Bitter Sweetness of Malignant Melanoma: Deciphering the Role of Cell Surface Glycosylation in Tumour Progression and Metastasis

Malignant melanoma is the sixth most commonly diagnosed cancer in developed countries. Like in many cancers, survival rates are closely associated with the time of melanoma detection. Regrettably, most cases of melanoma are caught at diffuse state and methods used in clinical practice and experimental trials are not effective. Thus, there is a great interest in discovering biomarkers that could be used for screening those with melanoma, as prognostic and prediction factors as well as new potential targets for melanoma treatment. For this purpose, many groups have examined alteration in the structure and expression of carbohydrate part of glycoconjugates to identify changes that occur with melanoma. The observed changes include increased β1,6 branching correlating with higher abundance of polylactosamine extension, increased sialylation accompanied by differences in the position of sialic acid residues, increased fucosylation, higher levels of T and Tn antigens as well as changes in the expression of ganglioside structures. As a consequence of glycan modification, the loosened matrix adhesion, increased motility, higher invasive potential and metastasis formation have been observed. Growth and migration of melanoma cells have been also found to be stimulated by advanced glycation end products. Biomarker discovery is a multi-step process and the recent glycomic data on melanoma are mostly related to the discovery phase, as the first one leading to validation and standardisation steps

Diverse expression of N-acetylglucosaminyltransferase V and complex-type \beta1,6-1,6-branched N-glycans in uveal and cutaneous melanoma cells

Although both uveal (UM) and cutaneous (CM) melanoma cells derive from the transformed melanocytes, their biology varies significantly in several aspects. Malignant transformation is frequently associated with alternations in cell glycosylation, in particular those concerning branched complex-type N-glycans. These changes occur principally in β1,4-N-acetylglucosaminyltransferase III (GnT-III) that catalyzes the synthesis of glycans with bisected N-acetylglucosamine (GlcNAc) and β1,6-N-acetylglucosaminyltransferase V (GnT-V) that is involved in forming β1,6-branched antenna in complex-type glycans. We searched for the reasons of a different behavior of CM and UM cells in the expression of GnT-III and GnT-V and their oligosaccharide products. Our study showed that UM cells have more β1,6-branched glycans than CM cells, what results from a higher expression of MGAT5 gene encoding GnT-V. The higher β1,6-branching of glycans in UM may contribute to their higher potential to migrate on fibronectin and weaker binding to main extracellular matrix proteins, observed in our previous studies

Cadherins and their Role in Malignant Transformation: Implications for Skin Cancer Progression

Cadherins are a large family of Ca2+dependent adhesion proteins. They are transmembrane or closely related to membrane glycoproteins localized in specialized adhesive junction. The expression of various cadherins may be concomitant with cancer progression steps and the term ‘cadherin switch’ has been created due to the observation of down-regulation of E-cadherin (suppressor of metastatic potential) and up-regulation of N-cadherin (promoter of metastatic potential) expression during tumour progression. These changes are thought to be closely related to epithelial-to-mesenchymal transition of cells of many different types of cancer including skin cancers, and accompany the increase of their motility and invasion abilities resulting in the metastasis formation. The cadherin polypeptide is a potential substrate for post-translational modification, for example, N-glycosylation, and its important role in the regulation of cadherin function has been described. The changed glycosylation of cadherins has been described in various skin cancers including melanoma and was consistent with cadherins’ role in epithelial-to-mesenchymal transition. The detailed analysis of cadherin expression and cadherin-related glycosylation changes taking place during malignant transformation could be a key for better understanding of the nature of this process and may open new opportunities for the creation of more effective anticancer therapeutics and diagnostic tools

Towards understanding the role of sialylation in melanoma progression

Aberrant expression of sialic acids or altered linkage types is closely associated with malignant phenotype and metastatic potential, and can have prognostic significance in human cancer. The present study was undertaken to evaluate whether expression of sialylated derivatives on melanoma cell surface is associated with tumour progression. Four cell lines (WM1552C, WM115, IGR-39 and WM266-4) were used in the study. Cell surface expression of sialic acids was evaluated by flow cytometry with the use of Maackia amurensis and Sambucus nigra lectins. Moreover, adhesion and migration potential of melanoma cells and involvement of sialic acids in these processes were analysed. We have demonstrated that WM266-4 cells have a significantly higher level of α2,3-linked sialic acid residues than other cells, whereas IGR-39 cells had lower expression of α2,6-linked sialic acids. The adhesion efficiencies of WM1552C and WM115 cells were significantly lower than that of IGR-39 and WM266-4 cells. In contrast, WM266-4 cells repaired scratch wounds at least twice as fast as other cells. Melanoma cell adhesion to fibronectin in the presence of Sambucus nigra agglutinin (SNA) was reduced only in IGR-39 and WM266-4 cells, whereas the impact of Maackia amurensis agglutinin (MAA) on this process was much more important. Migration efficiency of melanoma cells was reduced more strongly in the presence of MAA than SNA. In conclusion, our results show that melanoma progression is associated with the increased expression of α2,3-linked sialic acids on the cell surface and these residues could promote melanoma cell interaction with fibronectin

