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Melanoma metastasis to the brain is one of the most frequent extracranial brain tumors. Cell surface gangliosides are elevated in melanoma metastasis; however, the metabolic regulatory mechanisms that govern these specific changes are poorly understood in melanoma particularly brain metastases (MBM) development. We found ganglioside GD3 levels significantly upregulated in MBM compared to lymph node metastasis (LNM) but not for other melanoma gangliosides. Moreover, we demonstrated an upregulation of ST8SIA1 (GD3 synthase) as melanoma progresses from melanocytes to MBM cells. Using RNA‐ISH on FFPE specimens, we evaluated ST8SIA1 expression in primary melanomas (PRM) (n = 23), LNM and visceral metastasis (n = 45), and MBM (n = 39). ST8SIA1 was significantly enhanced in MBM compared to all other specimens. ST8SIA1 expression was assessed in clinically well‐annotated melanoma patients from multicenters with AJCC stage III B‐D LNM (n = 58) with 14‐year follow‐up. High ST8SIA1 expression was significantly associated with poor overall survival (HR = 3.24; 95% CI, 1.19–8.86, P = 0.02). In a nude mouse human xenograft melanoma brain metastasis model, MBM variants had higher ST8SIA1 expression than their respective cutaneous melanoma variants. Elevated ST8SIA1 expression enhances levels of cell surface GD3, a phenotype that favors MBM development, hence associated with very poor prognosis. Functional assays demonstrated that ST8SIA1 overexpression enhanced cell proliferation and colony formation, whereby ST8SIA1 knockdown had opposite effects. Icaritin a plant‐derived phytoestrogen treatment significantly inhibited cell growth in high GD3‐positive MBM cells through targeting the canonical NFκB pathway. The study demonstrates GD3 phenotype associates with melanoma progression and poor outcome., We demonstrated the upregulation of a ganglioside synthase, ST8SIA1 and GD3 expression in MBM, which can be suppressed by icaritin treatment. Icaritin inhibits NFκB‐mediated activation of ST8SIA1 by downregulating p50. These findings suggest that ST8SIA1 is a significant factor and potential target to MBM.

International audience; We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.

V600E being the most common mutation in BRAF, leads to constitutive activation of the MAPK signaling pathway. The majority of V600E BRAF positive melanoma patients treated with the BRAF inhibitor vemurafenib showed initial good clinical responses but relapsed due to acquired resistance to the drug. The aim of the present study was to identify possible biomarkers associated with the emergence of drug resistant melanoma cells. To this end we analyzed the differential gene expression of vemurafenib-sensitive and vemurafenib resistant brain and lung metastasizing melanoma cells. The major finding of this study is that the in vitro induction of vemurafenib resistance in melanoma cells is associated with an increased malignancy phenotype of these cells. Resistant cells expressed higher levels of genes coding for cancer stem cell markers (JARID1B, CD271 and Fibronectin) as well as genes involved in drug resistance (ABCG2), cell invasion and promotion of metastasis (MMP-1 and MMP-2). We also showed that drug-resistant melanoma cells adhere better to and transmigrate more efficiently through lung endothelial cells than drug-sensitive cells. The former cells also alter their microenvironment in a different manner from that of drug-sensitive cells. Biomarkers and molecular mechanisms associated with drug resistance may serve as targets for therapy of drug-resistant cancer.

Tumor progression is a multistep process in which alterations in the expression of numerous gene products may give rise to highly malignant cellular variants. In the present study, we analyzed the differential expression of several genes in cellular variants of mammary adenocarcinomas with high or low malignancy potential, which originated in a common ancestor. To assess the generality of our findings, high and low malignancy variants were derived from two different mammary adenocarcinoma cell lines, namely DA3 and CSML cells. Of major importance is the fact that the differences between high- and low-malignancy variants observed in one system of mammary adenocarcinoma cells (DA3 cells) were identically reproduced in the other system of mammary adenocarcinoma cells (CSML cells). The high malignancy variants of tumors both DA3-high and CSML-high (previously called CSML-100), expressed higher levels of factors that induce monocyte migration than the low malignancy DA3-low and CSML-low (previously called CSML-0) variants. In addition, it was found that DA3-high and CSML-high cell variants expressed higher levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and matrix metalloproteinases (MMPs) than the low malignancy variants (DA3-low and CSML-low). These results suggest that MCP-1, IL-6 and MMPs potentially contribute to mammary adenocarcinoma progression and that their expression is regulated by a common pathway. The expression of MCP-1, IL-6 and MMPs in both DA3-high and CSML-high cells was up-regulated by tumor necrosis factor alpha (TNFalpha). The fact that TNFalpha exerted similar effects on the expression of these three factors in both cell systems raises the possibility of a coordinated co-regulation of tumor-promoting factors.

