Molecular subtyping of metastatic melanoma based on cell ganglioside metabolism profiles

BMC Cancer

Cristina Tringali³  Ilaria Silvestri³  Francesca Testa³  Paola Baldassari⁴  Luigi Anastasia⁵  Roberta Mortarini⁴  Andrea Anichini⁴  Alejandro López-Requena¹  Guido Tettamanti²  Bruno Venerando³ 

[1] Immunobiology Department, Center of Molecular Immunology, Havana, Cuba

[2] Laboratory of Stem Cells for Tissue Engineering, IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy

[3] Department of Medical Biotechnology and Translational Medicine, University of Milan, Segrate, Milan, Italy

[4] Human Tumors Immunobiology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

[5] Department of Biomedical Sciences for Health, University of Milan, Segrate, Milan, Italy

关键词: Survival;    Sialidase;    N glycolyl GM3;    Melanoma;    Ganglioside;   

Others  :  1125250
DOI  :  10.1186/1471-2407-14-560

 received in 2014-03-18, accepted in 2014-07-28,  发布年份 2014

【 摘 要 】

Background

In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids). Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor. On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients.

Methods

Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients. Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-³H]sphingosine and by FACS. N-glycolyl GM3 was identified employing the 14 F7 antibody. Gene expression was assayed by Real Time PCR. Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [³H] thymidine incorporation. Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis.

Results

Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3. Moreover, our data demonstrated that: a) a correlation could be traced between patients’ survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients’ survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins.

Conclusions

Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness. Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.

