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.

Reciprocal feedback between the ECM and tumor cells influence the hallmarks of cancer by providing biological abilities to malignant cells that are required for growth, survival, and dissemination. The ECM is a dynamic network of macromolecules with distinctive biochemical and mechanical properties that plays a major role in establishing tumor niches (1). Increased ECM deposition, fiber alignment, and covalent cross-link between collagen molecules lead to tumor stiffening, which has been associated to an elevated risk of cancer and poor clinical outcome in patients with breast or pancreatic cancers (2, 3).

Cancer-associated fibroblasts (CAF) are the main producers of tumorigenic ECM and function like myofibroblasts during wound healing and fibrosis (4). Cells apply contractile forces to sense the physical environmental stiffness through integrin-based focal adhesion (FA) complexes that connect the actin–myosin cytoskeleton with the ECM (2, 5). Matrix rigidity also leads to enhanced nucleus localization and activity of the mechanical-responsive Yes-associated protein (YAP) transcriptional regulator of the Hippo pathway (6). In CAFs, YAP acts as a critical factor regulating force-mediated ECM remodeling towards increased stiffening (7). Similar to YAP, the SRF transcriptional coactivator MRTF is translocated to the nucleus upon actin polymerization and functionally interacts with YAP to coordinate mechanosignaling and CAF contractility (8, 9). Beside, YAP mainly through its interaction with TEAD transcription factors have been shown to promote resistance to RAF/MEK–targeted cancer therapies in tumor cells such as melanoma (10–12).

Because of its resistance to treatment and propensity for metastasis, cutaneous melanoma is one of the most aggressive human cancers (13). Melanoma comprises phenotypically heterogeneous subtypes of cancer cells that can switch between transcriptional programs and differentiation states (14–16). The majority of melanomas display genetic alterations in BRAF or NRAS, leading to constitutive activation of the MAPK pathway. MAPK pathway inhibitors, such as BRAF inhibitors (BRAFi), MEK inhibitors (MEKi), or their combination, achieve significant clinical benefits in patients with BRAFV600-mutant melanoma. However, most patients relapse within months due to the acquisition of drug resistance attributed to intrinsic genetic and nongenetic changes in melanoma cells. Although genetic resistance frequently result from the reactivation of the MAPK pathway through de novo mutations, such as NRAS mutations (17, 18), nongenetic mechanisms involve epigenetic and/or transcriptomic changes in tumor cells during the early phase of treatment (19, 20). Such mechanisms often result in a dedifferentiation cell state characterized by downregulation of the master regulator of melanocyte differentiation microphthalmia-associated transcription factor (MITF) and upregulation of receptor tyrosine kinases (RTK) such as AXL (21–23). In addition, the dedifferentiated resistant MITFlow/AXLhigh population was shown to display a mesenchymal invasive phenotype (24–26). Transcriptional reprogramming of proliferative drug-sensitive melanoma cells into invasive drug-resistant cell population is thus a critical event in acquired resistance to targeted therapies.

Beside tumor cell-autonomous events, there is evidence that extrinsic factors derived from the microenvironment contribute to melanoma resistance to MAPK pathway inhibition. Stromal cells including CAFs and macrophages secrete growth and inflammatory factors, and ECM components such as fibronectin, which contribute to drug tolerance (27–31). Interestingly, melanoma cells have the ability to secrete their own matrix, in particular upon cellular transition to a de-differentiated mesenchymal state occurring in response to BRAF inhibition (20, 32, 33). In this study, we asked whether melanoma cell-derived ECM impacts on tumor mechanics and contributes to resistance to targeted therapies. We show that both acquired resistance and early adaptation to MAPK signaling inhibition paradoxically induces a force-mediated ECM reprogramming in melanoma cells that increases intrinsic mechanical sensing properties and alters ECM composition and topography. This fuels a mechanical positive-feedback loop where melanoma cell-derived ECM and YAP/MRTF intracellular pathways play a pivotal role and that could favor the reservoir of therapy-resistant cells.

Cells and reagents

Melanoma cell lines 501Mel and MNT1 were obtained as described previously (34, 35). 1205Lu cells were from Rockland. Isogenic pairs of vemurafenib-sensitive (P) and -resistant (R) cells (M229, M238, M249) were provided by R. Lo (21). Cells were cultured in DMEM plus 7% FBS (Hyclone). Resistant cells were continuously exposed to 1 μmol/L vemurafenib. Cell lines were used within 6 months between resuscitation and experimentation. Cell lines were authenticated via STR profiling (Eurofins Genomics) and were routinely tested for the absence of Mycoplasma by PCR. For live imaging, M238P, 501Mel, and 1205Lu were transduced with NucLight Red lentivirus reagent (Essen Bioscience) and selected with puromycin (1 μg/mL). Culture reagents were purchased from Thermo Fisher Scientific. BRAFi (PLX4032, vemurafenib), MEKi (GSK1120212, trametinib), and ROCK inhibitor Y27632 were from Selleckem. YAP inhibitor verteporfin was from Sigma.

RNAi studies

siGENOME siRNA SMARTpools for YAP1, MRTFa, and nontargeting control were from Dharmacon (Horizon Discovery). Fifty nmol/L of either siRNA pool was transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific), following the manufacturer's protocol.

Immunoblot analysis and antibodies

Cell lysates were subjected to immunoblot analysis as described previously (35). The following antibodies were used at dilution of 1:1,000, unless otherwise stated: type I collagen and smooth muscle actin-α (αSMA) (Abcam); TAGLN2 (Genetex); PDGFRβ (Cohesion Biosciences); EGFR and LOXL2 (Bio-Techne); LOX (Novus Biological); MITF (Thermo Fisher Scientific); fibronectin, thrombospondin (TSP1), β1 integrin, FAK, paxillin, FAP, and MRTF (BD Biosciences); SPARC (Haematologic Technologies); ERK1/2, HSP90, HSP60, MLC2 (Santa Cruz Biotechnology); AXL, YAP, phospho-paxillin (Y118), phospho-ERK1/2 (T202/Y204), phospho-Rb (S807/811), Rb, p27KIP1, caveolin-1, survivin, and tubulin (Cell Signaling Technology).

Generation of cell-derived ECM and drug-protection assays

Three-dimensional (3D) ECMs were generated as described previously (36). Briefly, gelatin-coated culture dishes were seeded with cells and cultured for 8 days in complete medium, supplemented with 50 μg/mL ascorbic acid every 48 hours. Cell cultures were then washed with PBS and matrices were denuded following a 2-minute treatment with prewarmed extraction buffer (PBS 0.5% Triton X-100, 20 μmol/L NH4OH). Matrices were then gently washed several times with PBS. For drug-protection assays, melanoma cells were seeded onto decellularized matrices for 24 hours, and cultured for another 48-hour period in presence or not of indicated drugs.

Cell proliferation

Cell-cycle profiles were determined by flow cytometry as described previously (34). Proliferation was measured by a MTS conversion assay (34) or followed by live imaging of NucLight Red-stained cells using the IncuCyte ZOOM system (Essen BioScience) or by nuclei quantification of Hoescht-stained cells.

Cell contraction assay

A total of 5 × 104 melanoma cells were embedded in 100 μL of collagen I/Matrigel and seeded on a glass bottom 96-well plate (MatTek). Once the gel was set (1 hour at 37°C), cells were maintained in DMEM 10% FBS with or without indicated drugs. Gel contraction was monitored at day 3. The gel area was measured using ImageJ software and the percentage of contraction was calculated using the formula 100 × (well diameter − gel diameter)/well diameter as described (37).

Traction force microscopy

Contractile forces were assessed by traction force microscopy (TFM) as described (38) using collagen-coated polyacrylamide hydrogels with shear modulus of 4 kPa coated with red fluorescent beads (SoftTrac; Cell Guidance Systems). Cells were plated on bead-conjugated gels for 48 hours. Images were acquired before and after cell removal using a fluorescence microscope (Leica DMI6000, ×10 magnification). Tractions exerted by cells were estimated by measuring beads displacement fields, computing corresponding traction fields using Fourier transformation and calculating root-mean-square traction using the particle image velocity plugin on ImageJ. The same procedure was performed on a cell-free region to measure baseline noise.

