Detecting altered expression and activation of signaling pathways in Cisplatin resistant ovarian cancer by in Cell Westerns.

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The determination of drug resistance patterns for a tumor is important for selecting efficacious therapies and minimizing side effects from drugs to which tumors will not respond. In addition, understanding the signaling pathways that contribute to the resistant phenotype or provide the tumor with a growth advantage may also expose potential therapy choices. With so many signal transduction inhibitors in development, identification of critical pathways in tumors resistant to the primary therapy choices may provide new and successful second line therapies. The Extreme Drug Resistance (EDR) Assay^™ is a clinically validated assay used to test tumors for drug resistance. Based on response in the EDR assay, tumors are segregated into three groups: extreme drug resistant (EDR), intermediate drug resistant (IDR), and low drug resistant (LDR). In this study a set of ovarian tumors, fifteen of each Cisplatin drug response, was selected from chemotherapy naïve patients with LDR response to all other drugs tested. These tumors had also been analyzed on Affymetrix gene arrays. The In Cell Western protocol takes advantage of the LI-COR Odyssey, a sensitive flatbed scanner capable of detecting two fluorophores simultaneously, to quantitate total protein expression and phosphorylation in a rapid, moderate-throughput manner. The advantages of this system over traditional Western blotting are the ability to rapidly screen a large number of samples, reduced sample processing, reduced reagent costs, and simplicity of incorporation of replicates for higher quality data. Using the In Cell Western protocol, forty-five ovarian tumors were scored for EGFR, ERK, Akt, STAT3, β-catenin, and VEGFR2 expression and phosphorylation. The level of activation of each signaling protein is reported as a percentage of phosphoprotein to expressed protein. Traditional Western blot experiments, performed on a subset of the selected tumors, were used to corroborate the In Cell Western data. Results from the In Cell Westerns were analyzed by ANOVA and changes in EGFR, ERK, Akt, and STAT3 protein expression between drug responsive phenotypes, but not β-catenin and VEGFR2 expression, were found to be statistically significant. Analysis of the phosphorylation data is currently being done. The In Cell Western data was comparable to the traditional Western blot data. Moreover, analyses of the gene arrays data both supported the activation of these pathways and provided additional pathways to be examined. Our initial findings support the use of In Cell Westerns to rapidly screen for pathways that provide insight into drug resistance. These types of data may assist selection of more efficacious second line therapies based on tumor specific pathway profiling.