【 授权许可】

   
2014 Tringali et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin 2005, 55:74-108.
• [2]Miller AJ, Mihm MC Jr: Melanoma. N Engl J Med 2006, 355:51-65.
• [3]Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, Buzaid AC, Cochran AJ, Coit DG, Ding S, Eggermont AM, Flaherty KT, Gimotty PA, Kirkwood JM, McMasters KM, Mihm M, Morton DL, Ross MI, Sober AJ, Sondak VK: Final version of AJCC melanoma staging and classification. J Clin Oncol 2009, 2009(27):6199-6206.
• [4]Thompson JF, Scolyer RA, Kefford RF: Cutaneous melanoma. Lancet 2005, 365:687-701.
• [5]Haass NK, Smalley KS: Melanoma biomarkers: current status and utility in diagnosis, prognosis, and response to therapy. Mol Diagn Ther 2009, 13:283-296.
• [6]Gould Rothberg BE, Rimm DL: Biomarkers: the useful and the not so useful–an assessment of molecular prognostic markers for cutaneous melanoma. J Invest Dermatol 2010, 130:1971-1987.
• [7]Baruthio F, Quadroni M, Ruegg C, Mariotti A: Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage. Proteomics 2008, 8:4733-4747.
• [8]Brunner G, Reitz M, Heinecke A, Lippold A, Berking C, Suter L, Atzpodien J: A nine-gene signature predicting clinical outcome in cutaneous melanoma. J Cancer Res Clin Oncol 2013, 139:249-258.
• [9]Schramm SJ, Campain AE, Scolyer RA, Yang YH, Mann GJ: Review and cross-validation of gene expression signatures and melanoma prognosis. J Invest Dermatol 2012, 132:274-283.
• [10]Hakomori S: Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv Exp Med Biol 2001, 491:369-402.
• [11]Ruan S, Raj BK, Lloyd KO: Relationship of glycosyltransferases and mRNA levels to ganglioside expression in neuroblastoma and melanoma cells. J Neurochem 1999, 72:514-521.
• [12]Portoukalian J, Zwingelstein G, Dore JF: Lipid composition of human malignant melanoma tumors at various levels of malignant growth. Eur J Biochem 1979, 94:19-23.
• [13]Carubia JM, Yu RK, Macala LJ, Kirkwood JM, Varga JM: Gangliosides of normal and neoplastic human melanocytes. Biochem Biophys Res Commun 1984, 120:500-504.
• [14]Yu RK, Macala LJ, Taki T, Weinfield HM, Yu FS: Developmental changes in ganglioside composition and synthesis in embryonic rat brain. J Neurochem 1988, 50:1825-1829.
• [15]Welte K, Miller G, Chapman PB, Yuasa H, Natoli E, Kunicka JE, Cordon-Cardo C, Buhrer C, Old LJ, Houghton AN: Stimulation of T lymphocyte proliferation by monoclonal antibodies against GD3 ganglioside. J Immunol 1987, 139:1763-1771.
• [16]Yamashiro S, Okada M, Haraguchi M, Furukawa K, Lloyd KO, Shiku H, Furukawa K: Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes. Glycoconj J 1995, 12:894-900.
• [17]Furukawa K, Hamamura K, Nakashima H, Furukawa K: Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas. Proteomics 2008, 8:3312-3316.
• [18]Liu JW, Sun P, Yan Q, Paller AS, Gerami P, Ho N, Vashi N, Le Poole IC, Wang XQ: De-N-acetyl GM3 promotes melanoma cell migration and invasion through urokinase plasminogen activator receptor signaling-dependent MMP-2 activation. Cancer Res 2009, 69:8662-8669.
• [19]Blanco R, Rengifo E, Rengifo CE, Cedeno M, Frometa M, Carr A: Immunohistochemical Reactivity of the 14 F7 Monoclonal Antibody Raised against N-Glycolyl GM3 Ganglioside in Some Benign and Malignant Skin Neoplasms. ISRN Dermatol 2011, 2011:848909.
• [20]Hamamura K, Furukawa K, Hayashi T, Hattori T, Nakano J, Nakashima H, Okuda T, Mizutani H, Hattori H, Ueda M, Urano T, Lloyd KO, Furukawa K: Ganglioside GD3 promotes cell growth and invasion through p130Cas and paxillin in malignant melanoma cells. Proc Natl Acad Sci U S A 2005, 102:11041-11046.
• [21]Miyata M, Kambe M, Tajima O, Moriya S, Sawaki H, Hotta H, Kondo Y, Narimatsu H, Miyagi T, Furukawa K, Furukawa K: Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides. Cancer Sci 2011, 102:2139-2149.
• [22]Perotti V, Baldassari P, Bersani I, Molla A, Vegetti C, Tassi E, Dal Col J, Dolcetti R, Anichini A, Mortarini R: NFATc2 is a potential therapeutic target in human melanoma. J Invest Dermatol 2012, 132:2652-2660.
