title: Identification of MET and SRC activation in melanoma cell lines showing primary resistance to PLX4032.
abstract: PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF(V600E) and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.
authors.affiliation: Immunotherapy Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Vergani
authors.forename: Viviana
authors.initials: V
authors.lastname: Vallacchi
authors.forename: Simona
authors.initials: S
authors.lastname: Frigerio
authors.forename: Paola
authors.initials: P
authors.lastname: Deho
authors.forename: Piera
authors.initials: P
authors.lastname: Mondellini
authors.forename: Paola
authors.initials: P
authors.lastname: Perego
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cassinelli
authors.forename: Cinzia
authors.initials: C
authors.lastname: Lanzi
authors.forename: Maria Adele
authors.initials: MA
authors.lastname: Testi
authors.forename: Licia
authors.initials: L
authors.lastname: Rivoltini
authors.forename: Italia
authors.initials: I
authors.lastname: Bongarzone
authors.forename: Monica
authors.initials: M
authors.lastname: Rodolfo
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology*; D000077484:Vemurafenib; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000077484:Vemurafenib; D019859:Proto-Oncogene Proteins c-met; D019061:src-Family Kinases; D048493:Proto-Oncogene Proteins B-raf; D051059:PTEN Phosphohydrolase