Notch1 Signaling Promotes Primary Melanoma Progression by Activating Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-Kinase-Akt Pathways and Up-regulating N-Cadherin Expression

Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival, and differentiation. 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 enhances tumor cell survival when cultured as three-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)

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 γ-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-Jκ/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 transcription 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 can act as either a tumor promoter or a suppressor depending on the 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 experimental 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 epidermis. Using their dendrites, they interact with keratinocytes to distribute the pigment melanin. Melanoma development and progression is a stepwise process (25): (a) common acquired and congenital nevi with structurally normal melanocytes, (b) dysplastic nevi with structural and architectural atypia, (c) nontumorigenic primary melanomas without metastatic competence [radial growth phase (RGP)], (d) tumorigenic primary melanomas with competence for metastasis [vertical growth phase (VGP)], and (e) metastatic melanoma.

A recent study based on a high-throughput screening approach highlighted the activation of the Notch pathway as a novel mechanism of melanocytic transformation (26). We have further shown that the Notch1 pathway is activated in human melanoma, and activation of Notch1 signaling is required for β-catenin-mediated progression of primary melanoma (27). In this study, we provide extensive evidence and associated mechanisms to reveal that the Notch1 signaling exerts its oncogenic role by influencing melanoma cell proliferation, survival, and adhesion. Our data define a novel role for Notch signaling in primary melanoma progression and implicate the Notch pathway as a potential therapeutic target.

Reagents. LY294002, PD98059, and U0126 were purchased from Cell Signaling Technology (Beverly, MA), Calbiochem (San Diego, CA), and Promega (Madison, WI), respectively. SDS-polyacrylamide gels were obtained from Invitrogen (Carlsbad, CA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation kits were from American Type Culture Collection (Manassas, VA). All other chemicals and solutions were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

Cell culture. Human melanoma cell lines SBcl2, WM35 (RGP), WM278, WM3248 (VGP), 1205Lu, and WM164 (metastatic) derived from different progression stages were isolated as described (28). Melanoma cells were cultured in complete tumor medium W489, a 4:1 mixture of MCDB153 and L15 supplemented with 2% fetal bovine serum (FBS; Hyclone, Logan, UT), 5 μg/mL insulin, and 1.6 mmol/L CaCl₂. We cultured 293T cells, Phoenix cells (Ampho), and NIH/3T3 cells in DMEM (Invitrogen) supplemented with 10% FBS. All cells were incubated at 37°C in 98% humidified air containing 5% CO₂.

Cell growth, colony formation, and adhesion assays. Cell proliferation was measured by MTT assays according to the manufacturer's protocol. Colony formation in soft agar was carried out as described (29). Melanoma cell adherence to uncoated plates was done by seeding 1 × 10⁵ to an uncoated 24-well cell culture plate in basic W489 medium and culturing at 37°C for 30 minutes. After washing out unattached cells with PBS, attached cells were counted and photographed. Homotypic melanoma cell adhesion assays were carried out on semisolid agar. We seeded 5,000 cells to each well of a 96-well plate coated in 1.5% agar (Difco, Sparks, MD) and cultured in complete W489 medium. Plates were incubated for 48 hours, and cells stayed in nonadherent conditions on agar to form spheroids.

Three-dimensional spheroid growth/survival assays. Melanoma spheroids were prepared using the liquid overlay method as described (30). Briefly, 200 μL melanoma cells (25,000/mL) were added to a 96-well plate coated in 1.5% agar. Plates were incubated for 48 hours, at which time cells had organized into three-dimensional spheroids on agar. Spheroids were harvested and implanted into a gel of bovine collagen type I containing essential DMEM and l-glutamine. Basic W489 medium was overlain on top of the solidified collagen, and the spheroids were incubated for a further 72 or 96 hours. Spheroids were then washed twice in PBS and stained using a Live/Dead Viability kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Cells that lost membrane integrity and were no longer viable stained red. After this time, pictures of spheroids were taken using a Nikon 300 inverted microscope (Nikon, Melville, NY).