Immunofluorescence analysis

Cells were grown on collagen-coated polyacrylamide/bisacrylamide synthetic hydrogels with defined stiffness as described (39), then rinsed, fixed in 4% formaldehyde, and incubated in PBS 0.2% saponin 1% BSA in PBS for 1 hour with 1:100 dilution of the indicated primary antibodies. Following incubation with Alexa Fluor-conjugated secondary antibodies (1:1,000), hydrogels were mounted in Prolong antifade (Thermo Fisher Scientific). F-actin was stained with Texas Red-X or Alexa Fluor-488 phalloidin (1:100; Thermo Fisher Scientific). Nuclei were stained with DAPI. Images were captured on a widefield microscope (Leica DM5500B, ×40 magnification). Cell area and roundness and orientation of fibronectin fibers were assessed on immunofluorescence images using ImageJ. Nuclear/cytosolic ratio of YAP or MRTF was assessed by measuring the fluorescence intensity of nucleus and cytosol and quantified using ImageJ. The corresponding DAPI staining image was used to delimit nuclear versus cytosolic regions.

Collagen imaging

Collagen deposition and organization were visualized by standard Masson's trichrome staining or picrosirius red staining accordingly to (see Supplementary Materials and Methods for details; ref. 40). Second harmonic generation (SHG) and multiphoton-fluorescence images were acquired on a Zeiss 780NLO (Carl Zeiss Microscopy) with Mai Tai HP DeepSee (Newport Corporation). Acquisitions were achieved simultaneously in backward through 10× dry NA 0.45 objective and forward through condenser NA 0.55. Each side is equipped with dual NDD GaAspP detectors (BiG) with 440/10 (for SHG forward and backward) and 525/50 filter (for autofluorescence). Transmission images were acquired with 514 nm laser through the 525/50 filter.

Cell line–derived xenograft tumor models

Mouse experiments were carried out in accordance with the Institutional Animal Care and the local ethical committee (CIEPAL-Azur agreement NCE/2014-179). A total of 1 × 106 melanoma cells were subcutaneously implanted into both flanks of 6-week-old female athymic nude nu/nu mice (Envigo). When tumor reached 100 mm3, mice were randomly grouped into control and test groups. The BRAFi group received six intraperitoneal injections of vemurafenib (35 mg/kg) over a period of 2 weeks. Verteporfin was delivered intraperitoneally three times per week at 45 mg/kg. Mice in the control group were treated with vehicle. At the end of the experiment, mice were sacrificed, tumors were dissected, weighed, and either snap frozen in liquid nitrogen (for mRNA and protein analysis), in Tissue-Tek O.C.T. (VWR; for AFM analysis) or formalin fixed and paraffin embedded for picrosirius red or Masson's trichrome staining, SHG analysis, and IHC.

Patient-derived xenograft tumor models

Patient-derived xenograft (PDX) models treated or not with the BRAFi–MEKi combination as described previously (see Supplementary Materials and Methods for details; ref. 41) were established by TRACE (PDX platform; KU Leuven) using tissue from melanoma patients undergoing surgery at the University Hospitals KU Leuven. Written informed consent was obtained from all patients and all procedures were approved by the UZ Leuven Medical Ethical Committee (S54185/S57760/S59199) and carried out in accordance with the principles of the Declaration of Helsinki. All procedures involving animals were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of KU Leuven and within the context of approved project applications P147/2012, P038/2015, P098/2015, and P035/2016. Formalin-fixed and paraffin-embedded tumor biopsies were sectioned for picrosirius red staining.

Elastic modulus measurements

Mechanical properties of tumor sections were analyzed by atomic force microscopy (AFM) as described previously (42) with a Bioscope Catalyst operating in Point and Shoot (Bruker Nano Surfaces), coupled with an inverted optical microscope (Leica DMI6000B; Leica Microsystems Ltd.). The apparent Young's modulus (Eapp) was measured on unfixed frozen tumor sections using a Borosilicate Glass spherical tip (5 μm of diameter) mounted on a cantilever with a nominal spring constant of 0.06 N/m (Novascan Technologies). The force–distance curves were collected using a velocity of 2 μm/s, in relative trigger mode and by setting the trigger threshold to 1 nN. Eapp values were presented in a boxplot using GraphPad Prism (GraphPad software).

Gene expression omnibus data analysis

Public datasets of human melanoma cell lines developing drug resistance to vemurafenib (M229R and SKMel28R) and double resistance to vemurafenib and selumetinib (M229DDR and SKMel28DDR) were used to analyze gene levels compared with drug-naive parental cell lines (GSE65185; ref. 19). Differential gene expression was also examined in datasets derived from tumor biopsies from melanoma patients before and after development of drug resistance to BRAFi, MEKi, or BRAFi/MEKi combination [GSE50535 (25); Tirosh and colleagues (15)]. Normalized data were prepared using MeV software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism. Unpaired two-tailed Mann–Whitney test were used for statistical comparisons between two groups and Kruskal–Wallis test with Dunn posttests or two-way analysis of variance test with Bonferroni posttests to compare three or more groups. Error bars are ±SD.

MITFlow/AXLhigh mesenchymal BRAFi-resistant cells display increased mechanoresponsiveness and YAP/MRTF activation

To investigate whether acquired resistance to BRAFi modifies mechanosensing pathways, we exploited models of isogenic pairs of parental (P) and resistant (R) melanoma cells showing either reactivation of MAPK pathway through NRAS mutation (M249R) or upregulation of AXL, EGFR, and PDGFRβ RTKs associated with low levels of MITF and reduced differentiation of melanoma cells (M229R, M238R; Supplementary Fig. S1; ref. 20). Cells were cultured on collagen-coated hydrogels with stiffness ranging from 0.2kPa (low), 4kPa (medium) to 50kPa (high; ref. 39). In contrast to parental sublines, a dramatic modification of M238R (Fig. 1A and B) and M229R (Supplementary Figs. S2A and S2B) cell morphology measured by actin reorganization, cell roundness, and area was noticeable upon increased substrate stiffness. In contrast, the shape and actin cytoskeleton of the NRAS-mutated M249R subline and its parent M249P showed no significant changes in response to mechanical stimulation (Fig. 1C and D; Supplementary Figs. S2C and S2D). Importantly, MITFlow/AXLhigh BRAFi-resistant M229R or M238R cells, but not NRAS-mutated M249R cells, exhibited an enhanced capacity to proliferate on a collagen-coated stiff substrate (Fig. 1E; Supplementary Fig. S2E).

Figure 1.

Mesenchymal BRAFi-resistant melanoma cells display increased mechanosensitivity and proliferation on collagen stiff substrate. A, Images of parental (M238P) and BRAFi-resistant (M238R) cells after 48-hour culture on collagen-coated hydrogels of increasing stiffness. Staining represents F-actin (green) and nucleus (blue). Scale bar, 100 μm. Insets show higher magnification views. Scale bar, 50 μm. B, Quantification of cell morphologic changes. Data are represented as scatter plot with mean ± SD from a minimum of 10 cells/field from three random fields. Data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001; Kruskal–Wallis analysis. C, Morphology of mesenchymal BRAFi-resistant M229R and of BRAFi-resistant M249R harboring a secondary NRAS mutation cells compared with parental cells, 48 hours after plating on 4 kPa hydrogels. Staining represents F-actin (green) and nucleus (blue). Scale bar, 100 μm. Insets show higher magnification views. Scale bar, 50 μm. D, Quantification of cell morphological changes. Data are represented as scatter plot with mean ± SD from a minimum of 10 cells/field from three random fields. Data are representative of three independent experiments. *, P < 0.05; ***, P < 0.001, Kruskal–Wallis analysis. E, Bar plot of cell number quantification of parental and resistant cells cultured for 72 hours on low (0.2 kPa) versus high (50 kPa) stiffness. Cells were counted by Hoechst-labeled nuclei staining. Data were normalized to the parental cells on soft substrate. *, P < 0.05; ***, P < 0.001, two-way ANOVA analysis. NS, nonsignificant.

Figure 1.

