Human giant congenital melanocytic nevus exhibits potential proteomic alterations leading to melanotumorigenesis
- Hyoung Kyu Kim†1,
- Yong Kyu Kim†2,
- In-Sung Song1,
- Sung-Ryul Lee1,
- Seung Hun Jeong1,
- Min Hee Kim1,
- Dae Yun Seo1,
- Nari Kim1,
- Byoung Doo Rhee1,
- Kyoung Soo Ko1,
- Kwan Chul Tark3,
- Chul Gyoo Park4,
- Je-Yoel Cho5 and
- Jin Han1Email author
© Kim et al.; licensee BioMed Central Ltd. 2012
Received: 9 April 2012
Accepted: 26 July 2012
Published: 20 August 2012
A giant congenital melanocytic nevus (GCMN) is a malformation of the pigment cells. It is a distress to the patients for two reasons: one is disfigurement, and the other is the possibility of malignant changes. However, the underlying mechanisms of the development of GCMN and melanotumorigenesis in GCMN are unknown. Hence, the aim of this study was to identify the proteomic alterations and associated functional pathways in GCMN.
Proteomic differences between GCMN (n = 3) and normal skin samples (n = 3) were analyzed by one-dimensional-liquid chromatography-tandem mass spectrometry Relative levels of the selected proteins were validated using western blot analysis. The biological processes associated with the abundance modified proteins were analyzed using bioinformatic tools. Among the 46 abundance modified proteins, expression of 4 proteins was significantly downregulated and expression of 42 proteins was significantly upregulated in GCMN compared to normal skin samples (p < 0.05). More importantly, 31% of the upregulated proteins were implicated in various cancers, with five proteins being specifically related with melanoma. The abundance modified proteins in GCMN were involved in the biological processes of neurotrophin signaling, melanosome, and downregulated of MTA-3 in ER-negative breast tumors. In particular, an increase in the expression of the 14-3-3 protein family members appeared to be associated with key cellular biological functions in GCMN. Western blot analysis confirmed the upregulation of 14-3-3epsilon, 14-3-3 tau, and prohibitin in GCMN.
These findings suggest that GCMN exhibits potential proteomic alterations, which may play a role in melanotumorigenesis, and the significant alteration of 14-3-3 family proteins could be a key regulator of the biological pathway remodeling in GCMN.
KeywordsGiant congenital melanocytic nevi Melanotumorigenesis Proteomics 14-3-3 epsilon 14-3-3 tau Systemic analysis
Congenital melanocytic nevi (CMN) are pigment cell malformations that are visible at birth, or are nevi showing congenital features that become clinically obvious shortly after birth . CMN is caused by abnormal melanocyte differentiation, migration, and deposition in the dermis during the early stages of embryogenesis [2, 3]. It is a distress to patients for two reasons: one is disfigurement, and the other is the increased risk of developing malignant melanoma [3, 4], especially in individuals with giant congenital melanocytic nevi (GCMN; over 20 cm in diameter) . Several genomic and proteomic studies have been performed to elucidate the mechanism of melanotumorigenesis arising from CMN. Gene-based analyses have revealed that the oncogenic BRAF  and NRAS  mutations are frequently seen in CMN. Additionally, increased Bcl-2 expression in CMN has been suggested to suppress apoptosis, which otherwise plays an important role in the maintenance of nevocytes . In spite of these findings, the major biological processes and pathways of melanotumorigenesis remain unclear. Therefore, the complete proteomic characterization of GCMN and related biological pathways through comparative protein profiling is essential to understand the underlying mechanism of the origin of GCMN and the subsequent process of melanotumorigenesis.
