BMC Cancer 2008, 8:19 http://www.biomedcentral.com/1471-2407/8/19 Background Melanoma arises from melanocytes, specialized cells in the skin responsible for synthesizing and distributing the pigment melanin. This tumor is one of the most aggres- sive malignancies, marked by elevated capacity to metas- tasize and by high drug resistance [for review see [1-3]]. Melanoma often arises from inherited or simple acquired nevi, which are pigmentary melanocytic lesions that can progress through hyperplastic to dysplastic nevi and cul- minate in some cases in the radial growth phase melanoma (RGP), an early melanoma lesion that is con- fined to the epidermis [for review see [4]]. This lesion usu- ally further progress to a vertical growth phase (VGP) melanoma, in which the cells that were growing only lat- erally in the epidermis become able to invade into the der- mis and acquire metastatic potential [for review see [4]]. The establishment of metastasis is believed to require few additional genetic changes, once cells presenting meta- static phenotype can be readily selected from most VGP melanomas [5]. Cell lines derived from RGP, VGP and metastatic melanoma represent an interesting experimental model for identification and characterization of genes involved in melanoma development, since they sustain in vitro the characteristics representing the original state of the tumor stage from which they are derived [6,7]. RGP cell lines usually mimic early, less aggressive melanoma lesions, since they show low anchorage-independent growth, high growth factor dependency, and are non-tumorigenic or have limited ability to induce tumor in immunodefficient mice [6,8]. VGP lesions usually contain cells that have already acquired metastatic capacity and so they show behavior and expression profiles similar to cells from metastasis [4,9]. However, since the tumor lesions are het- erogeneous, some cells derived from VGP tumors can still sustain a less aggressive phenotype [5,10]. The heteroge- neity of VGP cells was strengthened by the result of a microarray study using melanoma cell lines, in which some VGP cells clustered with metastatic melanoma whereas others did so with RGP cells [11]. In spite of the fact that different lines of evidence support the notion that cancer progresses through discrete pheno- typic stages marked by a stepwise acquisition of oncogenic alterations, recent evidence from high-throughput gene expression studies in cancer [12] lead to an emerging par- adigm that tumor aggressiveness is intrinsically associated to the mechanisms of tumor birth [13]. From this point of view, higher proclivity towards a metastatic phenotype would be inherent to the initial set of genetic alterations that generate a tumor. The tendency of melanoma to gen- erate metastasis may as well be corroborated by the fact that this tumor derives from melanocytes, naturally migratory neural crest descendants, as suggested by Gupta et al. [14]. The notion that a metastatic melanoma may arise from an RGP lesion has been also supported by clin- ical and molecular evidence [15,16], strengthening the importance of determining molecular alterations that dis- tinguish particularly less aggressive melanoma cells from metastatic cells as an approach to identify molecular events that drive the selection towards one of these phe- notypes. Many genes with altered expression associated to melanoma progression have been identified [3,4]. A nota- ble example of a molecular marker of the transition from RGP to VGP melanomas is the β3 subunit of the αvβ3 integrin, a vitronectin receptor. The expression of β3 integrin is detected in most VGP and metastatic melano- mas, whereas normal melanocytes and RGP melanomas do not express this integrin subunit [17]. The expression of β3 integrin has been shown to contribute to metastatic phenotype by altering the adhesion and promoting sur- vival of melanoma cells [8,18,19]. Another example of a gene involved in the transition from RGP to VGP is KISS1, which has been postulated as a metastasis suppressor gene [20,21]. KISS1 encodes the protein kisspeptin-1/metastin that was identified as the endogenous ligand for the G protein-coupled receptor GPR54/KISS1R [22,23], and has been shown to play an anti-migratory role in vitro and to act as a metastasis inhibitor in vivo [20]. KISS1 expression is detected in normal melanocyte and RGP melanomas, but its expression is lost in VGP and metastatic cells [20,21]. Also, in other cancer types such as breast, bladder and pancreatic cancer, loss or reduced expression of KISS1 has been associated to the metastatic phenotype [24-26]. On the other hand, RHOC has been identified as an over- expressed gene in metastatic murine and human melanoma cells in comparison with the non-metastatic parental cells [27]. RHOC, like the other RHO family pro- teins, is involved in the regulation of the actin cytoskele- ton dynamics and overexpression of RHOC induces the cells to become highly metastatic by enhancing their migratory and invasive capacities [27]. Although many cancer-related genes have been character- ized, several lines of evidence suggest that many more remain to be identified. Present estimate has indicated that around 1% of the genes in the human genome are involved in cancer and there are predictions that 5–10% or more can contribute to oncogenesis [28]. Suppression subtractive hybridization is a widely used method for sep- arating mRNA sequences that distinguish two mRNA pop- ulations [29]. A key feature of the method is the simultaneous normalization and subtraction steps. The normalization step equalizes the abundance of mRNA within the target population, and the subtraction step excludes sequences that are common to the two popula- tions being compared. The SSH methodology allows the Page 2 of 18 (page number not for citation purposes)