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Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations.

Hammond DE, Mageean CJ, Rusilowicz EV, Wickenden JA, Clague MJ, Prior IA - J. Proteome Res. (2015)

Bottom Line: Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases.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).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.

View Article: PubMed Central - PubMed

Affiliation: Division 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.

No MeSH data available.


Related in: MedlinePlus

Responses within the local Ras signaling network. Nodesidentifiedin the proteome data set at highlighted in yellow. Phosphosites identifiedin the phosphosite data set are highlighted in red, those displayingat least a 2-fold change versus Parental cells in at least one ofthe KRAS mutant cell lines are highlighted in green.
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fig8: Responses within the local Ras signaling network. Nodesidentifiedin the proteome data set at highlighted in yellow. Phosphosites identifiedin the phosphosite data set are highlighted in red, those displayingat least a 2-fold change versus Parental cells in at least one ofthe KRAS mutant cell lines are highlighted in green.

Mentions: Our study represents the first unbiased global screen of signalingpathways downstream of endogenous oncogenic KRAS. Our experimentalapproach enabled differences in outputs emanating from each KRAS mutantto be identified without the confounding effects of significant differencesin genetic background. The majority of nodes within the immediateRas signaling network displayed differential responses at the proteomeand phosphoproteome level (Figure 8). The mechanistic basis for this is currently unclear; however, itvividly illustrates the importance of factoring precise mutation statusinto the designs and interpretation of experiments comparing Ras function.For example, several recent studies identified genes that are syntheticallylethal when depleted or inhibited in cells harboring oncogenic KRAS.20,53,56−59 Each study used a different panelof cell lines with a variety of codon 12 or codon 13 mutations andresponsiveness between cell types was inconsistent. Our data predictthat synthetic lethality would likely vary, depending upon which specificmutation is present, and suggest that an isogenic cell line approachwill be important for identifying contingencies of drug responsivenesson mutation status.


Differential reprogramming of isogenic colorectal cancer cells by distinct activating KRAS mutations.

Hammond DE, Mageean CJ, Rusilowicz EV, Wickenden JA, Clague MJ, Prior IA - J. Proteome Res. (2015)

Responses within the local Ras signaling network. Nodesidentifiedin the proteome data set at highlighted in yellow. Phosphosites identifiedin the phosphosite data set are highlighted in red, those displayingat least a 2-fold change versus Parental cells in at least one ofthe KRAS mutant cell lines are highlighted in green.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356034&req=5

fig8: Responses within the local Ras signaling network. Nodesidentifiedin the proteome data set at highlighted in yellow. Phosphosites identifiedin the phosphosite data set are highlighted in red, those displayingat least a 2-fold change versus Parental cells in at least one ofthe KRAS mutant cell lines are highlighted in green.
Mentions: Our study represents the first unbiased global screen of signalingpathways downstream of endogenous oncogenic KRAS. Our experimentalapproach enabled differences in outputs emanating from each KRAS mutantto be identified without the confounding effects of significant differencesin genetic background. The majority of nodes within the immediateRas signaling network displayed differential responses at the proteomeand phosphoproteome level (Figure 8). The mechanistic basis for this is currently unclear; however, itvividly illustrates the importance of factoring precise mutation statusinto the designs and interpretation of experiments comparing Ras function.For example, several recent studies identified genes that are syntheticallylethal when depleted or inhibited in cells harboring oncogenic KRAS.20,53,56−59 Each study used a different panelof cell lines with a variety of codon 12 or codon 13 mutations andresponsiveness between cell types was inconsistent. Our data predictthat synthetic lethality would likely vary, depending upon which specificmutation is present, and suggest that an isogenic cell line approachwill be important for identifying contingencies of drug responsivenesson mutation status.

Bottom Line: Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼ 16% of all cancer cases.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).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.

View Article: PubMed Central - PubMed

Affiliation: Division 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.

No MeSH data available.


Related in: MedlinePlus