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.

Precision medicine for human cancers with Notch signaling dysregulation (Review)

“…dLL, delta-like canonical Notch ligand; JAG, jagged canonical Notch ligand; NEcd, Notch extracellular domain; NTMd, Notch transmembrane domain; NIcd, Notch intracellular domain; AdAM10, disintegrin and metalloproteinase domain-containing protein 10; ATM, serine-protein kinase ATM; MAML, mastermind like protein; cSL, cBF1-suppressor of hairless-LAG1; BMI1, BMI1 proto-oncogene polycomb ring finger; CCND1, cyclin d1; HES1, hes family bHLH transcription factor 1; HEY1, hes related family bHLH transcription factor with YRPW motif 1; REST, RE1 silencing transcription factor; TCF7, transcription factor 7; RAc1, Ras-related protein Rac1. snail family transcriptional repressor 2 and twist family bHLH transcription factor 1 repression, and subsequent mesenchymal-to-epithelial transition (85); and iv) HEY1-mediated IL6 downregulation and subsequent depletion of cancer stem cells (86). Because Notch signals drive lateral induction as well as lateral inhibition to fine-tune organ development and homeostasis (17,87,88), bifunctional cellular responses are a common feature of Notch signaling during embryogenesis, adult tissue homeostasis and tumorigenesis.…”

Notch4+ cancer stem‐like cells promote the metastatic and invasive ability of melanoma

“…Multiple groups have reported that amplified Notch signaling contributes to melanoma growth in vitro and in vivo and promotes a more aggressive phenotype, at least in part through the inhibition of E‐cadherin expression . However, other researchers have found that the high expression of Notch4 increases E‐cadherin expression and suppresses malignant behavior of melanoma . The authors reported that Notch4 induces suppression of Snail2 and Twist1 by downstream targets Hey1 and Hey2 and is mediated in a non‐canonical fashion in melanoma cell lines WM9 and WM164.…”

Turn It Down a Notch

“…Moreover, the pleiotropic nature of the pathway means the various Notch receptors can act as tumor suppressors for example in epithelial tumors or as oncogenes in leukemia and a variety of solid cancers (Radtke and Raj, 2003; Miele et al, 2006; Lobry et al, 2014; Alketbi and Attoub, 2015; Habets et al, 2015; Bonyadi Rad et al, 2016). From this vast literature we will focus here on activating mutations in Notch1 which are predominantly located in the extracellular heterodimerization (HD) domain resulting in ligand-independent exposure of the S2 cleavage site (Malecki et al, 2006; Van Tetering et al, 2009), or in the PEST domain, leading to constitutive activation of the pathway through increased NICD stability or in FBXW7, in line with its fundamental role in restricting the signaling strength/duration of the Notch pathway (Oberg et al, 2001; Tetzlaff et al, 2004; O'neil et al, 2007; Thompson et al, 2007; Wang et al, 2012; Bolos et al, 2013).…”