Abstract 5126: Development of a high throughput quantitative phosphoproteomic method to study trastuzumab resistance

Her2 is a receptor tyrosine kinase that is overexpressed in approximately 20-30% of human breast cancers and affects clinical prognosis and outcome of these cancers. Treatment of Her2-positive breast cancer with the monoclonal antibody, trastuzumab, has improved the outcomes and prognosis for patients with this breast cancer subtype. However, development of resistance to trastuzumab is a major clinical problem in metastatic Her2-positive breast cancer. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns and therefore we have employed quantitative phosphoproteomics to further study trastuzumab resistance. Quantitative phosphoproteomics is a powerful method to comparatively identify phosphorylated proteins between different samples. Using electrospray tandem-mass spectrometry coupled with immunoaffinity purification and titanium dioxide (TiO2) phosphoenrichment, we wanted to identify differentially phosphorylated proteins in a pair of trastuzumab sensitive and resistant cells. We used stable isotope labeling of amino acids in cell culture (SILAC) to obtain quantitative information on the proteins identified by mass spectrometry. We have obtained quantitative data on two trastuzumab sensitive and resistant pairs of Her2 overexpressing human breast cancer cell lines (BT474 and SKBR3). 275 proteins were identified in the phosphoenriched fraction from the SKBR3 pair with 83 proteins reporting a ratio. 769 proteins were identified in the phosphoenriched fraction from the BT474 pair with 683 proteins reporting a ratio. Treatment of the trastuzumab resistant cells with Her2 siRNA drastically resulted in cell death indicating the sustained dependence of these cells on Her2 despite the resistance to Her2 targeted therapy. RNAi experiments, in combination with trastuzumab exposure, of candidate proteins expressing an increase in phosphorylation will be tested further to determine if trastuzumab resistance can be reversed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2011-5126