Immunoblotting and immunohistochemistry. Western blotting was done as described (31). Membranes were probed with antibodies to Notch1 (a rabbit polyclonal antiserum directly against residues 1,759-2,095) (32), phosphorylated mitogen-activated protein kinase (MAPK), p44/42 MAPK, Akt, phosphorylated Akt (Ser⁴⁷³/Thr³⁰⁸), Myc tag (9B11, New England Biolabs, Beverly, MA), phosphatidylinositol 3-kinase (PI3K) p85 subunit, focal adhesion kinase (FAK), phosphorylated FAK (pY397, BD Biosciences, San Diego, CA), mouse anti-E-cadherin antibodies, N-cadherin antibodies (SHE78-7 and 33-3900, Zymed Laboratories, San Francisco, CA), or β-actin (AC-15). This was followed by probing with horseradish peroxidase–conjugated second antibody (Jackson Immunoresearch, West Grove, PA) and subjected to enhanced chemiluminescence (Amersham, Piscataway, NJ). Membranes were stripped and reblotted as required in the individual experiment. For immunohistochemistry, 5-μm paraffin sections were processed as described (27), then incubated with anti–green fluorescent protein (GFP) antibody (ab6556, Abcam, Cambridge, MA) for 1 hour at room temperature, and then incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Immunoreactivity was detected using the avidin-biotin complex method Elite kit (Vector Laboratories). The nuclei were counterstained with hematoxylin. Negative controls for all antibodies were made by replacing the primary antibody with nonimmunogen IgG.

Recombinant lentiviruses, retroviruses, and adenoviruses. For gene transfer, we constructed various viral vectors. Generation of GFP/lentiviral vector, N^IC-GFP/lentiviral vector, recombinant retroviruses encoding a dominant-negative mutant of MAML1 (DN-MAML1)-Myc/pBabe, and pBabe (control) was described previously (27).

Production of pseudotyped lentivirus was achieved by cotransfecting 293T cells with three plasmids as described (33). Recombinant retroviruses were generated by transfection of vector into Phoenix (Ampho), helper-free retrovirus producer lines (obtained from Dr. G.P. Nalon, Stanford University, Stanford, CA) with calcium phosphate method. Both lentiviruses and retroviruses collected 48 hours after transfection displayed titers of ∼10⁷ transducing units/mL in NIH/3T3 cells.

Recombinant adenovirus encoding dominant-negative mutant of Akt (DN-Akt/Ad; ref. 34) was obtained from Dr. J.Q. Cheng (University of South Florida, Tampa, FL). Dominant-negative mutant of p85 by adenoviral vector (DN-Δp85/Ad) was provided by Dr. W. Ogawa (Kobe University, Kobe, Japan) and described elsewhere (35). LacZ/Ad was purchased from Gene Therapy Program (University of Pennsylvania, Philadelphia, PA).

Viral infection of targeting cells. To infect target cells by lentiviruses or retroviruses, melanoma cells were exposed overnight to virus with 5 multiplicities of infection in the presence of 4 μg/mL Polybrene. Cells were then washed, cultured with regular complete medium for 2 additional days, and analyzed for protein expression by Western blot or pooled for subsequent analysis as indicated in individual experiments. To infect cells by adenoviruses, we exposed cells to virus [100 plaque-forming units (pfu)/cell] in serum-free medium for 4 hours. Cells were washed, cultured with regular complete medium for 2 additional days, and analyzed for protein expression by immunoblot or pooled for subsequent analysis as indicated in individual experiments.

Animal experiments. Severe combined immunodeficient (SCID) CB-17 mice were purchased from Charles River Laboratories (Wilmington, MA). Animal experiments were approved by the Wistar Institute Animal Care and Use Committee. S.c. injection: WM278 melanoma cells transfected with GFP or N^IC-GFP lentiviruses were injected into SCID mice (3 × 10⁶ per mouse). Tumors were harvested after 18 weeks, weighed, photographed, and then either fixed in formalin and embedded in paraffin or frozen for future use. Lung colony formation assay: WM278 melanoma cells transfected with either GFP/lentiviral vector or N^IC-GFP/lentiviral vector were injected (2 × 10⁵ per mouse) into mice via the tail vein. The experiment was terminated at week 12, and the lung samples were harvested and subjected to immunohistochemistry analysis.

