Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 7;14(2):e11814.
doi: 10.15252/emmm.201911814. Epub 2021 Dec 27.

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

Ilona Berestjuk  1   2 , Margaux Lecacheur  1   2 , Alexandrine Carminati  1   2 , Serena Diazzi  1   2 , Christopher Rovera  1   2 , Virginie Prod'homme  1   2 , Mickael Ohanna  1   2 , Ana Popovic  1   2 , Aude Mallavialle  1   2 , Frédéric Larbret  1   2 , Sabrina Pisano  3 , Stéphane Audebert  4 , Thierry Passeron  1   5 , Cédric Gaggioli  3 , Christophe A Girard  1   2 , Marcel Deckert #  1   2 , Sophie Tartare-Deckert #  1   2
Affiliations

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

Ilona Berestjuk et al. EMBO Mol Med. .

Abstract

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

Keywords: DDR; NF-κB2; extracellular matrix; melanoma; therapeutic resistance.

PubMed Disclaimer

Conflict of interest statement

T.P. is the co‐founder of Yukin Therapeutics. The remaining authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Composition, topology, and mechanical properties of fibroblast‐derived 3D ECMs

  1. Images show collagen matrix gel contraction by HDF (human dermal fibroblasts), skin‐MAF (melanoma‐associated fibroblasts isolated from skin lesions), LN‐FRC (lymph node fibroblast reticular cells), and LN MAF (melanoma‐associated fibroblasts isolated from metastatic lymph node). Dashed circles represent the diameter of the gel. Data are representative of n = 3 independent experiments.

  2. Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de‐cellularized ECM produced by human fibroblasts. Fiber orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data are represented as mean ± s.d. (n = 10 random fields from 2 independent determinations). Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed images is shown for each condition.

  3. Atomic force microscopy (AFM) measurement of the elastic properties (apparent Young’s modulus, Εapp) of fibroblast‐derived ECMs. Each dot represents a specific Young's modulus obtained by fitting the corresponding individual force curve acquired on a determined point of the sample. A representative experiment from 2 independent experiments is shown. Scatter plots show mean ± SEM. The black bars represent the median and the interquartile range. ****P < 0.0001, two‐tailed Mann–Whitney test.

Figure 2
Figure 2. Fibroblast‐derived 3D ECM confers drug‐protective action to melanoma cells against anti‐BRAFV600 therapies
  1. A

    Scheme of the ECM‐mediated drug‐protective assay.

  2. B

    Illustration of the BRAFV600E pathway and the MAPK pathway inhibitors used in the study.

  3. C

    Time‐lapse imaging of proliferation of NucLight‐labeled 1205Lu cells plated on plastic (left panel), Coll‐1 (collagen 1; middle panel), or FRC‐derived ECM (right panel) and treated with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi using the IncuCyte ZOOM system. Each data point represents the mean of NucLight red nuclear objects per field ± SEM. ****P < 0.0001, two‐way ANOVA followed by Dunnett’s multiple comparisons test. Data are representative of n = 3 independent experiments.

  4. D, E

    Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll‐1, or the indicated fibroblast‐derived ECMs prior to a 96‐h treatment with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst‐labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P = 0.015, ***P = 0.0009, and ****P < 0.0001; and (E) *P = 0.0204, ***P = 0.0006, and ****P < 0.0001, the Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

  5. F

    Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic, Coll‐1, or the indicated fibroblast‐derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.

  6. G

    Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic, Coll‐1, or the indicated fibroblast‐derived ECMs in the presence or not of BRAFi or BRAFi/MEKi for 96 h, using antibodies against P‐ERK1/2, ERK2, or cell cycle markers (P‐Rb, Rb, cyclin D1, p27KIP1, survivin, and p53). HSP60, loading control.

Source data are available online for this figure.
Figure EV1
Figure EV1. Fibroblast‐derived 3D ECM confers drug‐protective action to melanoma cells against anti‐BRAFV600E therapies

  1. Time‐lapse imaging of proliferation of NucLight‐labeled 1205Lu cells using the IncuCyte ZOOM system. Cells were plated for 48 h on HDF‐ or MAF‐derived ECMs prior to a 96‐h treatment with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi. Each data point represents the mean of NucLight red nuclear objects per field ± SEM. **P = 0.0079 (left panel), **P = 0.0012 (right panel), and ****P < 0.0001, two‐way ANOVA followed by Dunnett’s multiple comparisons test. Data are representative of n = 3 independent experiments.

