In the present study, the proteomic composition of GCMN was compared with that of normal skin. A major aim of the study was the identification of proteins whose expression is altered in GCMN, which will help understand the altered biological processes in GCMN and help gain an insight into the mechanism of melanotumorigenesis in these malformations. LC-MS/MS analysis showed that 46 of the 438 identified proteins changed in their abundance levels between the normal skin and GCMN samples. In the GCMN samples, 92% of the abundance modified proteins were upregulated, but only 8% were downregulated (Figure 2 and Table 2). The use of different bioinformatic tools showed that GCMN clearly differed from normal skin in terms of protein expression patterns, which suggested that specific biological processes are altered in GCMN. As derived from the GO categories, KEGG pathways, and Reactome_biocarta, these processes were shown to encompass several major biological functions, namely the neurotrophin signaling pathway, downregulated of MTA-3 in ER-negative breast tumors, the cell cycle, phospholipase inhibitor activity, and glycolysis/gluconeogenesis. Strikingly, among these, neurotrophin signaling [17, 18], MTA-3 downregulation (Table 3) , cell cycle deregulation , and glycolysis/gluconeogenesis  have been implicated in the development and progression of melanoma and other cancers.
Comparison of systemic properties of the GCMN and metastatic melanoma proteomes revealed that these two different disease proteomes shared at least five proteomic alterations in common and their abundance modified proteins closely interacted with each other (Figure 5A). Because closely related diseases are known to share common proteins or common interactions , our results suggested the close relationship between GCMN and melanoma.
Our proteomic analysis also revealed the significantly increased expression of 14 cancer-related proteins in GCMN compared to normal skin samples. Among them, PHB is a molecular maker of malignant cancers, and overexpression of PHB has been reported in melanoma [11, 12] and various kinds of cancers, including gastric carcinoma , thyroid cancer , and hepatocellular carcinoma . This significant upregulation of cancer-related proteins in GCMN, specifically which of melanoma-implicated proteins, strengthened the possible risk of melanotumorigenesis in GCMN.
The 14-3-3 proteins comprise a highly conserved family of proteins whose members are found in both plants and mammals. They mediate signal transduction by binding to phosphoserine-containing proteins and are involved in many biological cellular processes, such as metabolism, protein trafficking, signal transduction, apoptosis, and cell cycle regulation, through interaction with various phosphoserine-containing proteins, such as CDC25 phosphatases, RAF1, and IRS1 proteins. In the present study, 14-3-3 family proteins were estimated to interact with 23 proteins in GCMN and melanoma (Figure 5B), and their average number of interactions was about 2-fold higher than the average number of interactions of other abundance modified proteins. These results suggested that 14-3-3 family proteins could play an important role in the alteration of biological processes in GCMN and melanoma.
The 14-3-3 family proteins consist of seven isoforms: beta, gamma, epsilon, sigma, zeta, tau, and eta. The alpha and sigma isoforms are the phosphoforms of 14-3-3 beta and zeta, respectively. All 14-3-3 proteins are ubiquitously expressed, with the exception of 14-3-3 sigma, which is exclusively expressed in epithelial cells . Among the 14-3-3 family members, the overexpression of the 14-3-3 sigma gene or its respective protein is frequently found in cancers such as ovarian carcinomas , pancreatic cancer , papillary thyroid carcinoma , hepatocellular carcinoma , and breast cancer . In our proteomic analysis, the expression of 14-3-3 proteins was significantly higher in GCMN than in normal skin samples, which strongly supports the greater tendency toward melanotumorigenesis in GCMN. In particular, the enhanced expression of 14-3-3 epsilon and tau proteins was clearly shown in western blot analysis (Figure 6).
Compared to the other isoforms, little is known about the molecular and biological role of 14-3-3 tau and epsilon proteins. Like other isoforms, 14-3-3 tau is also involved in cell death and survival processes. For example, 14-3-3 tau binds to ataxia telangiectasia-mutated (ATM)-phosphorylated E2F1 during DNA damage and promotes E2F1 stability, leading to the induction of apoptosis , and the deletion of 14-3-3 tau leads to embryonic lethality in a mouse model . Interestingly, a recent study suggested that 14-3-3 tau exhibits an oncogenic role by downregulating p21 in breast cancer .
14-3-3 epsilon has been shown to play an essential role in cell development. Studies in Drosophila showed that 14-3-3 epsilon is required for the correct timing of mitosis in undisturbed post-blastoderm cell cycle . More recently, defects in neuronal migration during the development of 14-3-3 epsilon-knockout mice were reported .
The phosphorylation-induced binding of 14-3-3 epsilon to the pro-apoptotic transcription factor forkhead transcription factor-like 1 (FKHRL1 or FOXO3a) leads to structural changes in 14-3-3 epsilon and inhibits its pro-apoptotic activity . In inflammation and carcinogenesis, 14-3-3 epsilon interacts with key molecules of the mitogen-activated protein kinase signaling module to selectively modulate tumor necrosis factor-alpha-induced nuclear factor-kappa-beta activity . The function and regulatory mechanism of 14-3-3 epsilon in carcinogenesis is controversial and appears to be tumor-specific. Expression of the protein is higher in renal cell carcinoma than that in normal kidney . Moreover, on the basis of their involvement in the tumorigenesis of meningioma, 14-3-3 epsilon, zeta, and theta are thought to be efficient markers for predicting the degree of malignancy of these tumors . In contrast, mRNA and protein expression of 14-3-3 epsilon in laryngeal squamous cell carcinoma tissues was shown to be significantly lower than that in normal tissues . An early role of 14-3-3epsilon in tumorigenesis is suggested by the observation that 14-3-3 epsilon expression is increased in intrinsically aged and photoaged human skin . Interestingly, we found even higher protein levels of 14-3-3 epsilon, 14-3-3 tau, and PHB in GCMN than those in aged skin samples. This result suggested that GCMN may have a higher risk of tumorigenesis than aged skin. Because of the limitation in sample availability, we could not directly determine the expression level of 14-3-3 proteins and PHB in malignant melanoma tissue; however, we demonstrated significantly increased protein expression of 14-3-3 epsilon and tau in two different melanoma cell lines, SK-MEL-2 and SK-MEL-28, compared to normal skin cell line (Detroit 551). This result might support the association of 14-3-3 epsilon and tau upregulation with clinical melanotumorigenesis (Figures 7A and B).
Nevertheless, further studies are needed to validate the functional role of 14-3-3 proteins in melanotumorigenesis through the proteomic comparison of different malignant melanoma patients with giant congenital melanocytic nevi. Furthermore, it is also necessary to carefully validate the biological meaning of the upregulation of melanoma-implicated proteins in GCMN and their role in melanotumorigenesis.