The hidden potential of glycomarkers: Glycosylation studies in the service of cancer diagnosis and treatment.

Changes in the glycosylation process appear early in carcinogenesis and evolve with the growth and spread of cancer. The correlation of the characteristic glycosylation signature with the tumor stage and the appropriate therapy choice is an important issue in translational medicine. Oncologists also pay attention to extracellular vesicles as reservoirs of new cancer glycomarkers that can be potent for cancer diagnosis/prognosis. In this review, we recall glycomarkers used in oncology and show their new glycoforms of improved clinical relevance. We summarize current knowledge on the biological functions of glycoepitopes in cancer-derived extracellular vesicles and their potential use in clinical practice. Is glycomics a future of cancer diagnosis? It may be, but in combination with other omics analyses than alone.

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma.

The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes-a commonly used approach-is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty .

Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus.

Circadian rhythmicity in mammals is sustained by the central brain clock-the suprachiasmatic nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks. Here, we use a combination of molecular, electrophysiological, and optogenetic tools to evaluate intrinsic clock properties of the main retinorecipient thalamic center-the lateral geniculate nucleus (LGN) in male rats and mice. We identify the dorsolateral geniculate nucleus as a slave oscillator, which exhibits core clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate leaflet and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the beginning of the projected night. Altogether, this study for the first time demonstrates autonomous timekeeping mechanisms shaping circadian physiology of the LGN.

Contribution of sialic acids to integrin α5ß1 functioning in melanoma cells.

PURPOSE: To establish the relationship between sialylation of integrin α5ß1 and possible alteration in the function of α5ß1 receptor in melanoma cells. MATERIALS AND METHODS: Integrin α5ß1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5ß1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5ß1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins. RESULTS: Both subunits of integrin α5ß1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5ß1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5ß1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5ß1 antibodies resulted in lower invasion abilities of primary WM115 cells. CONCLUSIONS: Our data suggest that in primary melanoma cells integrin α5ß1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.

RT-qPCR analysis of human melanoma progression-related genes - A novel workflow for selection and validation of candidate reference genes.

The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes. NormFinder software was used to identify the best reference gene or pair of reference genes from five candidate housekeeping genes, on the basis of expression stability in a given experimental model. The suitability of references was validated by normalizing the transcriptional activities of E-cadherin (CDH1), N-cadherin (CDH2) and endoplasmic reticulum aminopeptidase 1 (ERAP1) target genes. It has been shown that the relative expression of CDH2 and ERAP1 target genes in a given cell line may vary between experimental models, leading to biological misinterpretation. In view of this, we devised a strategy for improved selection of the best stable reference and for obtaining biologically consistent results. This strategy avoided experimental model- and normalizer-dependent conclusions concerning the relative expression of target gene, in the examined cell lines.

Stress and its molecular consequences in cancer progression.

Stress, caused by psychological, physiological and physical factors has an adverse impact on human body homeostasis. There are two kind of stress: short-term and chronic. Cancer patients usually live under chronic stress, caused by diagnosis-related strong emotional experience and depression, resulting from various difficulties associated with disease progression and treatment. At the molecular level, stress factors induce production and secretion of stress-related hormones, such as catecholamines, glucocorticoids and dopamine (as a part of adaptational body response), which influence both normal and transformed cells through their specific receptors. The particular effects exerted by these molecules on cancer cells have been also observed in in vitro cultures and include changes in proliferation, apoptosis susceptibility and migration/invasion potential. As a result, it has been suggested that stress hormones may be responsible for progression of malignancy and thus accelerate the metastasis formation in cancer patients. However, the clinical data on correlation between stress and the patients survival, as well as the molecular analysis of stress hormone receptors expression and action in cancer cell, have not yet provided an unequivocal answer. For this reason, extensive studies, on molecular and clinical level are needed to fully determine stress impact on cancer progression and on the effectiveness of anti-cancer treatment. Nowadays, it seems reasonable that the personalization of anti-cancer therapy should also focus on mental state of cancer patients, and provide them with psychological tools or techniques for stress management.

Diversified ß-2-adrenergic Receptor Expression and Action in Melanoma Cells.

