In order to define gene expression patterns during the course of melanoma development and progression, we evaluated a series of primary and metastatic melanoma cells derived from lesions of discrete phases of melanoma progression as well as pools of primary human melanocytes. Tumor cell lines derived from three radial growth phase (RGP) melanomas (WM35, SBC12, and WM1552C), four vertical growth phase (VGP) melanomas (WM902B, WM278, WM983A, and WM793), and three metastatic melanomas (WM852, WM983B, 1205Lu) were evaluated. These cell lines possess a notable ability to recapitulate the clinical stages of disease from which they were derived 
and have been characterized with respect to tumorigenicity and metastasis 
; cellular growth characteristics including life span, growth factor dependency, anchorage-independent growth 
; and pigmentation and morphology 
. In addition, cytogenetic analyses in these cell lines including non-random abnormalities such as deletions, translocations, and amplifications have been well-documented and suggest high relevance to the primary tumor of origin (reviewed in 
Global gene expression patterns were obtained using Affymetrix gene chips and comparison of gene expression profiles was performed using hierarchical clustering analysis. This clustering analysis identified two distinct groups of melanoma cell lines based on the similarity of their expression patterns, separating radial growth phase (RGP) and metastatic melanomas (MM) (); however, vertical growth phase (VGP) melanomas failed to form a distinctive cluster (). The first group, which we characterized as “less-aggressive” primary melanomas (designated as Group1), included all three RGP melanomas (WM35, Sbcl2, and WM1552C) and two VGP melanomas (WM902B and WM278). The second group, which we characterized as “more-aggressive” melanomas (designated as Group2), included all three metastatic melanomas (WM853, WM983B, and 1205Lu) and two VGP melanomas (WM983B and WM793). Additional cluster analyses with different linkage matrices produced similar results (data not shown). Of note, only 2 of the 10 cell lines (Sbcl2 and WM853) were found to be wildtype for BRAF kinase and this genotype failed to demonstrate a notable cluster in our hierarchical analyses.
Evaluation of gene expression profiles from melanoma cells lines of varying stages of progression identifies a signature for aggressive melanomas.
In order to identify a cohort of genes differentially expressed between our defined groups of melanomas, the gene expression array dataset was subjected to the microarray data analysis program Significance Analysis of Microarray (SAM) 
. This analysis resulted in 142 differentially expressed probesets with a 3.5 % false discovery rate (). In total, 89 probe sets representing 65 well-defined genes were found to be upregulated in more-aggressive (group 2) versus less-aggressive (group 1) melanomas and 53 probe sets representing 37 well-defined genes were found to be downregulated (Table S1
). When more stringent criteria were applied (well-characterized genes which are differentially expressed greater than 4-fold) to this signature, we identified 21 upregulated and 5 downregulated genes in the more-aggressive melanoma cells (). Of note, the set of genes highly expressed in more-aggressive melanomas includes many novel genes with reported functional roles in cell cycle regulation and proliferation such as ZWINT, CDCA2, NCAPH, NCAPG, NCAPG2, PBK, NUSAP1, BIRC5, ESCO2, HELLS, MELK, and CDKN2C 
as well as genes that are involved DNA replication and repair processes including GINS1, GINS4, RAD54L, TYMS
, and DHFR 
(). Differential expression of these genes was validated by quantitative real-time RT-PCR ().
Genes with altered expression in aggressive melanoma cells are involved in cell cycle control, cell proliferation, DNA repair and replicationa.
Since we were interested in defining melanoma progression signatures, and all melanomas are initiated in primary human melanocytes, we evaluated our expression profiling data in the context of cultured neonatal primary human melanocytes (). Surprisingly, when two pools of short-term cultured primary human melanocytes (HPM1 and HPM2) were included in the previously employed hierarchical clustering protocol, the global gene expression pattern of the normal melanocytes was found to be more similar to that of the more-aggressive melanomas (Group 2) than the less-aggressive melanomas (Group 1) (). Since early cultures of primary human melanocytes derived from neonatal foreskins divide rapidly yet possess a normal differentiation program, we reasoned that the similarities of these cells to more aggressive tumors was likely due to their proliferative potential. In order to test this hypothesis we compared gene expression profiles of more-aggressive melanoma cells (Group 2) to those of short-term cultured primary human melanocytes. Expression profiles were subjected to SAM analysis which identified a cohort of differentially expressed genes with a 0.85% false discovery rate. Remarkably, all differentially expressed genes were found to be down-regulated genes in aggressive melanoma cells versus primary human melanocytes suggesting that loss of specific gene signatures may be a key event in the development of advanced melanomas (). Further assessment of all melanoma expression profiles using TreeView revealed that the majority of these melanoma-associated genes are also down-regulated in the less-aggressive primary melanomas (Group1) (). This gene signature is comprised of critical mediators of cellular adhesion and melanocyte development and differentiation and includes: CDH3, CDH1, c-KIT, PAX3, CITED1/MSG-1, TYR, MELANA, MC1R,
and OCA2 
(). While such a loss of cellular adhesion by E-cadherin and P-cadherin has been extensively documented in melanoma (reviewed in 
), and the loss of differentiation-associated genes is not wholly surprising, this signature does notably identify specific defects in the intrinsic melanocyte development program that may contribute to melanoma development. In addition, genes with tumor suppressor and metastasis suppressor functions (DPP4, SYK) are included in this melanoma signature 
. Significant down-regulation of these genes in the aggressive metastatic melanoma cells was validated using semi-quantitative duplex RT-PCR (). Furthermore, this differentially expressed “melanoma signature” contains many genes whose functional roles in melanoma progression have not been well characterized and may provide novel insights into the early development of melanoma from primary melanocytes.
