Tailoring extracellular vesicles (EVs) as targeted drug delivery systems to enhance the therapeutic efficacy showed superior advantage over liposomal therapies. Herein, we developed a novel nanotool for targeting B16.F10 murine melanoma, based on EVs stabilized with Polyethylene glycol (PEG) and loaded with doxorubicin (DOX). Small EVs were efficiently enriched from melanoma cells cultured under metabolic stress by ultrafiltration coupled with size exclusion chromatography (UF-SEC) and characterized by size, morphology, and proteome. To reduce their clearance in vivo , EVs were PEGylated and passively loaded with DOX (PEG-EV-DOX). Our data suggested that the low PEG coverage of EVs might still favor EV surface protein interactions with target proteins from intratumor cells, ensuring their use as “Trojan horses” to deliver DOX to the tumor tissue. Moreover, our results showed a superior antitumor activity of PEG-EV-DOX in B16.F10 murine melanoma models in vivo compared to that exerted by clinically applied liposomal DOX in the same tumor model. The PEG-EV-DOX administration in vivo reduced NF-κB activation and increased BAX expression, suggesting better prognosis of EV-based therapy than liposomal DOX treatment. Collectively, our results highlight the promising potential of EVs as optimal tools for systemic delivery of DOX to solid tumors.

Eyes absent (EYA) proteins are unusual proteins combining in a single polypeptide chain transactivation, threonine phosphatase, and tyrosine phosphatase activities. They play pivotal roles in organogenesis and are involved in a variety of physiological and pathological processes including innate immunity, DNA damage repair or cancer metastasis. The molecular targets of EYA tyrosine phosphatase activity are still elusive. Therefore, we sought to identify novel EYA substrates and also to obtain further insight into the tyrosine-dephosphorylating role of EYA proteins in various cellular processes. We show here that Src kinase phosphorylates tyrosine residues in two human EYA family members, EYA1 and EYA3. Both can autodephosphorylate these residues and their nuclear and cytoskeletal localization seems to be controlled by Src phosphorylation. Next, using a microarray of phosphotyrosine-containing peptides, we identified a phosphopeptide derived from WD-repeat-containing protein 1 (WDR1) that is dephosphorylated by EYA3. We further demonstrated that several tyrosine residues on WDR1 are phosphorylated by Src kinase, and are efficiently dephosphorylated by EYA3, but not by EYA1. The lack of phosphorylation generates major changes to the cellular actin cytoskeleton. We, therefore, conclude that WDR1 is an EYA3-specific substrate, which implies that EYA3 is a key modulator of the cytoskeletal reorganization.

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold‐induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2‐induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.