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

Clavaín, Laura; Fernández‐Pisonero, Isabel; Movilla, Nieves; Lorenzo‐Martín, L. Francisco; Nieto, Blanca; Abad, Antonio; García-Navas, Rósula; Llorente-González, Clara; Sánchez-Martı́n, Manuel; Vicente‐Manzanares, Miguel; Santos, Eugenio; Alarcón, Balbino; Aznar, José Manuel García; Dosil, Mercedes; Bustelo, Xosé R.

doi:10.1038/s41388-022-02563-9

Cited by 4 publications

(7 citation statements)

References 53 publications

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“…It has recently been shown that endogenous R-RAS2 (upon R-RAS2 knock-out and knock-in of fluorescently labelled R-RAS2), unlike canonical Ras proteins, specifically localizes in focal adhesions [ 12 ]. However, in our experiments we did not observe that the sponge or the human homolog localizes in focal adhesions.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, in contrast to previous reports using ectopic expression systems that showed subcellular localization of R-RAS2 protein in the plasma membrane, Golgi apparatus, or endoplasmic reticulum [ 9 , 10 ], a recent study showed that endogenous wild-type R-RAS2 protein is specifically localized in both nascent and mature focal adhesions. The distribution of R-RAS2 overlaps with the focal adhesion marker vinculin and with F-actin present in these structures and regulates focal adhesion- and invasion-related functions [ 12 ]. This is in line with previous detection of R-RAS2 in proteomics experiments in myosin II-responsive focal adhesion complexes [ 58 ].…”

Section: Discussionmentioning

confidence: 99%

“…Our previous study confirmed the localization of R-RAS2 in the plasma membrane and vesicular membranes, possibly the endocytic vesicles, in HeLa and MJ90 fibroblast cell lines [ 11 ]. Endogenous wild-type R-RAS2 specifically localizes to focal adhesions in cancer cell lines from different cancer types (ovary, breast, fibrosarcoma) and species (human and mouse), suggesting its role in cell adhesion [ 12 ]. M-RAS plays an important role in cytoskeletal remodeling, cell migration, osteoblastic and neuronal differentiation, dendrite formation, and lymphocyte adhesion [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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The ancestral type of the R-RAS protein has oncogenic potential

Talajić,

Dominko,

Lončarić

et al. 2024

Cell Mol Biol Lett

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Background The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. Methods Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. Results We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. Conclusions This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The ancestral type of the R-RAS protein has oncogenic potential

Talajić,

Dominko,

Lončarić

et al. 2024

Cell Mol Biol Lett

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“…RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is located in the plasma membrane and functions as a signal transducer. Like other RAS proteins, it is considered to regulate cell proliferation and differentiation via the RAS/MAPK cascade, and somatic mutations in the RRAS2 gene are drivers of tumorigenesis; they have been detected in various tumors such as colon, lung, and skin cancers ( Aoki et al, 2016 ; Clavaín et al, 2023 ). Furthermore, germline RRAS2 mutations have been reported to cause NS ( Capri et al, 2019 ; Niihori et al, 2019 ; Weinstock and Sadler, 2022 ; Yu et al, 2023 ).…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype

Iida,

Igarashi,

Fukunaga

et al. 2024

Front. Genet.

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Introduction: RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis.Materials and methods: Wild-type (WT) and mutant RRAS2 genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed.Results: No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway.Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients.

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“…Moreover, other mutations (G23V/A/C/S, G24D/C/V, A70T, and Q72H) in human cancer have shown transforming potential when expressed in immortal cell lines [42].…”

Section: Introductionmentioning

confidence: 99%

Mice Overexpressing Wild Type RRAS2: A Novel Preclinical Model for Testing Anti-Chronic Lymphocytic Leukemia Therapies

Alarcón,

Hortal,

Villanueva

et al. 2023

Preprint

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B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the Western world. Mutations in different genes, such as TP53 and ATM, and deletions in specific chromosomal regions, including 11q or 17p, have been associated with a worse prognosis for the disease. Recent research from our group has demonstrated that, contrary to the usual cancer development process through missense mutations, B-CLL is driven by the overexpression of the small GTPase RRAS2 in its wild-type form, without activating mutations. While some mouse models of this disease have been developed to date and are commonly used in B-CLL research, they come with various disadvantages, such as a long waiting period until the leukemia fully develops, the need for cell engraftment, or, in some cases, a failure to replicate the alterations found in human patients. We have recently introduced Rosa26-RRAS2fl/flxmb1-Cre as a new mouse model of B-CLL with full disease penetrance. In this work, we have validated this mouse model as a novel tool for the development of new therapies for B-CLL by testing two of the most widely applied targeted agents: ibrutinib and venetoclax. This also paves the way for the development of new targeted agents against R-RAS2 itself, an approach that has not yet been explored in clinical practice.

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

Cited by 4 publications

References 53 publications

The ancestral type of the R-RAS protein has oncogenic potential

The ancestral type of the R-RAS protein has oncogenic potential

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype

Mice Overexpressing Wild Type RRAS2: A Novel Preclinical Model for Testing Anti-Chronic Lymphocytic Leukemia Therapies

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