Activated Notch1 selectively enables VGP melanoma cells to grow without mitogen. To test the effect of aberrant Notch signaling on melanoma progression, we transduced N^IC into malignant melanoma cell lines that were derived from melanoma lesions in different stages of tumor progression. Two cell lines from each stage were examined. Cells were either left untransduced or transduced with GFP/lentiviral vector or N^IC-GFP/lentiviral vector. Transductants (>95%) expressed GFP as observed by fluorescence microscopy (data not shown). Efficacy was comparable between the GFP/lentiviral vector and the N^IC-GFP/lentiviral vector in infecting melanoma cells (Fig. 1A). Expression of exogenous N^IC in transductants was also comparable as confirmed by Western blot assay (Fig. 1B). Many aggressive tumor cells acquire the ability to proliferate in a growth factor–independent manner. We therefore tested growth properties of different transductants in serum-free and growth factor–free conditions. Transductants were cultured in basic W489 medium: a mixture of MCDB153 and L15 at a ratio of 4:1. Of the melanoma cells derived from different stages of tumor progression, parental metastatic melanoma cells (WM164 and 1205Lu) were initially growth factor independent, and the introduction of N^IC did not further enhance their proliferation rates. Whereas N^IC-GFP-transduced RGP melanoma cells (WM35 and SBcl2) still required serum and growth factor for proliferation, as did their untransduced parental cells and GFP-transduced control cells, N^IC-GFP-transduced VGP melanoma cells (WM278 and WM3248) were able to proliferate in basic W489 medium compared with the GFP-transduced control cells and untransduced parental cells. Figure 1C shows cell growth/apoptosis curves of SBcl2, WM278, and 1205Lu cells in basic W489 medium. Very similar results were also observed in WM35, WM3248, and WM164 cells (data not shown). These data suggest that activated Notch1 specifically enhances VGP melanoma cell growth. Interestingly, in the presence of serum and growth factors, activation of the Notch1 pathway was able to accelerate both VGP and RGP melanoma cell proliferation (27), whereas, in the absence of serum and growth factors, Notch1 specifically rescued VGP cell growth. The differential effects of Notch1 on VGP and RGP cell growth in serum-free and growth factor–free medium suggest an inefficacy of another signaling pathway(s) that cooperates with Notch1 in driving melanoma cell cycle progression. Activated Notch1 also induced a clear cell morphologic change in VGP melanoma cells (WM278) but not in RGP (SBcl2) or metastatic (1205Lu) tumor cells (Fig. 1D). Similar results were observed in WM35 (RGP), WM3248 (VGP), and WM164 (metastatic; data not shown). Morphologic changes in VGP melanoma cells might reflect an increased cell adhesion capability and a more aggressive phenotype. Taken together, our results suggest a stage-specific effect of activated Notch1 on VGP melanoma cell growth in the absence of mitogenic signals.

Activated Notch1 is oncogenic in promoting VGP melanoma progression. To further investigate the function of activated Notch1 signaling in promoting VGP melanoma progression, we examined the in vitro and in vivo tumor growth of stably N^IC-GFP-transduced WM278 cells versus GFP-transduced cells. We examined tumor colony formation of transductants in soft agar. WM278-GFP cells, such as untransduced parental cells, did not readily form colonies in soft agar. In contrast, we observed a significantly improved capacity for colony formation for WM278-N^IC-GFP cells (Fig. 2A). For the in vivo study, we s.c. injected WM278-N^IC-GFP and WM278-GFP cells into SCID mice (n = 10 for each group). Tumor samples were harvested after 18 weeks. A remarkable increase in melanoma growth was achieved in WM278-N^IC-GFP cells versus WM278-GFP (Fig. 2B) cells. Both in vitro and in vivo data implied an oncogenic effect of aberrant Notch1 signaling in VGP melanoma. Similar results were obtained with WM3248 in vitro and in vivo (data not shown). Whether activated Notch1 signaling promotes the progression of VGP melanoma was determined by lung colony formation in an experimental metastasis model. We i.v. injected WM278-N^IC-GFP and WM278-GFP cells into SCID mice (n = 10 for each group). After 12 weeks, mice were sacrificed and lung samples were collected. Histologic examination with anti-GFP antibodies and anti-S100 (a melanocytic-specific maker; data not shown) showed that some tumor colonies formed in the lungs of WM3248-N^IC-GFP cell-injected mice (Fig. 2C), but no detectable tumor colonies formed in WM3248-GFP cell-injected mice. Tumor colonies were detectable in >50% of mice injected with WM278-N^IC-GFP cells, whereas all mice injected with WM278-GFP cells were negative for tumors. Similarly, a significantly increased metastatic ability was observed in WM3248-N^IC-GFP cells compared with control cells (data not shown). These data indicate that primary melanoma cells acquired an enhanced metastatic ability when the Notch1 pathway was constitutively activated. Taken together, these data implicate aberrant Notch1 signaling in oncogenesis and primary melanoma progression.

