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 1

Conditional Smad4 deletion in a genetic mouse model of melanoma prevents tumorigenesis.

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Conditional Smad4 deletion in a genetic mouse model of melanoma prevents...
(A) Schematic of the melanoma mouse model harboring Tyr::NrasQ61K, Ink4a−/−, Tyr::CreERT2, floxed Smad4, and R26R Cre-reporter alleles. (B) Experimental strategy used to analyze early loss of Smad4 before appearance of visible melanomas. Control mice either lacked the Tyr::CreERT2 allele or were not treated with TM. (C) Representative H&E staining of trunk skin sections of control and cKO mice at day of sacrifice showing ectopic dermal hyperpigmentation. (D) Quantification of the percentage of hair follicles exhibiting ectopic pigmentation in control (nontreated with TM) and cKO mice (n = 350 hair follicles quantified from 6 different mice). (E) Immunofluorescent staining for Pax3 (control, nontreated with TM) and Pax3+β-Gal+ (cKO) on back skin sections at 6 months to quantify extent of dermal hyperplasia. Open arrowheads indicate Pax3+ cells, white arrowheads Pax3+β-Gal+ cells. (F) Quantification of the percentage of dermal Pax3+ cells between hair follicles (n = 300 hair follicles from 6 different cKO and control mice). (G) Macroscopic pictures of a control and a Smad4-cKO littermate 6 months after Smad4 ablation. (H) Quantification of recombined primary tumor numbers per control (n = 12) and cKO (n = 11) mice at the day of sacrifice. (I) Quantification of lung macrometastases counts at day of sacrifice using macroscopic pictures and staining on sections (n = 12). (J and K) Kaplan-Meier curves displaying overall and melanoma-specific survival, respectively, of control (n = 12) and Smad4-cKO (n = 17) animals. Vertical bars (K) indicate mice censored because of melanoma-unrelated tumors developing due to constitutive loss of Ink4a. Data are represented as a mean of 3 independent experiments ± SEM (H and I) and ± SD (D and F). ***P < 0.001, unpaired Student’s t test (D, F, H, I), log-rank Mantel-Cox test (J and K). Ctrl, control; HF, hair follicle. Scale bars: 50 μm (E); 500 μm (C).