Notch1 Signaling Promotes Primary Melanoma Progression by Activating Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-Kinase-Akt Pathways and Up-regulating N-Cadherin Expression Zhao-Jun Liu, Min Xiao, Klara Balint, Keiran S.M. Smalley, Patricia Brafford, Ruihua Qiu, Chelsea C. Pinnix, Xueli Li, and Meenhard Herlyn The Wistar Institute, Philadelphia, Pennsylvania Abstract Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival, and differen- tiation. Aberrant Notch activation has been linked to a variety of human neoplasms. Here, we show that Notch1 signaling drives the vertical growth phase (VGP) of primary melanoma toward a more aggressive phenotype. Constitutive activation of Notch1 by ectopic expression of the Notch1 intracellular domain enables VGP primary melanoma cell lines to proliferate in a serum-independent and growth factor– independent manner in vitro and to grow more aggressively with metastatic activity in vivo . Notch1 activation also en- hancestumor cell survival when cultured asthree-dimensional spheroids. Such effects of Notch signaling are mediated by activation of the mitogen-activated protein kinase (MAPK) and Akt pathways. Both pathways are activated in melanoma cells following Notch1 pathway activation. Inhibition of either the MAPK or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway reverses the Notch1 signaling-induced tumor cell growth. Moreover, the growth-promoting effect of Notch1 depends on mastermind-like 1. We further showed that Notch1 activation increases tumor cell adhesion and up-regulates N-cadherin expression. Our data show regulation of MAPK/ PI3K-Akt pathway activities and expression of N-cadherin by the Notch pathway and provide a mechanistic basis for Notch signaling in the promotion of primary melanoma progression. (Cancer Res 2006; 66(8): 4182-90) Introduction Cutaneous melanoma has one of the fastest-rising incidence rates for malignancies in the past several decades. The high mortality rate of melanoma is linked to its resistance to standard therapies, such as chemotherapy and radiation, and its high propensity to metastasize (1). Development and progression of malignant melanoma are the pathologic consequences of environmentally initiated disruptions in the cellular control mechanisms, which are likely governed by specific genetic aberrations. Thus far, aberrations of several cellular signaling pathways, such as B-Raf (2), N-Ras (3), p16 INK4 (4), p53/ Apaf-1 (5), PTEN/Akt (6), cyclin D1/cyclin-dependent kinase 4 (7, 8), Wnt5a (9), and Grm-1 (10), have been suggested in melanocyte transformation and melanoma progression. However, each of these pathways only contributes to the development or progression of malignant melanoma under selected circumstances. The search is continuing for novel critical signaling pathways involved in this disease. It is hoped that a greater understanding of the molecular pathways involved in melanoma cell proliferation and survival will lead to more effective targeted therapies. Notch signaling controls a variety of processes, involving cell fate specification, differentiation, proliferation, and survival (11). The central components of the Notch pathway are evolutionarily conserved. In mammals, the Notch family consists of four transmembrane receptors (Notch1-4) and five ligands (Jagged1, Jagged2, Delta1, Delta3, and Delta4). Binding of ligand to its cognate receptor initiates metalloproteinase-mediated and g- secretase-mediated proteolysis. The Notch1 intracellular (N IC ) domain is cleaved from the plasma membrane and translocates into the nucleus, where it associates with transcription factors RBP-Jn/CSL [CBF1, Su(H), Lag-2] and mastermind-like (MAML) to form a heteromeric complex. This complex mediates the transcription of target genes in the hairy enhancer of split (HES) families and HES-related families of basic helix-loop-helix tran- scription factors, Deltex, and others (12). Notch signaling was initially known to be critical for organism development and tissue homeostasis. Over time, increasing evidence suggests its involvement in tumorigenesis, as deregulated Notch signaling is frequently observed in a variety of human cancers. Notch canactaseitheratumorpromoterorasuppressordependingonthe cell type and context. Involvement of Notch in tumorigenesis was originally found in a small subset of T-cell acute lymphoblastic leukemias (T-ALL), in which a chromosomal translocation of the NOTCH1 gene resulted in constitutive activation of Notch signaling (13). A recent study further identified novel types of activating mutations in the NOTCH1 gene in more than half of all T-ALL cases (14). Additionally, aberrant Notch signaling was observed in small cell lung cancer (15), neuroblastoma (16, 17), and cervical (18, 19) and prostate (20) carcinomas. Activated Notch was reported to transform primary Schwann cells (21). An association of Notch signaling with skin cancer has been recently implicated in exper- imental mouse models. Notch signaling induces cell growth arrest and differentiation in keratinocytes (22, 23). Deletion of Notch1 in murine epidermis causes epidermal hyperplasia and skin carcinoma and facilitates chemical-induced basal and squamous carcinomas, which suggests a role for Notch1 as a tumor suppressor (24). Human epidermis is composed mainly of three types of cells: keratinocytes, melanocytes, and Langerhans cells. Whereas basal cell and squamous cell carcinomas are derived from keratinocytes, melanomas originate from pigment-producing melanocytes. The melanocyte in human skin is normally embedded in the basal layer of keratinocytes and anchored to the basement membrane of the Note: K. Balint is a PhD student of the Department of Dermatology of the Medical and Health Science Center of the University of Debrecen, Hungary. Requests for reprints: Meenhard Herlyn, Molecular and Cellular Oncogenesis Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215- 898-3950; Fax: 215-898-0980; E-mail: herlynm@wistar.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3589 Cancer Res 2006; 66: (8). April 15, 2006 4182 www.aacrjournals.org Research Article Research. on February 13, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from