• [23]Tassi E, Zanon M, Vegetti C, Molla A, Bersani I, Perotti V, Pennati M, Zaffaroni N, Milella M, Ferrone S, Carlo-Stella C, Gianni AM, Mortarini R, Anichini A: Role of Apollon in human melanoma resistance to antitumor agents that activate the intrinsic or the extrinsic apoptosis pathways. Clin Cancer Res 2012, 18:3316-3327.
• [24]Daniotti M, Oggionni M, Ranzani T, Vallacchi V, Campi V, Di Stasi D, Torre GD, Perrone F, Luoni C, Suardi S, Frattini M, Pilotti S, Anichini A, Tragni G, Parmiani G, Pierotti MA, Rodolfo M: BRAF alterations are associated with complex mutational profiles in malignant melanoma. Oncogene 2004, 23:5968-5977.
• [25]Anichini A, Mortarini R, Nonaka D, Molla A, Vegetti C, Montaldi E, Wang X, Ferrone S: Association of antigen-processing machinery and HLA antigen phenotype of melanoma cells with survival in American Joint Committee on Cancer stage III and IV melanoma patients. Cancer Res 2006, 66:6405-6411.
• [26]Anichini A, Mazzocchi A, Fossati G, Parmiani G: Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction. J Immunol 1989, 142:3692-3701.
• [27]Sensi M, Nicolini G, Petti C, Bersani I, Lozupone F, Molla A, Vegetti C, Nonaka D, Mortarini R, Parmiani G, Fais S, Anichini A: Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma. Oncogene 2006, 25:3357-3364.
• [28]Tringali C, Lupo B, Cirillo F, Papini N, Anastasia L, Lamorte G, Colombi P, Bresciani R, Monti E, Tettamanti G, Venerando B: Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3. Cell Death Differ 2009, 16:164-174.
• [29]Scaringi R, Piccoli M, Papini N, Cirillo F, Conforti E, Bergante S, Tringali C, Garatti A, Gelfi C, Venerando B, Menicanti L, Tettamanti G, Anastasia L: NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1alpha. J Biol Chem 2013, 288:3153-3162.
• [30]Scandroglio F, Loberto N, Valsecchi M, Chigorno V, Prinetti A, Sonnino S: Thin layer chromatography of gangliosides. Glycoconj J 2009, 26:961-973.
• [31]Casadesus AV, Fernandez-Marrero Y, Clavell M, Gomez JA, Hernandez T, Moreno E, Lopez-Requena A: A shift from N-glycolyl- to N-acetyl-sialic acid in the GM3 ganglioside impairs tumor development in mouse lymphocytic leukemia cells. Glycoconj J 2013, 30:687-699.
• [32]Carr A, Mullet A, Mazorra Z, Vazquez AM, Alfonso M, Mesa C, Rengifo E, Perez R, Fernandez LE: A mouse IgG1 monoclonal antibody specific for N-glycolyl GM3 ganglioside recognized breast and melanoma tumors. Hybridoma 2000, 19:241-247.
• [33]Mortarini R, Gismondi A, Maggioni A, Santoni A, Herlyn M, Anichini A: Mitogenic activity of laminin on human melanoma and melanocytes: different signal requirements and role of beta 1 integrins. Cancer Res 1995, 55:4702-4710.
• [34]Tringali C, Cirillo F, Lamorte G, Papini N, Anastasia L, Lupo B, Silvestri I, Tettamanti G, Venerando B: NEU4L sialidase overexpression promotes beta-catenin signaling in neuroblastoma cells, enhancing stem-like malignant cell growth. Int J Cancer 2012, 131:1768-1778.
• [35]Tringali C, Lupo B, Silvestri I, Papini N, Anastasia L, Tettamanti G, Venerando B: The plasma membrane sialidase NEU3 regulates the malignancy of renal carcinoma cells by controlling beta1 integrin internalization and recycling. J Biol Chem 2012, 287:42835-42845.
• [36]Tringali C, Anastasia L, Papini N, Bianchi A, Ronzoni L, Cappellini MD, Monti E, Tettamanti G, Venerando B: Modification of sialidase levels and sialoglycoconjugate pattern during erythroid and erytroleukemic cell differentiation. Glycoconj J 2007, 24:67-79.
• [37]Papini N, Anastasia L, Tringali C, Croci G, Bresciani R, Yamaguchi K, Miyagi T, Preti A, Prinetti A, Prioni S, Sonnino S, Tettamanti G, Venerando B, Monti E: The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions. J Biol Chem 2004, 279:16989-16995.
• [38]Malykh YN, Schauer R, Shaw L: N-Glycolylneuraminic acid in human tumours. Biochimie 2001, 83:623-634.
• [39]Varki A: N-glycolylneuraminic acid deficiency in humans. Biochimie 2001, 83:615-622.
• [40]Irie A, Suzuki A: CMP-N-Acetylneuraminic acid hydroxylase is exclusively inactive in humans. Biochem Biophys Res Commun 1998, 248:330-333.