Mesenchymal BRAFi-resistant melanoma cells display increased mechanosensitivity and proliferation on collagen stiff substrate. A, Images of parental (M238P) and BRAFi-resistant (M238R) cells after 48-hour culture on collagen-coated hydrogels of increasing stiffness. Staining represents F-actin (green) and nucleus (blue). Scale bar, 100 μm. Insets show higher magnification views. Scale bar, 50 μm. B, Quantification of cell morphologic changes. Data are represented as scatter plot with mean ± SD from a minimum of 10 cells/field from three random fields. Data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001; Kruskal–Wallis analysis. C, Morphology of mesenchymal BRAFi-resistant M229R and of BRAFi-resistant M249R harboring a secondary NRAS mutation cells compared with parental cells, 48 hours after plating on 4 kPa hydrogels. Staining represents F-actin (green) and nucleus (blue). Scale bar, 100 μm. Insets show higher magnification views. Scale bar, 50 μm. D, Quantification of cell morphological changes. Data are represented as scatter plot with mean ± SD from a minimum of 10 cells/field from three random fields. Data are representative of three independent experiments. *, P < 0.05; ***, P < 0.001, Kruskal–Wallis analysis. E, Bar plot of cell number quantification of parental and resistant cells cultured for 72 hours on low (0.2 kPa) versus high (50 kPa) stiffness. Cells were counted by Hoechst-labeled nuclei staining. Data were normalized to the parental cells on soft substrate. *, P < 0.05; ***, P < 0.001, two-way ANOVA analysis. NS, nonsignificant.

Close modal

The β1 integrin/FA pathway is essential for ECM mechanosignaling (43). Consistently, when compared with drug-sensitive cells, M238R and M229R cells expressed higher levels of β1 integrin and increased phosphorylation of FA components, including FAK, p130Cas, and paxillin (Supplementary Fig. S1). In addition, M238R cells displayed higher number of FAs upon increased matrix rigidity compared with parental cells (Supplementary Fig. S3).

YAP and MRTF are critical transcriptional mediators of mechanical signals through partially overlapping signaling pathways and target genes (6, 7, 9, 44). Immunofluorescence analysis of melanoma cells plated on soft or rigid substrates revealed that in contrast to M238P cells, M238R cells showed higher levels of nuclear YAP (Fig. 2A and B) on low stiffness substrate (0.2kPa). Nuclear YAP and MRTF markedly increased in M238R cells plated on medium (4kPa) and high (50kPa) substrate stiffness, whereas translocation of YAP and MRTF was only apparent when parental cells were plated on stiff substrate. Consistently, expression of shared YAP/MRTF target genes paralleled increasing collagen rigidity in M238R, but not M238P cells (Fig. 2C). Furthermore, impairment of the actomyosin cytoskeleton with the ROCK inhibitor Y27632 reduced the nuclear localization of YAP and MRTF in M238R cells plated on high stiffness substrate (Fig. 2D). Accordingly, ROCK inhibition abrogated the expression of two shared YAP/MRTF target genes CTGF and CYR61 activated in M238R cells on stiff substrate (Fig. 2E).

Figure 2.

The mechanosensors YAP and MRTF are activated in mesenchymal BRAFi-resistant melanoma cells. A, Effect of collagen stiffening on YAP and MRTF nuclear translocation assessed by immunofluorescence in cells cultured for 48 hours on hydrogels of increasing stiffness. Insets show nuclei staining by DAPI. Scale bar, 40 μm. B, Bar graphs show the proportion of cells in which YAP or MRTF was located either in the nucleus (N) or in the cytoplasm (C; n ≥ 30 cells per condition). Data are representative of three independent experiments. C, qPCR analysis of expression of YAP/MRTF target genes in cells plated for 48 hours on hydrogels. Data are normalized to the expression in parental cells plated on soft substrate. Data are represented as mean ± SD from a technical triplicate representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA analysis. D, M238R cells plated on high stiffness substrate were treated with 10 μmol/L of Y27632 for 48 hours. Nuclear versus cytoplasmic location of YAP and MRTF was assessed by immunofluorescence. Top, data are represented as scatter plots with mean ± SD (n ≥ 30 cells per condition). Data are representative of three independent experiments. ***, P < 0.001, Kruskal–Wallis analysis. Top, immunofluorescence images of YAP and MRTF. Insets show nuclei staining by DAPI. Scale bar, 20 μm. E, qPCR analysis of CYR61 and CTGF expression in M238R cells cultured and treated as above. Data are normalized to the expression in vehicle-treated cells. Data are the mean ± SD from a technical triplicate representative of three independent experiments. ***, P < 0.001, two-way ANOVA analysis. F, Vemurafenib dose–response curves from MTS proliferation assays of M238R cells transfected with control siRNA (siCtrl), siYAP, or siMRTF. Right, lysates from transfected cells were immunoblotted with indicated antibodies. Densitometric quantification is shown.

Figure 2.

The mechanosensors YAP and MRTF are activated in mesenchymal BRAFi-resistant melanoma cells. A, Effect of collagen stiffening on YAP and MRTF nuclear translocation assessed by immunofluorescence in cells cultured for 48 hours on hydrogels of increasing stiffness. Insets show nuclei staining by DAPI. Scale bar, 40 μm. B, Bar graphs show the proportion of cells in which YAP or MRTF was located either in the nucleus (N) or in the cytoplasm (C; n ≥ 30 cells per condition). Data are representative of three independent experiments. C, qPCR analysis of expression of YAP/MRTF target genes in cells plated for 48 hours on hydrogels. Data are normalized to the expression in parental cells plated on soft substrate. Data are represented as mean ± SD from a technical triplicate representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA analysis. D, M238R cells plated on high stiffness substrate were treated with 10 μmol/L of Y27632 for 48 hours. Nuclear versus cytoplasmic location of YAP and MRTF was assessed by immunofluorescence. Top, data are represented as scatter plots with mean ± SD (n ≥ 30 cells per condition). Data are representative of three independent experiments. ***, P < 0.001, Kruskal–Wallis analysis. Top, immunofluorescence images of YAP and MRTF. Insets show nuclei staining by DAPI. Scale bar, 20 μm. E, qPCR analysis of CYR61 and CTGF expression in M238R cells cultured and treated as above. Data are normalized to the expression in vehicle-treated cells. Data are the mean ± SD from a technical triplicate representative of three independent experiments. ***, P < 0.001, two-way ANOVA analysis. F, Vemurafenib dose–response curves from MTS proliferation assays of M238R cells transfected with control siRNA (siCtrl), siYAP, or siMRTF. Right, lysates from transfected cells were immunoblotted with indicated antibodies. Densitometric quantification is shown.

Close modal

Finally, to evaluate the potential contribution of ECM stiffness-induced YAP/MRTF activation in MITFlow/AXLhigh associated resistance, M238R cells cultured on rigid collagen hydrogels were transfected with siRNA pool targeting YAP or MRTF and treated with increasing doses of BRAFi (vemurafenib). The sensitivity of M238R cells to BRAFi-induced cell proliferation arrest was partially restored upon YAP or MRTF knockdown, suggesting that collagen stiffening through YAP and MRTF activation contributes to acquired resistance (Fig. 2F). Together, these results indicate that the dedifferentiated MITFlow/AXLhigh-resistant cell state is associated with a mechanophenotype.

MITFlow/AXLhigh BRAFi-resistant cells display YAP and MRTF-dependent contractile activity and assemble an organized ECM

Further functional analysis of the dedifferentiated MITFlow mesenchymal resistant state revealed that M229R and M238R cells were characterized by high expression levels of typical CAF markers such as caveolin-1 (CAV1), myosin light-chain 2 (MLC2), αSMA, fibroblast activation protein (FAP), transgelin-2 (TAGLN2), in addition to ECM proteins collagen 1 (COL1) and fibronectin (Fig. 3A). In contrast, parental and mutant NRAS-driven resistance M249R cell lines showed low or no expression of such markers. We thus examined whether MITFlow/AXLhigh-resistant cells display CAF-associated features such as ROCK-dependent actomyosin contractility and force-mediated ECM remodeling leading to fibers organization (7, 37). We first compared traction stresses generated by sensitive and BRAFi-resistant cells using TFM and observed that M238R cells applied stronger forces on collagen-coated stiff matrices than their drug-sensitive parental counterparts (Fig. 3B). Next, we performed collagen gel contraction assays to assess cell contractility. Contractility in 3D collagen was observed for M238R, but not for M238P cells. Inhibition of ROCK by Y27632 or YAP by Verteporfin reduced the capacity of M238R cells to contract collagen gels to levels that were observed for drug-sensitive M238P cells (Fig. 3C). Moreover, siRNA-mediated knockdown of YAP or MRTF abrogated the contractile activity of drug-resistant M238R cells (Fig. 3D).

