SMAD signaling promotes melanoma metastasis independently of phenotype switching
Eylul Tuncer, … , Reinhard Dummer, Lukas Sommer
Eylul Tuncer, … , Reinhard Dummer, Lukas Sommer
Published April 30, 2019
Citation Information: J Clin Invest. 2019;129(7):2702-2716. https://doi.org/10.1172/JCI94295.
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Research Article Cell biology Dermatology

SMAD signaling promotes melanoma metastasis independently of phenotype switching

Abstract

The development of metastatic melanoma is thought to require the dynamic shifting of neoplastic cells between proliferative and invasive phenotypes. Contrary to this conventional “phenotype switching” model, we now show that disease progression can involve malignant melanoma cells simultaneously displaying proliferative and invasive properties. Using a genetic mouse model of melanoma in combination with in vitro analyses of melanoma cell lines, we found that conditional deletion of the downstream signaling molecule Smad4, which abrogates all canonical TGF-β signaling, indeed inhibited both tumor growth and metastasis. Conditional deletion of the inhibitory signaling factor Smad7, however, generated cells that are both highly invasive and proliferative, indicating that invasiveness is compatible with a high proliferation rate. In fact, conditional Smad7 deletion led to sustained melanoma growth and at the same time promoted massive metastasis formation, a result consistent with data indicating that low SMAD7 levels in patient tumors are associated with a poor survival. Our findings reveal that modulation of SMAD7 levels can overcome the need for phenotype switching during tumor progression and may thus represent a therapeutic target in metastatic disease.

Authors

Eylul Tuncer, Raquel R. Calçada, Daniel Zingg, Sandra Varum, Phil Cheng, Sandra N. Freiberger, Chu-Xia Deng, Ingo Kleiter, Mitchell P. Levesque, Reinhard Dummer, Lukas Sommer

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Figure 6

Loss of SMAD7 promotes emergence of proliferative-invasive MITFhiAXLhi melanoma cells in vitro.

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Loss of SMAD7 promotes emergence of proliferative-invasive MITFhiAXLhi m...
(A) Experimental design used in this study. Proliferative M010817 cells were exposed to combinatorial ligand treatment over 3 days. Cells were pulsed with EdU, followed by immunofluorescent staining (B) Representative IF images. White arrowheads mark ZEB1+EdU+ or MITFhiAXL+ cells. Open arrowheads mark ZEB1–EdU+ or MITFhiAXL– cells. (C) Quantification of single- and double-labeled cells for ZEB1 and EdU (n = 3). (D) Quantification of cells expressing AXL, MITFhi, or both (n = 3). (E) Experimental design and sorting approach for functional analysis of MITFhiAXL+ cells. (F) Quantification of EdU+ proliferative cells expressing MITFhiAXL and expressing MITFloAXL by FACS (n = 3). (G, I, K) Enrichment for the AXL+EdU+ subpopulation in BMP7/TGF-β2/siSMAD7–treated cells as shown by FACS (n = 3) (yellow quadrant [Q2)] AXL+EdU+, gray quadrant [Q4] AXL+EdU–). (H, J, L) Histograms show the MFI values for MITF expression in AXL+EdU+ versus AXL+EdU– cells. (M) FACS analysis of cells stained with an AXL antibody following TGF-β2 and BMP7 treatment with or without siRNA-mediated SMAD7 knockdown. (N) Matrigel assays of sorted AXL+ and AXL– cells. (O) MITF MFI of AXL+ and AXL– cells following combinatorial treatment. Mean of 3 (C, D, F) or 2 independent experiments (N and O) ± SD. ***P < 0.001, unpaired Student’s t test (C, D, F, N, O). Scale bars: 50 μm.