Quantitative proteomics reveals novel proteins and central pathways associated with endocrine resistance in breast cancer

Introduction

Acquired resistance to endocrine therapies remains a major clinical obstacle in hormone-sensitive breast tumors. The complexity of the underlying biological mechanisms remains poorly understood and the purpose of this study was to identify low abundant proteins and central pathways associated with tamoxifen resistance.

Methods

The global protein expression of the parental tamoxifen-sensitive MCF7S0.5 cell line and the tamoxifen-resistant TamR1 cell line were compared using SILAC labeling and quantitative mass spectrometry. Data were processed using MaxQuant, the global protein-protein interaction network was predicted using STRING and enriched pathways were identified with KEGG analysis. Selected proteins differentially expressed were validated using western blotting and immunocytochemistry.

Results

Proteomic analysis identified 5,370 proteins based on at least one unique peptide of which 4,448 proteins could be quantified based on at least two peptides. Forty-one proteins were found to be differentially expressed more than threefold and eight proteins were validated by western blotting and immunocytochemistry as potential biomarkers associated with tamoxifen resistance. In total 539 proteins were differentially expressed 1.5-fold or more and could be subgrouped into kinases, transcription factors, receptor activity proteins, cell adhesion proteins, cell cycle proteins and stress responder proteins. We identified several regulated proteins to be important in subnetworks that among others are involved in focal adhesion, DNA replication, apoptosis, and insulin and HER2 signaling pathway.

Conclusion

Novel low abundant proteins not previously associated with tamoxifen resistance have been identified and validated using biochemical methods. At present the proteins are being validated on a panel of primary breast cancer biopsies from patients treated with tamoxifen monotherapy and with known clinical outcome. Our data also revealed several pathways associated to tamoxifen resistance. The importance of these pathways needs to be studied further.