• [41]Hedlund M, Tangvoranuntakul P, Takematsu H, Long JM, Housley GD, Kozutsumi Y, Suzuki A, Wynshaw-Boris A, Ryan AF, Gallo RL, Varki N, Varki A: N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution. Mol Cell Biol 2007, 27:4340-4346.
• [42]Yamaguchi K, Koseki K, Shiozaki M, Shimada Y, Wada T, Miyagi T: Regulation of plasma-membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors. Biochem J 2010, 430:107-117.
• [43]Furukawa K, Horie M, Okutomi K, Sugano S, Furukawa K: Isolation and functional analysis of the melanoma specific promoter region of human GD3 synthase gene. Biochim Biophys Acta 2003, 1627:71-78.
• [44]Seyfried TN, Yu RK: Ganglioside GD3: structure, cellular distribution, and possible function. Mol Cell Biochem 1985, 68:3-10.
• [45]Ohkawa Y, Miyazaki S, Hamamura K, Kambe M, Miyata M, Tajima O, Ohmi Y, Yamauchi Y, Furukawa K, Furukawa K: Ganglioside GD3 enhances adhesion signals and augments malignant properties of melanoma cells by recruiting integrins to glycolipid-enriched microdomains. J Biol Chem 2010, 285:27213-27223.
• [46]Cheresh DA, Reisfeld RA, Varki AP: O-acetylation of disialoganglioside GD3 by human melanoma cells creates a unique antigenic determinant. Science 1984, 225:844-846.
• [47]Schauer R: Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol 2009, 19:507-514.
• [48]Nystedt J, Anderson H, Hirvonen T, Impola U, Jaatinen T, Heiskanen A, Blomqvist M, Satomaa T, Natunen J, Saarinen J, Lehenkari P, Valmu L, Laine J: Human CMP-N-acetylneuraminic acid hydroxylase is a novel stem cell marker linked to stem cell-specific mechanisms. Stem Cells 2010, 28:258-267.
• [49]Osorio M, Gracia E, Rodriguez E, Saurez G, Arango Mdel C, Noris E, Torriella A, Joan A, Gomez E, Anasagasti L, González JL, Melgares Mde L, Torres I, González J, Alonso D, Rengifo E, Carr A, Pérez R, Fernández LE: Heterophilic NeuGcGM3 ganglioside cancer vaccine in advanced melanoma patients: results of a Phase Ib/IIa study. Cancer Biol Ther 2008, 7:488-495.
• [50]Gabri MR, Otero LL, Gomez DE, Alonso DF: Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines. J Exp Clin Cancer Res 2009, 28:146.
• [51]Hayashi N, Chiba H, Kuronuma K, Go S, Hasegawa Y, Takahashi M, Gasa S, Watanabe A, Hasegawa T, Kuroki Y, Inokuchi J, Takahashi H: Detection of N-glycolyated gangliosides in non-small-cell lung cancer using GMR8 monoclonal antibody. Cancer Sci 2013, 104:43-47.
• [52]de Leon J, Fernandez A, Mesa C, Clavel M, Fernandez LE: Role of tumour-associated N-glycolylated variant of GM3 ganglioside in cancer progression: effect over CD4 expression on T cells. Cancer Immunol Immunother 2006, 55:443-450.
• [53]de Leon J, Fernandez A, Clavell M, Labrada M, Bebelagua Y, Mesa C, Fernandez LE: Differential influence of the tumour-specific non-human sialic acid containing GM3 ganglioside on CD4 + CD25- effector and naturally occurring CD4 + CD25+ regulatory T cells function. Int Immunol 2008, 20:591-600.
• [54]Wang J, Yu RK: Interaction of ganglioside GD3 with an EGF receptor sustains the self-renewal ability of mouse neural stem cells in vitro. Proc Natl Acad Sci U S A 2013, 110:19137-19142.
• [55]Dong Y, Ikeda K, Hamamura K, Zhang Q, Kondo Y, Matsumoto Y, Ohmi Y, Yamauchi Y, Furukawa K, Taguchi R, Furukawa K: GM1/GD1b/GA1 synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line. Cancer Sci 2010, 101:2039-2047.
• [56]Bonaventure J, Domingues MJ, Larue L: Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells. Pigment Cell Melanoma Res 2013, 26:316-325.
• [57]Kim SM, Jung JU, Ryu JS, Jin JW, Yang HJ, Ko K, You HK, Jung KY, Choo YK: Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors. Biochem Biophys Res Commun 2008, 371:866-871.
• [58]Ravindranath MH, Tsuchida T, Morton DL, Irie RF: Ganglioside GM3:GD3 ratio as an index for the management of melanoma. Cancer 1991, 67:3029-3035.
• [59]Kakugawa Y, Wada T, Yamaguchi K, Yamanami H, Ouchi K, Sato I, Miyagi T: Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc Natl Acad Sci U S A 2002, 99:10718-10723.
• [60]Valaperta R, Chigorno V, Basso L, Prinetti A, Bresciani R, Preti A, Miyagi T, Sonnino S: Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J 2006, 20:1227-1229.