Expression and subcellular localization of RAGE in melanoma cells

Abstract

The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression.

Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

“…Oligomerization of membrane receptors may be induced by ligands but can be constitutive. Apparently, RAGE belongs to the second group 38,39 , although the formation of the protein–ligand complex may further affect the structure and stoichiometry of the receptor complex. Structural studies of different truncated extracellular RAGE variants in the absence of ligands have shown different oligomerization modes in which different domains in different arrangements mediate self-association.…”

RAGE and its ligands in cancer – culprits, biomarkers, or therapeutic targets?

“…In melanoma RAGE silencing by RNAi selectively inhibited migration of melanoma cells [53]. Overexpresssion of RAGE on human melanoma cell line associated with the upregulation of S100B and downregulation of p53, ERK1/2, cyclin E and NF-κB.…”

“…Expression of RAGE and its splice variants increased [29] Gastric cancer A Gly82Ser polymorphism associated with gastric cancer and invasion of gatric cancer [34] Colorectal cancer RAGE highly expressed [22], ↑ RAGE expression in primary tumor correlates with the formation of metastases and reduced survival [35], Gly82Ser RAGE polymorphism associated in Chinese population with the risk and invasion of colorectal cancer [36] Inverse association between sRAGE and colorectal adenoma among pts without hypertension [13], inverse relation between sRAGE and colorectal cancer [65] Hepatocellular cancer RAGE highly expressed [22] sRAGE inversely associated with liver cancer [63], sRAGE predicted the response to transarterial chemoembolization of liver cancer [64], Pancreatic cancer In a murine model targeted ablation of RAGE delayed the development of pancreatic neoplasia [38], Absence of RAGE in mice delayed the development of pancreatic neoplasia [39], polymorphisms of RAGE not associated with pancreatic cancer [40] sRAGE levels inversely associated with pancreatic cancer [61], sRAGE levels ↓ in pacreatic cancer (↓↓ in diabetics with pancreatic cancer [40], no association between levels of esRAGE and pancreatic cancer with the exception of patients with shorter follow-up [ Estrogens induce RAGE expression on breast cancer cells and prolong their survival [49], RAGE highly expressed in breast cancer [22], RAGE expression correlated with the levels of AGE modified proteins [24], no difference in genetic polymorphisms of RAGE between breast cancer and healthy controls [50,51], rs184003 polymorphism and T-T-G-T haplotype more frequent in breast cancer compared to healthy controls [52] Lower sRAGE levels in breast [50], but increasing in pts with advanced breast cancer, lower grade, positive estrogen receptors, and intermediate positivity of Her2/neu, genetic polymorphisms of RAGE related to serum levels of RAGE [50] Melanoma RAGE silencing inhibits migration of melanoma cells [53], ↑ RAGE on human metastatic melanoma a cell line associated with metastatic phenotype [54], Anti-RAGE antibodies [55], or DNA-AGE ap...…”

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression

“…Subcellular Fractionation of Melanoma Cells-Obtaining subcellular fractions from MJS and SK28 cells was performed after the protocol described for MJS in detail elsewhere (30), which represented an adaptation of the method described originally elsewhere (31). Briefly, MJS or SK28 cells resulting from four 175-cm 2 flasks (cultured as semi-confluent cultures) were homogenized in Mg 2ϩ /Ca 2ϩ -containing base buffer and protease inhibitors on ice.…”