Phosphoproteomic study of breast cancer: high-throughput SH2 screening.

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Abstract Abstract #2031 Background: Protein tyrosine phosphorylation plays an important role in breast cancer cells. Tyrosine kinases such as Src and HER-2 have been shown to be overexpressed in invasive breast cancer tissues, and thus are used as prognostic markers and therapeutic targets. However, substrate proteins for tyrosine kinases in breast cancer tissues have not been extensively studied due to technical difficulties including low sensitivity and large sample requirement. Recently we established a high-throughput method using Src homology 2 (SH2) domain probes, termed SH2 profiling, to profile proteomes based on interaction between SH2 domains and tyrosine-phosphorylated proteins. In this study we aimed to optimize the assay for breast cancer tissues and evaluate SH2 profiling based classification. Material and Methods: Protein lysates from 10 breast cancer patient tissues as well as cancer cell lines were prepared and spotted in multiple duplicates on a nitrocellulose membrane. The blocked membrane was incubated with HRP-labeled GST-SH2 domain probes using a 96 well chamber apparatus. Bound probes were detected by chemiluminescence and signals were quantified using densitometry. Quantitative SH2 binding profiles of breast cancer samples were compared using a hierarchical clustering program. Results and discussion: 1. Although signal-to-noise ratio of breast cancer tissues was relatively low compared to cell lines, possibly due to non-cancerous material in clinical samples, specific signal was detectable using as little as 100 micrograms of sample protein for the entire assay. 2. Unsupervised clustering analysis based on SH2 binding patterns correctly separated HER-2 positive from HER-2 negative tumors, indicating the diagnostic potential of this method. Further validation studies with more samples are needed. 3. Although phosphoproteomics has not yet been adopted in the clinic because of issues related to sample requirements, reproducibility, and sensitivity, this pilot study strongly suggests that clinical applications of phosphoproteomics are feasible. This study is supported by the Breast Cancer Alliance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2031.