Blockade of the pro‐fibrotic reaction mediated by the miR‐143/‐145 cluster enhances the responses to targeted therapy in melanoma
2022; Springer Nature; Volume: 14; Issue: 3 Linguagem: Inglês
10.15252/emmm.202115295
ISSN1757-4684
AutoresSerena Diazzi, Alberto Baeri, Julien Fassy, Margaux Lecacheur, Oskar Marín-Béjar, Christophe A. Girard, Lauren Lefevre, Caroline Lacoux, Marie Irondelle, Carine M. Mounier, Marin Truchi, Marie Couralet, Mickaël Ohanna, Alexandrine Carminati, Ilona Berestjuk, Frédéric Larbret, David Gilot, Georges Vassaux, Jean‐Christophe Marine, Marcel Deckert, Bernard Mari, Sophie Tartare‐Deckert,
Tópico(s)Cancer Mechanisms and Therapy
ResumoArticle14 February 2022Open Access Source DataTransparent process Blockade of the pro-fibrotic reaction mediated by the miR-143/-145 cluster enhances the responses to targeted therapy in melanoma Serena Diazzi Serena Diazzi Université Côte d'Azur, INSERM, C3M, Nice, France Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Alberto Baeri Alberto Baeri Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Julien Fassy Julien Fassy Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Margaux Lecacheur Margaux Lecacheur orcid.org/0000-0002-2426-1534 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation, Methodology Search for more papers by this author Oskar Marin-Bejar Oskar Marin-Bejar Laboratory For Molecular Cancer Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium Department of Oncology, KU Leuven, Leuven, Belgium Contribution: Data curation, Investigation Search for more papers by this author Christophe A Girard Christophe A Girard Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation, Methodology Search for more papers by this author Lauren Lefevre Lauren Lefevre Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Caroline Lacoux Caroline Lacoux Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation Search for more papers by this author Marie Irondelle Marie Irondelle Université Côte d'Azur, INSERM, C3M, Nice, France Contribution: Formal analysis Search for more papers by this author Carine Mounier Carine Mounier Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France CYU Université, ERRMECe (EA1391), Neuville-sur-Oise, France Contribution: Investigation Search for more papers by this author Marin Truchi Marin Truchi Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis Search for more papers by this author Marie Couralet Marie Couralet orcid.org/0000-0002-4581-8418 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation Search for more papers by this author Mickael Ohanna Mickael Ohanna orcid.org/0000-0002-7751-9164 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Alexandrine Carminati Alexandrine Carminati Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Ilona Berestjuk Ilona Berestjuk Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Frederic Larbret Frederic Larbret Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author David Gilot David Gilot orcid.org/0000-0002-2646-671X INSERM U1242, University of Rennes, Rennes, France Contribution: Resources, Writing - review & editing Search for more papers by this author Georges Vassaux Georges Vassaux orcid.org/0000-0002-1149-7716 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation, Writing - review & editing Search for more papers by this author Jean-Christophe Marine Jean-Christophe Marine Laboratory For Molecular Cancer Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium Department of Oncology, KU Leuven, Leuven, Belgium Contribution: Resources, Writing - review & editing Search for more papers by this author Marcel Deckert Marcel Deckert orcid.org/0000-0003-2094-559X Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Bernard Mari Corresponding Author Bernard Mari [email protected] orcid.org/0000-0002-0422-9182 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France FHU-OncoAge, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sophie Tartare-Deckert Corresponding Author Sophie Tartare-Deckert [email protected] orcid.org/0000-0001-8680-5720 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France FHU-OncoAge, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Serena Diazzi Serena Diazzi Université Côte d'Azur, INSERM, C3M, Nice, France Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Alberto Baeri Alberto Baeri Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Julien Fassy Julien Fassy Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Margaux Lecacheur Margaux Lecacheur orcid.org/0000-0002-2426-1534 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation, Methodology Search for more papers by this author