RRAS2 Human

What is RRAS2 and how does it relate to other RAS family proteins?

RRAS2, originally known as teratocarcinoma clone 21 (TC21), is a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases. It received significant attention upon its discovery in 1990 due to several unique features that distinguished it from other RAS superfamily members:

• It shares 100% homology at the protein level in the effector regions (switch I and switch II) with classical RAS proteins (H-RAS, K-RAS, N-RAS)

• It binds to a similar spectrum of RAS regulators and proximal effectors, including PI3K, RAL GDS, and RAF family members

• Despite these similarities, endogenous RRAS2 has a distinct subcellular localization compared to classical RAS proteins, specifically concentrating in focal adhesions rather than plasma membrane, Golgi apparatus or endoplasmic reticulum

• It regulates cell proliferation and differentiation via the RAS/MAPK signaling pathway

These distinctive characteristics make RRAS2 a unique player in cellular signaling with non-redundant functions compared to classical RAS proteins.

What mutations in RRAS2 are found in human diseases?

Several mutations in RRAS2 have been identified in both cancer and developmental disorders:

Cancer-associated mutations:

• Hotspot mutations at Q72L and Q72H (analogous to Q61 in classical RAS proteins)

• Additional mutations targeting residues G23 (G23A/C/S/V), G24 (G24C/D/V), and A70 (A70T)

• These mutations are found at higher frequencies in specific cancer types including central nervous system germinomas (14.6% of cases) and KIT+ germ cell tumors (11.8% of cases)

Developmental disorder mutations:

• Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome

• Specific variants include p.Gly23Val and p.Gly24Glu, which can present with Noonan-like phenotypes

The Q72 residue is particularly important as mutations at this position impair GTP hydrolysis, maintaining the GTPase in an active state and promoting oncogenic effects .

What signaling pathways does RRAS2 activate?

RRAS2 activates multiple signaling pathways that contribute to its biological functions:

• PI3K-AKT pathway: Oncogenic RRAS2 mutants strongly activate the PI3K-AKT pathway, which appears to be critical for their transforming activity

• MEK-ERK1/2 pathway: RRAS2 also activates the ERK pathway, although activation of both PI3K-AKT and ERK pathways seems necessary for full transforming potential

• Transcriptional regulation: RRAS2 Q72L regulates gene expression programs linked to cell adhesion and inflammatory/immune-related responses

Interestingly, mutations that only activate the ERK1/2 pathway without concurrent PI3K-AKT activation (such as D44E, R147Q, K159Q and A167T) show minimal transforming activity, suggesting that PI3K-AKT engagement is essential for RRAS2's oncogenic functions .

How can researchers assess the transforming activity of RRAS2 mutations?

To evaluate the transforming potential of RRAS2 mutations, researchers can employ several methodological approaches:

Focus formation assays:

• This is the primary method used to assess transforming activity of RRAS2 mutants

• Express mutant versions in immortalized cell lines (such as NIH3T3) and quantify focus formation

• Compare results with known oncogenic mutations (Q72L serves as a positive control)

Signaling pathway activation analysis:

• Perform immunoblot analyses of phosphorylated ERK1/2 and AKT in serum-starved cells expressing RRAS2 mutants

• Establish correlation between signaling pathway activation and transforming activity

• A strong correlation exists between PI3K-AKT activation and transforming potential

In vivo tumorigenesis assays:

• Use inducible knock-in mouse models expressing RRAS2 mutations

• Monitor tumor development in multiple tissues

• The Q72L mutation, for example, drives rapid development of multiple tumor types when somatically expressed in mice

Table 1: Transforming activity and signaling pathway activation by selected RRAS2 mutations

What methodologies are effective for studying endogenous RRAS2 localization and function?

Several cutting-edge techniques can be employed to study endogenous RRAS2:

CRISPR-Cas9 gene editing approaches:

• Knockout studies: Generate RRAS2 knockout cell lines to assess its necessity for maintaining cancer cell properties

• Endogenous tagging: Insert fluorescent protein tags (such as EGFP) in-frame with the RRAS2 open reading frame to visualize endogenous protein without overexpression artifacts

• This approach revealed that endogenous RRAS2 specifically localizes to focal adhesions, contrary to earlier studies using ectopic expression systems

Confocal microscopy with co-localization markers:

• Use markers like vinculin to confirm localization in focal adhesions

• Compare localization of wild-type versus mutant RRAS2 proteins

• Assess dynamic changes in localization in response to cellular stimuli

Functional assays:

• Measure cell adhesion, proliferation, and invasiveness in RRAS2 wild-type versus knockout cells

• Analyze focal adhesion dynamics and turnover

• Evaluate mitochondrial respiration to assess metabolic effects

Why is RRAS2 essential in cancer cells that already harbor RAS pathway mutations?