BACKGROUND/AIM: Growing evidence links stress hormones with development and progression of various cancer types. The aim of this study was to assess susceptibility of cutaneous and uveal melanoma cells to adrenaline (AD). MATERIALS AND METHODS: The expression of ß-2-adrenergic receptor in primary cutaneous (FM-55-P), primary uveal (92-1, Mel202) and metastatic cutaneous (A375) melanoma cells was estimated at mRNA, protein and cell surface levels. The impact of AD on cell proliferation and migration was also studied. RESULTS: The expression of ß-2-adrenergic receptor was cell line-dependent. Adrenaline treatment caused a slight stimulation of melanoma cell proliferation and activation of matrix metalloproteinases. Adrenaline-treated uveal melanoma cells showed an increased migration rate, whereas, in cutaneous melanoma cells, no changes or even lower migration speed were observed. CONCLUSION: Melanoma cell susceptibility to AD varies depending on origin and progression stage. Metastatic cutaneous melanoma cells were found to be less responsive to AD than primary cutaneous and uveal melanoma cells.

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.

Gal-3 does not suppress cisplatin-induced apoptosis in A-375 melanoma cells.

Melanoma is the most aggressive of all skin cancers and is exceptionally resistant to therapies. During melanoma progression, cancer cells reprogram their proliferation and survival pathways and achieve resistance to treatment-induced apoptosis. Galectin-3 (gal-3) is a member of the lectin family and is involved in such biological processes as cell adhesion, growth and differentiation, the cell cycle, and apoptosis. Gal-3 also plays an important role in tumor development and metastasis. The relationship between gal-3 expression and these processes is specific to the tumor type and the stage of cancer progression. The biological functions of gal-3 depend on its localization in the cell. In the present study, human metastatic melanoma A-375 cells, characterized by weak endogenous expression of gal-3, were transfected with gal-3 cDNA and cisplatin-induced apoptosis was measured. Data from AnnexinV and mitochondrial membrane potential analysis revealed that gal-3 did not protect the A-375 melanoma cells against cisplatin. This result probably is associated with its nuclear localization in the cells.

Flutamide alters ß-catenin expression and distribution, and its interactions with E-cadherin in the porcine corpus luteum of mid- and late pregnancy.

This study examined whether flutamide-induced androgen deficiency during mid- and late pregnancy in pigs affected luteal expression of adherens junction protein, ß-catenin, and its interactions with E-cadherin. Flutamide (50 mg/kg body weight) was administered into pregnant gilts between days 43-49 (GD50F), 83-89 (GD90F) or 101-107 (GD108F) of gestation. Corpora lutea (CLs) were obtained on day 50, 90 or 108 of pregnancy (n=8-11 per each group). Total ß-catenin and E-cadherin expression was examined at mRNA (real-time PCR) and protein (Western blot) level. Moreover, subcellular ß-catenin fractions were extracted and immunoblotted. Immunohistochemistry was used for ß-catenin localization. To determine whether flutamide disturbs ß-catenin/E-cadherin mutual interactions, coimmunoprecipitation using anti-ß-catenin antibody was performed. Furthermore, phosphorylation of E-cadherin was assessed. Flutamide exposure led to decreased ß-catenin mRNA expression in all examined groups (p<0.001 or p<0.01), but protein level was lower only in the GD90F and GD108F groups (p<0.05). E-cadherin mRNA (p<0.05 or p<0.01) and protein (p<0.05) levels were up-regulated in all flutamide-treated groups when compared to controls. ß-catenin was predominantly found in membranes of luteal cells with no significant changes after antiandrogen treatment. ß-catenin/E-cadherin complexes were more abundant in the GD90F (p<0.05) and GD108F (p<0.01) groups than in controls due to enhanced E-cadherin phosphorylation at serine 838/840 in those animals (p<0.05). Overall, although androgen deficiency affected ß-catenin expression in the CL of pregnancy in pigs, a compensatory mechanism by enhanced interactions with E-cadherin is possible. Thus, androgen signaling via androgen receptors appears to be crucial in the regulation of luteal cells cross-talk.

Aberrant glycosylation of αvß3 integrin is associated with melanoma progression.

