Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells

Area of Science:

• Cancer Biology
• Molecular Oncology
• Drug Discovery

Background:

• Mutations in the KRAS gene are prevalent in human cancers, making it a critical therapeutic target.
• Despite its importance, mutant KRAS proteins have historically been challenging to target effectively with drugs.
• Recent approvals of RAS inhibitors represent progress, but challenges like limited efficacy and acquired resistance persist.

Purpose of the Study:

• To generate and characterize isogenic cell lines with specific oncogenic KRAS mutations (G12V, G12C, G12D) in a physiological context.
• To investigate the impact of these KRAS mutations on cell proliferation, motility, and signaling pathways.
• To evaluate the differential responses of these mutant cell lines to various targeted inhibitors.

Main Methods:

• Generation of near-diploid hTERT RPE-1 isogenic cell lines with endogenous KRAS alleles mutated at glycine 12 (G12V, G12C, G12D) or wild-type (WT).
• Assessment of cell proliferation, motility, and focal adhesion dynamics.
• Analysis of epidermal growth factor (EGF)-induced phosphorylation of ERK and AKT signaling pathways.
• Drug sensitivity assays using hydroxyurea, MEK inhibitors (U0126, trametinib), and a PI3K inhibitor (PIK-90).

Main Results:

• Oncogenic KRAS mutations (KRAS^G12X/+) increased cell proliferation and motility, with KRAS^G12V/+ cells showing reduced focal adhesions.
• EGF-induced ERK and AKT phosphorylation were comparable across all tested KRAS genotypes (mutant and WT).
• KRAS^G12X/+ cells exhibited varying drug sensitivities: KRAS^G12V/+ and KRAS^G12D/+ were more sensitive to hydroxyurea and MEK inhibitors but resistant to a PI3K inhibitor compared to WT cells.
• A combination of hydroxyurea and U0126 demonstrated additive growth inhibition, more pronounced in KRAS^G12V/+ cells.

Conclusions:

• The generated isogenic cell lines provide a valuable resource for studying oncogenic RAS signaling in a relevant cellular context.
• These models highlight differential cellular phenotypes and drug responses associated with specific KRAS mutations.
• Findings support the development of novel anti-KRAS reagents and combination therapies with improved efficacy and reduced off-target effects on wild-type cells.