Figure 3.

Mesenchymal BRAFi-resistant melanoma cells produce an organized ECM fibrillar network through increased contraction forces and contractility. A, Immunoblot analysis of myofibroblast markers on lysates from BRAFi-resistant cells and parental cells. B, Heat scale plot showing the traction forces applied by cells seeded on 4 kPa fluorescent bead-embedded collagen-coated hydrogels for 48 hours. Scale bar, 25 μm. Bottom, quantification of contractile forces. Data are the mean ± SD (n = 30 fluorescent bead displacement measured per cell from six cells. ***, P < 0.001, Kruskal–Wallis analysis. C, Collagen contraction assays of indicated cells in presence or not of Y27632 (10 μmol/L) or Verteporfin (1 μmol/L). Images of assays are shown. Bottom, quantification of gel contraction. Bar graph represents the mean ± SD of triplicate experiments. ***, P < 0.001, Kruskal–Wallis test. D, Collagen contraction assays of M238R transfected with a siRNA control (siCtrl), siYAP, or siMRTF. Images of assays are shown. Bottom, quantification of gel contraction. Bar graph is mean ± SD of triplicate experiments. **, P < 0.01; ***, P < 0.001. E, Fibronectin and collagen staining of decellularized 3D ECM derived from indicated cells. Top, anti-fibronectin immunofluorescence; bottom, picrosirius red staining. Scale bar, 50 μm. F, Quantification of fibronectin fibers orientation. Fibers were visualized as in E and their orientation angles plotted as a frequency distribution. Percentages indicate oriented fibers accumulated in a range of ±21° around the modal angle. Data are represented as mean ± SD (n = 10 random fields from a duplicate determination). ***, P < 0.001, Kruskal–Wallis analysis. G, Heatmap showing the differential expression of selected genes in cells or patient (Pt) biopsies upon BRAFi and/or MEKi treatment. Data were extracted from public datasets of human melanoma cells developing resistance to BRAFi (R) and double resistance to BRAFi/MEKi (DDR) compared with drug-naive cells and from datasets of melanoma biopsies from patients before and after development of resistance to BRAFi (*), MEKi (°), or BRAFi/MEKi combination (*°).

Figure 3.

Mesenchymal BRAFi-resistant melanoma cells produce an organized ECM fibrillar network through increased contraction forces and contractility. A, Immunoblot analysis of myofibroblast markers on lysates from BRAFi-resistant cells and parental cells. B, Heat scale plot showing the traction forces applied by cells seeded on 4 kPa fluorescent bead-embedded collagen-coated hydrogels for 48 hours. Scale bar, 25 μm. Bottom, quantification of contractile forces. Data are the mean ± SD (n = 30 fluorescent bead displacement measured per cell from six cells. ***, P < 0.001, Kruskal–Wallis analysis. C, Collagen contraction assays of indicated cells in presence or not of Y27632 (10 μmol/L) or Verteporfin (1 μmol/L). Images of assays are shown. Bottom, quantification of gel contraction. Bar graph represents the mean ± SD of triplicate experiments. ***, P < 0.001, Kruskal–Wallis test. D, Collagen contraction assays of M238R transfected with a siRNA control (siCtrl), siYAP, or siMRTF. Images of assays are shown. Bottom, quantification of gel contraction. Bar graph is mean ± SD of triplicate experiments. **, P < 0.01; ***, P < 0.001. E, Fibronectin and collagen staining of decellularized 3D ECM derived from indicated cells. Top, anti-fibronectin immunofluorescence; bottom, picrosirius red staining. Scale bar, 50 μm. F, Quantification of fibronectin fibers orientation. Fibers were visualized as in E and their orientation angles plotted as a frequency distribution. Percentages indicate oriented fibers accumulated in a range of ±21° around the modal angle. Data are represented as mean ± SD (n = 10 random fields from a duplicate determination). ***, P < 0.001, Kruskal–Wallis analysis. G, Heatmap showing the differential expression of selected genes in cells or patient (Pt) biopsies upon BRAFi and/or MEKi treatment. Data were extracted from public datasets of human melanoma cells developing resistance to BRAFi (R) and double resistance to BRAFi/MEKi (DDR) compared with drug-naive cells and from datasets of melanoma biopsies from patients before and after development of resistance to BRAFi (*), MEKi (°), or BRAFi/MEKi combination (*°).

Close modal

Given that increased cellular forces lead to matrix fiber organization and that BRAFi-resistant mesenchymal cells secrete high levels of ECM proteins (20, 21), we analyzed the topography of the fibronectin and collagen network generated by this resistant cellular state. We compared ECM proteins differentially produced and deposited by M238P and M238R cells. Cell-derived 3D matrices were generated, denuded of cells, and analyzed by quantitative mass spectrometry. Compared with M238P cells, M238R cells assembled a matrix that was enriched in ECM glycoproteins (fibronectin, fibrilin-1, thrombospondin-1, and fibulin-1/2), collagens, proteoglycans (versican and biglycan), as well as collagen-modifying enzymes such as transglutaminase 2 and LOXL2 (Supplementary Table S1). Furthermore, in contrast to parental cells, M238R cells assembled fibronectin and collagen fibers oriented in parallel patterns that resembled those produced by TGFβ-activated fibroblasts (Fig. 3E). Fibronectin fibers organization was quantified by measuring the relative orientation angle of fibers. The percentages were 16.5%, 23.7%, and 27.8% for M238P, M238R, and fibroblasts 3D ECM, respectively (Fig. 3F). Importantly, the lower degree of ECM production by parental cells was not due to a difference in proliferation as evidenced by nuclear and fibronectin stainings of M238P and M238R cell cultures before the decellularization process (Supplementary Fig. S4). Together, these results suggest that MITFlow/AXLhigh BRAFi-resistant cells display increased traction forces and contractility, leading to aligned organization of ECM fibers.

Given our observations so far, we explored publicly available expression array studies searching for mechanosignaling, and cell contractility gene expression in drug-resistant human melanoma cells. Data extracted from the GEO database (GSE65185; ref. 19) showed increased levels of several YAP/MRTF target genes (THBS1, CYR61, CTGF, AMOTL2, ANKRD1, and SERPINE1) together with high levels of ECM genes (COL1A1, COL1A2, and FN1) and mesenchymal markers (PDGFRB, MYL9, ACTA2, FAP, and TAGLN) in MITFlow/AXLhigh cells developing drug resistance to vemurafenib (BRAFi; M229R and SKMel28R) and double resistance to vemurafenib and selumetinib (BRAFi + MEKi; M229DDR and SKMel28DDR) as compared with drug-sensitive parental cells (Fig. 3G). Moreover, analysis of gene expression on tumor biopsies from patients progressing during therapy with BRAFi and/or MEKi [GSE50535 and Tirosh and colleagues (Supplementary Information); refs. 15, 25] revealed that expression of ECM and mechanosignaling genes markedly increased in a subset of relapsing patients with MITFlow/AXLhigh expression (Fig. 3G).

Early adaptation to MAPK pathway inhibition induces mechanotransduction pathways, contractility, and ECM fiber organization

We next questioned whether adaptive response to MAPK pathway inhibition involves mechanosensing pathways and ECM remodeling. BRAF-mutant melanoma cells (1205Lu and M238P) were plated on collagen-coated hydrogels and treated with the BRAFi vemurafenib or the MEKi trametinib (Fig. 4A; Supplementary Fig. S5). In both cases, drug-treated cells displayed pronounced morphological and actin cytoskeleton changes that were accompanied by increased YAP and MRTF nuclear localization (Fig. 4A; Supplementary Figs. S5A and S5B), and transcriptional activation of the YAP/MRTF shared target gene CYR61 relative to untreated cells (Supplementary Fig. S5C). Moreover, drug-treated M238P and 1205Lu cells displayed significantly higher number of FAs with larger size as compared with control cells (Supplementary Fig. S6). We further confirmed that a short-term treatment of cells with either BRAFi or MEKi increased the expression of collagen 1 (COL1) and fibronectin and of the YAP/MRTF target thrombospondin-1 (TSP1), along with reduced phosphorylation of RB and increased expression of p27KIP1, two cell-cycle markers that are modulated by MAPK pathway inhibition (Fig. 4B). Importantly, short-term treatment with BRAFi or MEKi was sufficient to increase the contractile activity of 1205Lu cells embedded in collagen gels (Fig. 4C). Consistently, when cultivated one week in the presence of vemurafenib, 1205Lu cells assembled an organized ECM composed of collagen and fibronectin fibers that were anisotropically oriented, as compared with untreated cells (Fig. 4D). A further indication of the involvement of mechanopathways in adaptation of melanoma cells to MAPK inhibition was brought by the observation that M238P, 1205Lu, and 501Mel cells cultivated on stiff collagen-coated substrates were significantly more resistant to increasing doses of BRAFi as compared with cells cultivated on soft substrates (Fig. 4E). Together, these results demonstrate that melanoma cells rapidly adapt to MAPK pathway inhibition by acquiring an ECM-remodeling contractile phenotype associated with increased mechanosignaling pathways.