  2. Quantification of proliferation of MM099 short‐term melanoma cell cultures plated for 48 h on plastic, Coll‐1, or the indicated fibroblast‐derived ECMs prior to a 96‐h treatment with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst‐labeled nucleus staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. *P = 0.0416 and ****P < 0.0001, two‐way ANOVA followed by Dunnett’s multiple comparisons test.

  3. Flow cytometry analysis of cell cycle distribution of MM099 cells cultured on indicated substrates and treated with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.

  4. Immunoblotting of protein extracts from 1205Lu cells (left panel) and MM099 cells (right panel) cultivated on indicated substrates in the presence or not of 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h, using antibodies against P‐ERK1/2, ERK2, P‐Rb, Rb, E2F1, survivin, p27KIP1, cyclin D1, and p53. HSP60, loading control.

Figure 3
Figure 3. Expression of DDR1 and DDR2 in human melanoma
  1. A

    Meta‐analysis of 363 cutaneous melanoma from TCGA (skin cutaneous melanoma, PanCancer Atlas) (http://www.cbioportal.org/ ) showing the percentage of samples with genetic alterations in DDR1 and DDR2. Cases with missense (green) and truncating (blue) mutations, amplification (red), and mRNA overexpression (pink) are indicated; gray, individual cases.

  2. B

    Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays. Representative IHC images and quantification (right bar histograms) of DDR1 and DDR2 expression in normal skin, nevus, primary melanoma (PM), and lymph node melanoma metastases (MM). Scale bar, 100 µm. Histological scoring of the samples was performed in a blinded fashion. Samples were scored as low, medium, or high for DDR1 or DDR2 expression (nevus, n = 12; PM, n = 30; and MM, n = 20).

  3. C–E

    Immunoblotting of equal amounts of protein extracts from melanoma cell lines (C), patient‐derived short‐term cell cultures (D), or isogenic pairs of parental‐sensitive and BRAFi‐resistant cell lines (E) using antibodies against DDR1, DDR2, or markers of the melanoma cell differentiation AXL, MITF, or SOX10. ERK2, loading control.

  4. F

    DDR1 and DDR2 levels increase in de‐differentiated melanoma cells. Box‐and‐whisker plots show DDR1, DDR2, MITF, AXL, and SOX10 expression among four differentiation melanoma cell states (U, undifferentiated, n = 10; NC, neural crest‐like, n = 14; T, transitory, n = 12; and M, melanocytic, n = 17) (GSE80829). The expression of AXL, MITF, and SOX10 is shown as control markers of cell differentiation. n, number of cell lines representative of each cell state. Central bars represent the median, and the whiskers, the 10th to 90th percentile of the boxplot. Multiple comparisons were performed using ordinary one‐way ANOVA.

Source data are available online for this figure.
Figure EV2
Figure EV2. Knockdown and pharmacological inhibition of DDR1 and DDR2 abrogate ECM‐mediated resistance to BRAFV600E pathway inhibition

  1. Immunoblotting of protein extracts from 1205Lu and MM099 cells cultivated on HDF‐ or MAF‐derived matrices using antibodies against P‐DDR1, P‐DDR1/P‐DDR2, DDR1, and DDR2. β‐actin, loading control.

  2. Immunoblotting of protein extracts of siCTRL‐, siDDR1#2‐, siDDR2#2‐, or siDDR1#2/siDDR2#2‐transfected 1205Lu cells plated on FRC‐derived ECM and treated or not with 5 µM BRAFi for 96 h, using antibodies against DDR1, DDR2, P‐MEK1/2, P‐ERK1/2, ERK2, P‐Rb, and survivin. HSP60, loading control.

  3. Immunoblotting of protein extracts of siCTRL‐, siDDR1#1/siDDR2#1, or siDDR1#2/siDDR2#2‐transfected MM099 melanoma short‐term cultures plated on FRC‐derived ECM and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi for 96 h, using antibodies against DDR1, DDR2, P‐ERK1/2, ERK2, and cleaved caspase‐3. HSP60, loading control.

  4. Immunoblot analysis of collagen I‐induced DDR1 and DDR2 tyrosine phosphorylation. 1205Lu cells were incubated with 10 µg/ml of Coll‐I (collagen I) in the presence or not of 7 µM imatinib or 1 µM DDR1‐IN‐1 for 18 h. After cell lysis, DDR2 phosphorylation was analyzed with anti‐P‐DDR2 following immunoprecipitation (IP) with anti‐DDR2 antibodies. DDR1 phosphorylation was analyzed in total cell lysates with anti‐P‐DDR1. HSP60, loading control.