One intriguing aspect of RRAS2 biology is that cancer cells can remain dependent on RRAS2 Q72L even when they contain concurrent gain-of-function mutations in classical RAS pathway genes. Researchers investigating this phenomenon have found:

• In the A2780 ovarian cell line (RRAS2 Q72L positive), knockout of RRAS2 significantly reduced cell proliferation and colony formation despite the presence of mutations in PIK3CA (H1047R/+)

• Similarly, in the CAL-51 breast cancer cell line (RRAS2 Q72L positive), RRAS2 knockout reduced malignant properties despite the cell line harboring mutations in PIK3CA (E542K/+) and PIK3R1 (A629S/+)

• RRAS2 Q72L knockout led to significant reductions in phosphorylation levels of both MEK-ERK1/2 and PI3K-AKT pathway components in these cell lines

These findings suggest that RRAS2 Q72L provides non-redundant oncogenic signaling that cannot be compensated by other RAS pathway mutations. The distinct subcellular localization of RRAS2 in focal adhesions likely contributes to its unique functions in regulating cell adhesion, invasion, and gene expression programs that are essential for maintaining the cancer phenotype .

How can RRAS2 pathogenic variants be functionally analyzed in developmental disorders?

For studying RRAS2 variants associated with developmental disorders like Noonan syndrome, researchers employ a multi-system approach:

In vitro cellular systems:

• Transiently express wild-type and mutant RRAS2 genes in human cell lines (such as HEK293 cells)

• Confirm expression and pathway activation through Western blotting for RRAS2 and phosphorylated ERK1/2

• Quantify RAS signaling pathway activity using reporter assay systems with serum response element-luciferase constructs

Model organism approaches:

• Drosophila models: Express wild-type and mutant RRAS2 in Drosophila eye using the glass multiple reporter-Gal4 driver to assess developmental effects

• Zebrafish models: Microinject mutant mRNA into zebrafish embryos and observe developmental phenotypes, particularly in craniofacial structures like the jaw

• These in vivo systems allow visualization of how RRAS2 variants affect tissue patterning and morphogenesis

Patient-derived studies:

• Correlate genotype with detailed phenotypic characterization of patients

• For example, patients with RRAS2 p.Gly23Val mutations may present with features including hypertelorism, down-slanted palpebral fissures, broad nasal root, low-set ears, macrotia, and cardiac abnormalities like ventricular septal defects

What databases and tools are useful for identifying RRAS2-dependent cancer cell lines?

To identify cancer cell lines dependent on RRAS2 or other RAS family members, researchers can utilize several resources:

Novartis Drive Data Portal:

• This database contains shRNA-mediated depletion data for hundreds of cell lines

• Analysis of this dataset revealed that only 2 out of 387 cell lines (A2780 ovarian and CAL-51 breast cancer cell lines) show RRAS2-dependency

• Both RRAS2-dependent cell lines harbor the Q72L mutation

cBioPortal database:

• Useful for identifying the frequency of RRAS2 mutations across different cancer types

• Provides information on co-occurring genomic alterations

• Helps identify potential cell line models for RRAS2 studies

Experimental validation approaches:

• CRISPR-Cas9 knockout to confirm dependency in candidate cell lines

• Proliferation, colony formation, and invasion assays to assess the impact of RRAS2 loss

• Analysis of signaling pathway activation in wild-type versus knockout cells

Table 2: Cell line dependency on RAS family members based on Novartis Drive Data Portal

What controls should be included when studying RRAS2 in cancer models?

When designing experiments to investigate RRAS2 in cancer, appropriate controls are essential:

• Cell line selection: Include both RRAS2-mutant and wild-type cell lines from the same tissue origin (e.g., A2780 with RRAS2 Q72L vs. COV362/COV504 with wild-type RRAS2)

• Genetic controls: Generate multiple CRISPR knockout clones and match with parental cells to account for clonal effects

• Pathway analysis controls: Compare effects of RRAS2 knockout with targeted inhibitors of downstream pathways (PI3K, MEK)

• Rescue experiments: Re-express wild-type or mutant RRAS2 in knockout cells to confirm specificity of observed phenotypes

When examining transformation potential, it's critical to compare multiple RRAS2 mutations simultaneously, as transforming activity can vary substantially even among mutations affecting the same residue (e.g., G24C/V vs. G24D) .

How can researchers distinguish RRAS2-specific functions from those of classical RAS proteins?

