title: Molecular mechanisms and modulation of key pathways underlying the synergistic interaction of sorafenib with erlotinib in non-small-cell-lung cancer (NSCLC) cells.
abstract: Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.
authors.affiliation: Dept. Medical Oncology, VUmc Cancer Center Amsterdam, VU University Medical Center, CCA room 1.52, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands. elisa.giovannetti@gmail.com
authors.forename: E
authors.initials: E
authors.lastname: Giovannetti
authors.forename: M
authors.initials: M
authors.lastname: Labots
authors.forename: H
authors.initials: H
authors.lastname: Dekker
authors.forename: E
authors.initials: E
authors.lastname: Galvani
authors.forename: J S W
authors.initials: JS
authors.lastname: Lind
authors.forename: R
authors.initials: R
authors.lastname: Sciarrillo
authors.forename: R
authors.initials: R
authors.lastname: Honeywell
authors.forename: E F
authors.initials: EF
authors.lastname: Smit
authors.forename: H M
authors.initials: HM
authors.lastname: Verheul
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D000069347:Erlotinib Hydrochloride; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D058990:Molecular Targeted Therapy; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D049488:Phosphatidylethanolamine Binding Protein; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology; D011799:Quinazolines / Q000008:administration & dosage / Q000494:pharmacology*; D000077157:Sorafenib; D053719:Tandem Mass Spectrometry
chemical_list: D010671:Phenylurea Compounds; D049488:Phosphatidylethanolamine Binding Protein; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D009536:Niacinamide; D000077157:Sorafenib; D000069347:Erlotinib Hydrochloride