Abstract 1996: Surface proteomics of KRASG12V transfected MCF10A cells reveals molecular phenotype of a model cell line expressing oncogenic KRAS

Abstract

Abstract Oncogenic RAS mutations play a major role in the most aggressive epithelial cancers in humans (i.e., pancreatic and lung cancer). Mutated K-Ras protein is found in ∼ 95% of pancreatic and ∼ 40% of lung and/or colon carcinomas, characterized by metastasis and a low average survival time after diagnosis. Plasma membrane proteins are top therapeutic targets due to their accessibility at the surface of cancer cells. In order to expand the treatment options of cancers driven by oncogenic RAS, new cell surface targets need to be identified and characterized. Here, we describe mass spectrometry based phenotyping of the KRASG12V cell surface using MCF10A-KRASG12V as a cell line model of constitutively activated KRAS. Extensive view of the MCF10A-KRASG12V surface proteome was achieved by applying concurrently targeted hydrazide-based cell surface capturing (CSC) technology and global shotgun membrane (GSM) proteomics. Our combined approach revealed 666 plasma membrane proteins unique to the MCF10A-KRASG12V cell surface. Of these, 104 were cell surface glycoproteins identified by CSC technology while 562 cell surface proteins were identified using GSM proteomics. K-Ras was exclusively identified by GSM proteomics in the membrane fraction of MCF10A-KRASG12V cells and subsequently cross-validated by Western blot (WB) analysis. Subtractive proteomics, spectral counting-based based quantitation of changes in protein abundances, and Ingenuity Pathway Analysis showed that this investigation reliably revealed expected K-Ras induced changes at the surface of MCF10A-KRASG12V cells as well as alterations in cell surface protein expression with very little prior information. This analysis uncovered a subset of eight proteins identified exclusively at the cell surface of MCF10A-KRASG12V cells by both CSC and GSM technologies. Of these, two were further cross-validated using immunofluorescence and WB analysis. Furthermore, scanning electron microscopy and functional cell assays showed extensive changes at the KRASG12V cell surface consistent with widespread epithelial to mesenchymal transformation (EMT). Taken together, this dataset greatly extends the known molecular phenotype of the MCF10A-KRASG12V cell surface and reveals the important role of EMT in the pathophysiology of the KRASG12V driven malignant transformation in MCF10A- KRASG12V model cell line. Citation Format: Xiaoying Ye, King C. Chan, Thomas J. Turbyville, Rachel Bagni, Alexander Martinko, Juan Diaz, Jim Wells, Franck McCormick, Gordon Whiteley, Josip Blonder. Surface proteomics of KRASG12V transfected MCF10A cells reveals molecular phenotype of a model cell line expressing oncogenic KRAS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2015-1996