Role of LOXL2 enzyme in melanoma cell pro-invasive behavior and matrix remodeling of tumor stroma

Le mélanome cutané est un cancer de la peau agressif, hétérogène et hautement métastatique. Malgré le succès des nouveaux traitements comme les thérapies ciblant la voie oncogénique BRAFV600, et les inhibiteurs des points de contrôle immunitaire, de nombreux patients restent en échec thérapeutique en raison de résistances innées ou acquises dues à des mutations génétiques ou des phénomènes non-génétiques de reprogrammation et plasticité phénotypique. Des études récentes ont identifié des sous-populations cellulaires tumorales qui se distinguent selon leur état de différenciation et leur signature transcriptionnelle. En réponse aux signaux du microenvironnement et aux pressions thérapeutiques, les cellules de mélanome peuvent passer d'un état mélanocytaire à des états dédifférenciés associés à une expression accrue de récepteurs tyrosine kinase (RTK), de marqueurs mésenchymateux ou de cellules souches de la crête neurale. Cette transition a été décrite comme un facteur d'adaptation et de résistance aux thérapies ciblées. La progression tumorale est influencée par les propriétés biochimiques et biophysiques du stroma matriciel environnant. Ainsi, une augmentation des dépôts de collagène et de la réticulation des fibres rigidifie la tumeur, promeut la progression maligne et protège des thérapies. Mon travail s'appuie sur des observations originales de l'équipe selon lesquelles les cellules dédifférenciées produisent une matrice extracellulaire (MEC) abondante riche en lysyl oxidase-like 2, LOXL2, une enzyme de réticulation du collagène également connue pour son implication dans la transition épithéliale-mésenchymateuse du cancer du sein. Son rôle dans la biologie des mélanomes n'est pas connu. Nous avons émis l'hypothèse que la production de LOXL2 par les cellules de mélanome pourrait influencer leurs propriétés mésenchymateuses ainsi que le remodelage matriciel et la rigidité tumorale observés in vivo au cours de la réponse adaptative au traitement.Nous montrons dans un premier temps que LOXL2 est préférentiellement exprimé par les cellules de mélanome dédifférenciées et que son expression est associée à un mauvais pronostic. LOXL2 est induit par la thérapie ciblée ou l'hypoxie, des facteurs connus pour favoriser la transition vers un état dédifférencié, et est régulé par des facteurs de transcription de la plasticité tumorale comme ZEB1. De façon intéressante, l'induction de LOXL2 par la thérapie ciblée est inversée par les inhibiteurs des voies RTK et AKT. Dans un deuxième temps, nous avons étudié la contribution spécifique de LOXL2 dans le phénotype tumoral dédifférencié par des approches perte de fonction. Nous révélons que LOXL2 joue un rôle important dans la formation d'adhésions focales et la morphologie des cellules mésenchymateuses et favorise leur migration, invasion et formation de métastases. Sur le plan mécanistique, une analyse protéomique comparative a permis d'identifier l'inhibiteur de l'activateur du plasminogène 2, PAI-2, un membre de la famille des serpines comme un effecteur potentiel de la migration dépendante de LOXL2. Enfin, mes données indiquent que le ciblage de LOXL2 dans les cellules dédifférenciées et les fibroblastes associés au mélanome réduit leur capacité à contracter une matrice de collagène et à assembler une MEC organisée, ce qui suggère l'implication de LOXL2 dans le dialogue entre cellules de mélanome et matrice tumorale.L'ensemble de mes résultats établit un lien original entre LOXL2, l'hétérogénéité phénotypique des cellules de mélanome et le remodelage matriciel stromal. Notre étude révèle qu'en plus de son rôle dans le remodelage du collagène, LOXL2 joue un rôle pro-invasif « cell autonomous » intrinsèque à la cellule de mélanome. De plus, mon travail améliore notre compréhension des signaux biomécaniques de la MEC qui affectent la plasticité tumorale et l'adaptation aux thérapies et souligne l'intérêt du ciblage de LOXL2 dans le traitement de la maladie métastatique et résistante.Cutaneous melanoma is an aggressive, heterogeneous and highly metastatic skin cancer. Despite successful therapies targeting the BRAFV600E oncogenic pathway or immune checkpoints, many patients relapse and remain in therapeutic failure. Innate or acquired resistances are due to genetic mutations or non-genetic phenotypic reprogramming and plasticity. Recent studies based on single cell RNA-seq analysis of cutaneous melanoma have identified tumor cell subpopulations that are classified according to their differentiation state and transcriptional signature. Upon microenvironment and therapeutic pressures, melanoma cells can switch from a melanocytic state to dedifferentiated states associated with increased expression of tyrosine kinase receptors (RTK) and mesenchymal or neural crest stem cell-like markers. Such adaptive plasticity was described as a driver of resistance to targeted therapies. Tumor progression is influenced by alterations in the biochemical and biophysical properties of the tumor microenvironment. Increased collagen fiber deposition and crosslinking are indeed known to stiffen tumors, promote malignant progression and confer resistance to treatments. Our study relies on original observations from the team that dedifferentiated cells produce an abundant extracellular matrix (ECM) enriched in the lysyl oxidase-like 2 LOXL2, a collagen crosslinking enzyme also known to drive the epithelial-to-mesenchymal transition in breast cancer. Its role in melanoma biology remains poorly explored. We hypothesized that LOXL2 production by melanoma cells could influence their mesenchymal properties as well as ECM remodeling and tumor stiffness observed in vivo during adaptive response and therapeutic resistance.We first showed that LOXL2 is preferentially expressed by dedifferentiated melanoma cells and that its expression is associated with poor prognosis in melanoma. LOXL2 is induced by the combination of BRAF and MEK inhibitors or hypoxia, cues known to promote the transition towards a dedifferentiated phenotype, and regulated by the cell plasticity transcription factor ZEB1. Interestingly, LOXL2 induction by the targeted therapy is reversed by inhibition of the RTK and AKT pathways. We then investigated the specific contribution of LOXL2 to the dedifferentiated mesenchymal phenotype using loss-of-function approaches. We revealed that LOXL2 plays a role in focal adhesion formation and mesenchymal cell morphology, and promotes melanoma cell migration, invasion and metastasis. Mechanistically, comparative proteomic analysis identified the plasminogen activator inhibitor 2, PAI-2, a member of the serpin family, as a potential effector of LOXL2-mediated migration. Finally, we showed that targeting LOXL2 in dedifferentiated cells and melanoma-associated fibroblasts impairs their ability to contract a collagen matrix and assemble an organized ECM, suggesting the implication of LOXL2 in the dialogue between melanoma cells and the tumoral matrix.Taken together, these results establish an original link between LOXL2, melanoma cell phenotypic diversity and stromal matrix remodeling. Our study reveals that, in addition to its conventional role in collagen remodeling, LOXL2 exerts a tumor cell-autonomous pro-invasive action in melanoma. In addition, my work provides a better understanding of the ECM biomechanical signals that affect tumor cell plasticity and adaptation to anti-melanoma therapies, and highlights the value of targeting LOXL2 in the treatment of metastatic and resistant disease

