Characterization of Signalling Pathways by Reverse Phase Protein Arrays

Reverse phase protein array (RPPA) is a very suitable technique to analyze large numbers of proteins in small samples like for example tumor biopsies. Beside their small size another major hindrance for the analysis of proteins from biopsies is the extraction of proteins from formalin-fixed and paraffin-embedded (FFPE) tissues. Here we describe a protocol, allowing quantitative extraction of large numbers of proteins from FFPE tissues and their subsequent analysis by RPPA. To elucidate the role of epidermal growth factor receptor (EGFR) signalling in ovarian cancer, we analyzed 23 primary tumors and corresponding metastases for the expression of 25 proteins involved in EGFR signalling with special emphasis on epithelial–mesenchymal transition (EMT). We found a significant correlation of Snail with EGFR(Tyr1086) and p38 MAPK(Thr180/Tyr182) in primary ovarian carcinoma and with EGFR(Tyr1086) in their corresponding metastases. Additionally, we showed that high expression levels of the E-cadherin repressor Snail in primary tumors combined with high expression levels of the pp38 MAPK(Thr180/Tyr182) in metastasis lead to an increased risk for death in ovarian carcinoma patients.