  5. Time‐lapse imaging of proliferation of NucLight‐labeled 1205Lu cells plated for 48 h on FRC‐ or MAF‐derived ECMs prior to treatment with 5 µM BRAFi in the presence or not of 7 µM imatinib (left panels) or 1 µM DDR1‐IN‐1 (right panels) for the indicated times. Each data point represents the mean of NucLight red nuclear objects per field ± SEM. One experiment representative of 3 independent experiments is shown. ****P < 0.0001, 2‐way ANOVA followed by Dunnett’s multiple comparisons test.

  6. Quantification of proliferation of SKMEL5 cells plated for 48 h on FRC‐ (left panel) or MAF‐derived ECMs (right panel) prior to a 96‐h treatment with vehicle or 5 µM BRAFi in the presence or not of 10 µM imatinib or 5 µM DDR1‐IN‐1. Cells were counted by Hoechst‐labeled nucleus staining. Data are represented as bar plots with mean ± SEM normalized to vehicle. ***P = 0.0002, the Mann–Whitney test (n = 3).

  7. Quantification of proliferation of MM099 melanoma short‐term cultures plated for 48 h on FRC‐ (left panel) or MAF‐derived ECMs (right panel) prior to a 96‐h treatment with vehicle or 5 µM BRAFi in the presence or not of 10 µM imatinib or 3 µM DDR1‐IN‐1. Cells were counted by Hoechst‐labeled nucleus staining. Data are represented as bar plots with mean ± SEM normalized to vehicle. ***P = 0.0002, the Mann–Whitney test (n = 3).

Figure 4
Figure 4. Inhibition of DDR1 and DDR2 by genetic or pharmacological approaches abrogates ECM‐mediated resistance to BRAFV600 pathway inhibition

  1. Immunoblotting of protein extracts from 1205Lu cells transfected with a siRNA control (CTRL) or two different sequences of siRNA (#1 and #2) directed against DDR1 or DDR2 alone or in combination prior to being cultivated on MAF‐derived ECM and treated or not with 5 µM BRAFi for 96 h, using antibodies against the indicated proteins. HSP60, loading control.

  2. Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination of siDDR1#2 and siDDR2#2 prior to being cultivated on FRC‐ or MAF‐derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi, or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control.

  3. Quantification of the time‐lapse imaging of the proliferation of NucLight‐labeled 1205Lu cells using the IncuCyte ZOOM system. Cells were plated for 48 h on FRC‐ or MAF‐derived ECMs prior to a 96‐h treatment with vehicle or 5 µM BRAFi in the presence or not of 7 µM imatinib or 1 µM DDR1‐IN‐1. The bar plots represent the mean normalized to vehicle of NucLight red nuclear objects per field ± SEM from 3 independent experiments performed in triplicate. ****P < 0.0001, two‐way ANOVA followed by Sidak’s multiple comparisons test.

  4. Immunoblotting of protein extracts from 1205Lu, SKMEL5, and MM099 cells plated for 48 h on FRC‐derived ECM prior to a 96‐h treatment with vehicle or 5 µM BRAFi in the presence or not of imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1‐IN‐1 (1 µM for 1205Lu, 5 µM for SKMEL5, and 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.

  5. Flow cytometry analysis of cell death (Annexin V/PI labeling) in 1205Lu cells plated on FRC‐derived ECM and treated as above. Right bar plots show the distribution of cells (% of total) across the different forms of death.

Source data are available online for this figure.
Figure EV3
Figure EV3. Pharmacological inhibition of DDR abrogates ECM‐mediated resistance to BRAFV600E pathway inhibition and induces cell death

  1. Immunoblotting of protein extracts from 1205Lu cells cultivated on MAF‐derived ECM, treated or not with 5 µM BRAFi and/or 7 µM imatinib using antibodies against P‐Rb, P‐ERK1/2, survivin, or cleaved caspase‐3. HSP60, loading control.

  2. Immunoblotting of protein extracts from SKMEL5 cells (left panel) and MM099 melanoma short‐term cultures (right panel) cultivated on MAF‐derived ECM, treated or not with 5 µM BRAFi in combination with imatinib (10 µM) or DDR1‐IN‐1 (5 µM for SKMEL5 and 3 µM for MM099) using antibodies against P‐ERK1/2, ERK2, P‐Rb, Rb, E2F1, survivin, or cleaved caspase‐3. HSP60, loading control.

