Quantitative proteomic analysis of HER2 normal and overexpressing MCF-7 breast cancer cells revealed proteomic changes accompanied with HER2 gene amplification

Abstract Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 ( ERBB2 ) gene, which leads to HER2 protein overproduction. While the presence of the overexpressed HER2 protein is a necessary precondition for sensitivity to anti-HER2 therapies, many patients develop resistance. Thus, identification of the downstream effectors of this pathway will help in understanding mechanism(s) of chemoresistance and further, the identified molecules themselves may have the potential to be used as therapeutic targets. In this work, we studied the proteomic changes that accompany the HER2 gene amplification to identify potential new therapeutic targets and biomarkers. We analyzed bio-triplicate proteome samples extracted from wild-type MCF-7 human breast cancer cells and their isogenic stably overexpressing HER2 (amplified) transfectants. In total, 2455 unique proteins were quantified with 1278 of them differentially expressed in HER2 normal and HER2 overexpressing MCF-7 cells. Select biomarker candidates of particular interest were validated by western blotting, and evaluated for clinical relevance by the immunohistochemical assessment of protein abundance in breast tumor biopsies. HER2 transfection produced marked changes in proteins related to multiple aspects of cancer biology, and the identified expression patterns were recapitulated in the clinical samples. Biological significance Breast cancer is a major cause of death in women. Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 ( ERBB2 ) gene, which leads to HER2 protein overproduction that triggers intracellular signaling events that drive proliferation, invasion, metastases, and resistance to apoptosis. While the presence of the overexpressed HER2 gene product, HER2 protein, is a necessary precondition for sensitivity to the therapeutic monoclonal antibody trastuzumab, the downstream effects of HER2 protein overexpression are incompletely understood. In this work, we applied quantitative proteomics to identify proteomic changes accompanying ERBB2 gene amplification. The significance of this work includes 1) identification of new biomarkers associated with the HER2 phenotype, 2) measurement of the magnitude of the proteomic changes triggered by the amplification of this single gene, and 3) better understanding of the downstream biological changes triggered by HER2 overexpression.