Antitumor Effects of Ral-GTPases Downregulation in Glioblastoma

Abstract

Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.

Keywords: Ral-GTPases; RalB; glioblastoma; glioma; recurrence; therapy.

Figure 1

Expression of RalA and RalB in glioblastoma. (A) Immunoblot to detect the levels of RalA and RalB in different primary glioblastoma (GBM), in low-grade astrocytoma (A1, A3) primary cultures, and in the glioblastoma cell line U251-MG. β-actin was used as a loading control. Numbers below the panel A are the estimated levels of RalA and RalB relative to β-actin and refer to the astrocytoma sample A1. (B) RalB-GTP pull down in two different primary GBM cells. Active RalB-GTP was affinity purified with Ral BP-beads from cell lysates. RalB-GTP and total RalB were detected by immunoblot. Ral BP-beads were used as a loading control. (C) Analysis of RalA and RalB mRNA expression levels by RNAseq from GlioVis database (161 low-grade astrocytoma and 152 glioblastoma samples). Tukey’s Honest Significant Difference (HSD), p < 0.001 (***) (http://gliovis.bioinfo.cnio.es/, accessed on 5 July 2022).

Figure 2

Knockdown of Ral GTPases promotes a reduction of cell growth without affecting cell viability. Primary GBM cells were infected with lentivirus, driving interference RNA against RalA (shRalA) or RalB (shRalB) or both. Scramble shRNA was used as a control. Three days after infection, cells were seeded and counted every 24 h for 3 days. (a) Graphics and bar diagrams representing growth of knockdown GBM cells. Data is represented as mean ± SEM (n = 3; three independent experiments). Scramble group was compared with the other three conditions by ANOVA and Tukey-HSD post-test (* p < 0.05; ** p < 0.01). (b) Western blot to determine the levels of RalA and B in primary GBM cells after knockdown. β-actin was used as a loading control. (c) Representative phase-contrast images of GBM65 cells were taken after 4 days of infection (50 μm bar).

Figure 3

Knockdown of Ral GTPases reduces soft-agar colony growth. U251-MG cell line, GBM65, and GBM6 were infected with lentivirus-mediating interference RNA against RalA or RalB and seeded in a soft agar layer. After 15 days, the growing colonies were stained by MTT reaction, and the plates were scanned. (a) Representative images of U251-MG and GBM65 plates. (b) Diagram representing the number of colonies quantified with the ImageJ program. Values represent mean ± SEM (n = 4). Significance was calculated by ANOVA and Tukey-HSD post-test. Significance of scramble versus shRalA and shRalB groups is represented (* p ≤ 0.05; ** p ≤ 0.01).

Figure 4

Downregulation of Ral GTPases promotes a senescence-like response in glioblastoma cells. Ral GTPases RalA and RalB were downregulated by RNA interference—shRalA and shRalB, respectively. Scramble shRNA was used as a control. GBM cells were infected by lentiviral vectors harboring the shRNAs. Five days after infection, cells were processed for senescence analyses. (a) Representative images of X-gal senescence assay. Blue cells indicate positive senescent cells (25 μm bar). (b) Quantification of senescence assay in (a) as a percentage of positive cells versus total cells. Values represent mean ± SEM. Significance was determined by ANOVA and Tukey post-test (p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). (c) Quantification of BrdU incorporation assay. Values represent the percentage of BrdU-positive nuclei. The number of total nuclei was determined by counting nuclei stained with Hoechst. The mean ± SD (n = 3) is shown. Significance was determined by ANOVA and Tukey post-test. Significance of scramble versus shRalA, shRalB, or shRalA + shRalB groups is represented (p ≤ 0.05, *; p ≤ 0.01, **).

Figure 5

RalB knockdown induces a senescent-like response in primary glioblastoma cells through non- canonical mechanisms. Primary GBM cells growing in the same conditions as in Figure 4 were processed for immunoblot. (a) Panels showing the levels of the indicated proteins in primary GBM cells. Actin was used as a loading control. Quantification of the protein levels for p53 (b), Rb1 (c), p27 (d), and Phosphop70S6K (e). The number of independent experiments (n) is shown in the panels. The mean ± SEM is shown. Significance was determined by ANOVA or t-test (p ≤ 0.05, *).

Figure 6

Ral GTPases downregulation inhibits the growth of GBM cells as tumorspheres. GBM65 and GBM6 were infected with lentivirus, mediating interference RNA against RalA and RalB or both, and cells were seeded under special conditions in order to form tumorspheres. After 48 h, tumorspheres were precipitated and stained with trypan blue. (a) Representative images of tumorspheres (150 μm bar). (b) Diagrams representing the number of spheres observed in (a). Spheres were counted using the ImageJ program. Values represent mean ± SEM (n = 3). Significance was calculated by ANOVA and Tukey-HSD post-test. Significance of scramble versus shRalA, shRalB, and shRalA + shRalB groups is represented (* p ≤ 0.05).

Figure 7

Ral GTPases downregulation prevents the growth of GBM cells in vivo. (a) Human U251-MG cells were infected with lentiviruses harboring scramble or shRalB. Infected cells were inoculated subcutaneously in immunodeficient SCID male mice. Tumor sizes were measured every week. Mice were euthanized seven weeks after injection and the tumors excised. (b) Quantification of tumor size at different weeks. Data is mean ± SEM. Significance was calculated by two-way ANOVA and Bonferroni-HSD post-test. Significance of scramble versus shRalB group is represented (** p ≤ 0.01; *** p ≤ 0.001). (c) Analysis of survival times (months) of glioma patients (TCGA cohort of diffuse gliomas, Gliovis) with a Kaplan–Meier plot comparing the effects of copy number increase of RalB (gain; n = 20), heterozygous loss of RalB (het loss; n = 18), and wild type RalB alleles (wt.; n = 600). The median survival in months was 15.1 (RalB gain), 18.1 (RalB wt.), and 22.25 (RalB het loss), with p = 0.048 (Mantel–Cox test). (d) Analysis of survival times (months) of glioma patients as in (c). RalA copy number increase (gain; n = 226) and wild type RalA alleles (wt.; n = 417). The median survival was 13.8 (RalA gain) and 21.55 (RalA wt.), with p < 0.0001 (Mantel–Cox test).

Figure 8

Effects of the treatment of primary glioblastoma cells with temozolomide (TMZ) and RalB downregulation. GBM6 (a) or GBM65 (b) cells infected with lentivirus driving scramble or shRalB were treated with TMZ (100 μM) or placebo. Growth was analyzed by MTT assay at different time points. Values represent mean ± SEM (n = 3). Significance was determined by ANOVA and Tukey-HSD post-test. Significance of scramble ± TMZ versus shRalB ± TMZ groups is represented (** p < 0.01; *** p < 0.001).