  3. Immunoblotting of protein extracts from 1205Lu cells cultivated on FRC‐derived ECM for 96 h in the presence of 5 µM BRAFi, 0.01 µM MEKi, or the combination of 2 µM BRAFi and 0.01 µM MEKi, in the presence or not of 10 µM imatinib or 5 µM nilotinib using anti‐P‐MEK1/2, P‐ERK1/2, P‐Rb, E2F1, survivin, or cleaved caspase‐3 antibodies (n = 2). HSP60, loading control.

  4. Flow cytometry analysis of cell death (Annexin V/PI labeling) in SKMEL5 cells (left) and MM099 cells (right) plated on FRC‐derived ECM and treated by the indicated drugs as described above. Data show the percentage of the different forms of cell death based on Annexin V/PI positivity.

Figure 5
Figure 5. Interaction of melanoma cells with 3D ECMs induces the linear clustering of phosphorylated DDR upon BRAFi/MEKi treatment

  1. Representative images of 1,205 cells cultivated on collagen I (Coll‐I) or FRC‐derived ECM for 48 h prior to treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for phospho‐DDR1 (P(Y792)‐DDR1) (red; left panels) and phospho‐DDR1/2 (P(Y796)‐DDR1/P(Y740)‐DDR2) (red; right panels), F‐actin (green), and nuclei (blue) is shown. Enlarged images of P‐DDR1 and P‐DDR1/2 immunostaining are shown. White arrows indicate P‐DDR1 and P‐DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).

  2. Quantification of globular versus linear clusters of phospho‐DDR1 (left panels) and phospho‐DDR1/2 (right panels) from immunofluorescence staining shown in (A) using ImageJ software. Prior to the quantification of DDR clusters, a “subtract background” function of ImageJ has been applied to all images. In order to quantify clusters, the IsoData threshold has been used. Clusters with circularity 0.3–1 have been defined as “globular”, and clusters with circularity 0–0.29 have been defined as “linear”. Data are from > 20 individual cells (n = 3). Error bars reflect mean ± s.d. Values for each treated condition are compared to the vehicle control. 2‐way ANOVA followed by Dunnett’s multiple comparisons test.

  3. Quantification of globular versus linear clusters of Phospho‐DDR1 and Phospho‐DDR1/2 from immunofluorescence staining shown in Fig EV4A of 1205Lu cells cultivated on HDF‐ or MAF‐derived ECM and treated with the indicated targeted drugs as described in (A). Data are from > 20 individual cells. Error bars reflect mean ± s.d. Values for each treated condition are compared to the vehicle control. 2‐way ANOVA followed by Dunnett’s multiple comparisons test.

Source data are available online for this figure.
Figure EV4
Figure EV4. Interaction of melanoma cells with 3D ECM induces the clustering of phosphorylated DDR upon BRAFi or BRAFi/MEKi treatment

  1. Representative images of 1205Lu cells cultivated on HDF‐ or MAF‐derived ECM for 48 h prior to treatment with vehicle, 5 µM BRAFi, or 2 µM BRAFi combined with 0.01 µM MEKi for 96 h. Immunofluorescence for phospho‐DDR1 (P(Y792)‐DDR1) (red; left panels) or phospho‐DDR1/2 (P(Y796)‐DDR1/P(Y740)‐DDR2) (red; right panels) is shown. Nuclei (blue) were stained with DAPI. Enlarged images of P‐DDR1 and P‐DDR1/2 immunostaining are shown. White arrows indicate P‐DDR1 and P‐DDR1/2 cell membrane linear clustering. Scale bar = 25 µm (enlarged images, scale bar = 10 µm).

  2. Analysis of co‐localization of phospho‐DDR with collagen 1 in 1205Lu cells cultivated on MAF‐derived ECM and treated with BRAFi/MEKi. Immunofluorescence for phospho‐DDR1 (P(Y792)‐DDR1) (red; upper panels) and phospho‐DDR1/2 (P(Y796)‐DDR1/P(Y740)‐DDR2) (red; lower panels), collagen 1 (green), and nuclei (blue) is shown. Enlarged images are shown. White arrows indicate co‐localization (yellow fluorescence). Images were captured on Nikon Eclipse Ti confocal microscope at 60x magnification. Scale bar, 20 µm.

