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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Identification of mutant K-Ras-dependent phenotypes using a panel of isogenic cell lines
Steffan Vartanian1, Carolyn Bentley, Matthew J Brauer
1Department of Discovery Oncology, Genentech Inc., South San Francisco, California 94080, USA.
Abstract:
To assess the consequences of endogenous mutant K-Ras, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. These include the cancer cell lines DLD1, HCT116, and Hec1A, in which either the WT or mutant K-ras allele has been disrupted, and SW48 colorectal cancer cells and human mammary epithelial cells in which a single copy of mutant K-ras was introduced at its endogenous genomic locus. We find that single copy mutant K-Ras causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and N-Ras occurs in some, but not all, of these cell lines. Mutant K-Ras also had relatively minor effects on cell proliferation and cell migration but more dramatic effects on cell transformation as assessed by growth in soft agar. Surprisingly, knock-out of the wild type K-ras allele consistently increased growth in soft agar, suggesting tumor-suppressive properties of this gene under these conditions. Finally, we examined the effects of single copy mutant K-Ras on global gene expression. Although transcriptional programs triggered by mutant K-Ras were generally quite distinct in the different cell lines, there was a small number of genes that were consistently overexpressed, and these could be used to monitor K-Ras inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant K-Ras signaling and phenotypes but that many depend on cell context and environmental cues.
Insights
Mutant K-Ras (K-Ras) has modest effects on cell signaling and growth but dramatically impacts cell transformation. Wild-type K-Ras showed tumor-suppressive properties in some contexts, highlighting context-dependent K-Ras functions.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- K-Ras is a key oncogene frequently mutated in human cancers.
- Understanding the precise consequences of endogenous mutant K-Ras activation is crucial for targeted cancer therapies.
Purpose of the Study:
- To investigate the signaling and biological effects of endogenous mutant K-Ras in isogenic cell lines.
- To determine the role of wild-type K-Ras in cancer development.
- To identify conserved transcriptional responses to mutant K-Ras.
Main Methods:
- Utilized isogenic cell lines with disrupted or introduced mutant K-Ras alleles (DLD1, HCT116, Hec1A, SW48, human mammary epithelial cells).
- Assessed downstream signaling pathways (ERK, Akt, EGFR, N-Ras).
- Evaluated biological phenotypes including proliferation, migration, and anchorage-independent growth (soft agar assay).
- Performed global gene expression analysis.
Main Results:
- Single-copy mutant K-Ras induced modest ERK and Akt activation but triggered feedback loops to EGFR and N-Ras in some cell lines.
- Mutant K-Ras had minor effects on proliferation and migration but significant effects on cell transformation (soft agar growth).
- Knock-out of wild-type K-Ras consistently enhanced soft agar growth, suggesting tumor-suppressive roles.
- Identified a small set of consistently overexpressed genes across cell lines, useful for monitoring K-Ras inhibition.
Conclusions:
- Mutant K-Ras signaling and phenotypes exhibit conserved elements but are highly dependent on cellular context and environment.
- Wild-type K-Ras may possess tumor-suppressive functions under specific conditions.
- Conserved gene expression signatures can serve as biomarkers for K-Ras pathway activity.
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