Figure 4.

MAPK signaling inhibition triggers mechanoactivation pathways, melanoma cell contractility activity, and ECM fibril alignment. A, Images of YAP and MRTF immunostaining of drug-sensitive 1205Lu cells plated for 48 hours on 2.8 kPa collagen-coated hydrogels and treated with vehicle, 3 μmol/L vemurafenib or 1 μmol/L trametinib. Scale bar, 40 μm. Insets show nuclei staining by DAPI. Bottom, quantification of the nucleocytoplasmic distribution of YAP and MRTF (n ≥ 30 cells per condition). Data are representative of three independent experiments. B, Immunoblot analysis of ECM proteins and proliferation markers on lysates from cells treated as above. C, Collagen contraction assays of 1205Lu pretreated for 72 hours with vehicle, 3 μmol/L vemurafenib, or 1 μmol/L trametinib. Right, quantification of gel contraction. Bar graph is the mean ± SD of triplicate experiments. ***, P < 0.001. D, Immunofluorescence analysis of fibronectin and collagen I fibers assembly in decellularized ECM generated from 1205Lu cells treated with vehicle or vemurafenib for 7 days. Scale bar, 40 μm. Histograms, quantification of fibronectin fibers orientation. Percentages indicate fibers accumulated in a range of ± 21° around the modal angle. E, Cells were cultured on low (0.2 kPa) versus high (50 kPa) stiffness substrate for 72 hours in the presence of the indicated dose of vemurafenib. Bar graphs show cell number quantification by Incucyte analysis of red-labeled nuclei. Data are normalized relative to the number of cells on soft substrate and 1 μmol/L vemurafenib. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA analysis.

Figure 4.

MAPK signaling inhibition triggers mechanoactivation pathways, melanoma cell contractility activity, and ECM fibril alignment. A, Images of YAP and MRTF immunostaining of drug-sensitive 1205Lu cells plated for 48 hours on 2.8 kPa collagen-coated hydrogels and treated with vehicle, 3 μmol/L vemurafenib or 1 μmol/L trametinib. Scale bar, 40 μm. Insets show nuclei staining by DAPI. Bottom, quantification of the nucleocytoplasmic distribution of YAP and MRTF (n ≥ 30 cells per condition). Data are representative of three independent experiments. B, Immunoblot analysis of ECM proteins and proliferation markers on lysates from cells treated as above. C, Collagen contraction assays of 1205Lu pretreated for 72 hours with vehicle, 3 μmol/L vemurafenib, or 1 μmol/L trametinib. Right, quantification of gel contraction. Bar graph is the mean ± SD of triplicate experiments. ***, P < 0.001. D, Immunofluorescence analysis of fibronectin and collagen I fibers assembly in decellularized ECM generated from 1205Lu cells treated with vehicle or vemurafenib for 7 days. Scale bar, 40 μm. Histograms, quantification of fibronectin fibers orientation. Percentages indicate fibers accumulated in a range of ± 21° around the modal angle. E, Cells were cultured on low (0.2 kPa) versus high (50 kPa) stiffness substrate for 72 hours in the presence of the indicated dose of vemurafenib. Bar graphs show cell number quantification by Incucyte analysis of red-labeled nuclei. Data are normalized relative to the number of cells on soft substrate and 1 μmol/L vemurafenib. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-way ANOVA analysis.

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Mesenchymal-associated resistance and early adaptation to MAPK pathway inhibition induce the production of a drug-protective ECM

The findings described above support the notion that a subset of BRAF-mutant melanoma cells in response to early and late MAPK pathway inhibition acquire the capacity to produce and remodel a matrix reminiscent to CAF-derived ECM. Because ECM plays a major role in mediating drug resistance, we hypothesized that melanoma cell-derived matrix functions as a supporting niche for melanoma cell behavior. To investigate the effect of melanoma cell-derived ECM on survival and resistance to targeted therapies, drug naïve BRAF-mutant melanoma cells were plated on 3D matrices generated from parental cells (M238P and M229P) or their BRAFi-resistant counterparts (M238R and M229R), and treated or not with vemurafenib alone or the combination vemurafenib/trametinib (Fig. 5; Supplementary Fig. S7). Time-lapse monitoring of 501Mel proliferation revealed that matrices derived from MITFlow/AXLhigh mesenchymal BRAFi-resistant cells significantly reduced the proliferation arrest induced by MAPK pathway inhibition in contrast to ECMs from BRAFi-sensitive cells, which had no impact on the cytostatic action of BRAF and MEK inhibition (Fig. 5A and B; Supplementary Figs. S7A and S7B). Cell-cycle analysis further confirmed the protective action of matrices from mesenchymal resistant, but not parental cells, over the G0–G1 cell-cycle arrest induced by BRAFi on drug-naive 501Mel and MNT1 cells (Fig. 5C; Supplementary Figs. S7C and S7D). At the molecular level, matrix-mediated therapeutic escape from BRAF inhibition was associated in both 501Mel and MNT1 cells with sustained levels of the proliferation marker phosphorylated-RB and of survivin, low levels of the cell-cycle inhibitor p27KIP1 together with maintained phosphorylation of ERK1/2 in presence of the drug (Fig. 5D; Supplementary Figs. S7E and S7F). Importantly, similar biochemical events were promoted in 501Mel cells escaping from the combination of BRAFi and MEKi upon adhesion to M238R-derived, but not M238P-derived ECM (Fig. 5E). Next we wondered if short-term MAPK pathway inhibition fosters a drug-protective ECM program in melanoma cells. 501Mel cells were plated on matrices generated from vehicle or vemurafenib-treated 1205Lu cells, and treated with or without BRAFi. Cell cycle and biochemical analysis showed that BRAF inhibition rapidly promoted the production by 1205Lu cells of an ECM that significantly counteracted the cytostatic action of vemurafenib in 501Mel cells (Fig. 5F and G).

Figure 5.

Early adaptation and mesenchymal-associated resistance to MAPK pathway inhibition is associated with the production of a drug-protective ECM. A, Proliferation curves of 501Mel cells cultured on decellularized M238P or M238R cell-derived matrices and treated with vehicle or with 2 μmol/L vemurafenib in combination or not with 0.1 μmol/L trametinib. Time-lapse analysis of cells using the IncuCyte system. Graphs show quantification of cell numbers from NucLight Red nuclear object counting. Data are the mean ± SD (n = 3). ***, P < 0.001, two-way ANOVA analysis. A.U., arbitrary unit. B, Representative images of nuclear labeling and red fluorescence at the end of the experiment shown in A. C, Cell-cycle distribution of 501Mel (top) or MNT1 (bottom) cells cultured on M238P or M238R-derived ECM for 48 hours and treated with vehicle or 2 μmol/L vemurafenib. Histograms represent the percentage of cells in different phases of the cell cycle. D, Immunoblot analysis of cell-cycle markers from experiments shown in C. E, Immunoblot analysis of cell-cycle markers on lysates from 501Mel cultured on M238P or M238R cell-derived ECM treated for 48 hours with a combination of 2 μmol/L vemurafenib and 0.1 μmol/L trametinib. F, Cell-cycle distribution of 501Mel cultured on cell-derived matrices generated from vehicle or vemurafenib-treated 1205lu cells and treated with 2 μmol/L vemurafenib for 48 hours. Cell-cycle profiles were analyzed as above. G, Immunoblot analysis of cell-cycle markers on lysates from 501Mel cells obtained from F.

