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Updated: Apr 18, 2026
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations
Dean E Hammond1, Craig J Mageean, Emma V Rusilowicz
1Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool , Crown Street, Liverpool L69 3BX, United Kingdom.
Abstract:
Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.
Insights
Ras mutations drive cancer, with KRAS being a frequent target. Different KRAS mutations, like G13D versus codon 12, impact colon cancer cell proteomes and phosphoproteomes distinctly.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Oncogenic Ras mutations, particularly in KRAS, are prevalent in approximately 16% of cancers.
- KRAS is the most frequently mutated Ras isoform, with specific codon mutations (12, 13, 61) leading to constitutive activation.
- Distinct codon-specific mutation patterns exist for KRAS across different cancer types, influencing patient outcomes and treatment responses, especially in colon cancer.
Purpose of the Study:
- To investigate the differential cellular effects of specific KRAS mutations (G12V, G12D, G13D) in colon cancer.
- To perform a quantitative proteome and phosphoproteome analysis of isogenic cell lines harboring these KRAS mutations.
Main Methods:
- Utilized isogenic SW48 colon cancer cell lines with edited endogenous KRAS genes (G12V, G12D, G13D).
- Performed quantitative proteomic and phosphoproteomic analysis to capture global protein and phosphorylation changes.
- Analyzed distinct mutational signatures generated by each KRAS mutation.
Main Results:
- Each KRAS mutation (G12V, G12D, G13D) resulted in a unique proteomic and phosphoproteomic signature.
- Significant variability was observed between the G13D mutation and codon 12 KRAS mutants.
- The short form of doublecortin-like Kinase 1 (DCLK1), a colon cancer stem cell marker, was specifically upregulated in KRAS codon 12 mutant cells.
- This DCLK1 upregulation could be reversed by KRAS suppression.
Conclusions:
- KRAS mutations at different codons induce distinct cellular states and molecular profiles.
- The G13D mutation presents a different molecular signature compared to codon 12 mutations in KRAS.
- Targeting KRAS or its downstream effectors may differentially impact cancer stem cell populations, as exemplified by DCLK1 regulation.
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