N-glycosylation of integrins plays an important role in cancer progression. Increased αvß3 integrin expression during melanoma progression is well-documented but the role of its glycans in tumorigenesis is still poorly understood. In the present study we used the WM793 primary melanoma cell line and its highly metastatic variant, WM1205Lu, to examine αvß3 glycosylation. Lectin precipitation, enzyme digestion and the use of swainsonine (SW) showed that αvß3 integrin glycosylation differs significantly between primary and metastatic melanoma cells. High-mannose structures and complex glycans with bisecting N-acetylglucosamine (GlcNAc) were more abundant in both subunits of primary cells. We also observed a shift in the sialylation of αvß3 integrin related to reduction of α2-6-linked sialic acid expression and an increase of α2-3 sialylation of both subunits in melanoma progression. Metastatic melanoma migration on vitronectin (VN) was reduced in the presence of antibody against αvß3 and the lectins phytohemagglutinin-L (PHA-L), Sambucus nigra agglutinin (SNA) and Maackia amurensis (MAA) in woundhealing assays. Our results show that the acquisition of metastatic competence by melanoma cells is accompanied by alteration of αvß3 integrin glycosylation and that both αvß3 and ß1-6-branched sialylated complex-type N-glycans promote metastatic melanoma migration on VN.

Is oestrogen an important player in melanoma progression?

The oestrogen-dependent regulation of cell behaviour is realised by stimulation of specific oestrogen receptors. The classical oestrogen receptors ERα and ERß are transcription factors, and they modulate expression of hormonally regulated genes, while the third one, GPER, is thought to be responsible for the observed rapid, non-genomic cellular response. Oestrogen dependency is attributed to a number of cancers, including breast, ovarian and endometrial cancer; however, there is still growing evidence that melanoma should also be cited as a hormonally dependent tumour. This comes from the observations of gender-related differences in melanoma progression and reports concerning the history of the malignant course of melanomas during pregnancy. Although, the observations of oestrogen regulation of melanoma progression are controversial, the effect of oestrogen should not be neglected, as the skin possesses its own hormonal microenvironment. This aspect of melanoma progression should be taken under careful consideration as it may offer new therapeutic possibilities.

Studies on primary uveal and cutaneous melanoma cell interaction with vitronectin.

We have examined the diversity between primary uveal (92-1 and Mel202) and cutaneous (FM55P and IGR-39) melanoma cells in their interaction with vitronectin, and established the effect of integrins and ß1,6-branched N-oligosaccharides on this process. The adhesion level of uveal melanoma cells to vitronectin was at least twice lower than that of cutaneous ones, but all cells tested repaired scratch wounds on vitronectin-coated surfaces with similar speed. Swainsonine treatment, by reducing the amount of ß1,6-branches, significantly decreased cell attachment in all cases, but reduction of wound healing efficiency was compromised only in cutaneous melanoma cell. Functional blocking antibodies used in adhesion and migration assays revealed that integrin αvß3 was strongly involved in adhesion and migration only in cutaneous melanoma cells, but its role here was less pronounced than that of integrin αvß5. However, in uveal melanoma the specific anti-αvß5 integrin antibody had no impact on migration speed. Therefore, the anti-α3ß1 integrin antibody was used in order to explain the nature of uveal melanoma interaction with vitronectin, which caused a mild decrease in adhesion efficiency and reduced their motility. Expression of αvß5 integrin differed between the cell lines, but there was no distinct pattern to distinguish uveal melanoma from cutaneous melanoma. In conclusion, αvß5, but not αvß3 integrin is heavily involved in uveal melanoma cell interaction with vitronectin. The role of ß1,6-branched N-glycans in the adhesion, but not during migration, of all cells to vitronectin has been confirmed.

Overexpression of N-acetylglucosaminyltransferases III and V in human melanoma cells. Implications for MCAM N-glycosylation.