Figure 5.

Early adaptation and mesenchymal-associated resistance to MAPK pathway inhibition is associated with the production of a drug-protective ECM. A, Proliferation curves of 501Mel cells cultured on decellularized M238P or M238R cell-derived matrices and treated with vehicle or with 2 μmol/L vemurafenib in combination or not with 0.1 μmol/L trametinib. Time-lapse analysis of cells using the IncuCyte system. Graphs show quantification of cell numbers from NucLight Red nuclear object counting. Data are the mean ± SD (n = 3). ***, P < 0.001, two-way ANOVA analysis. A.U., arbitrary unit. B, Representative images of nuclear labeling and red fluorescence at the end of the experiment shown in A. C, Cell-cycle distribution of 501Mel (top) or MNT1 (bottom) cells cultured on M238P or M238R-derived ECM for 48 hours and treated with vehicle or 2 μmol/L vemurafenib. Histograms represent the percentage of cells in different phases of the cell cycle. D, Immunoblot analysis of cell-cycle markers from experiments shown in C. E, Immunoblot analysis of cell-cycle markers on lysates from 501Mel cultured on M238P or M238R cell-derived ECM treated for 48 hours with a combination of 2 μmol/L vemurafenib and 0.1 μmol/L trametinib. F, Cell-cycle distribution of 501Mel cultured on cell-derived matrices generated from vehicle or vemurafenib-treated 1205lu cells and treated with 2 μmol/L vemurafenib for 48 hours. Cell-cycle profiles were analyzed as above. G, Immunoblot analysis of cell-cycle markers on lysates from 501Mel cells obtained from F.

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Finally, we investigated the involvement of the mechanoresponsive YAP and MRTF transcriptional pathways in ECM-mediated drug protection. 501Mel cells were cultured on matrices prepared from parental M238P or drug-resistant M238R cells and the subcellular location of YAP and MRTF was examined by immunofluorescence microscopy. In contrast to ECM from M238P cells, matrices derived from M238R cells promoted the nuclear translocation of YAP and MRTF (Fig. 6A), and their transcriptional activation as indicated by the increased expression of ANKRD1 and SERPINE1 genes (Fig. 6B). Consistently, drug protective action provided by matrices derived from therapy-resistant M238R cells against BRAFi or the combination BRAFi/MEKi was dramatically reduced in 501Mel cells in which either YAP (Fig. 6C) or MRTF (Fig. 6D) expression was knocked-down. Depletion of YAP or MRTF enhanced the efficacy of MAPK pathway inhibition as shown by reduced levels of phosphorylation of ERK1/2 and RB and increased expression of p27KIP1 (Fig. 6E and F). These suggest that melanoma cell-derived ECM mediates drug protection through YAP and MRTF regulation.

Figure 6.

Matrices generated by resistant melanoma cells induce YAP and MRTF activation to confer protection to MAPK pathway inhibition. A, 501Mel cells were cultured on M238P or M238R cell-derived matrices for 48 hours and subjected to immunofluorescence analysis of YAP and MRTF. Insets show nuclei staining by DAPI. Scale bar, 40 μm. Right, quantification of the nucleocytoplasmic distribution of YAP and MRTF (n ≥ 15 cells per condition). Data are representative of three independent experiments. ***, P < 0.001, Mann–Whitney. B, qPCR analysis of shared YAP/MRTF target genes in cells obtained from A. Data are represented as mean ± SD from a technical triplicate representative of three independent experiments. *, P < 0.05; **, P < 0.01, two-way ANOVA analysis. C and D, Bar graphs showing quantification of cell proliferation of 501Mel cells plated on cell-derived matrices and treated for 72 hours with or without 2 μmol/L vemurafenib in combination or not with 0.1 μmol/L trametinib following transfection with control siRNA (siCtrl) or YAP (siYAP) siRNA (C), or following transfection with siCtrl or MRTF (siMRTF) siRNA (D). Data are the mean ± SD (n = 3). *, P < 0.05, ***, P < 0.001, two-way ANOVA. Right panels show immunoblots of YAP and MRTF levels in transfected cells. E and F, Immunoblot analysis on lysates obtained from the experiments described in C and D. NS, nonsignificant.

Figure 6.

Matrices generated by resistant melanoma cells induce YAP and MRTF activation to confer protection to MAPK pathway inhibition. A, 501Mel cells were cultured on M238P or M238R cell-derived matrices for 48 hours and subjected to immunofluorescence analysis of YAP and MRTF. Insets show nuclei staining by DAPI. Scale bar, 40 μm. Right, quantification of the nucleocytoplasmic distribution of YAP and MRTF (n ≥ 15 cells per condition). Data are representative of three independent experiments. ***, P < 0.001, Mann–Whitney. B, qPCR analysis of shared YAP/MRTF target genes in cells obtained from A. Data are represented as mean ± SD from a technical triplicate representative of three independent experiments. *, P < 0.05; **, P < 0.01, two-way ANOVA analysis. C and D, Bar graphs showing quantification of cell proliferation of 501Mel cells plated on cell-derived matrices and treated for 72 hours with or without 2 μmol/L vemurafenib in combination or not with 0.1 μmol/L trametinib following transfection with control siRNA (siCtrl) or YAP (siYAP) siRNA (C), or following transfection with siCtrl or MRTF (siMRTF) siRNA (D). Data are the mean ± SD (n = 3). *, P < 0.05, ***, P < 0.001, two-way ANOVA. Right panels show immunoblots of YAP and MRTF levels in transfected cells. E and F, Immunoblot analysis on lysates obtained from the experiments described in C and D. NS, nonsignificant.

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Collectively, our findings demonstrate that both early and late adaptation to MAPK pathway inhibition involves the mechanical reprogramming of melanoma cells, leading to the assembly of an organized matrix that confers de novo resistance to targeted therapies in a YAP and MRTF-dependent manner.

In vivo MAPK pathway inhibition promotes melanoma cell-derived ECM accumulation and tumor stiffening

Exposure of BRAF-mutant melanoma cells to MAPK pathway inhibition promotes a mechanophenotype associated with drug tolerance in vitro, which could have important outcomes for disease progression in vivo. To address this, we first explored whether BRAF inhibition induces ECM remodeling in human melanoma xenograft models. BRAF-mutant melanoma cells 1205Lu or M229P were xenografted into nude mice (melanoma CDX), which were treated blindly with either vehicle or vemurafenib (Supplementary Fig. S8A). As expected, BRAF targeting induced a strong inhibition of tumor growth (Supplementary Figs. S8B and S8C). Histologic, transcriptomic, and biophysic analyses were then performed at the experiment end point. Vemurafenib treatment triggered a profound remodeling of the 1205Lu (Fig. 7A) and M229P (Supplementary Fig. S8D) tumor stroma, with a marked increase of collagen fibers area and thickness, as measured by polarized light of picrosirius red-labeled tumors and SHG microscopy (Fig. 7A and B; Supplementary Fig. S8D). We then examined gene expression on BRAFi-treated melanoma tumors by performing RT-qPCR analysis using human and mouse probes. Consistent with a previous study (31), vemurafenib was found to significantly activate tumor-associated host stromal cells. However, compared with untreated tumors, tumors exposed to BRAFi also dramatically upregulated human mesenchymal and ECM genes, including genes for collagens (COL1A1, COL3A1, COL5A1, COL15A1), fibronectin (FN1), collagen-modifying enzyme (LOX), and myofibroblast markers (SPARC, ACTA2), as well as YAP and/or MRTF target genes, such as AXL, CYR61, SERPINE1, AMOTL2, and THBS1 (Fig. 7C). This observation supports the notion that BRAF inhibition can promote a cancer cell-autonomous mechanism of ECM production in vivo. Consistent with the changes in ECM composition and assembly, vemurafenib treatment significantly increased tumor elastic modulus in the two CDX models when measured by AFM (Fig. 7D; Supplementary Fig. S8E), suggesting that ECM stiffening constitute an adaptive response of melanoma cells to MAPK pathway inhibition in vivo. We next wished to validate these observations in melanoma PDXs. PDX exposed or not to the combination of BRAFi and MEKi were stained with picrosirius red (Fig. 7E). Combined BRAF and MEK inhibition also resulted in a marked accumulation of collagen fibers in the tumor stroma of melanoma PDX (Fig. 7E and F). Finally, Verteporfin, a FDA-approved drug used in photodynamic therapy for macular degeneration and a known inhibitor of YAP was used to interrogate if YAP contributes to BRAFi-induced collagen remodeling and therapy response in vivo. Although Verteporfin alone did not affect 1205Lu tumor growth, cotreatment with vemurafenib plus Verteporfin had a greater antitumor effect than vemurafenib alone after 17 days of drug regimens (Fig. 7G and H). Thus, combined Verteporfin and vemurafenib therapy enhanced vemurafenib response in a preclinical melanoma model. Furthermore, Masson's trichrome and picrosirius red stainings revealed that Verteporfin treatment abrogated the accumulation of collagen fibers induced by BRAF inhibition in the stroma of melanoma xenografts (Fig. 7I and J). Together these data suggest that YAP mechanosensing pathway contributes to collagen reorganization in response to MAPK pathway inhibition and support the concept of a combinatorial approach to overcome ECM-mediated therapy resistance in BRAF-mutated melanoma models.

