran KD and inhibition impair oVSV-mediated cancer killing. Relative viability of (A) TOV112D, TOV1946, TOV3041G, and TOV2835EP cells transfected with control non-targeting (NT) or Ran-targeting siRNAs and infected with oVSV-YFP at various MOIs for 24 h (n = 3) or (B) TOV112D, TOV1946, and OV3331 cells pre-treated with M36 (40 µM) for 16 h and infected with oVSV-YFP at various MOIs for 24 h (n = 3). The dotted lines represent viability in non-infected control conditions. Unpaired multiple t-test: *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001.

Figure 6

Ran activation enhances oVSV. (A) Virus outputs and (B) cell viability of TOV1946 and OV3331 cells transfected with constitutive active (CA) or wild type (WT) Ran constructs and infected with oVSV-RFP 24 h later (n ≥ 3). Samples were collected 24 h post-infection. Dotted lines represent viral inputs (A) or viability in non-infected control conditions in (B). Unpaired multiple t-test: ns: p > 0.05; *: p ≤ 0.05, ***: p ≤ 0.001.