title: Afatinib resistance in non-small cell lung cancer involves the PI3K/AKT and MAPK/ERK signalling pathways and epithelial-to-mesenchymal transition.
abstract: The epidermal growth factor receptor (EGFR) signalling is one of the most deregulated pathways in non-small cell lung cancer (NSCLC). Recently, the development of novel irreversible tyrosine kinase inhibitors (TKI), such as afatinib, has significantly improved the survival of advanced NSCLC patients harbouring activated EGFR mutations. However, treatment with TKI is not always curative due to the development of resistance. In the present study, we investigated the sensitivity to afatinib in two NSCLC EGFR mutated cell lines (NCI-H1650 and NCI-H1975) by expression profile analysis of 92 genes involved in the EGF pathway. Thereafter, the established afatinib resistant clones were evaluated at different biological levels: genomic, by array comparative genomic hybridisation (aCGH) and deep sequencing; transcriptomic, by quantitative polymerase chain reaction (qPCR) and proteomic, by Western blot and immunofluorescence. The baseline gene expression of the two cell lines revealed that NCI-H1650, the less afatinib-responsive cell, showed activation of two main EGFR downstream pathways such as PI3K/AKT and PLCγ/PKC axes. Analysis of the afatinib-resistant cells showed PI3K/AKT and MAPK/ERK pathways activation together with a biological switch from an epithelial-to-mesenchymal phenotype might confer afatinib-resistant properties to this cell line. Our data suggest that the activation of EGFR-dependent downstream pathways might be involved in the occurrence of resistance to afatinib assuming that the EGFR mutational status should not be exclusively considered when selecting TKI treatments. In particular, the epithelial-to-mesenchymal transition might provide a new basis for understanding afatinib resistance.
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST, L.go R. Benzi 10, 16132, Genoa, Italy, simona.coco@hsanmartino.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Coco
authors.forename: Anna
authors.initials: A
authors.lastname: Truini
authors.forename: Angela
authors.initials: A
authors.lastname: Alama
authors.forename: Maria Giovanna
authors.initials: MG
authors.lastname: Dal Bello
authors.forename: Roberta
authors.initials: R
authors.lastname: Venè
authors.forename: Anna
authors.initials: A
authors.lastname: Garuti
authors.forename: Enrico
authors.initials: E
authors.lastname: Carminati
authors.forename: Erika
authors.initials: E
authors.lastname: Rijavec
authors.forename: Carlo
authors.initials: C
authors.lastname: Genova
authors.forename: Giulia
authors.initials: G
authors.lastname: Barletta
authors.forename: Claudio
authors.initials: C
authors.lastname: Sini
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
authors.forename: Francesco
authors.initials: F
authors.lastname: Boccardo
authors.forename: Francesco
authors.initials: F
authors.lastname: Grossi
mesh_terms: D000077716:Afatinib; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology*; D045744:Cell Line, Tumor / Q000187:drug effects; D055028:Comparative Genomic Hybridization; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D058750:Epithelial-Mesenchymal Transition; D066246:ErbB Receptors / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D020935:MAP Kinase Signaling System; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction
chemical_list: D000970:Antineoplastic Agents; D011799:Quinazolines; D000077716:Afatinib; D019869:Phosphatidylinositol 3-Kinases; C512478:EGFR protein, human; D066246:ErbB Receptors; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases