Abstract 5560: Biomarker Discovery and Pathway Mapping using Differential Phosphoprofiling of PI3K-Pathway Inhibitors: PRAS40 Correlates with AKT Activation, but not PTEN Expression in Lung and Breast Cancer

Abstract

Protein phosphorylation plays a key role in cell signaling and pathophysiological alterations in protein kinases and phosphatases contribute to human diseases. We used stable isotope labeling by amino acids (SILAC) in cell culture combined with phospho-antibody based enrichment for quantitative mass-spectrometry-based identification of differentially phosphorylated proteins in response to small-molecule PI3K-pathway inhibition. We quantified over 500 non-redundant serine/threonine phosphopeptides (NSTPs) containing either the AKT-, MAPK-substrate, or PDK1-docking motif. Of these NSTPs, 71 phosphoproteins were modulated by inhibitors targeting either AKT, PDK1 or PI3K/mTOR and a common set of 11 were modulated by all three drugs. Bioinformatics analysis of the regulated phosphoproteins identified core components of the canonical PI3K pathway and showed enrichment in adaptor and scaffolding molecules involved in cell polarity (PAK4), cytoskeletal reorganization (Filamin, Stathmin, Dynamin) vesicle transport (LARP1, VPS13D, SLC20A1), protein translation (S6RP and PRAS40), and transcription (EIF4BP-1). These results have guided the rational selection of antibodies for biomarker and patient stratification assays and a newly developed phosphor-specific antibody against PRAS40T246 shows specificity for oncogenic PI3K-pathway activation in PTEN-deficient mouse prostate tumor tissue by immunohistochemistry. In addition, the phospho-PRAS40T246 biomarker was evaluated across a panel of 67 breast and 96 lung cancer cell lines and in triple negative human breast tumor tissue. In these datasets, phopspho-PRAS40T246 positively correlates with phospho-AKTS473, but not PTEN protein expression. As such, we have positioned PRAS40T246 as a clinically relevant biomarker for the identification of PI3K-pathway activated tumors to enable individualized cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5560.