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Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

Umar A, Kang H, Timmermans AM, Look MP, Meijer-van Gelder ME, den Bakker MA, Jaitly N, Martens JW, Luider TM, Foekens JA, Pasa-Tolić L - Mol. Cell Proteomics (2009)

Bottom Line: Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients.ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease.Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002).

View Article: PubMed Central - PubMed

Affiliation: Erasmus Medical Center Rotterdam, Josephine Nefkens Inst., Dept. of Medical Oncology, Laboratory of Genomics and Proteomics of Breast Cancer, Dr. Molewaterplein 50, Be 430c, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands. a.umar@erasmusmc.nl

ABSTRACT
Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

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Hierarchical clustering of OR and PD samples. Red and blue colors indicate relative high and low protein abundance, respectively, and white equals median abundance. Gray bars represent sample and protein clusters. The length of the tree arms is inversely correlated with similarity. Proteins are listed vertically from top to bottom and numbered from 1 to 100 in the same order as in Table II.
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f4: Hierarchical clustering of OR and PD samples. Red and blue colors indicate relative high and low protein abundance, respectively, and white equals median abundance. Gray bars represent sample and protein clusters. The length of the tree arms is inversely correlated with similarity. Proteins are listed vertically from top to bottom and numbered from 1 to 100 in the same order as in Table II.

Mentions: For the discovery of proteins that were associated with tamoxifen resistance, the 1,713 overlapping proteins were subjected to statistical analysis. The univariate two-sample t test from BRB-ArrayTools was used to search for differentially abundant proteins between OR and PD samples (Fig. 2). Protein abundances from all OR and PD samples from the two sample sets were analyzed together and compared with each other. The BRB analysis resulted in a list of 153 discriminating proteins using a significance threshold of p < 0.05 (the complete BRB analysis list is provided in supplemental Table S3). These 153 proteins were subsequently subjected to a Wilcoxon rank sum test, which narrowed the list down to 100 proteins with a p value <0.05. These 100 differentially abundant proteins were designated as a putative protein profile associated with the type of response to tamoxifen therapy. In this putative protein profile, 46 proteins had higher relative abundance in PD, and 54 had higher abundance in OR tissues. Protein information as well as OR:PD ratios and p values are listed in Table II in which the order and numbering of proteins corresponds to the order in Fig. 4. Our top discriminating protein in the putative protein profile was splice isoform 2 of basigin precursor (number 13 in Table II), also described in the literature as CD147 or EMMPRIN.


Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

Umar A, Kang H, Timmermans AM, Look MP, Meijer-van Gelder ME, den Bakker MA, Jaitly N, Martens JW, Luider TM, Foekens JA, Pasa-Tolić L - Mol. Cell Proteomics (2009)

Hierarchical clustering of OR and PD samples. Red and blue colors indicate relative high and low protein abundance, respectively, and white equals median abundance. Gray bars represent sample and protein clusters. The length of the tree arms is inversely correlated with similarity. Proteins are listed vertically from top to bottom and numbered from 1 to 100 in the same order as in Table II.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690491&req=5

f4: Hierarchical clustering of OR and PD samples. Red and blue colors indicate relative high and low protein abundance, respectively, and white equals median abundance. Gray bars represent sample and protein clusters. The length of the tree arms is inversely correlated with similarity. Proteins are listed vertically from top to bottom and numbered from 1 to 100 in the same order as in Table II.
Mentions: For the discovery of proteins that were associated with tamoxifen resistance, the 1,713 overlapping proteins were subjected to statistical analysis. The univariate two-sample t test from BRB-ArrayTools was used to search for differentially abundant proteins between OR and PD samples (Fig. 2). Protein abundances from all OR and PD samples from the two sample sets were analyzed together and compared with each other. The BRB analysis resulted in a list of 153 discriminating proteins using a significance threshold of p < 0.05 (the complete BRB analysis list is provided in supplemental Table S3). These 153 proteins were subsequently subjected to a Wilcoxon rank sum test, which narrowed the list down to 100 proteins with a p value <0.05. These 100 differentially abundant proteins were designated as a putative protein profile associated with the type of response to tamoxifen therapy. In this putative protein profile, 46 proteins had higher relative abundance in PD, and 54 had higher abundance in OR tissues. Protein information as well as OR:PD ratios and p values are listed in Table II in which the order and numbering of proteins corresponds to the order in Fig. 4. Our top discriminating protein in the putative protein profile was splice isoform 2 of basigin precursor (number 13 in Table II), also described in the literature as CD147 or EMMPRIN.

Bottom Line: Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients.ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease.Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002).

View Article: PubMed Central - PubMed

Affiliation: Erasmus Medical Center Rotterdam, Josephine Nefkens Inst., Dept. of Medical Oncology, Laboratory of Genomics and Proteomics of Breast Cancer, Dr. Molewaterplein 50, Be 430c, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands. a.umar@erasmusmc.nl

ABSTRACT
Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

Show MeSH
Related in: MedlinePlus