Receptor tyrosine kinases alterations in cancer modulates mechano-properties and drug response in tumoral and endothelial cells

In this thesis we analyzed the role of some RTKs and in particular of FGFR1 and VEGFR2 in pathological processes, focusing on the mechanisms that regulate their activation. Among these we analyzed the effect of some mutations identified through a pan cancer analysis in patients affected by cancer. We evaluated how mutations of the kinase domain could alter the receptor membrane dynamics. The substitution in position 255 of the kinase domain reduces its flexibility and stabilizes the Activation loop, that is, for a longer time period, in its “out” conformation, constitutively activating the receptors. Moreover, these active receptors lost their dependence from ligand stimulation. Mutant receptors showed altered membrane dynamics regulation, different membrane recycling and a reduced later mobility. Constitutively active receptors affected the membrane dynamic of the Wild Type form by heterodimerization increasing the pool of mobile WT receptors. Although the substitution in position 236 of the kinase domain reduces the enzymatic activity of the KD receptors, they retain their pro-oncogenic potential when expressed in heterozygous state. Through molecular docking analysis and with the Microscale Thermophoresis (MST) we showed how the mutations modified the affinity for ATP, modulating the ability of the receptor to transduce intracellular signals, and identified potential specific small molecules endowed with inhibitor enzymatic activity. Our results confirmed the capacity of domain-based-approach for the analysis of NGS data to identify mutational hotspots over conserved domains, usually key sequences for protein activity. This approach allows the transfer of clinical and biological information from well-studied mutations to unknown ones, unlocking new therapeutic possibilities through drug repositioning or the discovery of new therapeutic targets. We utilized a syngeneic melanoma model differing solely in mutated receptor expression to evaluate the impact of ECM remodeling on tumor vascularization. We isolated ECM released from these cells and characterized its effects on EC behavior under mechanical stress conditions. FAs were altered in size and number when ECs were seeded onto mutated matrices. Also the Focal Adhesion Kinase (FAK) activity within the plaque was reduced suggesting a modified FA turnover and dissociation rate. The expression of mutant VEGFR2 influences ECM deposition, which in turn affects the ECM-ECs interaction, ECs activity and its response to external mechanical triggers. ECs seeded on mutant VEGFR2 matrices and exposed to shear stress induced by the flow exhibited a cytoskeletal reorganization of the actin filaments. This reorganization could affect the balance between basal and lateral forces that regulate the mechanotransduction machinery. Here we demonstrated the dual regulatory role of VEGFR2 in the regulation of mechanotransduction. Its activation state influences the extracellular matrix modification while its expression level directly modulates intracellular signaling pathways. Indeed the VEGFR2 knockdown supports a pro-motile mechanotransduction in 2D cultures, altering the size of FAs and FAK activity at FAs. This data highlights VEGFR2 as a novel regulator of mechanosensitivity and mechanotransduction in cancer models, highlighting its potential role in the regulation and translation of mechanical forces from external stimuli to biochemical signals inside the cell. The fine characterization of RTK activity in cancer will unlock new potential therapeutic targets and new therapeutic strategies in cancer treatment.

In questa tesi abbiamo analizzato il ruolo di alcuni recettori tirosin-chinasici (RTK), in particolare di FGFR1 e VEGFR2, nei processi patologici, concentrandoci sui meccanismi che regolano la loro attivazione. Tra questi, abbiamo studiato l’effetto di alcune mutazioni identificate attraverso un’analisi pan-cancer in pazienti affetti da tumore. Alcune di queste alterazioni del dominio chinasico riducono la sua flessibilità e stabilizzano il loop di attivazione, alterando l’attività dei recettori. I recettori mutanti hanno mostrato una regolazione alterata della dinamica di membrana, un diverso riciclo di membrana e una ridotta mobilità laterale. Attraverso analisi di docking molecolare e la Microscale Thermophoresis (MST) abbiamo dimostrato come le mutazioni abbiano modificato l’affinità per l’ATP, modulando la capacità del recettore di trasdurre segnali intracellulari, e abbiamo identificato potenziali piccole molecole specifiche dotate di attività inibitoria enzimatica. Utilizzando un modello singenico di melanoma che differiva esclusivamente per l'espressione del recettore mutato è stato valutato l'impatto del rimodellamento della matrice extracellulare (ECM) sulla vascolarizzazione tumorale. Abbiamo isolato l’ECM rilasciata da queste cellule e caratterizzato i suoi effetti sul comportamento delle cellule endoteliali (EC) in condizioni di stress meccanico. Le adesioni focali (FAs) erano alterate in dimensioni e numero quando le EC venivano seminate su matrici mutate. Inoltre, Le EC seminate su queste matrici ed esposte a stress indotto dal flusso laminare hanno mostrato una riorganizzazione del citoscheletro dei filamenti di actina. Questa riorganizzazione potrebbe influenzare l’equilibrio tra forze basali e laterali che regolano la meccano-trasduzione. Questi dati evidenziano VEGFR2 come un nuovo potenziale regolatore della meccano-sensibilità e della meccano-trasduzione nei modelli di cancro mentre il suo livello di espressione modula direttamente le vie di segnalazione intracellulari. La caratterizzazione dettagliata dell’attività dei RTK nel cancro aprirà nuove possibilità per bersagli terapeutici e strategie di trattamento oncologico.