Here, we demonstrate that Notch1 signaling is activated in melanoma cells but not melanocytes and that constitutive Notch1 activation confers transforming properties to primary melanocytes in vitro. Notch receptors 1, 2 and 4 are overexpressed in melanoma cell lines and lesions, particularly when compared against primary melanocytes or normal human skin. Notch and Notch-target genes are upregulated in both melanoma lesions and melanoma cell lines. Ectopic NIC expression induced gross morphological changes, increased growth, adhesion, migration, survival, and resulted in the loss of E-cadherin expression and upregulation of MCAM, two well-characterized events in melanoma development. We identify MCAM as a direct Notch target due to the presence of two high-affinity CSL binding sites present in the MCAM promoter. The NIC oncoprotein conferred anchorage-independent growth, increased survival, and loss of contact inhibition; suppression of Notch signaling decreased the growth of melanoma cell lines while primary melanocytes were unaffected. Taken together, these data suggest that deregulation of Notch signaling plays a specific role in promoting a transformed phenotype in human melanocytes and define the importance of Notch signaling in human melanoma.
Our microarray data describing Notch pathway activity is underscored by a recent report by Hoek et al.
that revealed upregulation of Notch2 and Hey1 in a separate, but distinct panel malignant melanoma cell lines, suggesting a role for Notch activation in the transformation of melanocytes (22
). Previous immunohistochemical studies on early phase melanoma lesions have demonstrated overexpression of full-length Notch1 protein in melanoma tissue compared against benign human nevi (21
) and normal human skin (8
). In our current study, we examined active Notch1 levels and found overexpression of this protein by immunohistochemical analysis of paraffin-embedded melanoma lesions and western blotting of melanoma cell lines. Furthermore, suppression of Notch signaling via a dominant negative Mastermind-like construct or γ-secretase inhibition did not affect melanocyte growth but inhibited melanoma proliferation in vitro
as well as melanoma tumorigenicity in SCID mice (21
). Our study focused on Notch1 because of its overexpression in melanomas; however, Notch2 and Notch4 transcripts were also increased in melanoma tissues and cell lines. Therefore, it is likely that other Notch receptors play a role in mediating the oncogenic effect of Notch1 signaling activation. Further studies will be useful in determining the contribution of the individual Notch receptors to melanocyte transformation and melanoma development.
In light of studies highlighting novel activating mutations in T-ALL (24
), it might be expected that such mutations exist in melanomas as well. However, sequencing of a panel of 17 melanoma lines did not reveal any genetic alterations within the heterodimerization or PEST domains of Notch1 that have previously been shown to harbor activating mutations in 50% of human T-ALLs. In the absence of genetic mutations, another mechanism must exist to account for enhanced Notch signaling in human melanoma. We favor a scenario in which overexpression of Notch receptors in melanoma cells results in robust Notch signaling activation in human melanoma. Indeed, we observed significant upregulation of Notch receptors 1, 2 and 4 at the mRNA level by real-time RT-PCR in melanoma cell lines and fresh melanoma specimens when compared to normal melanocytes. There are likely upstream factors that account for the increased transcriptional activity at the Notch receptor loci. One possibility was Ras, as it has been previously shown that oncogenic Ras activates Notch signaling and the wild-type Notch1 receptor is required to maintain the neoplastic phenotype of Ras-transformed cells (35
). Studies performed in our laboratory, however, do not implicate MAPK signaling in transcriptional regulation of Notch1, as inhibitors of both Raf and MEK were unable to abrogate expression of Notch1 (data not shown). Certainly, unraveling the mechanistic details responsible for Notch1 upregulation in melanoma will be of immense value in the near future.
E-cadherin is the key adhesion molecule expressed by keratinocytes and melanocytes that permits keratinocytes to communicate with and exert regulatory control over melanocytic cellular processes (12
). Loss of E-cadherin expression allows epidermal melanocytes to regulate their growth and adhesion independent of keratinocytes and is a key event in melanoma development (12
). Here, NIC
downregulated E-cadherin expression in melanocytes and also promoted robust upregulation of MCAM, a cell-adhesion molecule whose protein levels highly correlate with aggressive invasive behavior of melanoma cells in vitro
and in vivo
). Our data suggest that MCAM is a direct Notch target based on the identification of 2 high-affinity CSL binding sites within the MCAM promoter. We propose that in early melanoma tumorigenesis, Notch activation results in MCAM expression and may ultimately contribute to melanoma progression. The significance of these data is underscored because loss of E-cadherin and upregulation of MCAM are consistent with changes in gene expression that occur during the development of malignant melanoma.
There is only one report of active Notch1 protein acting alone to fully transform primary cells (9
). Forced NIC
expression in primary rat Schwann cells resulted in transformation and loss of Schwann cell differentiation markers. Active Notch1 induced transformation in rat kidney embryo (RKE) cells; however, this was in cooperation with adenoviral protein E1A. Synergy of activated Notch1 and papillomavirus oncogenes E6 and E7 has also been reported in the transformation of immortalized epithelial cells (37
). While transformation of immortalized melanocytes subsequent to overexpression of a single oncogene has been reported (38
), full transformation of primary melanocytes, as defined by inducing tumorigenicity in animal models, generally requires disruption of several pathways including Rb and p53 (40
). Oncogenic Ras is capable of promoting growth in soft agar as well as tumor formation in SCID mice in primary melanocytes, but only in the presence of the Simian Virus 40 early region (SV40ER), which encodes the viral large T (LT) and small T (st) oncoproteins, and the catalytic subunit of the telomerase holoenzyme (hTERT) (40
). Thus, although in our primary melanocyte cell lines NIC
overexpression alone was capable of inducing a transformed phenotype in vitro
, it is not alarming that NIC
-infected cells failed to form tumors in NOD-SCID mice; therefore, Notch1 overexpression alone is not sufficient for full neoplastic transformation due to a lack of in vivo
growth. However, our findings are nonetheless of significant importance in the consideration of signaling pathways that are deregulated and cooperate in the process of melanocyte transformation and melanoma development.
Our current studies strongly suggest that constitutive Notch signaling is associated with melanocyte transformation and melanoma tumorigenesis. Of particular significance, is the ability of a γ-secretase inhibitor to selectively inhibit the growth of melanoma cell lines. These findings are consistent with a recent report identifying a γ-secretase inhibitor that induced effective apoptosis in human melanoma cells while sparing melanocytes (27
). Multiple γ-secretase inhibitors have been developed and are presently in trials for use in the treatment of Alzheimer's patients (42
). Based on the recent identification of activating Notch mutations in roughly 50% of human T-ALLs (24
), therapies designed to interfere with Notch activation, such as γ-secretase inhibitors, will undoubtedly be explored as a treatment option. Further extending the potential of Notch inhibitors for the treatment of human disease, these experiments suggest that targeting the Notch signaling may be a viable strategy in the therapy of melanoma.