In this study, the proteomic alteration and systemic properties of GCMN were assessed, with the aim of gaining an insight into the functional association between GCMN and melanotumorigenesis. We used label-free liquid chromatography-mass spectrometry (LC-MS) and established bioinformatic tools to identify the proteins that may play a key role in the malignant transformation of GCMN. We found that the 46 proteins were changed in protein abundance levels between normal skin and GCMN samples, and these proteins belonged to tightly organized functional clusters. Moreover, we found that five of the identified proteins were implicated in melanoma. The results of this study will improve our understanding in the biological identification of GCMN and the possible mechanisms that give rise to GCMN-associated melanotumorigenesis.
Characteristics of the study population
Clinical characteristics of GCMN and normal skin samples
Proteomic alterations in GCMN
Abundance modified proteins in GCMN
Accession number 1
Post-translational modification, protein turnover, chaperones (15)
CTSD cathepsin D -
breast  ovarian  melanoma [14,15]
HSP90AA1 heat shock protein 90-kDa alpha (cytosolic), class A member 1 isoform 1
HSP90AB1 heat shock protein HSP 90-beta
HSPA1A;HSPA1B heat shock 70-kDa protein 1
HSPA8 Isoform 1 of heat shock cognate 71-kDa protein
HSPB1 heat shock protein beta-1
PDIA3 protein disulfide-isomerase A3
gastric carcinoma , thyroid cancer , hepatocellular carcinoma , melanoma [16,17]
PSMB1 proteasome subunit beta type-1
SFN Isoform 1 of 14-3-3 protein sigma
YWHAB long isoform of 14-3-3beta/alpha
CAPZA1 F-actin-capping protein subunit alpha-1
CAPZB cDNA, FLJ93598, highly similar to Homo sapiens capping protein (actin filament) muscle Z-line, beta
Isoform 2 of dynein heavy chain 2, axonemal
Isoform 2 of myosin light chain kinase, smooth muscle
General function prediction only (5)
CDC42 Isoform 2 of cell-division control protein 42 homolog
immune escape of cancer 
CSPG4 chondroitin sulfate proteoglycan 4
melanoma, human carcinoma, sarcoma 
HNRNPA2B1 isoform B1 of heterogeneous nuclear ribonucleoproteins A2/B1
glioblastoma lung cancer 
PEBP1 phosphatidylethanolamine-binding protein 1
prostate , breast , gastrointestinal stromal , melanoma , ovarian 
RAC1 isoform A of Ras-related C3 botulinum toxin substrate 1
skin tumor 
Carbohydrate transport and metabolism (3)
ALDOA fructose-bisphosphate aldolase A
Uncharacterized protein ENSP00000348237
Energy production and conversion (2)
ATP5B ATP synthase subunit beta, mitochondrial
ETFA electron transfer flavoprotein subunit alpha, mitochondrial
Signal transduction mechanisms (3)
ARHGDIA rho GDP-dissociation inhibitor 1
breast cancer 
RYR3 uncharacterized protein RYR3
Intracellular trafficking, secretion, and vesicular transport (3)
ANXA1 annexin A1
breast cancer 
ANXA2 annexin A2 isoform 1
breast cancer 
ANXA5 annexin A5
colorectal cancer 
Amino acid transport and metabolism (2)
PHGDH D-3-phosphoglycerate dehydrogenase
breast cancer 
Lipid transport and metabolism (2)
ECH1 delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, mitochondrial
FASN fatty acid synthase
breast cancer, endometrial cancer  prostate cancer 
Translation, ribosomal structure, and biogenesis (2)
EEF1A1 elongation factor 1-alpha 1
RPSA ribosomal protein SA, 33-kDa protein
RNA processing and modification (1)
HNRNPU short isoform of heterogeneous nuclear ribonucleoprotein U
Cell cycle control, cell division, chromosome partitioning (1)
KRT1 keratin, type II cytoskeletal 1
Inorganic ion transport and metabolism (1)
CLIC1 chloride intracellular channel protein 1
Secondary metabolites biosynthesis, transport, and catabolism (1)
ADH1B alcohol dehydrogenase 1B
Function unknown (1)
Systemic properties of the altered GCMN proteome
Enriched functional group in GCMN
% Genes 1
Phospholipase inhibitor activity
Neurotrophin signaling pathway
Downregulated of MTA-3 in ER-negative breast tumors
Comparison of systemic properties between GCMN and metastatic melanoma
Validation of the abundance modified proteins
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.
