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Germline EPHB2 receptor variants in familial colorectal cancer.

Zogopoulos G, Jorgensen C, Bacani J, Montpetit A, Lepage P, Ferretti V, Chad L, Selvarajah S, Zanke B, Hudson TJ, Pawson T, Gallinger S - PLoS ONE (2008)

Bottom Line: Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I).The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases.In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

ABSTRACT
Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.

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Biochemical characterization of EPHB2 variants.Panel A: Diminished autophosphorylation of EPHB2 G787R variant in response to ephrinB1 stimulation. DU145 cells were transiently transfected with cDNA constructs (empty vector; wild-type, wt; A438T; G787R) and either left unstimulated (−) or stimulated (+) with preclustered ephrinB1-Fc for 30 min. EPHB2 was immunoprecipitated (IP) and immunoblotted (IB) with antiphosphotyrosine (4G10) to evaluate receptor autophosphorylation. The cell lysate was immunoblotted with antiEPHB2 to ascertain that there was equal transfection efficiency. Panel B: Abolished kinase activity of EPHB2 G787R variant. In vitro kinase assays were performed using wild-type EPHB2 or G787R immunoprecipitates and enolase as the exogenous substrate. Prior to imaging or immonoblotting against EPHB2, phosphorylated proteins were separated by gel electrophoresis and stained with coomassie. Autoradiogram showing 32PγATP incorporation in EPHB2 and enolase (upper panel), anti-EPHB2 immunoblot (middle panel) and equal loading of enolase is shown (lower panel). The table shows the relative kinase activity of the wild-type EPHB2 receptor (set to 100%) vs. the G787R variant.
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pone-0002885-g003: Biochemical characterization of EPHB2 variants.Panel A: Diminished autophosphorylation of EPHB2 G787R variant in response to ephrinB1 stimulation. DU145 cells were transiently transfected with cDNA constructs (empty vector; wild-type, wt; A438T; G787R) and either left unstimulated (−) or stimulated (+) with preclustered ephrinB1-Fc for 30 min. EPHB2 was immunoprecipitated (IP) and immunoblotted (IB) with antiphosphotyrosine (4G10) to evaluate receptor autophosphorylation. The cell lysate was immunoblotted with antiEPHB2 to ascertain that there was equal transfection efficiency. Panel B: Abolished kinase activity of EPHB2 G787R variant. In vitro kinase assays were performed using wild-type EPHB2 or G787R immunoprecipitates and enolase as the exogenous substrate. Prior to imaging or immonoblotting against EPHB2, phosphorylated proteins were separated by gel electrophoresis and stained with coomassie. Autoradiogram showing 32PγATP incorporation in EPHB2 and enolase (upper panel), anti-EPHB2 immunoblot (middle panel) and equal loading of enolase is shown (lower panel). The table shows the relative kinase activity of the wild-type EPHB2 receptor (set to 100%) vs. the G787R variant.

Mentions: Biochemical characterization of the A438T and G787R isoforms revealed that that the G787R variant is functionally impaired, whereas the A438T change likely represents a neutral polymorphism. DU145 cells, which do not express an endogenous functional EPHB2 receptor [11], were transiently transfected with either the wild-type EPHB2 receptor or one of the two variants. We found diminished autophosphorylation of the G787R receptor, but not the A438T variant, following ephrinB1 stimulation compared to the wild-type receptor (Figure 3A). We confirmed that the G787R receptor has reduced catalytic activity by using an in vitro kinase assay. The ability of the G787R receptor to autophosphorylate or to phosphorylate the enolase substrate was approximately 9-fold lower than that of the wild-type receptor (Figure 3B), demonstrating that the G787R mutation alters receptor activity and is not a rare neutral polymorphism.


Germline EPHB2 receptor variants in familial colorectal cancer.

Zogopoulos G, Jorgensen C, Bacani J, Montpetit A, Lepage P, Ferretti V, Chad L, Selvarajah S, Zanke B, Hudson TJ, Pawson T, Gallinger S - PLoS ONE (2008)

Biochemical characterization of EPHB2 variants.Panel A: Diminished autophosphorylation of EPHB2 G787R variant in response to ephrinB1 stimulation. DU145 cells were transiently transfected with cDNA constructs (empty vector; wild-type, wt; A438T; G787R) and either left unstimulated (−) or stimulated (+) with preclustered ephrinB1-Fc for 30 min. EPHB2 was immunoprecipitated (IP) and immunoblotted (IB) with antiphosphotyrosine (4G10) to evaluate receptor autophosphorylation. The cell lysate was immunoblotted with antiEPHB2 to ascertain that there was equal transfection efficiency. Panel B: Abolished kinase activity of EPHB2 G787R variant. In vitro kinase assays were performed using wild-type EPHB2 or G787R immunoprecipitates and enolase as the exogenous substrate. Prior to imaging or immonoblotting against EPHB2, phosphorylated proteins were separated by gel electrophoresis and stained with coomassie. Autoradiogram showing 32PγATP incorporation in EPHB2 and enolase (upper panel), anti-EPHB2 immunoblot (middle panel) and equal loading of enolase is shown (lower panel). The table shows the relative kinase activity of the wild-type EPHB2 receptor (set to 100%) vs. the G787R variant.
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Related In: Results  -  Collection

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pone-0002885-g003: Biochemical characterization of EPHB2 variants.Panel A: Diminished autophosphorylation of EPHB2 G787R variant in response to ephrinB1 stimulation. DU145 cells were transiently transfected with cDNA constructs (empty vector; wild-type, wt; A438T; G787R) and either left unstimulated (−) or stimulated (+) with preclustered ephrinB1-Fc for 30 min. EPHB2 was immunoprecipitated (IP) and immunoblotted (IB) with antiphosphotyrosine (4G10) to evaluate receptor autophosphorylation. The cell lysate was immunoblotted with antiEPHB2 to ascertain that there was equal transfection efficiency. Panel B: Abolished kinase activity of EPHB2 G787R variant. In vitro kinase assays were performed using wild-type EPHB2 or G787R immunoprecipitates and enolase as the exogenous substrate. Prior to imaging or immonoblotting against EPHB2, phosphorylated proteins were separated by gel electrophoresis and stained with coomassie. Autoradiogram showing 32PγATP incorporation in EPHB2 and enolase (upper panel), anti-EPHB2 immunoblot (middle panel) and equal loading of enolase is shown (lower panel). The table shows the relative kinase activity of the wild-type EPHB2 receptor (set to 100%) vs. the G787R variant.
Mentions: Biochemical characterization of the A438T and G787R isoforms revealed that that the G787R variant is functionally impaired, whereas the A438T change likely represents a neutral polymorphism. DU145 cells, which do not express an endogenous functional EPHB2 receptor [11], were transiently transfected with either the wild-type EPHB2 receptor or one of the two variants. We found diminished autophosphorylation of the G787R receptor, but not the A438T variant, following ephrinB1 stimulation compared to the wild-type receptor (Figure 3A). We confirmed that the G787R receptor has reduced catalytic activity by using an in vitro kinase assay. The ability of the G787R receptor to autophosphorylate or to phosphorylate the enolase substrate was approximately 9-fold lower than that of the wild-type receptor (Figure 3B), demonstrating that the G787R mutation alters receptor activity and is not a rare neutral polymorphism.

Bottom Line: Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I).The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases.In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism.

View Article: PubMed Central - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

ABSTRACT
Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.

Show MeSH
Related in: MedlinePlus