Phosphoproteomic study of breast cancer: quantitative analysis of SH2 binding proteins.

Abstract

Abstract Abstract #2034 Background: An important biological process involved in breast cancer cell is tyrosine phosphorylation. Particularly, two tyrosine kinases, Src and HER2, are over expressed in invasive breast cancer tissues and both are considered excellent prognostic markers and therapeutic targets. Since tyrosine kinases transduce downstream signals by phosphorylating substrate proteins, leading to cellular responses, we assume the status of tyrosine-phosphorylated proteins in tumors can also provide molecular markers for breast cancer. Recently we established a method termed SH2 profiling, in which a battery of Src Homology 2 (SH2) domains are used to profile proteomes based on interaction between SH2 domains and tyrosine phosphorylated proteins. In this study we developed a semi-automated method to quantify SH2 binding proteins on multiple blots, and explored the feasibility of SH2 binding patterns as potential molecular markers for breast cancer. Material and Methods: Protein lysates from 10 breast cancer patient tissues as well as cancer cell lines were prepared and loaded into multiple precast gels. Proteins were separated using a SDS-PAGE multigel apparatus and transferred onto nitrocellulose membranes. Blocked membranes were incubated with HRP-labeled GST-SH2 domain probes, and SH2 binding patterns were detected by chemiluminescence with a phosphorimager. To correct blot-to-blot variation, we produced a semi-automated imaging tool for multiple blots. SH2 binding proteins were individually quantified based on their molecular sizes on blots, and different breast cancer samples were compared using the SH2 binding profiles. Results and discussion: 1. We have developed a method to quantify and incorporate data from multiple protein blots, a task that is always challenging due to blot-to-blot variation. 2. Using the semi-automated quantitation approach, large numbers of SH2 binding proteins in breast cancer samples were analyzed. SH2 domains including Vav2 and Eat2 bound strongly to proteins even in HER-2 negative tumors suggesting activation of HER-2 independent tyrosine kinase signaling pathways in breast cancer. 3. Further development of the quantitation method is underway to determine if specific SH2 binding patterns are potential prognostic molecular markers for breast cancer. This study is supported by the Connecticut Breast Health Initiative, Inc. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2034.