Activated Notch1 increases melanoma cell survival/growth and promotes tumor invasion in three-dimensional spheroids. Our observation that activated Notch1 enables VGP melanoma cells to grow in growth factor–independent conditions suggests that Notch1 signaling antagonizes cell apoptotic signals. We thus examined the effect of N^IC on WM278 melanoma cell survival and/or growth in three-dimensional spheroids implanted in collagen I gel. The tumor three-dimensional spheroid is a novel, well-defined experimental system that mimics actual tumor growth conditions more closely than previous models (36). We implanted three-dimensional spheroids formed on agar by WM278-N^IC-GFP and WM278-GFP cells into collagen I gels and cultured them in serum-free and growth factor–free basic W489 medium for 96 hours. WM278-GFP spheroids revealed many apoptotic cells during cell spreading, whereas WM278-N^IC-GFP spheroids had comparatively few cells undergoing apoptosis and actually grew and invaded the collagen matrix (Fig. 3). These data indicate that Notch1 signaling enhances melanoma cell survival and promotes cell proliferation and invasion into surrounding matrix in three-dimensional experimental settings.

Notch1-induced melanoma cell growth is mediated by regulating MAPK and PI3K-Akt pathways. To determine the intracellular signaling pathways that mediate the effect of Notch signaling on growth factor–independent cellular proliferation, we tested the role of activated Notch1 in regulating two growth-prone signal transduction pathways in WM278 cells, MAPK and PI3K-Akt. WM278-N^IC-GFP and WM278-GFP cells were starved for 5 days in basic W489 medium and harvested for subsequent analysis. At day 5, cell viability was ∼100% for each type of transductant. Phosphorylation of both extracellular signal-regulated kinase (ERK1/2) and Akt was up-regulated in N^IC-GFP-transduced WM278 melanoma cells compared with GFP-transduced control cells (Fig. 4A), which indicates an activation of MAPK and Akt pathways by Notch1 signaling in VGP melanoma cells.

To investigate whether changes in the activity of the MAPK pathway account for Notch1-induced cell growth control, we tested the effect of suppressing MAPK activity on cell growth by using specific MAPK inhibitor, PD98059. As shown in Fig. 4B, treatment of WM278-N^IC-GFP cells with 10 μmol/L PD98059 substantially inhibited cell proliferation compared with the untreated cells, suggesting that MAPK pathway is responsible for mediating Notch-induced melanoma cell growth. Involvement of the PI3K pathway in mediating the effect of Notch on melanoma cell growth was tested using specific inhibitor, LY294002. Treatment cells with 10 μmol/L LY294002 significantly suppressed cell growth of N^IC-GFP-transduced WM278 cells.

