Abstract 230: Proteomic approaches in the discovery of novel drug targets or potential biomarkers in breast cancer
Authors: Orfanou Ioanna-Maria , Karampelas Theodoros , Mermelekas George , Vougas Konstantinos , Tamvakopoulos Constantin 
Published: July 2017

Abstract
The aim of this study is the detection, identification and quantification of known and new candidate cell membrane receptors that are overexpressed in Breast Cancer (BC) cells. In order to achieve this goal we have developed State-of-the-Art Proteomic approaches. These novel membrane target receptors can be used either as biomarkers or for the design of targeted drugs against aggressive disease subtypes with poor prognosis and therapeutic outcomes, such as Triple Negative BC (TNBC) and HER2 overexpressing BC.
For our study, four well characterized BC epithelial cell lines were selected: HCC-1954 and SKBR3 (HER2 overexpressing), MDA-MB-231 (TNBC) and MCF-10A (benign control). We employed a combination of subcellular fractionation and membrane enrichment protocols and combined those with quantitative, tandem Mass-Spectrometry (MS) based Proteomics. The discovery phase of our approach included: the GeLC-MS/MS technique, where protein fractions were first separated by 1D-gel electrophoresis followed by In-Gel enzymatic digestion prior to identification by a high resolution Orbitrap mass analyser. A complementary approach for known proteins associated with BC that cannot be readily detected in the discovery phase, such as GPCR receptors, was based on nano LC-MRM analysis. Proof of Concept experiments, with the use of a Triple Quadrupole (QqQ) instrument, were based on the detection of standard peptides derived from the protein of interest after following an in solution tryptic digestion protocol.
Using GeLC-MS/MS approach we identified the well-known BC involved receptors, EGFR and HER2, as well as other potential protein targets in concordance with the literature (e.g. TFR1, EPHA2, GPCR5A). We further confirmed these results by Western blot analysis. Certain GPCRs expected to be present in the cell lines tested were not detected in the studies used on the GeLC-MS/MS analysis, thus we proceeded to the more sensitive and quantitative nanoLC-MRM approach. As a paradigm target we chose the GnRH receptor, an established target for personalized therapy known to be expressed in BC. The quantitative nanoLC-MRM approach revealed GnRHR significant expression in the MDA-MB-231 BC line and in the WPE-NB26-3 Prostate cancer cell line (used as an overexpressing GNRHR positive control).
Our results indicate that the strategy of combining and applying various Proteomic approaches into selected cell lines for the identification of new protein targets will add more information to the genetic and histological classification of the tumor in order to achieve our goal, which is a holistic view of a patient’s molecular profiling that can ultimately lead to treatment in the most effective way.
Citation Format: Ioanna-Maria Orfanou, Theodoros Karampelas, George Mermelekas, Konstantinos Vougas, Constantin Tamvakopoulos. Proteomic approaches in the discovery of novel drug targets or potential biomarkers in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2017-230