Biomarker Discovery and Evaluation of Response to Anti-Cancer Therapeutics in Breast Cancer Using a Novel Nanofluidic Immunoassay Platform.

Abstract

Our research efforts focus on the identification and detection of fundamental molecular differences between normal and tumor cells in breast, as well as differences among distinct breast cancer subtypes, especially in terms of signal transduction pathways that control cell cycle, apoptosis and cell growth. Cancer subtype specific molecular variations dramatically affect patient responses to already existing treatments. For example, the phosphorylation status of many proteins that are involved in signal transduction pathways perturbed in cancer cells is extremely important in determining whether these cells are susceptible to killing by available cancer therapeutics. Therefore, differentially phosphorylated protein isoforms can be a particularly useful prognostic biomarker of drug response in the clinic. However, accurate detection and quantitative analysis of cancer-related phosphoproteins in tumors is limited by current technologies.Using a novel, fully automated nanocapillary electrophoresis technology (FireFlyTM) designed to separate protein molecules based on their isoelectric point (pI), we are currently developing highly sensitive assays for reliable assessment of the phosphorylation status of cancer-related phosphoproteins in tumors, before and during drug treatment. We have already developed and optimized assays measuring AKT1, AKT2, AKT3, ERK1 and ERK2, and their respective phosphoisoforms. Using these assays, we were able to measure levels of activated ERK1/2 and AKT1/2/3 in a breast cancer cell line panel developed in our lab, using protein extracted from as few as 125 cells. Based on RNA expression data, cell lines in this panel have previously been categorized in two distinct subtypes (Basal and Luminal) and their molecular phenotypes closely resemble the respective profiles of tumors obtained from breast cancer patients (CITATION). This cell line panel is extensively used to measure cellular responses to breast cancer therapeutics, including drugs that target MEK, ERK, PI3K and AKT. Using FireFly assays, we are currently measuring changes in the phosphorylation states of these targets during drug treatment, in order to completely characterize pharmacodynamic changes in these cells during treatment, and develop molecular profiles that predict response in breast cancer tumors. Since this technology enables accurate detection and quantification of protein isoforms and post-translational modifications from only very small amounts of tumor samples or serum, it promises to propel cancer biomarker discovery and enable the development of clinically useful prognostic and diagnostic assays that predict responses to drugs targeting cancer-specific molecular networks. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3172.