We further examined the effect of inhibition of PI3K pathway activation on Notch1-induced cell growth control by introduction of DN-Δp85 into WM278-N^IC-GFP cells using adenoviral vectors for gene transfer. The LacZ/Ad was used as a control. At a titer of 100 pfu/cell, >80% of cells express exogenous genes without observable cellular toxicity according to our predetermined experiments (data not shown). Expression of mutants of DN-Δp85 was examined by Western blot. Suppression of endogenous Akt phosphorylation by DN-Δp85 was also confirmed (Fig. 4C). Forty-eight hours after adenovirus infection, cells were harvested for subsequent analyses. The DN-Δp85-transduced cells showed decreased phosphorylation of Akt. Cell proliferation assay showed that gained growth rate enhancement of WM278-N^IC-GFP cells was lost if the PI3K-Akt pathway was inhibited (WM278-N^IC-GFP cells/DN-Δp85; Fig. 4C , right). Hence, our data suggest that the PI3K-Akt pathway is responsible for mediating Notch-induced melanoma cell growth.

Introduction of DN-Δp85 or treatment of WM278-GFP control cells with PD98059 also suppressed their growth rates (data not shown). This is not surprising because Akt and MAPK are not Notch pathway-specific targets but are general signaling molecules required for cell proliferation/survival. Our study places MAPK and PI3K-Akt pathways under the control of Notch signaling and suggests certain practically useful “ex-Notch signaling pathway” targets to municipalize the effect of Notch.

We also tested whether the MAPK and Akt activities accounted for Notch1-induced cell survival/growth and invasion in three-dimensional spheroids. We applied the MAPK inhibitor U0126 or introduced DN-Akt/Ad to WM278-N^IC-GFP cells to suppress MAPK and Akt activities. Expression of exogenous DN-Akt was confirmed by immunoblot (Fig. 4D,, left). Suppression of both Akt and MAPK activities inhibited cell aggregation (Fig. 4D,, right, top), suggesting that gained adhesion capability depends on these two pathways. Treatment of spheroids implanted in collagen I gels with 10 μmol/L U0126 for 48 hours dramatically inhibited cell proliferation and invasion (Fig. 4D,, right, middle) and resulted in cell apoptosis (Fig. 4D , right, bottom). A weak but clear effect was also observed in spheroids formed by DN-Akt/Ad-infected cells after 48 hours in collagen gels. The weakness in Akt pathway suppression might have been caused by the efficacy of the dominant-negative mutant, which is likely unable to completely block the Akt pathway. Taken together, our results support that both MAPK and Akt pathways are responsible for mediating the effect of Notch1 on melanoma survival and growth.

Role of Notch signaling in cell growth is MAML1 dependent. As one step toward elucidation of the mechanism of regulation of cell growth by the Notch signaling pathway, we tested whether this is a transcription-dependent or transcription-independent process because there is mounting evidence pointing to the existence of a transcription-independent mechanism in mediating multiple effects of Notch signaling (37, 38). We investigated a potential role of MAML1 in controlling melanoma cell growth. For this purpose, WM278-N^IC-GFP cells were transduced with DN-MAML1 to see whether it could block function of Notch. Expression of DN-MAML1-Myc in cells was shown by Western blot (Fig. 5A). As expected, DN-MAML1 altered Notch1-induced cell growth rates (Fig. 5B). To address whether the MAPK and PI3K-Akt pathways mediate effect on cell growth of MAML1, we examined the capacity of DN-MAML1 in alteration of the N^IC-induced activation of MAPK and PI3K-Akt pathways in melanoma cell lines. Phosphorylation of both ERK1/2 and Akt was significantly suppressed in WM278-N^IC-GFP/DN-MAML1 melanoma cells compared with WM278-N^IC-GFP/control cells (Fig. 5C and D). Thus, our data imply that the effect of Notch signaling on controlling cell growth and MAPK and PI3K-Akt pathways is an indirect event that is dependent on Notch/MAML cascade-mediated transcription.

