The membrane-targeted alkylphosphocholine erufosine interferes with survival signals from the extracellular matrix.

Integrin-dependent adhesion of tumor cells to extracellular matrix proteins provides anchorage-dependent protection from cell death. In the present investigation we aimed to understand whether and how the paradigmatic membrane-targeted synthetic phospholipid analog erufosine is relevant for tumor cell adhesion to extracellular matrix proteins, cell survival and migration. The antineoplastic action of erufosine was analyzed with glioblastoma and prostate cancer cells adhering to fibronectin or collagen I using proliferation, adhesion and migration assays. The composition of adhesion contacts containing activated ß1 integrins was studied using immunofluorescence. The importance of ß1 integrins for the observed effects was analyzed in fibroblasts proficient or deficient in ß1 integrin expression. Adhesion to collagen I and fibronectin increased the death threshold in serum-deprived tumor cells. Moreover, ß1 integrin-deficient cells were more sensitive to erufosine-treatment compared to ß1 integrin proficient cells suggesting a role of ß1 integrins for matrix-mediated death resistance. Most importantly, erufosine disturbed the maturation of the cell adhesion complexes containing paxillin, activated ß1 integrins and phosphorylated FAK, leading to a reduction of survival signals and inhibition of tumor cell adhesion and migration. These findings suggest that membrane-targeted synthetic phospholipids analogs may be of value for counteracting matrix-mediated treatment resistance in combined treatment approaches with radiotherapy or chemotherapy.

Migration of epithelial cells on laminins: RhoA antagonizes directionally persistent migration.

Spatial and temporal expression of laminin isoforms is assumed to provide specific local information to neighboring cells. Here, we report the remarkably selective presence of LM-111 at the very tip of hair follicles where LM-332 is absent, suggesting that epithelial cells lining the dermal-epidermal junction at this location may receive different signals from the two laminins. This hypothesis was tested in vitro by characterizing with functional and molecular assays the comportment of keratinocytes exposed to LM-111 and LM-332. The two laminins induced morphologically distinct focal adhesions, and LM-332, but not LM-111, elicited persistent migration of keratinocytes. The different impact on cellular behavior was associated with distinct activation patterns of Rho GTPases and other signaling intermediates. In particular, while LM-111 triggered a robust activation of Cdc42, LM-332 provoked a strong and sustained activation of FAK. Interestingly, activation of Rac1 was necessary but not sufficient to promote migration because there was no directed migration on LM-111 despite Rac1 activation. In contrast, RhoA antagonized directional migration, since silencing of RhoA by RNA interference boosted unidirectional migration on LM-332. Molecular analysis of the role of RhoA strongly suggested that the mechanisms involve disassembly of cell-cell contacts, loss of the cortical actin network, mobilization of α6ß4 integrin out of stable adhesions, and displacement of the integrin from its association with the insoluble pool of intermediate filaments.

Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cells.

Laminin-binding integrins form a complex with CD151, a member of the tetraspanin family suggested to be involved in the regulation of cell migration. In the epidermis, CD151 is localized with alpha3beta1 and alpha6beta4 integrins at cell-cell and cell-matrix contacts, respectively, characteristic structures of non-migrating cells. Taking advantage of a monoclonal antibody against CD151, TS151r, which epitope overlaps with the tetraspanin integrin-binding site, we have investigated the role of CD151 in epithelial cell migration. Under standard culture conditions, the migratory capacity of epithelial HaCaT cells on laminins is low, apparently due to endogenous laminin 5. However, challenging HaCaT cells with TS151r allows a re-arrangement of the actin cytoskeleton, dismantling of cell-cell and beta4 integrin-mediated cell-matrix contacts and cell migration. In vivo, free CD151 is absent in resting epithelial cells of interfollicular epidermis, and all CD151 is bound to integrins in intercellular and cell-matrix contacts. By contrast, free CD151 is present at intercellular contacts in the epithelial sheet lining the deeper region of anagen hair follicles, which is considered to contain migrating cells. Together, these results strongly suggest that dissociation of the CD151-integrin complex permits remodeling of epithelial cell interactions with the extracellular matrix and cell migration.

Keratinocytes from patients lacking collagen XVII display a migratory phenotype.

Acquired or inherited junctional epidermolysis bullosa are skin diseases characterized by a separation between the epidermis and the dermis. In inherited nonlethal junctional epidermolysis bullosa, genetic analysis has identified mutations in the COL17A1 gene coding for the transmembrane collagen XVII whereas patients with acquired diseases have autoantibodies against this protein. This suggests that collagen XVII participates in the adhesion of basal keratinocytes to the extracellular matrix. To test this hypothesis, we studied the behavior of keratinocytes with null mutations in the COL17A1 gene. Initial adhesion of mutant cells to laminin 5 was comparable to controls and similarly dependent on alpha3beta1 integrins. The spreading of mutant cells was, however, enhanced, suggesting a propensity to migrate, which was confirmed by migration assays. In addition, laminin 5 deposited by collagen XVII-deficient keratinocytes was scattered and poorly organized, suggesting that correct integration of laminin 5 within the matrix requires collagen XVII. This assumption was supported by the co-distribution of the two proteins in the matrix of normal human keratinocytes and by protein-protein-binding assays showing that the C-terminus of collagen XVII binds to laminin 5. Together, the results unravel an unexpected role of collagen XVII in the regulation of keratinocyte migration.