Distinguishing RRAS2-specific functions requires strategic experimental approaches:

Subcellular localization studies:

• Use endogenous tagging approaches rather than overexpression systems, as the latter may give misleading results regarding localization

• Compare localization of endogenously tagged RRAS2 with classical RAS proteins in the same cells

• Investigate protein-protein interactions at specific subcellular locations like focal adhesions

Signaling pathway analysis:

• Perform detailed time-course experiments to identify potential differences in signaling dynamics

• Use phospho-proteomics to identify unique downstream targets of RRAS2 versus classical RAS proteins

• Investigate context-dependent signaling differences in various cell types

Genetic interaction studies:

• Perform combinatorial knockout/knockdown of RRAS2 with classical RAS proteins

• Assess synthetic lethality or rescue effects between family members

• Examine differential dependencies across various cancer contexts

What are the advantages and limitations of different model systems for studying RRAS2 function?

Each model system offers unique advantages and limitations for RRAS2 research:

Cell culture systems:

• Advantages: Easy manipulation, human origin, amenable to high-throughput studies

• Limitations: Lack organismal context, potential adaptation to culture conditions

• Best applications: Molecular signaling studies, drug screening, initial characterization of mutants

Mouse models:

• Advantages: Physiological context, ability to study tissue-specific effects and tumor development in vivo

• Limitations: Species differences, time and resource intensive

• Best applications: Tumor initiation studies, evaluation of therapeutic strategies, investigation of systemic effects

Drosophila models:

• Advantages: Rapid generation time, powerful genetics, conservation of key signaling pathways

• Limitations: Evolutionary distance from humans

• Best applications: Initial functional validation of variants, genetic interaction studies

Zebrafish models:

• Advantages: Vertebrate system, transparent embryos allow direct visualization of development

• Limitations: Partial genome duplication can complicate genetic studies

• Best applications: Studying developmental phenotypes, craniofacial abnormalities, early patterning defects

How might understanding RRAS2 biology inform therapeutic strategies?

The unique properties of RRAS2 suggest several therapeutic opportunities:

• Targeted inhibition: The distinct localization of RRAS2 in focal adhesions might allow for specific targeting without affecting classical RAS proteins

• Synthetic lethality: Cancer cells with RRAS2 Q72L mutations might have unique vulnerabilities that could be exploited therapeutically

• Combination strategies: Since RRAS2 contributes to cancer cell properties even in the presence of other RAS pathway mutations, combining RRAS2-targeted therapies with existing RAS pathway inhibitors might overcome resistance mechanisms

The finding that RRAS2 Q72L is essential even in cells with concurrent mutations in classical RAS pathway genes suggests that targeting RRAS2 might be effective in cancers that are resistant to existing RAS pathway inhibitors .

What methods can determine if RRAS2 represents a therapeutic vulnerability in patient tumors?

Assessing RRAS2 as a therapeutic vulnerability requires multiple approaches:

Genomic profiling:

• Screen patient tumors for RRAS2 mutations, particularly Q72L/H, G23V, and other transforming variants

• Analyze co-occurring mutations in RAS pathway genes to identify potential synthetic lethal interactions

Functional assays:

• Develop patient-derived xenograft models or organoids from RRAS2-mutant tumors

• Test sensitivity to pathway inhibitors in these models

• Perform CRISPR screens to identify genetic dependencies specific to RRAS2-mutant cells

Biomarker development:

• Identify transcriptional or proteomic signatures associated with RRAS2 dependency

• Develop immunohistochemical methods to detect activated RRAS2 signaling in tumor samples

• Correlate RRAS2 status with clinical outcomes to identify patient populations most likely to benefit from targeted therapies

What are the outstanding questions regarding RRAS2 biology in human disease?

Despite recent advances, several key questions remain unanswered:

• How does the unique localization of RRAS2 in focal adhesions contribute to its specific functions in cell adhesion, invasion, and metastasis?

• What are the precise mechanisms by which RRAS2 regulates gene expression programs related to cell adhesion and inflammatory/immune responses?

• Are there tissue-specific functions of RRAS2 that explain the pattern of cancers associated with RRAS2 mutations?

• How do germline RRAS2 mutations in developmental disorders differ functionally from somatic mutations in cancer?

Addressing these questions will require integrative approaches combining structural biology, advanced imaging, genomics, and proteomics techniques.

What novel technologies might advance RRAS2 research?

Emerging technologies that could accelerate RRAS2 research include:

• Proximity labeling proteomics: BioID or APEX2-based approaches to identify the interactome of RRAS2 at focal adhesions

• Live-cell super-resolution microscopy: To visualize dynamics of RRAS2 signaling complexes in real time

• Single-cell multi-omics: To understand heterogeneity in RRAS2 signaling within tumors

• CRISPR base editing: For precise introduction of specific RRAS2 mutations without double-strand breaks

• Cryo-EM: To determine structures of RRAS2 in complex with effectors and regulators

These advanced technologies will help resolve the molecular mechanisms underlying RRAS2's unique functions in both physiological and pathological contexts.