We thank Luca Azzolin and Stefano Piccolo for YAP antibody and for their feedback on our work. that aid amyloid maturation into fibrils. We also confirm the accumulation of amyloid\like aggregates, much like those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta\secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta\secretase inhibitors as potential therapeutic approach for metastatic melanoma. model of metastatic and primitive melanoma With the aim to understand the functional pathways TIAM1 that differentiate tumor microenvironment of metastatic and primitive phenotype, we investigated two pairs of matched melanoma cell lines. In particular, IGR39 and IGR37 were derived from primitive tumor and lymph node metastasis, respectively, collected from your same 26\12 months\aged male patient. Similarly, WM115 and WM266.4 matched cell lines were derived from cutaneous primitive tumor and skin metastasis, respectively, from your same NVX-207 55\12 months\old female patient (Fig?1A). Despite their common origin, these cell lines display different phenotypes. NVX-207 In both cases, metastasis\derived cell lines showed a faster growth rate and increased ability NVX-207 to undergo rapid division compared to the matched primitive tumor\derived cell collection (Figs?1B and EV1A). Differences in morphology were also denoted between the two matched cell lines: Metastasis\derived IGR37 appeared with a short, elongated shape with a spontaneous predisposition to form clusters, while primitive tumor\derived IGR39 remained generally isolated, displaying higher quantity of branches and branch elongations (Fig?1C). On the other hand, IGR39 experienced higher mobility compared with IGR37 when monitored live using time\lapse microscopy (Fig?1D, Movie EV1 and EV2). All these data suggest that cells isolated from metastatic tumors grow faster, but move slower than primitive tumors, symptomatic of a proliferative phenotype (Hoek the ones present in the serum from cultured conditions, a triple SILAC was performed. We labeled the proteins coming from primitive and metastatic cell collection, respectively, with medium and heavy amino acids (Fig?2A). For each sample, we analyzed the conditioned medium (CM) after 24?h of serum deprivation to avoid contamination of high abundant proteins as albumin. We checked for the absence of proteins derived from lifeless cells by measuring the viability of the cell lines upon starvation, and we confirmed that none of them suffered that condition (viability ?95%, Fig?EV1B). As far as secreted proteins are highly glycosylated and this modification might mask proteolytic sites hampering protein digestion, we set up a novel method, named Key3D (Secretome De\glycosylation Double Digestion protocol), where a de\glycosylation step (PNGase) was added prior to protein digestion performed with double proteolysis to increase protein protection. Our method enables the unambiguous identification of secreted proteins with high efficiency and quantitative accuracy (Dataset EV1 and EV2). As reported in Fig?2A, by examining an equivalent of 500,000 cells, we identified 2,356 proteins in the SILAC IGR37/IGR39, and 2,157 proteins in the SILAC WM266.4/WM115, increasing four occasions the yield compared to digestion without de\glycosylation (Fig?EV1C, Dataset EV3 and EV4, Peptide Atlas repository; glycosylated peptides represent about one\fourth of the entire dataset, Fig?EV1D), and improving the proteome protection if compared to existing methods (Liberato primitive cell lines (Figs?1 and EV1A). BACE inhibition impairs the amyloidogenic machinery in metastatic melanoma cell lines After demonstrating the enrichment of protein aggregates in metastatic tissues, we wondered if it would be possible to interfere with their production and impact metastasis behavior. The beta\secretase (BACE 1 and 2) enzymes are known to be involved in the formation of protein amyloids. Indeed, PMEL and APP are cleaved by BACE 2 and 1, respectively, and are able to form mature amyloid fibrils through an APOE\mediated process (Rochin primitive cells. LXR activation was observed also in AD (Abildayeva (preprint: Kleffman reported that PMEL silencing affects mouse melanoma cells proliferation similarly to what we observed by BACE inhibition (Wang ECM production is mostly fibroblastic dependent, while ECM remodeling is usually both tumor cell and fibroblastic cell dependent (Lamar in 10?kDa molecular excess weight cutoff concentrating columns. Then, 500?l of concentrated medium was filtered by microcon filters with 10?kDa cutoff (Millipore) and buffer was exchanged with 8?M Urea 100?mM Tris or PBS. Protein aggregates NVX-207 detection Aggregates Fluorescence measurement was performed following manufacturer’s instructions (http://www.enzolifesciences.com/fileadmin/files/manual/ENZ-51023_insert.pdf). Briefly, 2?l of the.
We thank Luca Azzolin and Stefano Piccolo for YAP antibody and for their feedback on our work