RESUMEN
Tyrosine kinase inhibitors, such as erlotinib, display reliable responses and survival benefits for the treatment of human non-small cell lung cancer (NSCLC) patients. However, primary or acquired resistance limits their therapeutic success. In this study, we conducted in-depth mass spectrometric analyses of NSCLC cell secretomes. To identify secreted proteins that are differentially regulated in erlotinib-sensitive (PC-9) and -resistant (PC-9ER) NSCLC cell lines, SILAC experiments were performed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins. Fourteen proteins were found to be differently regulated among erlotinib-sensitive and -resistant NSCLC cell lines, with five proteins (tissue-type plasminogen activator, epidermal growth factor receptor, urokinase-type plasminogen activator, platelet-derived growth factor D, and myeloid-derived growth factor) showing the most prominent regulation. Tissue-type plasminogen activator (t-PA) was up to 10-times upregulated in erlotinib-resistant NSCLC cells compared with erlotinib-sensitive cells. T-PA is an established tumor marker for various cancer types and seems to be a promising prognostic marker to differentiate erlotinib-sensitive from erlotinib-resistant NSCLC cells. To gain further insights into t-PA-regulated pathways, a t-PA variant was expressed in E. coli cells and its interactions with proteins secreted from erlotinib-sensitive and -resistant NCSLC cells were studied by a combined affinity enrichment chemical cross-linking/mass spectrometry (MS) approach. Fourteen proteins were identified as potential t-PA interaction partners, deserving a closer inspection to unravel the mechanisms underlying erlotinib resistance in NSCLC cells.