Search for Breast Cancer-related Biomarker Proteins for Drug Discovery

Abstract

  The identification of biomarkers is a promising approach for the diagnosis and effective therapy of cancer. In particular, disease proteomics is a potentially useful method for identifying such biomarkers. However, very few biomarker proteins for drug development have been discovered using this approach. The main difficulty is to efficiently select potential biomarkers from the many candidate proteins identified by the proteomics approach. To circumvent this problem, we have developed “antibody proteomics technology” that can screen for biomarker proteins by isolating antibodies against each candidate in a rapid and comprehensive manner. Here, we applied “antibody proteomics technology” to breast cancer-related biomarker discovery and evaluated the utility of this novel technology. Cell extracts derived from breast tumor cells (SKBR3) and normal cells (184A1) were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE) to identify proteins over-expressed in the tumor cells. Candidate proteins were extracted from the gel pieces, immobilized onto a nitrocellulose membrane using a dot blot apparatus and then used as target antigens in scFv-phage enrichment and selection. Following this in vitro phage selection procedure, scFvs binding to 21 different over-expressed proteins in tumor cells were successfully isolated within several weeks. The expression profiles of the identified proteins were then determined by tissue microarray analysis using the scFv-phages. Consequently, we identified three breast tumor-specific proteins. Our data demonstrates the utility of an antibody proteomics system for discovering and validating tumor-related proteins in pharmaceutical proteomics. Currently, we are analyzing the functions of these proteins to use them as diagnostic markers or therapeutic targets.