No abstract

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRASmutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRASmutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnibtreated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

The urokinase plasminogen activator (uPA) and its receptor (uPAR) provide a cell surface integrated multimolecular complex that exerts pleiotropic functions influencing the development of inflammatory, immune, coagulation and fibrinolytic responses. Here we review the evidences indicating a role of the uPA/uPAR system in the regulation of the innate immune system in the inflammation process, of the adaptive immune response, as well as the role of fibrin and fibrin degradation products at the cross-road between coagulation and inflammation. Comparative studies have clearly highlighted the notion that coagulation and immunity are co-regulated and intertwined. The implication is that the vertebrate blood clotting system is evolutionarily by product of the innate immune system, where the blood clotting proteases have diverged from those comprising the complement system. Differences have emerged gradually, as shown by the acquisition of unique protein structures, such as kringle domains and gla (glutammic acid) domains, in order to comply with the increasingly complex vertebrate systems and to defend higher organisms against a range of infections and injuries. Plasminogen activation also controls the formation of complement anaphylotoxins (responsibe for vasodilatation, increase of venular permeability and leukocyte chemotaxis) and of bradykinin (which accounts for vasodilatation, increase of venular permeability and pain) by regulating the plasma contact system. The urokinase plasminogen activator and its cellular receptor, expressed on the surface of human leukocytes, provide a functional unit that, by regulating interaction of leukocytes with extracellular matrix, as well as its degradation, is critical for the migration of leukocytes and for their movement in the damaged tissues.

BackgroundBRAF 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.MethodsWe 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.FindingsWe 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.InterpretationWe 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.FundsAssociazione Italiana Ricerca sul Cancro (AIRC); Ente Cassa di Risparmio di Firenze.