ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves

Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential.

BackgroundSeveral protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas.PurposeOur purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior.MethodsWe evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis.ResultsIn A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblotting, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070-10 and C1070-14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity.ConclusionThese findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines

Hypoxia and extracellular matrix remodeling

International audienceHypoxia regulates composition of both the vascular basement membrane (BM) and the extracellular matrix (ECM) by modulating deposition, cross-linking, posttranslational modifications, and rearrangement events but also degradation. Hypoxia-driven remodeling of the ECM includes highly temporally and spatially coordinated processes that eventually affect angiogenesis leading to blood vessel formation from existing blood vessels. Hypoxia thereby affects the mechanical properties of the vascular milieu as well as matricellular proteins expression and function and availability of angiogenesis-regulating growth factors such as vascular endothelial growth factor (VEGF). ECM composition and stiffness may be required for optimal VEGFR2 expression and vascular development in vitro and in vivo (Mammoto et al. Nature 2009), but how it might control signaling pathways such as VEGFR2 signaling is not fully appreciated yet. Thus, vascular BM and ECM composition affects vascular microenvironment architecture and interaction with angiogenic growth factors but also exerts mechanical forces controlled by physical interactions between vascular cells and the ECM that cooperate in regulating angiogenesis