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

Extracellular matrix stiffness determines the phenotypic behavior of dedifferentiated melanoma cells through a DDR1/2-dependent YAP mechanotransduction pathway

Abstract Extracellular matrix (ECM) stiffening, resulting from increased collagen deposition and cross-linking, is a key biophysical factor of the tumor microenvironment. Cutaneous melanoma is a deadly metastatic cancer. Its aggressiveness stems from high intratumoral heterogeneity, resulting from the plasticity of melanoma cells, which transit from a melanocytic state to dedifferentiated therapy-resistant and invasive phenotypes, characterized by mesenchymal and/or neural crest stem cell-like features. Phenotypic plasticity is regulated by stroma-derived soluble factors, but the functional impact of ECM stiffening on melanoma cell phenotypes remains ill defined. Here, we found that melanoma cell subpopulations display difference in mechanical responsiveness. Compared to melanocytic cells, mesenchymal dedifferentiated cells showed increased proliferation, migration and resistance to MAP kinase-targeted therapy when seeded on stiff collagen. By contrast, a soft ECM impaired their proliferation and migration and sensitized them to targeted therapy. In addition, extracellular mechanical signals are required to sustain melanoma cell identity and dedifferentiation features. Further analyses indicated that the mechanosensitivity nature of dedifferentiated cells relies on the expression and activation of collagen receptors DDR1 and DDR2 that control actomyosin cytoskeleton reorganization and YAP mechanotransduction pathway. Inhibiting both DDR in dedifferentiated melanoma cells abrogated their mechano-induced behavior and drug-resistant phenotype, while forcing their expression in melanocytic cells induced mechanical responsiveness and a less differentiated phenotype. Our results reveal that phenotypic reprogramming endows dedifferentiated melanoma cells with increased sensitivity and addiction to ECM stiffness. We propose that mechano-addiction mediated by DDR collagen receptors may represent a novel vulnerability for aggressive dedifferentiated cancer cells that can be exploited for therapeutic benefits

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