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.
View: Text | PDF
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

×

Figure 2

Loss of Smad4 leads to decreased proliferation in established skin tumors.

Options: View larger image (or click on image) Download as PowerPoint
Loss of Smad4 leads to decreased proliferation in established skin tumor...
(A) Experimental strategy used to analyze the deletion of Smad4 in already established skin melanomas. Mice carrying Tyr::NrasQ61K Ink4a–/– Tyr::CreERT2 Smad4fl/WT R26R::LacZ or Tyr::NrasQ61K Ink4–/– Smad4fl/fl R26R::LacZ were used as controls. (B) Recombination efficiency was calculated by counting percentages of β-gal+Pax3+ cells on primary tumor sections (n = 6, cKO and control; Tyr::NrasQ61K Ink4a–/– Tyr::CreERT2 Smad4fl/WT R26R::LacZ treated with TM). (C) Representative immunofluorescent costaining of Ki67 with β-gal on skin melanoma sections 72 hours after conditional deletion of Smad4. White arrowheads indicate Ki67+β-Gal+ cells; open arrowheads indicate Ki67–β-Gal+ cells. (D) Quantification of Ki67+β-Gal+ cells in control (Tyr::NrasQ61K Ink4a–/– Tyr::CreERT2 Smad4fl/WT treated with TM) and cKO mice (n = 4). (E) Quantification of apoptotic cells by IHC for activated caspase-3 on skin sections of control and cKO mice (n = 4). (F) Quantitative reverse transcription PCR (RT–qPCR) for expression of multiple G1 cell cycle inhibitors in RIM3 (n = 3) and RIM4 (n = 2) cell lines after siRNA treatment. (G) Western blot performed on nuclear extracts from RIM3 for cell cycle regulator Cdkn1a (p21Waf1), Cdkn2c (p18Ink4c), and Cdkn2a (p16Ink4a) protein expression. (H) Percentage of S phase cells upon SMAD4 knockdown in various human melanoma cell lines. (I) RT-qPCR analysis of the same cell cycle regulators in M010817 human melanoma cell line. Data are represented as the mean ± SEM (D) and ± SD (B, E, F, H, I). **P < 0.01; ***P < 0.001. RT-qPCR results are shown as mean ± SD of 3 biological replicates. For Western blots, representative examples are shown. P values calculated with unpaired Student’s t test (B, D, E, F, H, I). Mel, Melan-A mouse melanocyte line. Scale bar: 50 μm.