SF1: Phenotypic and functional changes of 451Lu, WM9 and WM164 following N4ICD overexpression SF2: Immunoblots and immunohistochemistry of EMT markers and Notch4 SF3: Details of CSL binding to Snail2 and Twist1 promoter regions SF4: Immunoblots of overexpression and silencing experiments confirming the Notch4-Hey1/2-Snail2/Twist1 axis (SF4) SF5: Immunoblots of pLNCX-N4ICD and siHey1/2 transduced cells and details of Snail2 and Twist1 promoter regions ST1: Plasmids and siRNAs ST2: Antibodies ST3: Primers for qPCR and ChIP ST4: Sequence of EMSA fragments ST5: Sequence of luciferase fragments ST6: Fold regulation of EMT markers determined by PCR-array ST7: Details of immunohistochemical analyses of melanoma samples Supplementary references
Funding
Austrian Science Fund
EU Marie Curie Initial Training Network (ITN); Biomedical engineering for cancer and brain disease diagnosis and therapy development, EngCaBra
PhD program “Molecular Medicine” of the Medical University of Graz
ARTICLE 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.
