Extraction of Phosphorylated Proteins from Formalin-Fixed Cancer Cells and Tissues

Deregulated signal pathways are promising targets for cancer therapy as they can drive tumour growth, sur- vival, invasion, and metastasis. In most hospitals around the world, histopathological cancer diagnosis is based upon for- malin-fixed and paraffin-embedded (FFPE) tissues. In this study, we evaluated whether phosphorylated signaling proteins can be extracted from cells and tissues after formalin fixation. Three epidermal growth factor (EGF) receptor-positive cell lines were stimulated with EGF for various time points and then either fixed with formalin or left unfixed before protein extraction. Using Western blot, we were able to detect all phosphorylated proteins analyzed, including activated (phos- phorylated) extracellular signal-regulated kinase (ERK) 1 and 2. There were no differences between formalin-fixed and unfixed cells. Because ERK1/2 kinases are involved in HER2-mediated signal transduction, we applied our methodology to study ERK1/2 and phospho-ERK1/2 in 20 primary breast cancers using protein lysate microarray technology. We found a statistically significant correlation between HER2 status and ERK1/2 expression; however, activation (phosphory- lation) of ERK1/2 was independent of HER2 expression. Our study demonstrates the successful extraction of phosphory- lated proteins from FFPE tissue samples. Monitoring phosphorylated signaling proteins in FFPE tissues, as applied here, may allow us in the near future to infer the activity levels of proteins in particular pathways as starting point for the design of individual combination therapy regimens without changing the routine clinical workflow for tissue analysis.