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Background Neuropilin-1 (NRP1) is a transmembrane protein involved in surface receptor complexes for a variety of extracellular signals. NRP1 expression in human cancers is associated with prominent angiogenesis and advanced progression stage. However, the molecular mechanisms underlying NRP1 activity in the tumor microenvironment remain unclear. Notably, diffusible forms of NRP1 in the extracellular space have been reported, but their functional role is poorly understood. Methods Extracellular vesicles (EV) were isolated from conditioned media of diverse cancer cells. The quality of exosome-enriched preparations was validated by the presence of specific markers in western blotting, as well as by light scattering and nanoparticle tracking analysis. Wound healing, transwell, and digital real-time migration assays were carried out to assess the activity of cancer cell-derived exosomes in the regulation of endothelial cells. RNA interference was applied to obtain NRP1 knock-down, and cDNA transfer to achieve its overexpression, in exosome-releasing cells. The micro-RNA profile carried by exosomes was investigated by Next Generation Sequencing. miRNA-Scope in situ hybridization was used to assess the transfer of miRNA exosome cargo to target cells, and immunofluorescence analysis revealed expression regulation of targeted proteins. miRNA activity was blocked by the use of specific antago-miRs. Results In this study, we show that diverse human cancer cells release NRP1 embedded in exosome-like small extracellular vesicles, which mediate a previously unknown NRP1-dependent paracrine signaling mechanism regulating endothelial cell migration. By transcriptomic analysis of the cargo of NRP1-loaded exosomes, we found a significant enrichment of miR-210-3p, known to promote tumor angiogenesis. Gene knock-down and overexpression experiments demonstrated that the loading of miR-210-3p into exosomes is dependent on NRP1. Data furthermore indicate that the exosomes released through this NRP1-driven mechanism effectively transfer miR-210-3p to human endothelial cells, causing paracrine downregulation of the regulatory cue ephrin-A3 and promotion of cell migration. The mechanistic involvement of miR-210-3p in this pathway was confirmed by applying a specific antago-miR. Conclusions In sum, we unveiled a previously unknown NRP1-dependent paracrine signaling mechanism, mediated by the loading of pro-angiogenic miR-210-3p in exosomes released by cancer cells, which underscores the relevance of NRP1 in controlling the tumor microenvironment.

Drugs targeting mutant BRAF and MEK oncogenes are effective in melanoma, even though resistance rapidly develops. This complex picture includes acquired intrinsic tumor and tumor microenvironmental-mediated mechanisms. Here we show that melanoma cells resistant to BRAF inhibitors (BRAFi) overexpress the rate-limiting enzymes involved in nicotinamide (NAM) metabolism nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide N-methyltransferase (NNMT). Remarkably, these cells release NAMPT and NNMT both in the free-form or loaded into extracellular vesicles (EVs). NAMPT is emerging as a key mediator of resistance to BRAFi in melanoma, primarily due to its established role in NAD biosynthesis. Although previously identified as a soluble extracellular factor in this tumor, its presence within EVs released by melanoma cells has not been reported until now, highlighting a previously unrecognized mechanism through which NAMPT may influence the tumor microenvironment (TME). NNMT was revealed to increase in melanoma lesions compared to benign nevi. Here, we report for the first time its overexpression in resistant melanoma cell lines at intracellular and extracellular levels (secreted both as a soluble factor and into EVs). NNMT expression is increased in BRAF-mutated melanoma patients, suggesting a link between its upregulation and the BRAF oncogenic signaling. Moreover, NNMT levels positively correlate with gene signatures associated with pro-inflammatory signaling, immune cell migration, and chemokine-mediated pathways. NNMT pharmacological inhibition and genetic silencing significantly reduce resistant melanoma cell growth. In addition, we found that BRAFi-resistant cells are more sensitive to NNMT inhibition, highlighting a trait of vulnerability of BRAFi-resistant melanomas. Lastly, we proposed for the first time a tetrameric NNMT:TLR4 binding model offering a plausible structural and mechanistic basis for their association. Our functional results indicated that exogenous NNMT treatment is able to trigger NF-κB pathway, one of the main TLR4-dependent signaling, sharing this cytokine-like properties with NAMPT, and opening a future deeper exploration of its functional role in the extracellular space. Overall, the identification of NAMPT and, surprisingly also NNMT, included in EVs and abundantly released from resistant melanoma cells supports the impact of these moonlighting proteins involved in nicotinamide metabolism as mediators of BRAF/MEK inhibitors resistance with tumor intrinsic and potentially tumor microenvironment-mediated mechanisms. Interfering with nicotinamide metabolism could be a valid strategy to counteract drug resistance acting on the multifactorial tumor-host interactions.