Emerging Role of Deubiquitinating Enzymes (DUBs) in Melanoma Pathogenesis

Metastatic melanoma is the leading cause of death from skin cancer. Therapies targeting the BRAF oncogenic pathway and immunotherapies show remarkable clinical efficacy. However, these treatments are limited to subgroups of patients and relapse is common. Overall, the majority of patients require additional treatments, justifying the development of new therapeutic strategies. Non-genetic and genetic alterations are considered to be important drivers of cellular adaptation mechanisms to current therapies and disease relapse. Importantly, modification of the overall proteome in response to non-genetic and genetic events supports major cellular changes that are required for the survival, proliferation, and migration of melanoma cells. However, the mechanisms underlying these adaptive responses remain to be investigated. The major contributor to proteome remodeling involves the ubiquitin pathway, ubiquitinating enzymes, and ubiquitin-specific proteases also known as DeUBiquitinases (DUBs). In this review, we summarize the current knowledge regarding the nature and roles of the DUBs recently identified in melanoma progression and therapeutic resistance and discuss their potential as novel sources of vulnerability for melanoma therapy

USP9X is a mechanosensitive deubiquitinase that controls tumor cell invasiveness and drug response through YAP stabilization

Post-translational modification by ubiquitin is crucial for protein turnover. Deubiquitinases (DUBs) remove ubiquitin chains from target proteins to prevent their degradation by the proteasome, thus acting as gatekeepers of protein homeostasis alongside the ubiquitin-proteasome system (UPS). Tumor cells exhibit remarkable plasticity, enabling them to adapt to anticancer treatments and the conditions of the tumor microenvironment, including mechanical cues from the extracellular matrix (ECM). However, the role of DUBs in mechanotransduction remains unexplored. To identify DUBs involved in cancer cell mechanosignaling, we used melanoma cells grown on collagen matrices with varying stiffnesses and an activity-based ubiquitin probe to profile DUB activities. Our approach, combined with quantitative proteomics, revealed that ubiquitin-specific protease 9X (USP9X) is sensitive to ECM stiffness through discoidin domain receptors (DDR)/actomyosin signaling pathway. In silico analysis further indicated that the mechanosensor YAP is part of the USP9X interactome, and USP9X expression correlates with the YAP transcriptional signature in melanoma. We hypothesized that mechanical signals regulate YAP levels through USP9X DUB activity. Consistently, low collagen stiffness reduced YAP expression, and siRNA-mediated depletion or pharmacological inhibition of USP9X decreased YAP protein expression in tumor cells. Conversely, knockdown of the ubiquitin E3 ligase βTrCP increased YAP protein levels. Affinity purification of polyubiquitinated proteins using Tandem Ubiquitin Binding Entities (TUBEs) showed that combined USP9X and proteasome inhibition increased YAP poly-ubiquitination, revealing that USP9X deubiquitinates YAP to prevent its proteasomal degradation. Targeting USP9X impaired stiffness-mediated responses, including YAP nuclear translocation and transcriptional activity, cell migration and invasion, and drug resistance. An experimental metastasis assay showed that stable knockdown of USP9X impaired melanoma cell lung colonization. Finally, targeting USP9X in a syngeneic BRAF-mutant melanoma model counteracted targeted therapy-induced ECM remodeling, enhanced treatment efficacy, and delayed tumor relapse. Our findings reveal a novel role of USP9X in cancer cell mechanobiology and drug resistance through stiffness-dependent stabilization of the oncoprotein YAP, proposing USP9X as a targetable "mechano-DUB" in cancer

Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix‐mediated tumor cell adaptation and tolerance to BRAF‐targeted therapy in melanoma

International audienceResistance to BRAF/MEK inhibitor therapy in BRAFV600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma

Blockade of the pro‐fibrotic reaction mediated by the miR‐143/‐145 cluster enhances the responses to targeted therapy in melanoma

International audienceLineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy