Molecular subtyping of metastatic melanoma based on cell ganglioside metabolism profiles
- Cristina Tringali†1,
- Ilaria Silvestri†1,
- Francesca Testa1,
- Paola Baldassari2,
- Luigi Anastasia3, 4,
- Roberta Mortarini2,
- Andrea Anichini2,
- Alejandro López-Requena5,
- Guido Tettamanti3 and
- Bruno Venerando1Email author
© Tringali et al.; licensee BioMed Central Ltd. 2014
Received: 18 March 2014
Accepted: 28 July 2014
Published: 1 August 2014
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.
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.
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.
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.
KeywordsGanglioside Melanoma N glycolyl GM3 Sialidase Survival
Melanoma is the most lethal form of skin cancer and accounts for at least 48,000 deaths worldwide annually [1, 2]. Clinical outcome of melanoma largely depends on the tumor stage upon first diagnosis. Currently, melanoma staging is based on the guidelines published by the American Joint Committee on Cancer (AJCC) in 2009  and advises the employment of histopathological and clinical criteria. Nevertheless, this system is limited in its ability to provide a precise prognosis: a large number of patients with similar or identical clinical and histopathological features has different clinical outcome, from being cured to death . Also, the identification of some mutations concerning oncogenes, including BRAF, NRAS, KIT, has been shown to be very useful to predict the response to therapy, but it is not clear whether the determination of the mutational status could offer any prognostic value . Many attempts have been done to identify molecular markers or gene and protein signatures predicting clinical outcome in melanoma but, so far, none of them has been sufficiently validated [6–9].
Among the distinctive molecular markers displayed by melanoma cells, many studies identified specific gangliosides not detectable in normal melanocytes . A pivotal role played by gangliosides was recognized in the development of melanoma and in its biological features . In particular, GD3 and derivatives [12, 13], which are also present at high levels in proliferating cells [14–16], have been demonstrated to promote melanoma cell proliferation and invasion . Moreover, de-N-acetyl GM3 (d-GM3) (a variant of GM3 with a free amino group at the 5 position of sialic acid instead of the acetyl group) was found in melanoma and it was demonstrated that it enhanced cell migration and invasion . Also, N glycolyl GM3 (Neu5Gc-GM3) (a variant of GM3 that contains N-glycolylneuraminic acid instead of N-acetylneuraminic acid) was recognized in melanoma, even its role in this disease is still obscure . The alteration of the expression/catalytic activity of glycosyltransferases and enzymes involved in gangliosides metabolism could be critical factors in determining the melanoma cell ganglioside composition. In particular, the activation of GD3 synthase is linked to the increment of GD3 in melanoma cells [11, 20]. Similarly, the plasma membrane sialidase NEU3 has been shown to be highly expressed in human melanoma cell lines .
Prompted by these data, we sought to investigate the ganglioside metabolism profile of metastatic melanoma cell lines established from patients. Our results demonstrated that: a) melanomas displayed different ganglioside patterns and three clusters of tumors could be identified; b) a correlation could be traced between patients’ survival and melanoma ganglioside profiles; c) the expression of several enzymes involved in ganglioside metabolism was associated with patients’ survival; d) melanoma clusters identified on the basis of ganglioside profile exhibited different features determining melanoma malignancy.
Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients admitted to Fondazione IRCCS Istituto Nazionale dei Tumori, Milan [22, 23]. Molecular and biological characterization of the cell lines has been reported previously . All cell lines were maintained as described . All patients were informed about the scope and methods and delivered a written informed consent for the use of the surgical samples to establish cell lines. The study was approved by the Ethics Committee of the University of Milan and was performed according to the Declaration of Helsinki.
Clones 2/14 and 2/21 were isolated from a single human metastatic melanoma cell line, as described [26, 27]. NHEM-Ad and NHEM-Neo were purchased by Lonza (Basel, Switzerland) and PromoCell (Heidelberg, Germany), and maintained in mMGM-4 medium (Lonza).
Sphingolipid analysis was carried out through cell metabolic labeling with [3-3H]sphingosine (PerkinElmer, Waltham, MA, USA) . In order to assay the hypothesis that Neu5Gc-glicans could be incorporated from the culture medium and then employed for the synthesis of GM3, before [3-3H]sphingosine labeling, melanoma L6 cells were pre-incubated in the reduced-serum medium OptiMEM (Life Technology, Carlsbad, CA, USA) for 5 days.
Ganglioside and neutral sphingolipid extracts were analyzed by HPTLC carried out with the solvent systems chloroform/methanol/0.2% CaCl2 55:45:6 (v/v) and chloroform/methanol/water 110:40:6 (v/v), respectively. To separate Neu5Gc-GM3 from Neu5Ac-GM3, HPTLC was carried out using the solvent system chloroform/methanol/0.2% CaCl2/5 N NH3 50:42:6:4 (v/v). The sphingolipid pattern was determined and quantified by radiochromatoscanning (Betaimager 2000, Biospace, Paris, France) [28, 29].
Endogenous sphingolipid analysis performed to standardize metabolic labeling was performed as previously described .
Ganglioside standards were kindly given by Prof. Sonnino, University of Milan.
Immunostaining of HPTLC
After the chromatographic separation of gangliosides, the plates were soaked in acetone plus 0.1% polyisobutylmethacrylate. After drying and blocking with PBS-4% milk, the plates were incubated with 5 μg/ml of anti-Neu5Gc-GM3 murine 14 F7 antibody [31, 32], overnight, and, then, with a horseradish peroxidase-conjugated anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The reaction was stained with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Dallas, MA, USA).
Cytospin preparations of melanoma cell lines were fixed with formalin for 10 min at room temperature and then incubated with 14 F7 mAb, as described . The slides were then incubated with goat anti-mouse biotinylated secondary antibody, followed by streptavidin/HPR (Dako, Glostrup, Denmark), for 30 min at each step. The enzyme activity was detected with a commercial solution of 3, 3′-Diaminobenzidine (Sigma-Aldrich, St Louis, MO, USA). Cells were counterstained with Mayer’s Hematoxylin (Bio-Optica, Milan, Italy). Slides subjected to the same treatments, but without incubation with primary mAb, were used as negative controls. Images were acquired at × 20 with an Axiovert 100 microscope (Zeiss, Carl Zeiss, Thornwood, NY) equipped with a digital camera (AxioCam MrC5, Zeiss).
Phenotypic profile of melanoma cell lines
Cell surface expression of GM3, GD2 and GD3 was determined in representative melanoma cell lines belonging to different clusters, by staining with the following mAbs: anti GM3 (Neu5Ac-GM3) M2590 (Cosmo Bio Co, LTD, Tokyo, Japan), anti-human disialoganglioside GD2 (BD Pharmingen, San Diego, CA, USA), anti-ganglioside GD3 (R24) (Abcam Inc., Cambridge, UK), followed by a FITC-conjugated F(ab’)2 fragment goat anti-mouse Ab (Jackson ImmunoResearch, West Grove, PA) as described . Samples were acquired by a fluorescence-activated cell sorting (FACS)-Calibur cytofluorimeter (BD Biosciences) and analyzed by the FlowJo software (Tree Star, Ashland, OR, USA). Results were expressed as percentage of positive cells and as mean fluorescence intensity after subtraction of mean fluorescence intensity in cells stained with secondary antibody only.
In order to compare the phenotype of different tumors, all main FACScan operative settings were kept constant throughout the whole study.
PCR and Real-time RT-PCR
Primers used for gene expression
GD3 synthase (ST8SIAI)
GM3 synthase (ST3GAL5)
GM1 synthase (B3GALT4)
GM2 synthase (B4GALNT1)
GD1a synthase (ST6GALNAC4)
ACTB (β actin)
Melanoma cell invasiveness was assayed through a Matrigel assay, as previously reported .
Soft agar, thymidine incorporation, and MTT assays were performed as previously reported .
Sialidase activity toward endogenous cell gangliosides
To test the ability of melanoma plasma membrane sialidase to act on endogenous cell gangliosides, melanoma cell particulate fractions, obtained as previously described , were incubated with [3-3H]sphingosine labeled cell gangliosides at pH 3.8, overnight, at 37°C. Then, gangliosides were fractionated by HPTLC and visualized by radiochromatoscanning.
Hierarchical clustering was done using XLSTAT software. Survival analysis was carried out by the Kaplan-Meier method; survival curves were compared by the log-rank (Mantel-Cox) test; hazard ratios were computed by the Mantel-Haenszel test. Ganglioside composition, Real Time PCR data, [3H]thymidine incorporation were compared using Student t-test and 1-way ANOVA.
Melanoma cells can be clustered on the basis of their ganglioside profile
Sphingolipid pattern was determined in 23 short-term cell lines established from 5 primary VPG (vertical growth phase) melanomas and from 18 lymph nodes metastases as well as in adult (NHEM-Ad) and neonatal (NHEM-Neo) melanocytes. To this end, [3-3H]sphingosine was administrated to the cells leading to an extensive labeling of all sphingolipids . The treatment was performed in order to reach a metabolic steady state that corresponded to endogenous non-radiolabelled cell sphingolipid pattern. NHEM-Ad and NHEM-Neo melanocytes ganglioside profile was mainly composed of GM3 and GD3 (in traces in NHEM-Ad cells and more abundant in NHEM-Neo cells) with traces of sialyl-paragloboside (SPG) (Additional file 1).
A high amount of N-glycolyl GM3 characterizes melanoma cluster 1 cells
Correlation between glycosyltransferases expression and patients’ survival
We also analyzed the expression of other glycosyltransferases involved in ganglioside metabolism such as GD1a synthase, ST3GalVI, ST6GalNAc6 (data not shown) but we did not identify any correlation with patients’ survival.
Correlation between melanoma cell ganglioside profile and growth/survival and invasive potential
The resistance to apoptosis evidenced by the expression of BCL-2 and BCL-XL was clearly different between the three clusters: melanoma cells grouped in cluster 1 showed a higher expression of BCL-2 than melanocytes and also than melanoma cells grouped in clusters 2 and 3 (+150% versus adult melanocytes, P < 0.001; +150% versus cluster 2, P < 0.001; +900% versus cluster 3, P < 0.001) and BCL-XL (+38% versus adult melanocytes, P < 0.01; +57% versus cluster 2, P < 0.001; +38% versus cluster 3, P < 0.01) (Figure 8E).
Melanoma cluster 2 cells completely revolutionized the usual pathways of ganglioside biosynthesis present in melanocytes and acquired the expression of GM1 and GM2 synthases. Their ganglioside pattern was enriched by the presence of complex gangliosides such as GD1a and GT1b, belonging to the “a” pathway, which were not revealed in melanocytes, and to the “b” pathway. It was previously demonstrated that the transfection of GM1 synthase cDNA into the melanoma cell line SK-MEL-37 gave rise to the neo-expression of GD1b, GT1b, and GM1 and, in parallel, reduced cell growth and invasion . In melanoma cluster 2 cells, the expression of GM3 synthase was similar to adult melanocytes and, also, the content of GM3 and Neu5Gc-GM3 was significantly less than melanoma cluster 1 cells. Melanomas classified in cluster 3 represented a middle situation between that displayed by melanoma cluster 1 and melanoma cluster 2 with low Neu5Gc-GM3 levels and GM3 synthase expression and the presence of GD1a. Accordingly to these different ganglioside profiles, also the in vitro malignant behavior showed by these two clusters was clearly weakened. Thus, it appears realistic that the synthesis of different gangliosides, altering the recruiting and activation of signal molecules, could reflect on the biological properties of melanoma. Significantly, also the clinical behavior was very different: we traced a correlation between melanoma clusters and patients’ survival. In particular, the synthesis of GM3/GD3 and of “a” complex gangliosides appeared to have a worst and good prognostic value, respectively. Intriguingly, melanomas that retrieved a ganglioside metabolism typical of immature melanocytes (cluster 1), showed the worst outcome (median survival lower than 10 months). It could be supposed that this phenotype corresponds to a higher motile behavior: in fact, neural crest-derived melanoblasts are highly migratory cells . Instead, complex gangliosides, such as GD1a, usually denote more differentiated cells . In the past, pioneering studies have indicated that the GM3: GD3 ratio could be followed for the prognosis and therapeutic management of melanoma . Our results demonstrated that the overall cell ganglioside profile and metabolism could be more important than the presence of a single ganglioside type like GD3 or N-glycolyl GM3 for cell biologic features and, therefore, for prognosis.
Sialidase NEU3 and GD3 synthase genes were significantly up-regulated in melanomas in comparison to melanocytes, possibly as a direct consequence of the increase expression of the transcriptional factor Sp1, but we cannot record any significant correlation with patients’ survival. Sialidase NEU3 could be involved in melanoma malignancy decreasing the levels of Neu5Ac-GM3. It should also be noted that previous papers demonstrated that NEU3 is able to modulate cell signaling also inducing minimal changes in ganglioside profile [21, 59] and that NEU3 up-regulation could be counterbalanced by other modifications concerning the expression of enzymes involved in ganglioside metabolism, including GM3 synthase, inducing not expected effects on gangliosides . Therefore, the impact of NEU3 up-regulation in melanoma could be complex and should be further investigated in forthcoming works. In summary, the following factors appeared to be determinant for melanoma behavior and could have a significant prognostic value: a) the levels of GM3 and GD3; b) the levels of gangliosides GM1, GD1a, GT1b; c) GM3, GM1, and GM2 synthases expression. Therefore, the determination of ganglioside pattern could significantly improve the prediction of clinical outcome and possibly help the design of the most appropriate therapeutic strategy.
In addition to be classified on the basis of characteristic genetic mutations, different types of melanomas could also be distinguished on the basis of ganglioside profile. This analysis that could be performed in histochemistry also on surgical specimens, could indicate the aggressiveness of the tumor and, therefore, improve the prediction of the clinical outcome.
American Joint Committee on Cancer
N glycolyl GM3
N acetyl GM3
Vertical growth phase
- GM3 synthase:
- GD3 synthase:
- GM2 synthase:
- GM1 synthase:
- GD1a synthase:
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase.
Grant supports: Fondazione Cariplo (2010–0700), AIRC (IG-13131) to BV, and AIRC (IG-6036) to RM.
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