Evaluation of differential gene expression from aggressive melanomas (Group 2) vs. primary human melanocytes identifies a signature characterized by loss of differentiation-associated genes.
Differential expression of genes that are downregulated in aggressive melanoma cells (Group2) compared to primary human melanocytes. Genes with greater than five-fold differential expression are shown a.
A current melanoma progression model suggests the sequential evolution of primary in situ tumors and minimally invasive tumors which are termed “radial growth phase”, followed by a subsequent conversion to a more aggressive “vertical growth phase”, in which tumor cells are programmed to cross the epidermal basement membrane and invade vertically into the dermis. It has been postulated that the VGP is the critical stage in which a tumor gains metastatic capacity. We therefore compared the gene expression profiles of RGP and VGP melanomas using a uniquely designed data reduction algorithm in order to identify genes that are likely to be relevant to this critical invasive phenotype (). Our melanoma invasion-specific signature is notably characterized by the inclusion of several genes involved in chemotaxis and the inflammatory response (CXCL1, CXCL2, IL8, and IL6), cell adhesion (HNT, ITGA4, ITGB8, CSPG2, ZP4, and FLRT3), and extracellular matrix organization (MMP1, COL4A1, COL4A2, and COL5A2) (). These genes and their relative expression profiles are depicted in . These cellular processes have previously been implicated in tumor progression for a wide variety of malignancies including melanoma and are felt to be essential components of tumor invasion and metastasis. In addition, many of these invasion-specific signature genes are also upregulated in metastatic melanomas (). The differential expressions were validated on the selected genes using semi-quantitative duplex-PCR analysis ().
Identification of an invasion-specific gene signature for melanoma.
Melanoma invasion-specific signature genes that are upregulated in VGP compared to RGP melanoma cells a.
Since the melanoma invasion-specific signature was associated with common functions of matrix invasion/inflammation/cell migration we sought to determine whether a common upstream regulatory pathway might link these signature genes. The eight most highly up-regulated genes from our melanoma invasion-specific signature were selected for further evaluation and gene promoter sequences were analyzed to identify transcription factor binding cis
elements. This promoter analysis yielded a profile of transcription factors with common sequence elements in the signature genes. The most ubiquitous cis
elements among the gene promoters evaluated were E12, E47, GCN4, GR, HES-1, IL-6, MEF-2, NF-κB, N-Oct-3, PU.1, RAR-alpha1, SRF, and the basal gene transcriptional complex components of TFIID, TBP, and TBF1. We notably identified the NF-κB binding sequence in 7 out of 8 of the most upregulated invasion-specific signature genes (). We were particularly interested in the NF-κB pathway as a mediator of melanoma invasion since our most highly upregulated invasion-specific genes, CXCL-1
, have previously been reported to be activated by NF-κB and had previously been implicated in melanoma progression (reviewed in 
). Given the consequences of NF-κB activation in a cell, NF-κB function is highly regulated by specific cytosolic inhibitory activities which prevent inappropriate NF-κB activation and shuttling to the nucleus. Thus, only nuclear NF-κB is considered to be functionally activated. In order to evaluate NF-κB function in our tumor cell lines, we used NF-κB cellular localization as a surrogate marker for NF-κB activity. We find that invasive (VGP) melanomas posses both cytosolic (inactive) and nuclear (active) localization of NF-κB, while non-invasive (RGP) melanomas possess NF-κB confined to the cytosolic compartment suggesting specific activation of NF-κB during melanoma progression ().