Notch4 Signaling Induces a Mesenchymal-Epithelial-like Transition in Melanoma Cells to Suppress Malignant Behaviors

Abstract

The effects of Notch signaling are context-dependent and both oncogenic and tumor-suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the reexpression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting. Cancer Res; 76(7); 1690-7. ©2016 AACR.

Figure 1

Phenotypic switch following N4ICD overexpression in human melanoma. A, morphology of N4ICD-transduced cells. Expression of N4ICD was confirmed by immunoblotting. β-Actin was used as a loading control. B, chemokinesis (left) and migration (right) of N4ICD-transduced cells. C, invasion of N4ICD-transduced cells. D, tumor volume induced by N4ICD or empty vector–transduced 451Lu. Expression of N4ICD was confirmed by immunoblotting. β-Actin was used as a loading control. n.s., not significant; *, P ≤ 0.05; ***, P ≤ 0.01; ***, P ≤ 0.001.

Figure 2

Notch4 regulates EMT markers. A, mRNA levels of WM9 transduced with pLNCX-empty vector or pLNCX-N4ICD were analyzed using a qPCR array. Significant results are relative to empty vector–transduced cells and plotted as fold increase. B, immunoblots (left) and mRNA levels (right) of WM9 and WM164 transduced with pLNCX-empty vector or pLNCX-N4ICD C, E-cadherin and Notch4 staining of primary (n = 30) and metastatic (n = 30) melanoma samples plotted against each other (top). The size of the square indicates the number of samples, showing the respective expression levels. IHC of a primary melanoma and a subcutaneous metastasis for E-cadherin and Notch4 and unspecific secondary IgG antibody (×40; bottom). D, immunoblots (left) and mRNA levels (right) of WM9 and WM164 transfected with siRNA targeting CSL. The error bars represent SDs from the mean. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 3

Notch effector genes Hey1/2 suppress Snail2 and Twist1 expression. A, immunoblots (left) and mRNA levels (right) of WM9 and WM164 transduced with pLNCX-empty vector or pLNCX-N4ICD. B, immunoblots (top) and mRNA (bottom) of WM9 and WM164 transfected with siRNA-targeting Hey1 (left) or plasmids encoding Hey1 cDNA (right). C, immunoblots (top) and mRNA levels (bottom) of WM9 and WM164 transfected with siRNA targeting Hey2 (left) or plasmids encoding Hey2 cDNA (right). D, immunoblots of WM9 and WM164 transduced with pLNCX-empty vector, pLNCX-N4ICD, or pLNCX-N4ICD and siRNA targeting CSL. Error bars, SDs from the mean. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 4

Notch4-mediated activation of Hey1/2 suppress Snail2 and Twist1. A, immunoblots of WM9 and WM164 cotransfected with siRNA targeting Notch4 and plasmids encoding Hey1 or Hey2 cDNA. B, EMSA of WM164 incubated with Cy3-labeled promoter fragments and probed for Hey1 or Hey2. C, luciferase assay of WM164 cells cotransfected with the indicated plasmids. Error bars, SDs from the mean. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. D, proposed model of Notch4 signal transduction leading to a MET-like transition.