The c-Cbl-mediated non-canonical TGFBR2 neddylation drives BRAF V600E melanoma metastasis

<title>Abstract</title> <p> Background Metastatic dissemination is the main cause of death in patients with cutaneous melanoma, particularly those with the BRAF ^V600E subtype, in whom therapeutic resistance often limits durable disease control. Aberrant neddylation has been implicated in cancer progression. However, the contribution of non-canonical neddylation to melanoma metastasis remains poorly defined. The current study examined whether c-Cbl-mediated non-canonical neddylation stabilizes transforming growth factor-β receptor 2 (TGFBR2) and drives metastatic progression in BRAF ^V600E melanoma. Methods Data from The Cancer Genome Atlas were analyzed to assess the prognostic relevance of NEDD8 in melanoma. Gain-of-function and loss-of-function approaches were used to define the roles of NEDD8 and TGFBR2 in melanoma cell migration. Neddylation was pharmacologically inhibited using MLN4924 and TAS4464. Transcriptomic analyses, Smad-responsive luciferase assays, cycloheximide chase assays, and His-NEDD8 pulldown assays were conducted to characterize the link between neddylation and transforming growth factor-β (TGF-β) signaling. Site-directed mutagenesis and rescue experiments were used to identify functionally relevant neddylation sites on TGFBR2. In vivo significance was evaluated using xenograft tumor growth and orthotopic metastasis models. Results Elevated NEDD8 expression was associated with a poor prognosis, particularly in BRAF ^V600E melanoma, and correlated with enhanced migratory capacity in melanoma cells. Genetic depletion or pharmacologic inhibition of neddylation markedly suppressed melanoma cell migration. Mechanistically, neddylation blockade attenuated TGF-β/Smad signaling, reduced Smad-dependent transcription, accelerated TGFBR2 degradation, and diminished NEDD8 conjugation of TGFBR2. Functional studies revealed that TGFBR2 is required for melanoma cell migration, and rescue experiments indicated that the prometastatic activity of TGFBR2 depends on non-canonical neddylation at K556 and K567. In vivo, TGFBR2 depletion suppressed xenograft tumor growth, reduced tumor burden and Ki-67 expression, and abolished lymph node and brain metastasis in orthotopic models. Notably, c-Cbl was identified as the upstream E3 ligase required for this pathway, with c-Cbl silencing reducing TGFBR2 neddylation, impairing Smad2/3 activation, and suppressing melanoma cell motility. Conclusions In this study, we identified a novel c-Cbl–TGFBR2 non-canonical neddylation axis that stabilizes TGFBR2, sustains prometastatic TGF-β signaling, and drives BRAF ^V600E melanoma progression and metastasis. Targeting this posttranslational regulatory pathway may represent a promising therapeutic strategy for metastatic BRAF-mutant melanoma. </p>