Characterization of mutant versions of the R-RAS2/TC21 GTPase found in tumors

The R-RAS2 GTP hydrolase (GTPase) (also known as TC21) has been traditionally considered quite similar to classical RAS proteins at the regulatory and signaling levels. Recently, a long-tail hotspot mutation targeting the R-RAS2/TC21 Gln72 residue (Q72L) was identified as a potent oncogenic driver. Additional point mutations were also found in other tumors at low frequencies. Despite this, little information is available regarding the transforming role of these mutant versions and their relevance for the tumorigenic properties of already-transformed cancer cells. Here, we report that many of the RRAS2 mutations found in human cancers are highly transforming when expressed in immortalized cell lines. Moreover, the expression of endogenous R-RAS2Q72L is important for maintaining optimal levels of PI3K and ERK activities as well as for the adhesion, invasiveness, proliferation, and mitochondrial respiration of ovarian and breast cancer cell lines. Endogenous R-RAS2Q72L also regulates gene expression programs linked to both cell adhesion and inflammatory/immune-related responses. Endogenous R-RAS2Q72L is also quite relevant for the in vivo tumorigenic activity of these cells. This dependency is observed even though these cancer cell lines bear concurrent gain-of-function mutations in genes encoding RAS signaling elements. Finally, we show that endogenous R-RAS2, unlike the case of classical RAS proteins, specifically localizes in focal adhesions. Collectively, these results indicate that gain-of-function mutations of R-RAS2/TC21 play roles in tumor initiation and maintenance that are not fully redundant with those regulated by classical RAS oncoproteins.


SUPPLEMENTARY FIGURE 3. Impact on the transcriptome of endogenous R-RAS2 Q72L
(A) Heatmap of transcripts up-(red) and downregulated (blue) in RRAS2 Q72L knockout (KO#20) versus control (Parental) A2780 cells. Rows represent independent replicates. Total number of transcripts is indicated at the top. The level of expression is shown in a gradient from dark blue (lowest) to dark red (highest) as indicated in the scale on the right.
(B) Volcano plot of transcripts up-(red) and downregulated (blue) in RRAS2 Q72L knockout (KO#20) versus control (parental) A2780 cells according to data generated in A. Transcripts that show no statistically significant variations over the established fold-change threshold are shown in grey.
(C) KRAS oncogene-related gene signature that is preferentially enriched in the downregulated transcriptome of RRAS2 Q72L knockout A2780 cells. The FDR q value and NES (normalized enrichment score) for the downregulated gene signatures are indicated inside the plot.
(D and E) Examples of gene signatures enriched in the transcriptome of RRAS2 Q72L knockout A2780 cells. The FDR q value and NES (normalized enrichment score) for the downregulated gene signatures are indicated in each plot using either blue (gene signatures enriched in the downregulated transcriptome; panel E, glycolysis) or red (gene signatures positively enriched in the upregulated transcriptome, panel D and E, rest of signatures) or color fonts.

SUPPLEMENTARY FIGURE 4. Impact on the depletion of endogenous wild-type R-RAS2 in the expression of indicated genes in COV368 cells (A)
Expression levels of indicated transcripts in parental (P) and wild-type RRAS2 knockout COV362 cells (clone KO#41) were determined using qRT-PCR analyses. Points represent independent experiments. Bars represent mean ± SEM. Values are given in arbitrary units, taking the lowest mean as 1. Statistical significance was tested by the Student's t-test (*, p < 0.05; ***, p < 0.001).
(B) Representative immunoblot analysis of the phosphorylation (p-) of the p65 NFkB subunit upon the stimulation of serum-starved parental and wild-type RRAS2-knockout COV362 cells (clone KO#41) cells with TNFa for the indicated periods of time (upper panel). As control, we evaluated the level of total p65 (middle panel) and tubulin (bottom panel) in each sample. R, resting cells. n = 2 independent experiments.
(C) Expression levels of indicated transcripts in parental (P) and wild-type RRAS2 knockout COV362 cells (clone KO#41) were determined using qRT-PCR analyses. Points represent independent experiments. Bars represent mean ± SEM. Values are given in arbitrary units, taking the lowest mean as 1. Statistical significance was tested by the Student's t-test.
(D) Expression levels of indicated transcripts in parental (P) and RRAS2 Q72L knockout A2780 cells (clone KO#20) were determined using qRT-PCR analyses. Points represent independent experiments. Bars represent mean ± SEM. Values are given in arbitrary units, taking the lowest mean as 1. Statistical significance was tested by the Student's ttest.

SUPPLEMENTARY FIGURE 5. R-RAS2 regulates mitochondrial activity in cancer cells (A to E)
Mitochondrial respiration profile of the RRAS2 knockout cell lines A2780 (A), CAL-51 (B) or COV362 (C), indicated R-RAS2-expressing NIH3T3 cells (D), and the R-Ras2 Q72 -expressing mouse fibrosarcoma cell line FS#2 (E). In E, we used NIH3T3 cells as controls. ALR, ATP-linked respiration; BR, basal respiration; MR, maximal respiration; NM, non-mitochondrial O2 consumption; PL, proton leak; SC, spare capacity. Data represent mean ± SEM. n = 3 (for A and C), 4 (for B and D) and 7 (E). Statistical significance was tested by two-way ANOVA and Tukey's multiple comparisons test (**, p < 0.01; ***, p < 0.001). Due to this, the asterisks do not match those presented in Fig.  7 (which were calculated using unpaired t-test or Mann-Whitney test depending on the normality of each dataset).