Oskar Marin-Bejar Oskar Marin-Bejar Laboratory For Molecular Cancer Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium Department of Oncology, KU Leuven, Leuven, Belgium Contribution: Data curation, Investigation Search for more papers by this author Christophe A Girard Christophe A Girard Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation, Methodology Search for more papers by this author Lauren Lefevre Lauren Lefevre Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Caroline Lacoux Caroline Lacoux Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation Search for more papers by this author Marie Irondelle Marie Irondelle Université Côte d'Azur, INSERM, C3M, Nice, France Contribution: Formal analysis Search for more papers by this author Carine Mounier Carine Mounier Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France CYU Université, ERRMECe (EA1391), Neuville-sur-Oise, France Contribution: Investigation Search for more papers by this author Marin Truchi Marin Truchi Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Formal analysis Search for more papers by this author Marie Couralet Marie Couralet orcid.org/0000-0002-4581-8418 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation Search for more papers by this author Mickael Ohanna Mickael Ohanna orcid.org/0000-0002-7751-9164 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Alexandrine Carminati Alexandrine Carminati Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Ilona Berestjuk Ilona Berestjuk Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author Frederic Larbret Frederic Larbret Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Investigation Search for more papers by this author David Gilot David Gilot orcid.org/0000-0002-2646-671X INSERM U1242, University of Rennes, Rennes, France Contribution: Resources, Writing - review & editing Search for more papers by this author Georges Vassaux Georges Vassaux orcid.org/0000-0002-1149-7716 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France Contribution: Investigation, Writing - review & editing Search for more papers by this author Jean-Christophe Marine Jean-Christophe Marine Laboratory For Molecular Cancer Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium Department of Oncology, KU Leuven, Leuven, Belgium Contribution: Resources, Writing - review & editing Search for more papers by this author Marcel Deckert Marcel Deckert orcid.org/0000-0003-2094-559X Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Bernard Mari Corresponding Author Bernard Mari [email protected] orcid.org/0000-0002-0422-9182 Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France FHU-OncoAge, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Sophie Tartare-Deckert Corresponding Author Sophie Tartare-Deckert [email protected] orcid.org/0000-0001-8680-5720 Université Côte d'Azur, INSERM, C3M, Nice, France Equipe labellisée Ligue Contre le Cancer, Nice, France FHU-OncoAge, Nice, France Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Serena Diazzi1,2,3, Alberto Baeri2, Julien Fassy2, Margaux Lecacheur1,3, Oskar Marin-Bejar4,5, Christophe A Girard1,3, Lauren Lefevre1,3, Caroline Lacoux2, Marie Irondelle1, Carine Mounier2,6, Marin Truchi2, Marie Couralet2, Mickael Ohanna1,3, Alexandrine Carminati1,3, Ilona Berestjuk1,3, Frederic Larbret1,3, David Gilot7, Georges Vassaux2, Jean-Christophe Marine4,5, Marcel Deckert1,3, Bernard Mari *,2,8,† and Sophie Tartare-Deckert *,1,3,8,† 1Université Côte d'Azur, INSERM, C3M, Nice, France 2Université Côte d'Azur, CNRS, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Sophia Antipolis, France 3Equipe labellisée Ligue Contre le Cancer, Nice, France 4Laboratory For Molecular Cancer Biology, VIB Center for Cancer Biology, VIB, Leuven, Belgium 5Department of Oncology, KU Leuven, Leuven, Belgium 6CYU Université, ERRMECe (EA1391), Neuville-sur-Oise, France 7INSERM U1242, University of Rennes, Rennes, France 8FHU-OncoAge, Nice, France † These authors contributed equally to this work *Corresponding author. Tel: +33 493 957 719; E-mail: [email protected] *Corresponding author. Tel: +33 489 153 851; E-mail: [email protected] EMBO Mol Med (2022)14:e15295https://doi.org/10.15252/emmm.202115295 See also: B Sanchez-Laorden & MA Nieto (March 2022) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lineage 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. Synopsis This study identifies a critical miRNA-mediated regulatory axis controlling melanoma cell phenotypic adaptation and resistance to MAPK inhibitors (MAPKi) and targetable by the anti-fibrotic drug nintedanib. The fibrosis-associated miR-143/-145 cluster