Similarities and differences in the protein composition of cutaneous melanoma cells and their exosomes identified by mass spectrometry

Intercellular transport of proteins mediated by extracellular vesicles (EVs)—exosomes and ectosomes—is one of the factors facilitating carcinogenesis. Therefore, the research on protein cargo of melanoma-derived EVs may provide a better understanding of the mechanisms involved in melanoma progression and contribute to the development of alternative biomarkers. Proteomic data on melanoma-derived EVs are very limited. The shotgun nanoLC-MS/MS approach was applied to analyze the protein composition of primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) cutaneous melanoma cells and exosomes released by them. All cells secreted homogeneous populations of exosomes that shared a characteristic set of proteins. In total, 3514 and 1234 unique proteins were identified in melanoma cells and exosomes, respectively. Gene ontology analysis showed enrichment in several cancer-related categories, including cell proliferation, migration, negative regulation of apoptosis, and angiogenesis. The obtained results broaden our knowledge on the role of selected proteins in exosome biology, as well as their functional role in the development and progression of cutaneous melanoma. The results may also inspire future studies on the clinical potential of exosomes

Adhesion properties of human bladder cell lines with extracellular matrix components : the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the α2, a5 and β1 integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the β1 integrin subunit. Antibodies to α3 integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with colagen. It seems that α3 subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy

Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines

BACKGROUND: The aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins. RESULTS: Human non-malignant epithelial cell of ureter HCV29, v-raf transfected HCV29 line (BC3726) and transitional cell cancers of urine bladder Hu456 and T24 were grown in cell culture. Equal amounts of protein from each cell extracts were separated by SDS-PAGE electrophoresis and were blotted on an Immobilon P membrane. Cadherins were immunodetected using anti-pan cadherin mAb and lectin blotting assays were performed, in parallel. N-oligosaccharides were analysed by specific reaction with Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Datura stramonium agglutinin (DSA), Aleuria aurantia agglutinin (AAA), Phaseolus vulgaris agglutinin (PHA-L) and wheat germ agglutinin (WGA). The cadherin from HCV29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans, some of which were α2,6-sialylated. The cadherin from BC3726 cell line exhibited exclusively high mannose type glycans. Cadherins from Hu456 and T24 cell lines expressed high mannose type glycans as well as β1,6-branched oligosaccharides with poly-N-acetyllactosamine structures and α2,3-linked sialic acid residues. Additionally, the presence of fucose and α2,6-sialic acid residues on the cadherin from T24 cell line was detected. CONCLUSIONS: These results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis

Transcriptomic data analysis of melanocytes and melanoma cell lines of LAT transporter genes for precise medicine

Background: Boron Neutron Capture Therapy (BNCT) is a two-step treatment that can be used in some types of cancers. It involves administering a compound containing boron atoms to the patient and irradiating the affected area of the body with a neutron beam. The success of the therapy depends mainly on the delivery of the boron isotope ($^{10}$B) to the tumor using an appropriate boron carrier. One of the boron carriers used is boronophenylalanine (BPA). Therefore, in research on the use of boron carriers, it is also important to know the mechanisms of its uptake by cells. Aim: To study the expression of LAT family genes in two melanoma (high melanotic WM$_{115}$ and low melanotic WM$_{266-4}$) cell lines and melanocytes (HEMa-Lp) which are responsible for the transport the BPA into cells. Methods: To normalize data from the transcriptomic analysis, the ratio of the median method was used. This allowed the samples to be compared with each other. Comparison metrics included log-fold change (LFC) values. The heatmap of LFC values and the cluster map were created. These graphs show the similarities and differences between the samples. Results: Transcriptomic data show that in melanocytes, LFC for SLC7A5 (LAT$_{1}$) and SLC3A2 ($_{4}$Fhc) was higher than in melanoma cell lines, which corresponded with their melanin content. Conclusion: Our results indicate overexpression of BPA transporter genes in normal cells (melanocytes), which may suggest the highest level of these proteins in melanocytes compared to less melanotic melanoma. Therefore, for BNCT, the use of BPA as the $^{10}$B carrier will require additional qualifying tests of amino acid transporter expression for patients and specific tumors to develop a personalized BNCT