Marcelina Janik

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.

Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 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, β1,6-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), β1,6 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 β1,6-branched glycans on the main fibronectin receptors - integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.

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 ERβ 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.

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.

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 β1,6‐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 β1,6‐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 αvβ3 was strongly involved in adhesion and migration only in cutaneous melanoma cells, but its role here was less pronounced than that of integrin αvβ5. However, in uveal melanoma the specific anti‐αvβ5 integrin antibody had no impact on migration speed. Therefore, the anti‐α3β1 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 αvβ5 integrin differed between the cell lines, but there was no distinct pattern to distinguish uveal melanoma from cutaneous melanoma. In conclusion, αvβ5, but not αvβ3 integrin is heavily involved in uveal melanoma cell interaction with vitronectin. The role of β1,6‐branched N‐glycans in the adhesion, but not during migration, of all cells to vitronectin has been confirmed.

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.

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.

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.

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

Cadherins are a large family of Ca2+dependent adhesion proteins. They are transmem‐ brane 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‐regula‐ tion of N‐cadherin (promoter of metastatic potential) expression during tumour progression. These changes are thought to be closely related to epithelial‐to‐mesenchy‐ mal 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.