is upregulated by targeted therapy and highly expressed in MAPKi-resistant BRAF-mutated melanoma displaying a pro-fibrotic dedifferentiated mesenchymal-like phenotype. The two mature miRNAs miR-143-3p and miR-145-5p promote a dedifferentiated, slow-cycling and invasive phenotype associated with altered ECM production and therapy resistance. F-acting bundling protein Fascin1 (FSCN1) appears as a key functional target of both miRNAs to promote transition towards this therapy-resistant phenotype. The miR-143/-145/FSCN1 axis regulates actin cytoskeleton dynamics and YAP/MRTF-dependent mechanopathways. Nintedanib prevents miR-143/-145 cluster upregulation and combination therapy with MAPKi and nintedanib normalizes the tumor ECM niche and delays relapse in an allograft melanoma model. The paper explained Problem Despite recent improvements in targeting metastatic melanoma, resistance to inhibition of the BRAFV600 oncogenic pathway occurs in most patients treated with MAPK-inhibiting drugs. Melanoma cells adopt various means to evade therapy, including transcriptional reprogramming leading to phenotypic dedifferentiation and acquisition of mesenchymal and pro-fibrotic features. This state of cellular resistance is highly invasive and displays an increased ability to produce and remodel the extracellular matrix (ECM), creating a drug-tolerant microenvironment. However, the molecular networks that define this pro-fibrotic cellular behavior and promote resistance are still unclear. Results We show that the anti-fibrotic drug nintedanib prevents the fibrotic reaction and improves MAPK-targeting therapy efficacy, retarding the onset of resistance in a mouse melanoma model. Expression screening and mechanistic studies identified the pro-fibrotic miR-143/-145 cluster as a driver of nintedanib-sensitive mesenchymal resistant phenotype. Using a combination of gain- and loss-of-function approaches, we dissected the molecular and cellular processes regulated by these FibromiRs and demonstrate that during drug adaptation, melanoma cells upregulate the miRNA cluster, which drives a phenotypic switch toward a dedifferentiated therapy-resistant state. The miR-143/-145 cluster also induces ECM production and promotes cell migration and invasion through the activation of focal adhesion dynamics and mechanotransduction pathways. Finally, Fascin actin-bundling protein 1 (FSCN1) was identified as a key functional target of miR-143-3p and miR-145-5p for the acquisition of the pro-fibrotic therapy-resistant phenotype. Impact Our study highlights non-genetic mechanisms of therapeutic resistance in melanoma and deciphers a regulatory cascade involving the miR-143/-145/FSCN1 pro-fibrotic axis in the acquisition of a therapy-resistant cellular state. It also provides a scientific rationale for designing clinical trials with nintedanib and potentially other anti-fibrotic agents to overcome resistance in patients with BRAF-mutated melanoma. Finally, our findings might have implications for other MAPK-driven cancers and fibrosis-related diseases. Introduction Because of its high mutational burden, metastasis propensity, and resistance to treatment, cutaneous melanoma is one of the most aggressive human cancers and the deadliest form of skin cancer (Shain & Bastian, 2016). Melanoma is a non-epithelial tumor that originates from neural crest-derived and pigment-producing melanocytes in the skin. Genetic alterations in the BRAF, NRAS, or NF1 genes define melanoma subtypes and lead to the MAPK pathway hyperactivation (Flaherty et al, 2012; Cancer Genome Atlas, 2015). Current therapeutic options for BRAFV600E/K metastatic melanoma include MAPK-targeted therapies, which show remarkable efficacy during the first months of treatment (Chapman et al, 2011; Robert et al, 2019). However, the majority of patients treated with a combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) inevitably relapse within months (Long et al, 2017). Genetic mechanisms of resistance cannot singly explain the acquisition of therapy resistance in melanoma, and non-genetic heterogeneity actively participates in drug tolerance (Rambow et al, 2019; Marine et al, 2020). Extensive studies have been carried out to dissect the non-mutational mechanisms of resistance (Rambow et al, 2018; Tsoi et al, 2018). Genetic and non-genetic mechanisms of resistance are frequently linked and not mutually exclusive (Marine et al, 2020). Non-genetic resistance is due to the intrinsic melanoma cell phenotypic plasticity, i.e., ability to undergo transcriptional and epigenetic reprogramming in response to environmental challenges or upon therapy (Arozarena & Wellbrock, 2019). These adaptive mechanisms exploit the developmental plasticity of melanoma cells and often result in an undifferentiated state characterized by upregulation of receptor tyrosine kinases (RTK) such as PDGFRβ or AXL, downregulation of melanocyte differentiation transcription factors MITF and SOX10 (Sun et al, 2014), and acquisition of mesenchymal and invasive features (Nazarian et al, 2010; Villanueva et al, 2010; Girotti et al, 2013; Muller et al, 2014; Fallahi-Sichani et al, 2017; Rambow et al, 2018; Tsoi et al, 2018; Rathore et al, 2019). Tumors are shaped dynamically by reciprocal crosstalk between cancer cells and the extracellular matrix (ECM) through cellular–ECM interactions and stromal matrix remodeling. Recent findings indicated that elevated ECM production and remodeling contribute to adaptive and acquired resistance to BRAFi therapy by conferring a drug-protective niche to melanoma cells (Fedorenko et al, 2016; Titz et al, 2016; Girard et al, 2020; Marusak et al, 2020). Moreover, we recently reported that undifferentiated mesenchymal-like BRAFi-resistant cells exhibit myofibroblast/cancer-associated fibroblast (CAF)-like features leading to pro-fibrotic ECM reprogramming in vitro and in vivo (Diazzi et al, 2020; Girard et al, 2020). Cell-autonomous ECM deposition and remodeling abilities adopted by melanoma cells after MAPKi treatment result in cross-linked collagen matrix and tumor stiffening fostering a feedforward loop dependent on the mechanotransducers YAP and MRTFA and leading to therapy resistance (Girard et al, 2020). Thus, this pro-fibrotic-like response, typical of the early adaptation and acquired resistance to MAPK inhibition, provides a therapeutic escape route through the activation of alternative survival pathways mediated by cell-matrix communications. However, the signaling networks underlying the acquisition of this undifferentiated, mesenchymal-like melanoma cell state and drug-resistant behavior remain unclear. We reasoned that therapeutic approaches aimed at preventing this targeted therapy-induced abnormal pro-fibrotic reaction could represent rationale combination strategies to normalize the fibrous stroma and overcome non-genetic resistance in BRAFV600E-mutated melanomas. We show here that the anti-fibrotic drug nintedanib (BIBF1120, Ofev®) improves the response of the BRAFi/MEKi-targeted therapy in a preclinical model of melanoma and in BRAF-mutated cell lines by preventing MAPKi-induced lineage dedifferentiation, ECM reprogramming, and mesenchymal traits. We also identified the master regulator associated with the acquisition of this pro-fibrotic and dedifferentiation program, pointing the miR-143/-145 cluster as a driver of the phenotype switching to a drug-resistant mesenchymal-like cell state. Results Nintedanib/BIBF1120 prevents MAPKi-induced pro-fibrotic-like response, enhances targeted therapy efficiency, and delays tumor relapse In order to limit ECM reprogramming and collagen remodeling associated with therapy resistance and relapse in melanoma, we tested the effect of the anti-fibrotic drug nintedanib (BIBF1120), a triple inhibitor of PDGFR, VEGFR, and FGFR used to treat idiopathic pulmonary fibrosis (IPF) in combination with BRAFi/MEKi in a syngeneic model of transplanted murine YUMM1.7 Braf-mutant melanoma (Meeth et al, 2016). YUMM1.7 cells were subcutaneously injected, and tumors were treated with vehicle, BIBF1120 alone, a combination of BRAFi plus MEKi, or the triple combination (Fig 1A). BIBF1120 did not display any anti-melanoma effect when administered alone, slightly slowing down tumor growth but not triggering tumor volume decrease. Administration of the BRAFi/MEKi initially reduced tumor growth, but after three weeks of treatment, tumor growth resumed and 100% of tumors relapsed. Importantly, combination of MAPK-targeted therapies and BIBF1120 significantly delayed relapse and led to complete remission in 33% of mice (2 out of 6; Figs 1B and C, and EV1A). Overall, the combined treatment significantly improved mouse survival (Fig 1C) without body weight loss or sign of toxicity throughout the study (Fig 1D). As previously described in melanoma xenograft models (Girard et al, 2020), an extensive deposition of collagens and increased expression of ECM remodeling and myofibroblast markers were observed in YUMM1.7 tumors treated with the combination of BRAFi and MEKi as revealed by picrosirius red staining of collagen fibers and qPCR analysis of typical molecular markers of tumor fibrosis. This response was significantly reduced by the co-administration of BIBF1120 (Figs 1E–G and EV1B). Thus, combination of targeted therapy with the anti-fibrotic drug nintedanib prevents the appearance of a pro-fibrotic matrix observed upon MAPK-targeted therapy exposure and significantly delays the onset of resistance in vivo. Figure 1. Nintedanib/BIBF1120 prevents MAPKi-induced ECM remodeling, decreases resistance to targeted therapy, and delays tumor relapse A–G. Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice, and when tumors reached 100 mm3, mice were treated with vehicle (Ctrl), nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, vemurafenib and MEKi, trametinib), or BRAFi/MEKi plus BIBF (n = 6). (B) Representative median graphics showing tumor growth following treatment (n = 6). Two-way ANOVA was used for statistical analysis. **P ≤ 0.01. (C) Kaplan–Meier survival curves of mice treated with the indicated therapies (n = 6). The log rank (Mantel–Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P ≤ 0.0001. (D) Mouse body weight was measured at the indicated times. Data shown are mean ± SD (n = 6). (E, F) Tumor sections were stained with picrosirius red and imaged under polarized light. (E) Representative image of collagen fiber network in tumors from mice under the different treatments. Scale bar 200 μm. (F) Quantification of collagen fiber thickness (n = 6 for control, BIBF, and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fiber thickness quantification. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated. (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5). H–J. Human M238R cells and/or parental M238P cells were analyzed for different parameters. (H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in M238R compared to M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in M238R treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3). (J) Crystal violet viability assay of M238R cells treated with BRAFi/MEKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1 µM), BIBF (2 μM) or with BRAFi/MEKi (1 μM) plus BIBF (2 μM) for 72 h. Paired Student t-test was used for statistical analysis. ****P ≤ 0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated. Data is represented as mean ± SD from a triplicate representative of 3 independent experiments. Source data are available online for this figure. Source Data for Figure 1 [emmm202115295-sup-0003-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Administration of nintedanib/BIBF1120 resensitizes melanoma cells to MAPK-targeted therapies, delays tumor relapse, and normalizes MAPKi-induced ECM remodeling and miR-143/-145 expression A, B. YUMM1.7 cells were subcutaneously inoculated into C57BL/6 mice, and when tumors reached 100 mm3, mice were treated with the indicated therapies. (A) Individual graphics showing tumor growth following treatment. (B) Normalized expression of myofibroblast/CAF and ECM-related genes assessed by RT-qPCR in individual tumors treated as indicated. Data are represented as median with range (n = 5). One-way ANOVA was used for statistical analysis. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. C–E. Human M238P cells were treated with BRAFi (vemurafenib) + MEKi (trametinib; 1 μM), BIBF1120 (2 µM) or BRAFI + MEKi (1 µM) plus BIBF (2 µM) for 72 h. (C) Heatmap showing the expression of ECM, myofibroblast/CAF markers, and phenotype switch markers by RT-qPCR (n = 3). (D) Crystal violet viability assay of M238P cells treated with MAPK-targeted therapies as above. Paired Student's t-test was used for statistical analysis. ****P ≤ 0.0001. Data are represented as mean ± SD from a triplicate representative of three independent experiments. (E) Western blot showing the expression of ECM, myofibroblast/CAF markers, and activation levels of signaling pathways (AKT and ERK1/2) in the different conditions. F–H. Human M238R cells were treated with BIBF1120 (2 µM) or with CP673451 (2 µM) for 72 h. (F) Western blot showing activation levels of signaling pathways (PDGFR and AKT) in the different conditions. (G) Heatmap showing the expression of ECM, myofibroblast/CAF markers, and phenotype switch markers by RT-qPCR in M238R cells treated with the indicated inhibitors (n = 3). (H) Crystal violet viability assay of M238R cells treated with the indicated inhibitors. Paired Student's t-test was used for statistical analysis. ****P ≤ 0.0001. Data are represented as mean ± SD from a triplicate representative of three independent experiments. Download figure Download PowerPoint We next examined the impact of nintedanib on ECM reprogramming and cell phenotype switching in the context of early adaptation and resistance to MAPK-targeted therapy i
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