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Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells
Naushin L Hindul1, Lauren R Abbott1, Sumaya M D Adan1
1Department of Molecular and Cell Biology, University of Leicester, Leicester, UK.
Abstract:
About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anticancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. It also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have used the near-diploid hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with WT KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor -induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than WT cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signaling and developing effective anti-KRAS reagents with minimum cytotoxicity on WT cells.
Insights
New isogenic cell lines with KRAS mutations (KRASG12V/+, KRASG12C/+, KRASG12D/+) were created to study oncogenic RAS signaling. These models reveal distinct cellular behaviors and drug responses, aiding the development of targeted cancer therapies.
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 (KRASG12X/+) increased cell proliferation and motility, with KRASG12V/+ cells showing reduced focal adhesions.
- EGF-induced ERK and AKT phosphorylation were comparable across all tested KRAS genotypes (mutant and WT).
- KRASG12X/+ cells exhibited varying drug sensitivities: KRASG12V/+ and KRASG12D/+ 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 KRASG12V/+ 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.
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