Gα13 Promotes Clonogenic Growth by Increasing Tolerance to Oxidative Metabolic Stress in Prostate Cancer Cells

Abstract

The oncogenic role of the G12 family in many human solid cancers has been extensively studied, primarily through the effects of constitutively active mutants of these proteins on cell migration and invasion. However, these mutations are not seen in cancers, and the biological role of Gα13 in prostate cancer tumorigenesis is largely unexplored. Here, we report that Gα13 promotes anchorage-independent colony formation, spheroid formation, and xenograft tumor growth in human prostate cancer cell lines. Transcriptome analyses suggest that Gα13 modulates genes in the mitochondria and are involved in the oxidative stress response. Silencing of GNA13 increased mitochondrial superoxide levels when prostate cancer cells were cultured in galactose medium and increased the sensitivity to oxidative metabolic stress when the cells were cultured in media containing non-glycolytic metabolites. Furthermore, Gα13 levels impacts the abundance of superoxide dismutase 2 (SOD2) in the mitochondria, as well as SOD2 promoter activity and mRNA expression. Importantly, expression of SOD2 could rescue the effect of Gα13 loss on suppression of anchorage-independent growth. Likewise, stable knockdown of SOD2 decreased anchorage-independent cell growth, which was enhanced by overexpression of Gα13. These results outline a novel biological function of Gα13 mediated via SOD2 in prostate cancer tumorigenesis and highlight it as a potential treatment target.

Keywords: GNA13; Gα13; LNCaP; PC3; SOD2; mitochondria; oxidative metabolic stress; prostate cancer; superoxide; superoxide dismutase.

Figure 1

Modulation of Gα13 expression impacts 3D anchorage-independent growth in PC3 and LNCaP cells. (A,C) Gα13 protein levels in (A) PC3 and (C) LNCaP cells. (B,D) Number of colonies formed in 3D soft agar upon Gα13 modulation in (B) PC3 and (D) LNCaP cells. (E,G) Number of tertiary spheroids and (F,H) 3D colonies from tertiary spheroids upon Gα13 modulation in (E,F) PC3 and (G,H) LNCaP cells. Data points represent independent experiments. (A,C) p values calculated by unpaired, two-tailed t-tests or unpaired one-way ANOVA with Dunnett’s post hoc test. (B,D,E–H) p values calculated by paired, two-tailed t-tests or paired, one-way ANOVA with Dunnett’s post hoc test.

Figure 2

The association between Gα13 and mitochondrial SOD2 expression. (A,B) Effect of Gα13 on mitochondrial SOD2 protein levels in (A) PC3 and (B) LNCaP cells in representative Western blots. (C) Correlation plot of GNA13 against SOD2 mRNA expression from TCGA database of prostate cancer patient (PRAD) tumor samples extracted from cBioPortal. Correlations were calculated by Spearman’s Rank Correlation for the correlation coefficient rho. (D) Effect of Gα13 on SOD2 mRNA expression in PC3 cells. (E) Effect of Gα13 on SOD2 promoter activity in PC3 cells. The ratio of Firefly to Renilla luciferase activity was calculated, and fold change relative to sh-control cells is shown. (A,B,D,E) Data points represent independent experiments. p values calculated by unpaired, two-tailed t-tests or unpaired one-way ANOVA with Dunnett’s post hoc test.

Figure 3

Loss of Gα13 increases sensitivity to oxidative metabolic stress and decreases 3D colony formation. (A) Effect of Gα13 on mitochondrial superoxide levels in PC3 cells in 11 mM glucose or 10 mM galactose media. Relative mitochondrial superoxide to mitochondrial mass ratio was calculated by median fluorescence intensity (MFI) of MitoSOX Red divided by MFI of MitoView Green. PC3 sh-control cells were treated with 1 mM rotenone as a positive control and 100 µM MitoTempo as a negative control. (B) Effect of Gα13 loss on long-term colony formation in non-glycolytic metabolites media in PC3 cells. (C) Western blot of stable expression of SOD2 and catalytically inactive SOD2 (Q143A) in monolayer PC3 cells. (D,E) Effect of SOD2 or SOD2 (Q143A) expression on (D) colony formation and (E) tertiary spheroid colony formation in PC3 cells after silencing Gα13. (F) Western blot of stable knockdown of SOD2 in monolayer LNCaP cells. (G–I) Effect of SOD2 knockdown on (G) colony formation, (H) spheroid replating and (I) tertiary spheroid colony formation after overexpression of Gα13 in LNCaP cells. All data points represent independent experiments. (A–G) p values calculated by matched, one-way ANOVA with Tukey’s post hoc test. (H,I) p values calculated by paired, two-tailed t-tests.

Figure 4

Loss of Gα13 suppresses PC3 cell tumor growth in NOD SCID mouse xenograft model. (A) Panel 1, schematic of mouse xenograft injection. Panel 2–4, Western blot and relative densitometry of PC3 cells on the day of xenograft injections. (B) Tumor growth rate (n = 7). (C) Relative tumor weight per mouse. (D) Relative tumor volume per mouse. p values calculated by unpaired, two-tailed t-tests.