  3. Representative images of SKMEL5 cells cultivated on collagen I (Coll‐I) or on indicated fibroblast‐derived ECMs for 48 h prior to treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi combined with 0.01 µM MEKi for 96 h. Immunofluorescence for phospho‐DDR1 (P(Y792)‐DDR1) (red; left panels) or phospho‐DDR1/2 (P(Y796)‐DDR1/P(Y740)‐DDR2) (red; right panels) is shown. Nuclei (blue) were stained with DAPI. Enlarged images of P‐DDR1 and P‐DDR1/2 immunostaining are shown. Scale bar = 25 µm (enlarged images, scale bar = 10 µm).

  4. Quantification of globular (left panels) and linear (right panels) clusters of phospho‐DDR1 and phospho‐DDR1/2 from immunofluorescence staining shown in (B) using ImageJ software. Prior to the quantification of DDR clusters, a “subtract background” function of ImageJ has been applied to all images. In order to quantify clusters, the IsoData threshold has been used. Clusters with circularity 0.3–1 have been defined as “globular”, and clusters with circularity 0–0.29 have been defined as “linear”. Data are from > 20 individual cells. Error bars reflect mean ± s.d. Values for each treated condition are compared to the vehicle control. 2‐way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 6
Figure 6. Targeting DDR by imatinib sensitizes melanoma tumors to BRAFV600E inhibition

  1. Outline of the experimental setup and treatment regimens.

  2. 1205Lu cells were s.c.‐inoculated into nude mice, and when tumors reached 75 mm3, mice were treated with the indicated mono‐ or combo‐therapy for 30 days. Graphs show tumor growth following treatment by indicated drugs. Data shown are mean ± SEM of tumor volume. Vehicle and imatinib groups, n = 10 tumors from 5 mice; vemu and vemu/imatinib groups, n = 20 tumors from 10 mice). ****P < 0.0001, two‐way ANOVA followed by Tukey's multiple comparisons test.

  3. Scatter plot graphs showing the tumor weight upon treatment by the indicated mono‐ or combo‐therapy. Error bars show mean ± SEM of tumor weight (n = 10–11 tumors from 6 mice per condition). ***P = 0.0002, the Kruskal–Wallis test followed by Dunn’s multiple comparisons test; ns, non‐significant.

  4. Immunofluorescence staining using anti‐cleaved caspase‐3 (red), anti‐Ki67 (green), and DAPI in tumor sections of 1205Lu‐derived xenografts from (B). Scale bar, 100 µm. A microphotograph of the tumor size in each treated group is shown (representative of n = 5–10 mice).

  5. Kaplan–Meier survival curves of mice treated with vehicle, imatinib, BRAFi, or BRAFi plus imatinib. Median time to progression was 18, 20, 36, and 48 days, respectively. Log rank (Mantel–Cox) for BRAFi vs BRAFi/imatinib mesylate. ****P < 0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123–0.7106).

  6. Mouse body weight was measured at the indicated day. Data shown are mean ± SEM (n = 5–10 mice).

Figure 7
Figure 7. Imatinib normalizes collagen deposition and remodeling induced upon BRAFi treatment

  1. Sections of 1205Lu xenografts from Fig 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono‐ or combined regimens.

  2. Quantification of collagen maturity and fiber thickness in 1205Lu xenografts stained with picrosirius red using polarized light microscopy. Birefringence hue and amount of collagen fibers were quantified as a percent of total tissue area (2–4 fields per tumor section, n = 4 tumors per condition).

  3. Quantification of collagen fibers using SHG microscopy in tumor sections from (A). Error bars represent mean ± s.d. of 4 independent fields per section, n = 2 tumors per condition. ***P = 0.0002, the Mann–Whitney test.

Figure 8
Figure 8. Targeting the non‐canonical NF‐κB2 pathway overcomes DDR‐dependent MMDR to BRAFV600E pathway inhibition
  1. A

    Immunoblot analysis of protein extracts from siCTRL‐ or siDDR1/2‐transfected SKMEL5 cells plated on FRC‐ or MAF‐derived ECMs in the presence or not of 5 µM BRAFi, 2 µM BRAFi, plus 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P‐ERK1/2, ERK2, RelB, p100/p52, and HSP60 as loading control.

  2. B

    Immunoblot analysis of protein extracts from siCTRL‐ or siDDR1/2‐transfected MM099 short‐term cultures plated on FRC‐ or MAF‐derived ECMs in the presence or not of 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies as above. Note that data come from the same immunoblot.

  3. C

    Illustration of the non‐canonical p52/RelB NF‐κB2 pathway and inhibitors used in the study.

