Abstract 1829: Comparative surface proteomics of NCI-H2122 cells reveals distinct cell surface phenotype of a metastatic NSCLC cell line expressing oncogenic KRASG12C

Abstract

Abstract Oncogenic KRAS mutations are found in 40% of non-small cell lung carcinomas (NSCLC). In order to expand the treatment options for NSCLC harboring oncogenic K-Ras, new therapeutic cell surface targets need to be identified and characterized. Towards this goal we carried out comparative cell surface analysis of the NSCLC cell line H2122-KRASG12C and the BL2122 cell line (i.e., control), which has been established from the peripheral blood lymphocytes of the same NSCLC patient. Here, we describe optimized hydrazide-based glycoproteomics for mapping of the cell surface proteome of the NSCLC H2122 cell line harboring oncogenic KRASG12C. Our comparative glycoproteomics revealed 632 proteins identified by LC-MS at the surface of both H2122-KRASG12C and BL2122 cell lines. Subtractive proteomics revealed 215 proteins detected solely at the H2122-KRASG12C cell surface while 214 proteins were found germane to the cell surface of BL2122 cells. A total of 203 proteins were commonly identified at the surface of both cell lines. Spectral counting based quantitation revealed 44 proteins showing ≥ 3-fold increase in their relative concentration at the cell surface of H2122-KRASG12C cells. Subsequent meta-analysis via Ingenuity Pathway Analysis (IPA) revealed significant activation of canonical pathways known to be involved in NSCLC biology (e.g., EGFR/neuregulin, and PI3K/AKT signaling). From a subset of proteins showing significant up-regulation at the surface of H2122-KRASG12C cells, we further cross-validated CD147 using immunofluorescence analysis (IFA) and Western blotting (WB). Interestingly, subsequent IFA confirmed the over-expression of CD147 at the cell surface of pancreatic KP-3, lung H2444, and colon SW620 cancer cell lines, each harboring constitutively activated KRAS. Importantly, amongst 215 proteins identified solely at the cell surface of H2122-KRASG12C cells, proteins upstream of K-Ras, epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (ERBB2), receptor tyrosine-protein kinase erbB-3 (ERBB3), and disintegrin metalloproteinase domain-containing protein 17 (ADAM17) were unambiguously identified. Using WB analysis, we first confirmed the expression K-Ras in the membrane preparation of H2122-KRASG12C cells. Interestingly, insulin-like growth factor 1 receptor, (IGF1R), and mesothelin (MSLN) were also detected exclusively at the cell surface of the H2122-KRASG12C. We further cross-validated the expression of mesothelin using WB. Taken together, present approach greatly extends the known cell surface phenotype of the NSCLC H2122-KRASG12C cells and can be readily employed as a primary proteomic screen to provide the basis for discovery and characterization of novel cell surface therapeutic targets or diagnostic assays in cells/tissues harboring oncogenic K-Ras. Citation Format: Xiaoying Ye, Thomas J. Turbyville, Rachel Bagni, Franck McCormick, Gordon Whiteley, Josip Blonder. Comparative surface proteomics of NCI-H2122 cells reveals distinct cell surface phenotype of a metastatic NSCLC cell line expressing oncogenic KRASG12C. [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 1829. doi:10.1158/1538-7445.AM2015-1829