Activated Notch1 enhances melanoma cell adhesion and up-regulates the expression of N-cadherin. Morphologic changes observed in WM278-N^IC-GFP cells suggest an increased adhesive capacity. We therefore examined the adhesion of WM278-N^IC-GFP cells versus WM278-GFP cells. An equal number of cells were seeded on uncoated 24-well cell culture plates and cultured at 37°C for 30 minutes. After washing out unattached cells with PBS, attached cells were counted. As shown in Fig. 6A, there were more WM278-N^IC-GFP cells than WM278-GFP cells attached on the plates, indicating an increased adhesion capability in melanoma cells when Notch1 signaling is activated. Similar results were obtained when seeded cells were cultured in complete W489 medium. We also tested cell homotypic adhesion by forcing cell spheroids to form on semisolid agar. We added 5,000 cells to each well of a 96-well plate coated with 1.5% agar and cultured in complete W489 medium. Plates were incubated for 48 hours, and cells remained in nonadherent conditions on agar to form spheroids. Compared with WM278-GFP cells, WM278-N^IC-GFP cells formed larger and better-organized spheroids (Fig. 6B), which suggests an increased cell-cell adhesion among WM278-N^IC-GFP cells. Increased cell adhesion might generate adhesion signaling to support cell survival and proliferation, two tasks closely related with tumor progression. We further detected FAK activities in WM278 cells. Consistent with the increased cell adhesion capability, phosphorylation of FAK in WM278-N^IC-GFP cells was significantly enhanced compared with that in WM278-GFP control and untransduced parental cells (Fig. 6C). These data show that activated Notch1 enhances melanoma cell adhesion.

Increased cell adhesion suggests the induction or up-regulation of cell surface adhesion molecules in WM278-N^IC-GFP cells. We examined the expression of E-cadherin and N-cadherin on WM278 cells because cadherins are adhesion molecules, and their expression is directly related to tumorigenicity (39). Expression of E-cadherin was undetectable in both WM278-N^IC-GFP and WM278-GFP cells, whereas expression of N-cadherin was significantly up-regulated in WM278-N^IC-GFP cells compared with WM278-GFP control and untransduced parental cells (Fig. 6D). As we have shown previously, N-cadherin is able to mediate melanoma cell adhesion, migration, survival, and growth and may also play a role in accelerating metastasis (40). N-cadherin-mediated cell adhesion can activate the Akt pathway. Thus, our observations not only link Notch signaling to N-cadherin expression but also provide a substantial foundation for explaining the Notch signaling-induced phenotype of primary melanoma cells.

Here, we have shown that the Notch1 signaling plays an important role in melanoma progression by promoting the growth of VGP primary melanoma cells. Expression of truncated, constitutively active Notch1 selectively enables VGP melanoma cell lines to proliferate in a serum-independent and growth factor–independent manner similar to metastatic melanoma cells. Correspondingly, activated Notch1 significantly promotes colony formation on soft agar and xenograft growth in SCID mice. In addition, Notch1 signaling enhances tumor cell adhesion and increases N-cadherin expression. The oncogenic effect of activated Notch1 is at least partially mediated through regulation of the MAPK and PI3K-Akt pathways. Notch signaling-induced FAK activation may also contribute to the aggressive phenotype of VGP melanoma cells.

Promotion of Notch1 of melanoma cell growth in serum-free and growth factor–free conditions is stage specific. It selectively works on VGP primary melanoma cells but has little effect, for example, on either RGP primary or metastatic melanoma. Such specificity of Notch signaling in melanoma agrees with the well-known temporal and spatial properties of Notch signaling during development. However, the mechanism remains obscure. Because expression levels of N^IC are comparable in different transductants, the contrasting effects of Notch1 cannot be explained by gene dose. In primary or metastatic melanoma, the ultimate effect of Notch1 may be determined by other pathways that coincide or collaborate with the Notch pathway. Notch1 activation fails to rescue RGP cells; the inability of these cells to grow in serum-free and growth factor–free medium suggests that the hypothetical pathway provides an element of support that derives from serum and growth factor stimulation. On the other hand, growth of metastatic melanoma cells is initially independent of serum and growth factors, potentially all required oncogenic signals have been triggered, and downstream elements, such as cell cycle machinery, have been maximally activated. In this scenario, further activation would not enhance downstream pathways. Alternatively, as-yet-unknown negative feedback regulators may be activated in metastatic cells that antagonize the effect of Notch signaling.