Figure 7.

In vivo MAPK inhibition drives melanoma cell biomechanical reprogramming and tumor stiffening in melanoma tumors. A, Sections of 1205Lu melanoma CDX treated with vehicle or with vemurafenib (BRAFi) were stained with picrosirius red and imaged under original bright field (parallel) or polarized light (orthogonal). Scale bar, 500 μm. Collagen fibers area was quantified with ImageJ. Values represent mean ± SD of four independent fields. *, P < 0.05; **, P < 0.01. B, SHG microscopy from samples described in A. Scale bar, 500 μm. SHG intensity was quantified with ImageJ. Values represent mean ± SD of four independent fields. *, P < 0.05; **, P < 0.01. C, Heatmap showing the differential expression of human and mouse ECM genes, dedifferentiation markers, and YAP/MRTF target genes in untreated versus vemurafenib-treated tumors. Gene expression was assessed by RT-qPCR. D, Scatter plot with mean ± SD showing Young's modulus (Eapp) measurements of vehicle and vemurafenib-treated tumors. ****, P < 0.0001. E, Sections obtained from melanoma PDX were treated or not with BRAFi and MEKi, stained with hematoxylin and eosin (H&E) or picrosirius red, and imaged under transmission (parallel) or polarized light (orthogonal) microscopy. Scale bar, 150 μm. F, Collagen fibers area was quantified from picrosirius red stainings with ImageJ. Values represent mean ± SD of four independent fields. G, 1205Lu cells were injected into nude mice and when tumors reached 100 mm3, mice were administered (i.p. injection) vehicle, vemurafenib (BRAFi), Verteporfin (a YAP/TEAD inhibitor), or the combination of vemurafenib and Verteporfin. Data shown are mean ± SD. Photographs of mice and tumors taken at day 19 are shown. H, Bar graphs showing tumor weights at day 19. Data are means ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.01, Kruskal–Wallis test. I, Sections of 1205Lu melanoma CDX from the experiment shown in G were stained with Masson's trichrome or picrosirius red and imaged under transmission (parallel) or polarized light (orthogonal) microscopy. Scale bar, 50 μm. J, Collagen fibers area was quantified from picrosirius red stainings with ImageJ. Values are the mean ± SD of four independent fields. **, P < 0.01; ***, P < 0.001, Kruskal–Wallis test. K, Proposed model for the biomechanical reprogramming of melanoma cell induced by MAPK-targeted therapies. The scheme shows the reciprocal YAP/MRTF-dependent feed-forward loop between drug-exposed or -resistant cells and ECM remodeling to increase tumor stiffening, mechanosensing, and resistance.

Figure 7.

In vivo MAPK inhibition drives melanoma cell biomechanical reprogramming and tumor stiffening in melanoma tumors. A, Sections of 1205Lu melanoma CDX treated with vehicle or with vemurafenib (BRAFi) were stained with picrosirius red and imaged under original bright field (parallel) or polarized light (orthogonal). Scale bar, 500 μm. Collagen fibers area was quantified with ImageJ. Values represent mean ± SD of four independent fields. *, P < 0.05; **, P < 0.01. B, SHG microscopy from samples described in A. Scale bar, 500 μm. SHG intensity was quantified with ImageJ. Values represent mean ± SD of four independent fields. *, P < 0.05; **, P < 0.01. C, Heatmap showing the differential expression of human and mouse ECM genes, dedifferentiation markers, and YAP/MRTF target genes in untreated versus vemurafenib-treated tumors. Gene expression was assessed by RT-qPCR. D, Scatter plot with mean ± SD showing Young's modulus (Eapp) measurements of vehicle and vemurafenib-treated tumors. ****, P < 0.0001. E, Sections obtained from melanoma PDX were treated or not with BRAFi and MEKi, stained with hematoxylin and eosin (H&E) or picrosirius red, and imaged under transmission (parallel) or polarized light (orthogonal) microscopy. Scale bar, 150 μm. F, Collagen fibers area was quantified from picrosirius red stainings with ImageJ. Values represent mean ± SD of four independent fields. G, 1205Lu cells were injected into nude mice and when tumors reached 100 mm3, mice were administered (i.p. injection) vehicle, vemurafenib (BRAFi), Verteporfin (a YAP/TEAD inhibitor), or the combination of vemurafenib and Verteporfin. Data shown are mean ± SD. Photographs of mice and tumors taken at day 19 are shown. H, Bar graphs showing tumor weights at day 19. Data are means ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.01, Kruskal–Wallis test. I, Sections of 1205Lu melanoma CDX from the experiment shown in G were stained with Masson's trichrome or picrosirius red and imaged under transmission (parallel) or polarized light (orthogonal) microscopy. Scale bar, 50 μm. J, Collagen fibers area was quantified from picrosirius red stainings with ImageJ. Values are the mean ± SD of four independent fields. **, P < 0.01; ***, P < 0.001, Kruskal–Wallis test. K, Proposed model for the biomechanical reprogramming of melanoma cell induced by MAPK-targeted therapies. The scheme shows the reciprocal YAP/MRTF-dependent feed-forward loop between drug-exposed or -resistant cells and ECM remodeling to increase tumor stiffening, mechanosensing, and resistance.

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A major resistance program in melanomas exposed to MAPK-targeting therapies is linked to a dedifferentiated, mesenchymal transcriptional cell state characterized by low levels of the melanoma differentiation factor MITF and high levels of AXL (15, 19, 21–24). MITFlow/AXLhigh-resistant cells exhibit multiple traits of the Hoek's invasive gene signature (14), including prominent expression of ECM proteins (20, 21). Here we showed that this resistant cell population also exhibits key aspects of CAFs involved in ECM remodeling: they acquired a mechanical phenotype associated with an actomyosin/YAP/MRTF-dependent contractile activity, and the ability to deposit ECM to create a tumor-permissive environment. In contrast, drug-naive cells and a population of MITFhigh/NRAS-mutant resistant cells displayed no such mechanoresponsive features and ECM remodeling activities. Importantly, we also found that early adaptation to MAPK pathway inhibition promotes de novo acquisition of a CAF-like phenotype, leading to biomechanical reprogramming both in vitro and in vivo. We thus uncover a previously unidentified feed-forward loop between drug-exposed or resistant MITFlow/AXLhigh melanoma cells and ECM remodeling to increase tumor tissue stiffness, mechanosensing and resistance through YAP and MRTF regulation (Fig. 7K).

Short-term treatment of melanoma cells with targeted drugs induced actin dynamics, mechanosensitive regulation of YAP and MRTF and increased cell contractility. This differs from another early adaptation state to BRAF inhibition characterized by the emergence of a slow-cycling NGFR/CD271high persistent cell population (20). However, our results are in line with the observation that BRAFi modulates actin reorganization and YAP/TAZ activation (11) as well as Rho GTPase signaling (45). Thus, our findings underscore the exquisite phenotypic plasticity of melanoma cells and the notion that their biomechanical reprogramming may actively participate to intratumor heterogeneity and therapeutic escape.

