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CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases.

Tan YH, Krishnaswamy S, Nandi S, Kanteti R, Vora S, Onel K, Hasina R, Lo FY, El-Hashani E, Cervantes G, Robinson M, Hsu HS, Kales SC, Lipkowitz S, Karrison T, Sattler M, Vokes EE, Wang YC, Salgia R - PLoS ONE (2010)

Bottom Line: In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL.The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples.Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC.

Methods and findings: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility.

Conclusions: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

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Ubiquitination, viability, expression and cell cycle analysis of various c-CBL mutants.(A) c-CBL mutants do not alter ubiquitination of EGFR. Cells were co-transfected with EGFR and different c-CBL mutants were stimulated with EGF, immunoprecipitated with anti-EGFR antibody and blotted with anti-ubiquitin antibody. Immunoblot with anti-EGFR antibody served as the IP control, while the anti-HA blot was used as the input control. (B) Cell viability was measured by Trypan blue exclusion and compared to empty vector control. c-CBL wild-type (WT) and mutants S80N/H94Y, Q249E, and W802* showed 66.7%, 132.3%, 120.8%, and 147.9% cell viability respectively in A549 cells 48h after transfection. Experiments were done in triplicates and the mean data is shown. Error bars indicate Standard Deviation. (C) Protein expression levels of the various mutants were analyzed by Western blots using c-CBL antibody. (D) Cell cycle analysis of different c-CBL mutants 48 h after transfection in A549 cells.
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pone-0008972-g003: Ubiquitination, viability, expression and cell cycle analysis of various c-CBL mutants.(A) c-CBL mutants do not alter ubiquitination of EGFR. Cells were co-transfected with EGFR and different c-CBL mutants were stimulated with EGF, immunoprecipitated with anti-EGFR antibody and blotted with anti-ubiquitin antibody. Immunoblot with anti-EGFR antibody served as the IP control, while the anti-HA blot was used as the input control. (B) Cell viability was measured by Trypan blue exclusion and compared to empty vector control. c-CBL wild-type (WT) and mutants S80N/H94Y, Q249E, and W802* showed 66.7%, 132.3%, 120.8%, and 147.9% cell viability respectively in A549 cells 48h after transfection. Experiments were done in triplicates and the mean data is shown. Error bars indicate Standard Deviation. (C) Protein expression levels of the various mutants were analyzed by Western blots using c-CBL antibody. (D) Cell cycle analysis of different c-CBL mutants 48 h after transfection in A549 cells.

Mentions: To investigate whether the different c-CBL mutations affect the E3 activity, EGFR was chosen as a model substrate for c-CBL E3 function. All of the c-CBL mutants tested enhanced ubiquitination of the activated EGFR similar to the wild-type c-CBL protein. This result demonstrates that the catalytic activity of the c-CBL mutants is not impaired when EGFR was the substrate. (Figure 3A).


CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases.

Tan YH, Krishnaswamy S, Nandi S, Kanteti R, Vora S, Onel K, Hasina R, Lo FY, El-Hashani E, Cervantes G, Robinson M, Hsu HS, Kales SC, Lipkowitz S, Karrison T, Sattler M, Vokes EE, Wang YC, Salgia R - PLoS ONE (2010)

Ubiquitination, viability, expression and cell cycle analysis of various c-CBL mutants.(A) c-CBL mutants do not alter ubiquitination of EGFR. Cells were co-transfected with EGFR and different c-CBL mutants were stimulated with EGF, immunoprecipitated with anti-EGFR antibody and blotted with anti-ubiquitin antibody. Immunoblot with anti-EGFR antibody served as the IP control, while the anti-HA blot was used as the input control. (B) Cell viability was measured by Trypan blue exclusion and compared to empty vector control. c-CBL wild-type (WT) and mutants S80N/H94Y, Q249E, and W802* showed 66.7%, 132.3%, 120.8%, and 147.9% cell viability respectively in A549 cells 48h after transfection. Experiments were done in triplicates and the mean data is shown. Error bars indicate Standard Deviation. (C) Protein expression levels of the various mutants were analyzed by Western blots using c-CBL antibody. (D) Cell cycle analysis of different c-CBL mutants 48 h after transfection in A549 cells.
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getmorefigures.php?uid=PMC2813301&req=5

pone-0008972-g003: Ubiquitination, viability, expression and cell cycle analysis of various c-CBL mutants.(A) c-CBL mutants do not alter ubiquitination of EGFR. Cells were co-transfected with EGFR and different c-CBL mutants were stimulated with EGF, immunoprecipitated with anti-EGFR antibody and blotted with anti-ubiquitin antibody. Immunoblot with anti-EGFR antibody served as the IP control, while the anti-HA blot was used as the input control. (B) Cell viability was measured by Trypan blue exclusion and compared to empty vector control. c-CBL wild-type (WT) and mutants S80N/H94Y, Q249E, and W802* showed 66.7%, 132.3%, 120.8%, and 147.9% cell viability respectively in A549 cells 48h after transfection. Experiments were done in triplicates and the mean data is shown. Error bars indicate Standard Deviation. (C) Protein expression levels of the various mutants were analyzed by Western blots using c-CBL antibody. (D) Cell cycle analysis of different c-CBL mutants 48 h after transfection in A549 cells.
Mentions: To investigate whether the different c-CBL mutations affect the E3 activity, EGFR was chosen as a model substrate for c-CBL E3 function. All of the c-CBL mutants tested enhanced ubiquitination of the activated EGFR similar to the wild-type c-CBL protein. This result demonstrates that the catalytic activity of the c-CBL mutants is not impaired when EGFR was the substrate. (Figure 3A).

Bottom Line: In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL.The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples.Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

ABSTRACT

Background: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC.

Methods and findings: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility.

Conclusions: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

Show MeSH
Related in: MedlinePlus