An important role in cancer pathogenesis is attributed to N-glycans with "bisecting" N-acetylglucosamine and beta1-6 branches but the exact mechanisms still remain to be elucidated. Two structures are formed by Golgi beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC = 2.4.1.144, GnT-III) and alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A (EC = 2.4.1.155, GnT-V) respectively. The enzymes are encoded by MGAT3 and MGAT5 genes. The aim of this study was to establish two human melanoma cell lines with induced overexpression of GnT-III or GnT-V and to perform a preliminary functional characterization. WM-266-4 cells were stably transfected with human MGAT3 or MGAT5 cDNAs. GnT-III and GnT-V activities were assayed with a novel HPLC method based on labeling of N-glycan acceptor with 2-aminobenzamide (adapted from Taniguchi et al., 1989). Higher expression and activities of glycosyltransferases were detected. Increased amounts of "bisected" and beta1-6 branched N-glycans were present on melanoma cell adhesion molecule (known as MCAM/MUC18). However, cells did not display significant differences in viability and capabilities to migrate through an endothelial layer. To the best of our knowledge, the result of our study is the first to demonstrate that "bisected" N-glycans can be carried by MCAM. Moreover, increased modification of this protein by the two glycosyltransferases in WM-266-4-GnT-III cells was the consequence of the overexpression of only one enzyme. The obtained model can be useful for studying mechanisms of N-glycans branching and better understanding of their role in cancer progression. The proposed modification of the glycosyltransferase activity assay has shown to be a good alternative for 2-aminopyridine based HPLC systems.

[cAMP cascade in regulation of protein glycosylation]. / Regulacja procesu glikozylacji bialek przez kaskade cAMP.

O- and N-glycosylation are the most common and complex of the post-translational modifications. Both are enzymatic processes and it was suggested that both could be regulated by cAMP cascade at the early stages. N-glycosylation starts with the formation of lipid-linked oligosaccharides and this process is catalysed by crucial glycosyltransferase - dolichol phosphate mannose synthase. The results of several studies strongly suggest that the cAMP acting through a cAMP-dependent protein kinase A-mediated protein phosphorylation/dephosphorylation cycle may modulate activation of this enzyme. It was shown that cAMP can also up regulate another enzyme involved in phosphodolichole synthesis - cis-prenyltransferase. The mechanism acting here is the alteration of the rate of its gene expression. cAMP cascade is also involved in regulation of O-glycosylation since phosphorylation of human glutamine:fructose-6-phosphate amidotransferase results in depletion of O-GlcNAc structure formation. These observation suggested an important role of GPCRs and their ligand in regulation of N- and O-glycan synthesis.

Expression of integrins α3ß1 and α5ß1 and GlcNAc ß1,6 glycan branching influences metastatic melanoma cell migration on fibronectin.

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, ß1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), ß1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc ß1,6-branched glycans on the main fibronectin receptors - integrins α5ß1 and α3ß1. Our results show that GlcNAc ß1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.

Cell migration-the role of integrin glycosylation.

BACKGROUND: Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell-ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding. SCOPE OF REVIEW: The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation. GENERAL SIGNIFICANCE: A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.

Effect of alpha3beta1 and alphavbeta3 integrin glycosylation on interaction of melanoma cells with vitronectin.

The metastatic transformation of melanocytes is associated with altered expression of adhesion molecules, including alpha(v)beta(3) and alpha(3)beta(1) integrins. Integrin alpha(v)beta(3) is a primary vitronectin (VN) receptor, while both integrin types take part in adhesion to VN when they are in complex with uPAR. Although their role in melanoma cell interaction with VN is of great interest, the influence of N-oligosaccharides attached to these glycoproteins is still unappreciated. The present study assesses the role of alpha(v)beta(3) and alpha(3)beta(1) integrins and the influence of their glycosylation status on WM9 and WM239 metastatic melanoma cell interactions with VN. Cell adhesion to and migration on VN were selected as the studied cell behaviour parameters. Function-blocking antibodies and swainsonine (SW) treatment were used in these tests. Both cell lines interacted with VN in an integrin-mediated but cell-line-specific manner. In WM9 cells, migration was not completely inhibited by antibodies against alpha(3)beta(1) or alpha(v)beta(3) integrins, suggesting the participation of other VN receptors. In both cell lines in coprecipitation test the formation of an integrins/uPAR complex was shown. In the presence of SW formation of the complex did not occur, suggesting the participation of glycosylation in this process. Additionally, the adhesion properties of WM9 cells were changed after SW treatment. Our results suggest that in these two metastatic cell lines integrin-linked N-oligosaccharides influence the VN adhesion receptor activity and function.