BRAF inhibitor (BRAF-I) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months.,Understanding the molecular mechanisms behind BRAF-I responsiveness and acquired resistance is therefore an important issue.,Here we assessed the role of urokinase type plasminogen activator receptor (uPAR) as a potentially valuable biomarker in the acquisition of BRAF-I resistance in V600E mutant melanoma cells.,We examined uPAR and EGFR levels by real time PCR and western blot analysis. uPAR loss of function was realized by knocking down uPAR by RNAi or using M25, a peptide that uncouples uPAR-integrin interaction.,We investigated uPAR-β1integrin-EGFR association by co-immunoprecipitation and confocal immuno-fluorescence analysis.,Acquired resistance to BRAF-I was generated by chronic exposure of cells to vemurafenib.,We proved that uPAR knockdown in combination with vemurafenib inhibits melanoma cell proliferation to greater extent than either treatment alone causing a decrease in AKT and ERK1/2 phosphorylation.,Conversely, we demonstrated that uPAR enforced over-expression results in reduced sensitivity to BRAF inhibition.,Moreover, by targeting uPAR and EGFR interaction with an integrin antagonist peptide we restored vemurafenib responsiveness in melanoma resistant cells.,Furthermore, we found significant detectable uPAR and EGFR levels in tumor biopsies of 4 relapsed patients.,We disclosed an unpredicted mechanism of reduced sensitiveness to BRAF inhibition, driven by elevated levels of uPAR and identified a potential therapeutic strategy to overcome acquired resistance.,Associazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze.
Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations.,Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes.,Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS.,In this study we show that β-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi.,We further demonstrate that β-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo.,Unexpectedly, β-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of β-catenin in melanoma progression and therapy resistance.,We further demonstrate that β-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.,Scheme of the change of interacting proteins with β-catenin during the acquisition of resistance to BRAFi from TCF/LEF family members to novel interactants like Stat3.,Binding of ligands to different receptor tyrosine kinases like c-met and Egfr family members activate the MAPK signaling pathway and PI3K signaling pathway independently of mutated BRAF.,Additional MAPK signaling reactivating mechanisms like the acquisition of novel mutations (asterisk), the formation of Raf heterodimers to the BRAFi.,In this environment a switch in the interaction partners of β-catenin to Stat3 occurs further enhancing resistance and contributing to the relapse of the tumor.,Dotted lines indicated weak activating potential or indirect mechanisms.Image 1