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 5

Loss of Smad7 boosts proinvasive TGF-β/NODAL signaling in the presence of proproliferative BMP7.

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Loss of Smad7 boosts proinvasive TGF-β/NODAL signaling in the presence o...
(A) Growth curves of M010817 cells treated either with TGF-β2 (10 ng/μl), BMP7 (100 ng/μl), or both, with or without siRNA-mediated SMAD7 knockdown (siS7) (n = 1 independent experiment and 3 technical replicates). (B) Bar plots show the number of cells at day 3 after various treatments (n = 1 independent experiment and 3 technical replicates). (C) Percentage of S phase cells measured 3 days after ligand treatment based on propidium iodide and EdU staining. Treatment with BMP7 resulted in escape of cells from the TGF-β2–mediated cell cycle arrest and increased proliferation. (n = 3 independent experiments). (D) Quantification of substrate adherence capacity of M010817 cells upon different treatments. TGF-β2–mediated cell detachment was suppressed by BMP7. The percentage of BMP7/TGF-β2/siSMAD7–treated cells in suspension increased as compared with BMP7/TGF-β2–treated cells (n = 2 independent experiments). (E) Matrigel-based invasion assays of siControl and siSMAD7-depleted M010817 cells with combinatorial treatments. After invading cells had been counted in 5 random microscopic fields in each assay, the results were normalized and are presented as an invasion index (n = 2 independent experiments). (F) Photographs of cells invading the membrane, stained with Hoechst. (G) Heatmap shows qRT-PCR analysis for selected EMT genes under ligand treatments (n = 2 independent experiments and 3 technical replicates). The gene expression levels are represented as fold-change values transformed to log2 format compared with control. Color represents expression after normalization to nontreated control cells. Data are represented as a mean of 3 (C), 2 (D and E), or 1 (A and B) independent experiments ± SD. **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Tukey’s multiple comparison test.