Taken together, our data suggest that proteomic modifications with tumorigenic potential are present in GCMN, and these proteomic alterations possibly modify six important biological processes or pathways that include melanosome, neurotrophin signaling pathway, downregulated of MTA-3 in ER-negative breast tumors, cell cycle, phospholipase inhibitor activity, and glycolysis/gluconeogenesis These pathways may be significantly altered in GCMN skins. The intensive alteration of 14-3-3 family proteins and PHB possibly acts as a central regulator of GCMN biological pathway remodeling, which may have an important role in the development of GCMN and could be associated with melanotumorigenesis.
Materials and methods
A total of 10 normal and GCMN skin samples, which were defatted, were obtained from patients who underwent excision procedures at the Department of Plastic Surgery, Inje University Ilsan Paik Hospital, Korea. The collection and use of the samples were approved by the Institutional Review Board of Inje University Ilsan Paik Hospital (IRB No. IB-0902-015). The present study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.
Cell lines and culture conditions
The human embryo skin cell line Detroit 551 and the human malignant melanoma cell lines SK-MEL-2, SK-MEL-5, and SK-MEL-28 were obtained from the American Type Culture Collection (ATCC; Rockville, MD). The culture medium used throughout these experiments was RPMI-1640 (Lonza, Verviers, Belgium) containing 10% fetal bovine serum (PAA, Pasching, Austria) and 100 μg/ml penicillin-streptomycin (Lonza). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
Sample preparation for proteomics
Three paired normal and GCMN skin samples were selected for 1D-LC-MS/MS proteomic analysis to exclude environmental bias. Excised skin samples were ground to a powder in liquid nitrogen, dissolved in lysis buffer (9 M urea, 2 M thiourea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 40 mM dithiothreitol (DTT), and 1% protease inhibitor cocktail), vortexed, and incubated on ice for 1 h. The mixture was then centrifuged (10,000 × g, 30 min, 4°C), and the total proteins contained in the supernatant were used for the experiments. The total protein content of the solution was determined using the 2D Quant kit (GE Healthcare, Milwaukee, WI), with bovine serum albumin (0–50 mg/ml) as the standard.
Protein separation and LC-MS analysis were performed as previously described . Briefly, dissolved skin proteins were separated on a 12% polyacrylamide gel by SDS-PAGE. The gels were washed three times with ddH2O for 5 min each and stained with Bio-Safe Coomassie stain solution (Coomassie G250 stain; Bio-Rad, Hercules, CA) for 1 h, with gentle shaking at room temperature. The Coomassie-stained gels were evenly sliced into 15 slices and then destained by incubation in 75 mM ammonium bicarbonate/40% ethanol (1:1). Disulfides were reduced by treatment with 5 mM DTT/25 mM ammonium bicarbonate at 60°C for 30 min, followed by alkylation with 55 mM iodoacetoamide at room temperature for 30 min. The gel pieces were then dehydrated in 100% acetonitrile (ACN), dried, and swollen overnight at 37°C in 10 μl 25 mM ammonium bicarbonate buffer containing 20 μg modified sequencing-grade trypsin (Roche Applied Science, Indianapolis, IN) per ml. The tryptic peptide mixture was eluted from the gel using 0.1% formic acid. LC-MS/MS analysis was performed using a ThermoFinnigan ProteomeX workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA) equipped with a nanospray ionization (NSI) source (Thermo Electron). Briefly, 12 μl peptide sample obtained from the in-gel digestion was injected and loaded onto a peptide trap cartridge (Agilent, Palo Alto, CA). Trapped peptides were eluted onto a 10-cm reversed-phase PicoFrit column packed in-house with 5-μm, 300-Å pore size C18 and separated by gradient elution. The mobile phases consisted of H2O and ACN, both containing 0.1% v/v formic acid. The flow rate was maintained at 200 nl/min. The gradient started at 2% ACN, then reached 60% ACN in 50 min, 80% ACN in the next 5 min, and 100% H2O in the final 15 min. Data-dependent acquisition (m/z 400–1800) was enabled, and each MS survey scan was followed by five MS/MS scans within 30 s, with the dynamic exclusion option enabled. The spray voltage was 1.9 kV, the temperature of the ion transfer tube was 195°C, and the normalized collision energy was 35% .