Selectin ligands are crucial components in the interaction between endothelial cells and extravasating cancer cells and, thus, play an important role in metastasis formation. Head-and-neck squamous cell carcinoma (HNSCC) variants expressing high levels of E48, a human Ly-6 protein (E48hi), expressed higher levels of the fucose-generating FX enzyme and of the fucosylated E-selectin ligand sLea than cells expressing low levels of E48 (E48lo). Signaling through E48 upregulated expression levels of these molecules in HNSCC. In this work, we provide further evidence supporting the E48-FX-sLea link by showing that FX antisense oligonucleotides reduced sLea expression levels in HNSCC. We also show that E48 may be causally involved in regulating expression levels in HNSCC of 2 additional enzymes involved in the biosynthesis of sLea, namely, ST-30 and FucTIII. Also, selectin-mediated adhesion of E48hi variants to activated HUVECs was significantly higher than that of E48lo variants. Transfection experiments utilizing sense or antisense E48 cDNA indicated that E48 may be causally involved in this adhesion. Chemokines are involved in the extravasation process of tumor cells. The release of chemoattractants from HNSCC variants differing in E48 expression was therefore analyzed. HNSCC did not release any chemoattractants but induced the release of such factors from HUVECs. Supernatants from E48hi variants were significantly more efficient than E48lo cells at inducing the release of chemoattractants from HUVECs. Transfection experiments indicated that E48 may be causally involved in the induction of chemoattractant release from HUVECs. Angiogenesis is an important manifestation of cancer-endothelium interactions. We therefore assayed for the presence of angiogenic factors in culture supernatants of HNSCC. Supernatants from E48lo variants contained significantly higher amounts of PDGF than E48hi cells. Transfection experiments indicated that E48 may be causally involved. Taken together, our results suggest that E48 controls important interaction parameters between HNSCC and endothelial cells.

By differential display we demonstrated that antibody-mediated ligation of the GPI-linked protein product of E48, a newly discovered human Ly-6 gene, up-regulates the expression of the FX enzyme in 3 lines of head and neck squamous carcinoma cells. FX is responsible for the last step in the synthesis of GDP-l-fucose. The up-regulation of FX was E48 ligand-specific. 22AWT head and neck squamous carcinoma cells expressing high levels of E48 expressed significantly higher levels of FX than the E48 antisense transfected 22AWT cells (8-3 cells). The former cells also expressed higher levels of two major fucosylated glycans (the selectin ligand, Sialyl Lewis a, and VIM-2) than the E48 antisense transfectants. Conversely, transfection of cells from the 14CWT line expressing very low levels of E48 with E48 cDNA caused an up-regulated expression of FX and of the two fucosylated glycans in the 14C-CMV16 transfectants. Moreover, the expression levels of Sialyl Lewis a was significantly up-regulated on HNSCC upon ligation of E48 by anti-E48 antibodies. The functional significance of the E48-mediated up-regulation of Sialyl Lewis a was demonstrated in rolling experiments on E-selectin bearing surfaces under physiological conditions of shear flow and on tumor necrosis factor α-activated human umbilical venous endothelial cells. Only high E48/FX/Sialyl Lewis a expressing 14C-CMV16 cells could roll on purified E-selectin or establish E-selectin dependent rolling on the activated human umbilical venous endothelial cells. Low E48/FX/Sialyl Lewis a expressing 14CWT cells did not roll. These results show that E48 controls the expression of the FX enzyme and of certain fucosylated E-selectin ligands by HNSCC. E48 may thus function as a key regulator of the adhesiveness of these tumor cells to inflamed vessel walls expressing E-selectin.

Tissue-specific leukocyte localization is a requirement for immune surveillance and plays a key role in the pathogenesis of various inflammatory diseases. Leukocyte adhesion to endothelial cells is crucial for their extravasation to sites of inflammation. Extravasating leukocytes migrate through the endothelial barrier by first adhering to the endothelial cell surface and then transmigrating through the endothelial cell layer. Adhesion to the endothelium usually requires a cascade of steps mediated by the selectins, leukocyte activating chemotactic factors, and endothelial activating proinflammatory cytokines as well as by integrins. These molecules act in concert and regulate the sequence of distinct steps. In order for cancer cells to form metastases at specific organ sites, pre metastatic cells must exit the vasculature, migrate into these sites and be able to survive and proliferate there. Molecules involved in transendothelial migration are therefore key players in metastasis formation. Does the extravasation of pre-metastatic tumor cells from blood vessels follow the pathway of leukocyte extravasation? The molecular basis of cancer cell extravasation is poorly understood. A comprehensive and thorough understanding of the mechanisms underlying these processes is an absolute requirement for the development of therapeutic modalities aimed to block or retard metastasis. This presentation is focused on the interactions between endothelial (E)-selectin with its ligands on colorectal cancer (CRC) cells. Fucosylated selectin ligands initiate the interaction between extravasating cells and endothelium via their binding to endothelial selectins. We will discuss 2 distinct aspects of such an interaction. The first is the regulation of CRC adhesive capacity to endothelial cells and CRC extravasation. We will demonstrate that the fucose-generating FX enzyme plays a key role in the biosynthesis of selectin ligands and in their interaction with selectins. The second aspect is the regulation of gene expression in CRC by signals delivered to such cells by E-selectin.

In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long noncoding RNAs in melanoma progression. We hypothesized that copy number alterations (CNAs) of intergenic nonprotein-coding domains could help identify long intergenic noncoding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 (cancer susceptibility candidate 15) lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and upregulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC (American Joint Committee on Cancer) stage III lymph node metastasis. Moreover, small interfering RNA (siRNA) knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma.