Role of extracellular matrix and discoidin domain receptors, DDR1 and DDR2, in the resistance of melanoma cells to targeted therapies

Le mélanome est un cancer de la peau très agressif de part son fort potentiel métastatique et sa résistance aux traitements. La progression tumorale est accompagnée par des mutations « drivers » qui touchent principalement les gènes BRAF et NRAS et conduisent à l’activation constitutive de la voie des MAP kinases. De nouvelles options thérapeutiques comme les thérapies ciblées associant un inhibiteur de BRAF (vémurafénib) et un inhibiteur de MEK (tramétinib), et les immunothérapies sont indiquées mais ces traitements ne bénéficient qu'à certains patients et leur utilisation est limitée du fait de l'émergence de résistances primaires et secondaires. Il est donc fondamental de mieux comprendre les mécanismes de l'échappement aux thérapies ciblées dans le but d'en améliorer l'efficacité.La capacité des cellules cancéreuses à résister aux traitements repose en partie sur leur pouvoir d’adaptation au microenvironnement tumoral. Au sein d’une tumeur, la cellule cancéreuse interagit avec un écosystème complexe composé de cellules stromales dont des fibroblastes activés et de la matrice extracellulaire (MEC). La MEC, majoritairement produite par les fibroblastes est composée de molécules d’adhérence comme la fibronectine et de structure comme les collagènes. Elle constitue un réseau dynamique ayant des propriétés biochimiques et mécaniques qui influencent la progression tumorale. Ainsi, le stroma dans lequel sont ancrées les mélanomes et les interactions qu’ils entretiennent avec la MEC peuvent avoir un rôle prépondérant dans la résistance aux thérapies ciblées. Cette résistance induite par l’adhérence des cellules à une matrice est définie comme la MM-DR (Matrix Mediated-Drug Resistance).Dans un premier temps mes études ont montrés, a l’aide d’un modèle 3D de MEC, que les FRCs (Fibroblasts Reticular Cells) ou différentes populations de MAFs (Melanoma Associated Fibroblasts) isolées de biopsies de métastases lymphatiques produisent et remodèlent une MEC riche en fibres de collagènes, d’enzymes d’échafaudage et de protéines matricielles qui est plus rigide qu’une MEC produite par des fibroblastes d’origine dermique. J’ai également observé un effet « protecteur » de ces matrices sur les mélanomes face aux thérapies. L’adhérence des cellules de mélanome conduit à une résistance en réponse aux effets anti-prolifératifs des inhibiteurs de BRAF et de MEK. Dans un deuxième temps j’ai identifiée les DDRs (Discoidin Domain Receptors 1 & 2), deux récepteurs de collagène à l’activité tyrosine kinase et qui fonctionnent indépendamment des intégrines, comme étant impliqués dans la MM-DR. Par une approche d’interférence ARN et par une approche pharmacologique, j’ai montré que l’inhibition de DDR1 et de DDR2 ré-sensibilise les cellules cultivées sur matrice aux effets cytostatiques et cytotoxiques du vémurafénib. Ces résultats ont étaient également confirmés par une approche in vivo en utilisant un modèle de xénogreffe. En effet, le traitement des souris avec la combinaison Vémurafénib + Imatinib (un inhibiteur de tyrosine kinase utilisé en clinique dans le traitement de certaines leucémies) diminue drastiquement la croissance tumorale, avec une médiane de survie de 48 jours contre 36 jours pour le traitement de Vémurafénib seul. En conclusion, mes données ont mis en évidence les propriétés structurelles, biophysiques et fonctionnelles remarquables des MECs produites par les fibroblastes lymphatiques et un rôle de la MM-DR dans la résistance aux traitements ciblés. Mais également travaux ont permis d’identifier les DDRs comme étant des nouvelles cibles thérapeutiques impliquées dans la résistance des cellules de mélanome aux thérapies ciblées.Melanoma is a very aggressive skin cancer because of its intratumoral heterogeneity, high metastatic potential and resistance to treatment. Driver mutations mainly affect the BRAF and NRAS genes leading to the constitutive activation of the MAP kinase pathway (MAPK). New therapeutic options such as targeted therapies that combine BRAF (Vemurafenib) and MEK (Trametinib) inhibitors as well as immunotherapies are available, but these treatments only benefit certain patients and their use is limited because of the emergence of resistance. It is therefore essential to better understand the escape mechanisms in response to targeted therapies, in order to improve their effectiveness.The ability of cancer cells to resist treatment depends in part, on their ability to adapt to the tumour microenvironment. Within the primary tumour and various metastatic niches, the cancer cell interacts with a complex ecosystem composed of stromal cells, including activated fibroblasts and extracellular matrix (ECM). ECM, mainly produced by fibroblasts, is composed of adhesion and structural molecules such as fibronectin and collagen. It constitutes a dynamic network with biochemical and mechanical properties that influence tumour progression. Thus, the stroma in which melanoma cells are anchored and their interactions with the ECM contribute to anti-cancer treatment resistance. This resistance provided by the matrix is defined as MM-DR (Matrix Mediated-Drug Resistance).Firstly, my study showed, using a 3D model of ECM produced by fibroblasts, that FRCs (Fibroblast Reticular Cells) or different populations of MAFs (Melanoma Associated Fibroblasts) isolated from biopsies of lymphatic metastases produce and remodel an ECM rich in collagen fibres, scaffolding enzymes and matrix proteins and these ECMs are more rigid than those produced by dermal fibroblasts. Furthermore, I observed a protective effect of these matrices on melanoma cells carrying the BRAFV600E mutation to targeted therapies. Adherence of melanoma cells to the ECM lead to resistance in response to the antiproliferative effects of BRAF and MEK inhibitors.Additionally, I identified the discoidin domain receptors, DDR1 and DDR2, which have tyrosine kinase activity and interact with collagen, thereby playing a key role in MM-DR. Using the RNA interference and pharmacological approaches which targeted DDR kinase activity, I showed that inhibition of DDR1 and DDR2 re-sensitised melanoma cells cultured on ECM to the cytostatic and cytotoxic effects of Vemurafenib. These results were confirmed with an in vivo approach using a preclinical tumour xenograft model. Treatment of mice with the combination of Vemurafenib / Imatinib (a DDR inhibitor clinically used in the treatment of certain leukemias) greatly reduces tumour growth, prevented the development of resistance in response to Vemurafenib and increased mice survival.In conclusion, my thesis has highlighted the remarkable structural, mechanical and functional properties of the ECM produced by metastatic lymphatic niche fibroblasts in the survival and therapeutic resistance of melanoma and demonstrated a new role of DDRs in their escape from targeted therapies. Thus, inhibition of DDRs in combination with inhibitors of the MAPK pathway may represent a new therapeutic strategy for BRAF mutant melanoma

Effects on Melanoma Cell Lines Suggest No Significant Risk of Melanoma Under Cladribine Treatment

International audienceCladribine is an oral synthetic purine analog that depletes lymphocytes and induces a dose-dependent reduction of T and B cells. It was approved for the therapy of highly active relapsing-remitting multiple sclerosis. Given cladribine's mechanism of action, an increased risk of malignancies was suspected from the number of cancers that occurred in the 3.5 mg/kg-treated arm (CLARITY study). We showed that cladribine inhibits cell proliferation on three melanoma cell lines tested, irrespectively of their mutational oncogenic status and invasive/metastatic potential. Aggregated safety data demonstrated that the risk of melanoma is not confirmed

Invasive dedifferentiated melanoma cells inhibit JAK1-STAT3-driven actomyosin contractility of human fibroblastic reticular cells of the lymph node

Abstract Fibroblastic reticular cells (FRC) are immunologically specialized fibroblasts controlling the size and microarchitecture of the lymph node (LN), partly through their contractile properties. Swelling is a hallmark of tumor-draining LN in lymphophilic cancers such as cutaneous melanoma, a very aggressive and heterogeneous tumor with high risk of early metastasis. Melanoma cells can dynamically switch between melanocytic proliferative and dedifferentiated mesenchymal-like invasive phenotypes, which are characterized by distinct transcriptional signatures. Melanoma secreted cues, such as extracellular vesicles, growth factors or proinflammatory cytokines, promote LN stroma remodeling and metastatic spreading. But how FRC integrate these pro-metastatic signals and modulate their contractile functions remains poorly characterized. Here, we show that factors secreted by dedifferentiated melanoma cells, but not by melanocytic cells, strongly inhibit FRC actomyosin-dependent contractile forces by decreasing the activity of the RHOA-ROCK pathway and the mechano-responsive transcriptional co-activator YAP, leading to a decrease in F-actin stress fibers and cell elongation. Transcriptional profiling and biochemical analyses indicate that FRC actomyosin cytoskeleton relaxation is driven by inhibition of JAK1 and its downstream transcription factor STAT3, and is associated with increased FRC proliferation and activation. Interestingly, dedifferentiated melanoma cells reduce FRC contractility in vitro independently of extracellular vesicle secretion. These data show that FRC are specifically modulated by proteins secreted by invasive dedifferentiated melanoma cells and suggest that melanoma-derived cues could modulate the biomechanical properties of distant LN before metastatic invasion. They also highlight that JAK1-STAT3 and YAP signaling pathways contribute to the maintenance of the spontaneous contractility of resting human FRC

Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy

Resistance to BRAF and MEK inhibitors in BRAF V600E mutant melanomas remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapies, however the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MM-DR) in response to BRAF inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MM-DR is mediated by the drug-induced clustering of DDR1 and DDR2, two tyrosine kinase collagen receptors. Genetic depletion and pharmacological inhibition of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF inhibition. In melanoma xenografts, targeting DDRs by Imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling and delays tumor relapse. Mechanistically, DDR-mediated MM-DR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. Our study reveals a novel role of collagen-rich matrix and DDRs in tumor cell adaptation and therapy resistance, thus providing important insights into environment-mediated drug resistance and a pre-clinical rationale for targeting DDR1/2 signaling in combination with BRAF-targeted therapy in melanoma

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

Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3-Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

International audienceFibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node, in part through their contractile properties. Swelling of tumor-draining lymph nodes is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote premetastatic lymph node reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA-RHO-kinase (ROCK) pathway and the mechano-responsive transcriptional coactivator Yes1 associated transcriptional regulator (YAP). Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant lymph node in premetastatic mouse models. Finally, IL1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the premetastatic lymph node niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease.Significance: Communication between dedifferentiated melanoma cells and lymph node fibroblasts reprograms the biomechanical properties of the premetastatic lymph node niche to promote tumor invasion. See related commentary by Lund, p. 1692

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

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma

International audienceAberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg

A Feed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg.status: publishe

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