Systems Biology Approach to Evaluate Ezrin as a Therapeutic Target in Breast Cancer

摘要

The complexity of breast cancer demands a systems level understanding of aberrant biochemical signaling in order to develop smart therapeutic targets. This study was undertaken to identify the binding partners of the ERM family protein, ezrin, in normal and metastatic breast cancer cells. We report the generation of ezrin constructs that were tagged at the C- and N- terminus to enable tandem affinity purification of ezrin protein complexes. Mutagenesis of ezrin at key phosphorylation sites was performed to create inactive and constitutively active versions of the protein (T567D, T567A, Y353F, Y477F, S66A) that are expected to differ in their ability to bind to cellular proteins. Overexpression of mutant ezrin protein was tested in the highly metastatic breast cancer cell line 4T1. Overexpression of ezrin mutants did not affect the basal activity of MAPK and PI3K in 4T1 cells. We have completed the optimization of tandem affinity purification protocol for purification of TAP- tagged ezrin and identification by mass spectrometry. In future, we aim to generate 4T1 transfectants stably expressing wild type or mutant versions of ezrin, and purify ezrin complexes to identify their components in normal breast cells and the 4T1 breast cancer cells. The identification of ezrin-interacting proteins will allow elucidation of aberrant signaling pathways in breast cancer cells and offer novel therapeutic targets for the treatment of breast cancer.