Another indication of the ability of targeted therapies to switch melanoma cells towards a CAF-like phenotype is based on our findings that BRAFi induces melanoma cells to autonomously remodel a fibrillar and drug-protective ECM, an additional trait typical of CAFs. A previous study has shown that short-term BRAF inhibition upregulates adhesion signaling and drug tolerance in BRAF-mutant/PTEN-null melanoma cells (46). Extending this observation, our data demonstrate that short-term MAPK pathway inhibition induces the assembly by melanoma cells of an aligned ECM containing collagens, fibronectin and thrombospondin-1, indicating that targeted therapies have the capacity to rapidly exacerbate the intrinsic ability of melanoma cells to produce a pro-invasive ECM (32, 47). Vemurafenib treatment was shown to activate CAFs to generate a drug-tolerant niche through fibronectin-mediated integrin β1/FAK signaling (31). In this study, cell death following BRAF inhibition was reduced when melanoma cells were cultured on stiff substrates containing the combination of fibronectin, thrombospondin-1, and tenascin-C (31). A part from ECMs assembled by therapy-activated fibroblasts, our study reveals a crucial role of fibronectin and collagen-rich ECMs derived from either drug-resistant or drug-exposed melanoma cells in driving tolerance. The protection against the cytostatic effect of MAPK inhibition brought by melanoma-derived matrices is evidenced by the persistence of cycling cells, with sustained levels of proliferative markers and YAP/MRTF nuclear translocation. Remarkably, tolerance to BRAFi was achieved when BRAF-mutant melanoma cells were plated on collagen-coated stiff matrices, supporting the notion that, in addition to fibronectin (31, 46), the collagen network and ECM stiffening are major mediators of melanoma drug resistance. Interestingly, previous studies with bioengineered materials have shown the impact of substrate stiffness on targeted drugs responses in melanoma (48) and carcinoma cell lines (49).

YAP-TEAD and MRTF-SRF pathways functionally interact to coordinate mechanosignaling required for the maintenance of the CAF phenotype in solid tumors (7–9, 50). Similarly, we showed that the contractile behavior of the dedifferentiated resistant melanoma cells requires YAP and MRTF expression. Importantly, we found that YAP and MRTF are activated upon mechanical stress and contribute to ECM-mediated drug resistance. This is in agreement with recent reports demonstrating the contribution of the YAP pathway in BRAFi resistance (10–12). However, these studies were conducted on rigid plastic dishes that do not reflect tissue mechanical compliance. In contrast, we demonstrated the exacerbated ability of dedifferentiated resistant and BRAFi-exposed melanoma cells to adapt to substrate rigidity using cell-derived 3D ECMs and collagen-coated hydrogels with defined stiffness, which model more accurately the activation of YAP and MRTF mechanosensors. In contrast to YAP-TEAD pathway, the role of MRTF-SRF pathway in melanoma therapeutic resistance remains less defined. MRTF controls several cytoskeletal genes, including αSMA and MLC2 (8) that we found enriched in the MITFlow/AXLhigh resistant cells and in MITFlow tumor biopsies from progressing patients with melanoma. Moreover, several components of the matrisome from MITFlow resistant cells, such as tenascin-C, CYR61, thrombospondin-1, and serpine1 are known YAP and/or MRTF targets (9). Remarkably, a YAP1 enrichment signature has also been identified as a driver event of melanoma acquired resistance (19). This is in line with our in silico gene expression analyses that revealed a similar trend towards an increased expression of YAP/MRTF target genes in MITFlow tumor biopsies from patients relapsing from therapy. Of note, a recent study identified AXL, a RTK required to maintain the resistant phenotype in melanoma (24), in a YAP/TAZ target gene signature (51). Accordingly, we found several YAP/MRTF target genes including AXL induced upon BRAFi treatment in our xenograft model. This raises the possibility that the reservoir of AXLhigh resistant cells is promoted by biomechanical adaptation of melanoma cells to oncogenic BRAF inhibition. Interestingly, collagen stiffening has been recently shown to promote melanoma differentiation via YAP/PAX3-mediated MITF expression (52). This study and our present report support the emerging notion that collagen density and rigidity is a key microenvironmental factor that governs melanoma cell plasticity and intratumor heterogeneity. How YAP and MRTF actually coordinate mechanical signals from tumor microenvironments to drive melanoma differentiation, invasive behavior or drug resistance is currently unknown and requires further investigations.

Importantly, our data reveal a targetable vulnerability of vemurafenib-induced mechanical reprograming of melanoma in vivo. Tumors treated with BRAFi or combined BRAFi/MEKi therapy displayed an intense remodeling of the tumor niche associated to increased collagen fibers organization and YAP/MRTF-mediated gene expression. Earlier studies have underscored the critical role of CAFs activated by BRAF inhibition for the development of resistant niches (27, 28, 31, 53). Accordingly, we found that host stromal cells that likely include fibroblasts produce some ECM genes in response to vemurafenib. However, we demonstrated that the molecular changes associated with the dramatic remodeling of the tumor niche in response to MAPK pathway inhibition also results from the activation of human melanoma cells, thereby promoting an autocrine production of a rigid ECM enriched in collagen fibers. In line with the key role of the YAP pathway during melanoma relapse (19) and phenotypic heterogeneity (12), we found that YAP-TEAD inhibition by Verteporfin reverses vemurafenib-induced excessive collagen deposition. Consequently, treatment with Verteporfin cooperated with vemurafenib to reduce melanoma growth. Whether targeting MRTF-SRF signaling pathway may also demonstrate therapeutic efficiency is currently under investigation.

In conclusion, our findings disclose a novel mechanism of BRAF-mutant melanoma cells adaptation to MAPK-targeted therapies through the acquisition of an auto-amplifying CAF-like phenotype in which melanoma cell-derived ECM modulates mechanosensing pathways to promote tumor stiffening. In addition to therapy-induced tumor secretomes (54), therapy-induced mechanical phenotypes could endow cancer cells with unique cell-autonomous abilities to survive and differentiate within challenging tumor-associated microenvironments, thereby contributing to drug resistance and relapse. Our results suggest that cancer cell-ECM interactions and tumor mechanics provide promising targets for therapeutic intervention aimed at enhancing targeted therapies efficacy in melanoma.

No potential conflicts of interest were disclosed.

Conception and design: C.A. Girard, R. Ben Jouira, J.-C. Marine, M. Deckert, S. Tartare-Deckert

Development of methodology: C.A. Girard, M. Lecacheur, R. Ben Jouira, I. Berestjuk, S. Diazzi, C. Gaggioli, M. Deckert

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Ben Jouira, V. Prod'homme, A. Mallavialle, F. Larbret, M. Gesson, S. Schaub, S. Pisano, S. Audebert, B. Mari, E. Leucci, J.-C. Marine

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.A. Girard, M. Lecacheur, R. Ben Jouira, V. Prod'homme, M. Gesson, S. Schaub, S. Audebert, B. Mari, M. Deckert, S. Tartare-Deckert

Writing, review, and/or revision of the manuscript: C.A. Girard, M. Lecacheur, R. Ben Jouira, J.-C. Marine, M. Deckert, S. Tartare-Deckert

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Deckert, S. Tartare-Deckert

Study supervision: M. Deckert, S. Tartare-Deckert

We thank R.S. Lo for melanoma cells. We acknowledge the iBV, IRCAN, and C3M partners of “Microscopie Imagerie Côte d'Azur” (MICA) GIS-IBISA multisites platform supported by the GIS IBiSA, Conseil Départemental 06, and Région PACA. We also acknowledge the C3M animal facility and we thank TRACE (PDX platform at the KULeuven University) for providing the PDX models. This work was supported by institutional funds from Institut National de la Santé et de la Recherche Médicale (Inserm), Université Côte d’Azur, the Ligue Contre le Cancer (Equipe labellisée Ligue Contre le Cancer 2016 to S. Tartare-Deckert), and Institut National du Cancer (INCA_12673 to S. Tartare-Deckert). Funding from the Fondation ARC, National Research Agency (#ANR-18-CE14-0019-01 to M. Deckert), ITMO Cancer Aviesan within the framework of the Cancer Plan, and the French Government through the “Investments for the Future” LABEX SIGNALIFE (#ANR-11-LABX-0028-01) is also acknowledged. We also thank financial supports by Conseil Départemental 06 and Canceropôle PACA. R. Ben Jouira was a recipient of a doctoral fellowship from Fondation ARC. I. Berestjuk is a recipient of a doctoral fellowship from La Ligue Contre le Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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