Data-analyzed tandem mass spectra were extracted, and the charge state was deconvoluted and deisotoped using the Sorcerer 3.4 beta2 platform (Sorcerer software 3.1.4, Sorcerer Web interface 2.2.0 r334, and Trans-, Proteomic Pipeline 2.9.5). All MS/MS samples were analyzed using SEQUEST (version v.27, rev. 11; ThermoFinnigan, San Jose, CA), which was set to search the ipiHuman 3.29 database (IPI ver.3.29, 40131 entries), with semitrypsin as the digestion enzyme. The search used a fragment-ion mass tolerance of 1.00 Da and a parent-ion mass tolerance of 1.5 Da. Iodoacetamide-derivatized cysteine was specified as a fixed modification. Methionine oxidation, iodoacetamide derivatizion of cysteine, and phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. The Scaffold software (version Scaffold-2.0; Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if their probability was >95.0%, as specified by the Peptide Prophet algorithm, and if they contained at least one identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins containing similar peptides such that they could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. After identifying the proteins, each dataset was used for a subtractive analysis by semi-quantitative normalized spectral counts, which were normalized by total spectral counts in the Scaffold program .
A systemic bioinformatics analysis of the GCMN proteome was conducted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 8.3) , the Protein Analysis Through Evolutionary Relationships classification system (PANTHER 7.0) , the National Center for Biotechnology Information (NCBI) COG database , Cytoscape, and ClueGO .
Western blot analysis
Protein expressions of 14-3-3 alpha + beta (30 kDa), 14-3-3 epsilon (29 kDa), 14-3-3 zeta (28 kDa), 14-3-3 sigma (25 kDa), 14-3-3 tau (31 kDa), and prohibitin (30 kDa) in normal (n = 7) and GCMN (n = 7) samples were analyzed by western blots to confirm the proteomic results. In addition, protein expressions of 14-3-3 epsilon, 14-3-3 tau, and prohibitin in a normal cell line (Detroit 551) and melanoma cell lines (SK-MEL-2, SK-MEL-5, and SK-MEL-28) were analyzed by western blots. Relative expression of each protein was normalized to an internal standard protein, β-tubulin (55 kDa) or glyceraldehyde-3-phosphate dehydrogenase (37 kDa) (Abcam, Cambridge, MA). The values were expressed as mean ± standard error.
Two-tailed unpaired Student’s t-test in the Scaffold (version 2.06.02) software was used to compare abundance of each protein in the normal and GCMN skin samples. Using the Origin software (version 7.0220, OriginLabs, MA, USA), Bonferroni correction was applied to control the rate of false positives in the comparison of means of each protein’s abundance. A two-tailed unpaired Student’s t-test was used to compare the results of western blot analysis between the normal and GCMN skin samples. p < 0.05 was considered statistically significant.
Giant congenital melanocytic nevus
Protein disulfide-isomerase A3 precursor
Heat shock protein 70
ATP synthase subunit beta
Alcohol dehydrogenase 1B
Chondroitin sulfate proteoglycan 4
Ribosomal protein SA
Clusters of Orthologous Groups
Search Tool for the Retrieval of Interacting Genes/Proteins
Protein Analysis Through Evolutionary Relationships
Kyoto Encyclopedia of Genes and Genomes
This work was supported by Priority Research Centers Program and Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (353-2009-1-E00002 and 2010–0020224).
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