Abstract P1-05-06: A novel mutation in the tyrosine kinase domain of ErbB2: molecular and proteomic investigation of its role in breast cancer invasion

Abstract Introduction: A landmark genomic study revealed 17 somatic coding mutations in a lobular metastatic breast tumor, none of which were present in the patient's primary tumor. Among these was a novel mutation in the tyrosine kinase domain of ErbB2, a gene often amplified in breast cancer. Other mutations in this domain have been shown to stimulate transformation, growth and invasion in breast cancer cells. We hypothesize that an accumulation of mutations in the primary tumor over time preceded the development of an invasive clone, which allowed it to metastasise. Therefore, we investigated the specific effects of ErbB2 on cellular signalling and invasion, in conjunction with proteomic analyses to show its effects on global protein expression. Methods: Wild-type and mutant ErbB2 cDNAs were cloned into V180 pLP 3X FLAG-tagged expression vectors. T47D breast cancer cells were transfected with one of these constructs. Protein was detected by immunoblotting with an anti-ErbB2 antibody. Membranes were then immunoblotted with an anti-phospho-ERK1/2 antibody. Transfected cells were seeded onto Matrigel; cells that migrated through the matrix were stained and counted after 48h. To identify global proteomic changes caused by mutant ErbB2, T47D cells transfected with mutant/wild-type ErbB2 or vector control, were differentially labeled by growing them in medium containing various lysine/arginine isotopes (known as stable isotope labeling of amino acids in cell culture; SILAC). Protein lysates were mixed and subjected to LC-MS/MS mass spectrometry. Proteomic changes were analysed using MaxQuant and DAVID software. Results: Overexpression of the wild-type or mutant ErbB2 genes produced protein expression in T47D, a non-invasive breast cancer cell line, confirmed by western blotting. To measure signalling downstream of ErbB2, we measured levels of the MAP kinase phospho-ERK1/2. Increased phosphorylation of ERK1/2 was demonstrated in cells expressing mutated ErbB2, compared with cells transfected with wild-type. This indicates increased activation of mutant ErbB2, demonstrating distinct phenotypic effects between wild-type and mutant ErbB2 in breast cancer cells. To analyze whether increased activation was accompanied by enhanced invasive properties, invasion assays were performed using Matrigel. A greater number of cells (1.5-fold increase) expressing mutant ErbB2 migrated across this matrix compared with wild-type-expressing cells. Global proteomic analysis of T47D cells overexpressing wild-type or mutant ErbB2 yielded 1,357 total proteins, of which 543 could be quantified. Two subsets of proteins were identified, whose levels were either increased or decreased, in the mutant versus wild-type-transfected lysates (compared to control ratios). Proteins elevated in the mutant-expressing cells included candidates involved in cell migration, inhibition of apoptosis and promotion of cellular survival. Proteins showing reduced expression were involved in pathways including adhesion and DNA damage response. Conclusion: This previously undescribed mutation in the tyrosine kinase domain of ErbB2 differentially effects downstream signalling pathways and may play a role in invasion of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-06.

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