The current study has established a functional link between the pathways of Notch, MAPK, and PI3K-Akt and provide a possible explanation for the growth factor–independent proliferation induced by Notch1 signaling in VGP melanoma cells. The signaling cascades delivered from both MAPK and PI3K-Akt pathways ultimately regulate cell cycle machinery. Moreover, cellular signaling (e.g., FAK signaling) initiated by cell adhesion molecules also contributes to cell survival and proliferation. With the activation of multiple signaling pathways, cell cycle progression is practically guaranteed even in the absence of serum-induced and growth factor–induced mitotic signals. Complex cross-talk between Notch and the MAPK or PI3K-Akt pathways manifests in different ways. Not only is Notch signaling able to control the MAPK and PI3K-Akt pathways, but MAPK activation also helps induction of Jagged1 expression in endothelial cells and subsequently stimulates the Notch receptor in head and neck squamous cell carcinoma cells (41). In addition, as we have shown previously, PI3K-Akt pathway activation by vascular endothelial growth factor induces Notch1 and Dll4 expression in endothelial cells (42). Thus, depending on the circumstances, cross-talk between Notch and the MAPK or PI3K-Akt pathways determines various cell behaviors.

Notch signaling is transmitted via the Notch/MAML cascade. Although a transcription-independent mechanism may exist, Notch signaling regulates the MAPK and PI3K-Akt pathways post-transcriptionally as evidenced by the fact that DN-MAML1 can antagonize ability of Notch to regulate the MAPK and PI3K-Akt pathway-mediated cellular proliferation. Consistently, we could not detect a direct association between N^IC and MAPK or Akt,¹

which supports the notion that N^IC itself does not regulate the MAPK and PI3K-Akt pathways directly. The Notch/MAML cascade simultaneously controls the MAPK and PI3K-Akt pathways, and these pathways are usually initiated by receptor tyrosine kinase/phosphatase or adaptor-mediated kinase/phosphatase. Therefore, the Notch/MAML cascade likely regulates the MAPK and PI3K-Akt pathways by controlling common kinases/phosphatases or soluble factors that can stimulate these kinases/phosphatases. The upstream regulatory machinery that determines the status of MAPK and PI3K-Akt pathway activities might be cell type specific because Notch signaling can inhibit cell growth in other types of cells, such as keratinocytes (22) and endothelial cells (42). Future studies to identify target molecules of Notch signaling will help to define the mechanism underlying the cross-talk between Notch signaling and the MAPK and PI3KAkt pathways.

In normal skin homeostasis, melanocyte growth is regulated by the surrounding keratinocytes through a variety of paracrine growth factors and cell-cell adhesion molecules (39). During oncogenesis, this keratinocyte control is lost after down-regulation of E-cadherin (43). Loss of E-cadherin is typically accompanied by increased N-cadherin expression. This phenomenon is known as the cadherin switch, and it enables melanoma cells to communicate among themselves and with the surrounding dermal fibroblasts (40). The switch from E-cadherin to N-cadherin most likely provides the early tumor with important motility and survival advantages. Notch1-induced up-regulation of N-cadherin provides us with at least one of the mechanisms underlying increased melanoma cell adhesion, survival, growth, and tumor progression when Notch signaling is activated. Cadherins are part of large protein complexes involved in myriad events: cell adhesion, control of cell morphology, motility, and intracellular signaling (44). Direct roles for cadherin-based adhesion in signaling have been shown by the activation of the small GTPases Rho, Rac, Cdc42 (45), and PI3K-Akt (46) after homotypic cadherin adhesion. Increased expression of N-cadherin may initiate the Akt activation observed in WM278-N^IC-GFP cells, but these events can also be mutually exclusive. Further studies elucidating the detailed downstream signaling mechanisms will help us understand how Notch signaling regulates melanoma adhesion as well as survival, growth, and progression.

Grant support: NIH grants CA47159, CA76674, CA25874, and CA10815 and Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. A.J. Capobinaco for DN-MAML1/pbabe, Dr. W. Ogawa for DN-Δp85/Ad, Dr. J.Q. Cheng for DN-Akt/Ad, Dr. G.P. Nalon for Phoenix cells, and G. Ascione for editing the article.