Identification of Mutant K-Ras-dependent Phenotypes Using a Panel of Isogenic Cell Lines

Genentech, Inc, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2012; 288(4). DOI: 10.1074/jbc.M112.394130
Source: PubMed


In order to assess the consequences of endogenous mutant KRas, 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 KRas allele has been disrupted, and SW48 colorectal cancer cells and HMECs in which a single copy of mutant KRas was introduced at its endogenous genomic locus. We find that single copy mutant KRas causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and NRas occurs in some, but not all, of these cell lines. Mutant KRas 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, knockout of the wild type KRas 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 KRas on global gene expression. While transcriptional programs triggered by mutant KRas 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 KRas inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant KRas signaling and phenotypes, but that many depend on cell context and environmental cues.

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    • "To determine if overexpression of RasGAP had any effect on KRAS activity, a RAS activity assay was performed, using Raf-RBD-linked agarose beads to immunoprecipitate active KRAS (Figure 4D). As has been shown previously [40], KRAS overall was less active in the DKO4 cells compared to the DLD1 cells. We saw a slight but significant decrease in KRAS activity in DKO4 cells after RasGAP overexpression, indicating that RasGAP is able to regulate the wild-type KRAS in DKO4. "
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    ABSTRACT: KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.
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    • "Changes in the expression of IP 3 Rs in cancer cells have been reported previously. Most notably, an increase in IP 3 R3 expression at the mRNA level has been detected in a recent microarray analysis of K-Ras-deleted cells lines (Vartanian et al., 2013). In gastric cancer cells, an increase in IP 3 R3 was observed in the ascites, but not in cancer cells established from primary tumours. "

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    ABSTRACT: The mutant forms of K, N and HRas are known to drive the initiation and progression of a number of human cancers, but less is known about the role of wild-type (WT) Ras alleles and isoforms in this process. We used zinc finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth factor-dependent signaling through the ERK pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signaling. This was associated with decreased proliferation, increased apoptosis, and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumor growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumor growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. This study demonstrates that the WT copies of H, N and KRas play unique roles in the context of mutant KRas-driven tumors.
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