  4. D

    Immunoblot analysis of protein extracts from 1205Lu cells cultivated on plastic, Coll‐1, or indicated fibroblast‐derived ECMs for 48 h using antibodies against RelB, p100/p52, and ERK2 as loading control.

  5. E

    Immunoblot analysis of protein extracts from 1205Lu (left panels) or SKMEL5 (right panels) cells cultivated on FRC‐derived ECMs for 96 h in the presence of 5 µM BRAFi and/or a pan‐IKK inhibitor (IKKi, BMS‐345541 3 µM) using antibodies against P‐ERK1/2, P‐Rb, survivin, RelB, and HSP60 as loading control.

  6. F, G

    Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC‐ or MAF‐derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1‐IN‐1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P‐ERK1/2, ERK2, P‐Rb, Rb, survivin, caspase‐3, cleaved caspase‐3, RelB, p100/p52, and HSP60 as loading control were used.

Source data are available online for this figure.
Figure EV5
Figure EV5. DDR and NF‐κB2 pathway targeting overcomes MMDR in response to BRAFV600E inhibition

  1. Immunoblot analysis of protein extracts from siCTRL‐ or siDDR1/2#2‐transfected 1205Lu cells plated on MAF‐derived ECM in the presence or not of 5 µM BRAFi or 2 µM BRAFi and 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P‐ERK1/2, ERK2, RelB, and p100/p52. HSP60, loading control (n = 2).

  2. Immunoblotting of protein extracts from 1205Lu cells cultivated on MAF‐derived ECM treated with vehicle or 5 µM BRAFi and/or 7 µM imatinib, for the indicated time using antibodies against P‐ERK1/2, P‐Rb, Rb, survivin, caspase‐3, cleaved caspase‐3, and RelB. HSP60, loading control.

  3. Immunoblot analysis of protein extracts from MM099 melanoma short‐term cultures cultivated on FRC‐derived ECM for 96 h in the presence of BRAFi and/or a pan‐IKK inhibitor (IKKi, BMS‐345541 3 µM) using antibodies against P‐ERK1/2, P‐Rb, survivin, RelB and HSP60, loading control.

  4. Immunoblot analysis of protein extracts from 1205Lu (left panel) or SKMEL5 (right panel) cells cultivated on MAF‐derived ECM for 96 h in the presence of BRAFi and/or a pan‐IKK inhibitor (IKKi, BMS‐345541 3 µM) using antibodies against P‐ERK1/2, P‐Rb, survivin, RelB, and HSP60, loading control.

  5. Immunoblotting of protein extracts obtained from 1205Lu cells that were plated on FRC‐derived (left panel) and MAF‐derived (right panel) ECM and treated with 5 µM BRAFi in combination or not with 1 µM DDR inhibitor (DDR1‐IN‐1) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P‐ERK1/2, P‐Rb, Rb, survivin, cleaved caspase‐3, RelB, p100/p52, and HSP60 as loading control were used.

Figure 9
Figure 9. Model of DDR‐dependent matrix‐mediated drug resistance (MMDR) to MAPK‐targeting therapies in melanoma
BRAF‐mutated melanoma cells adapt to BRAF/MEK inhibition by turning on a drug‐tolerant pathway that is initiated by collagen‐rich environments interacting with cancer cell DDR. Clustered DDR activates the non‐canonical NF‐κB2 (p52/RelB) pathway that is therapeutically targetable with clinically approved DDR inhibitor such as imatinib or with preclinically tested NIK inhibitors.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agarwal G, Mihai C, Iscru DF (2007) Interaction of discoidin domain receptor 1 with collagen type 1. J Mol Biol 367: 443–455 - PubMed
    1. Albrengues J, Bourget I, Pons C, Butet V, Hofman P, Tartare‐Deckert S, Feral CC, Meneguzzi G, Gaggioli C (2014) LIF mediates proinvasive activation of stromal fibroblasts in cancer. Cell Rep 7: 1664–1678 - PubMed
    1. Ambrogio C, Gómez‐López G, Falcone M, Vidal A, Nadal E, Crosetto N, Blasco RB, Fernández‐Marcos PJ, Sánchez‐Céspedes M, Ren X et al (2016) Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS‐driven lung adenocarcinoma. Nat Med 22: 270–277 - PubMed
    1. Badiola I, Villace P, Basaldua I, Olaso E (2011) Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability. Oncol Rep 26: 971–978 - PubMed
    1. Beacham DA, Amatangelo MD, Cukierman E (2007) Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr Protoc Cell Biol Chapter 10: Unit 10 19 - PubMed

Publication types