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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

Increased DCLK1 expression is observed across a panelof codon12 mutant KRAS isogenic cell lines. (A) Increased expression of selectedhits from our SILAC proteome analysis were confirmed using Westernblotting. ERK1/2 is an example of a responsive controls. (B) Westernblotting of a wider panel of isogenic SW48 cells, including linesnot directly analyzed by proteomics, shows that DCLK1 and MET followthe same patterns of codon 12 specific up-regulation whereas ZO-2is coupled to KRAS G13D signaling. (C) QPCR analysis indicates significantup-regulation of DCLK1 isoform 3/4 expression in codon 12 mutant KRAScells. (D) Schematic diagram of DCLK1 isoforms expressed in humanand distribution of peptides observed in our data set indicates thatall of the DCLK1 peptides detected by mass spectrometry are in theC terminus. n ≥ 3 for each panel.
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fig6: Increased DCLK1 expression is observed across a panelof codon12 mutant KRAS isogenic cell lines. (A) Increased expression of selectedhits from our SILAC proteome analysis were confirmed using Westernblotting. ERK1/2 is an example of a responsive controls. (B) Westernblotting of a wider panel of isogenic SW48 cells, including linesnot directly analyzed by proteomics, shows that DCLK1 and MET followthe same patterns of codon 12 specific up-regulation whereas ZO-2is coupled to KRAS G13D signaling. (C) QPCR analysis indicates significantup-regulation of DCLK1 isoform 3/4 expression in codon 12 mutant KRAScells. (D) Schematic diagram of DCLK1 isoforms expressed in humanand distribution of peptides observed in our data set indicates thatall of the DCLK1 peptides detected by mass spectrometry are in theC terminus. n ≥ 3 for each panel.

Mentions: A total of 274 phosphopeptides from 198proteins representing 17%of the total number of unique sites were observed in clusters 5 and6 of the phosphoproteome data set (Figure 3A). In total, 56 out of 274 phosphopeptides are associated with 27proteins linked by GO analysis to cell adhesion or cytoskeletal functionin clusters 5 and 6 of the phosphoproteome data set (Figure 3B). Among these, MAP1B and TJP2/ZO-2 are representedby multiple phosphopeptides (Figure 3C). Othernotable phosphopeptide representatives of clusters 5 and 6 are theHGF-receptor MET Thr995 and Caveolin-1 Ser37 sites that both exhibit>10-fold increases in abundance in G12D versus G13D cells and theRas effector BRAF Ser729 site that is decreased in G12D versus G13Dcells. In the case of caveolin and MET, a component of this changeis due to the higher levels of protein expression observed in G12Dand G12V cells as judged by Western blotting (Figure 6).


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)

Increased DCLK1 expression is observed across a panelof codon12 mutant KRAS isogenic cell lines. (A) Increased expression of selectedhits from our SILAC proteome analysis were confirmed using Westernblotting. ERK1/2 is an example of a responsive controls. (B) Westernblotting of a wider panel of isogenic SW48 cells, including linesnot directly analyzed by proteomics, shows that DCLK1 and MET followthe same patterns of codon 12 specific up-regulation whereas ZO-2is coupled to KRAS G13D signaling. (C) QPCR analysis indicates significantup-regulation of DCLK1 isoform 3/4 expression in codon 12 mutant KRAScells. (D) Schematic diagram of DCLK1 isoforms expressed in humanand distribution of peptides observed in our data set indicates thatall of the DCLK1 peptides detected by mass spectrometry are in theC terminus. n ≥ 3 for each panel.
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Related In: Results  -  Collection

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fig6: Increased DCLK1 expression is observed across a panelof codon12 mutant KRAS isogenic cell lines. (A) Increased expression of selectedhits from our SILAC proteome analysis were confirmed using Westernblotting. ERK1/2 is an example of a responsive controls. (B) Westernblotting of a wider panel of isogenic SW48 cells, including linesnot directly analyzed by proteomics, shows that DCLK1 and MET followthe same patterns of codon 12 specific up-regulation whereas ZO-2is coupled to KRAS G13D signaling. (C) QPCR analysis indicates significantup-regulation of DCLK1 isoform 3/4 expression in codon 12 mutant KRAScells. (D) Schematic diagram of DCLK1 isoforms expressed in humanand distribution of peptides observed in our data set indicates thatall of the DCLK1 peptides detected by mass spectrometry are in theC terminus. n ≥ 3 for each panel.
Mentions: A total of 274 phosphopeptides from 198proteins representing 17%of the total number of unique sites were observed in clusters 5 and6 of the phosphoproteome data set (Figure 3A). In total, 56 out of 274 phosphopeptides are associated with 27proteins linked by GO analysis to cell adhesion or cytoskeletal functionin clusters 5 and 6 of the phosphoproteome data set (Figure 3B). Among these, MAP1B and TJP2/ZO-2 are representedby multiple phosphopeptides (Figure 3C). Othernotable phosphopeptide representatives of clusters 5 and 6 are theHGF-receptor MET Thr995 and Caveolin-1 Ser37 sites that both exhibit>10-fold increases in abundance in G12D versus G13D cells and theRas effector BRAF Ser729 site that is decreased in G12D versus G13Dcells. In the case of caveolin and MET, a component of this changeis due to the higher levels of protein expression observed in G12Dand G12V cells as judged by Western blotting (Figure 6).

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