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1.
Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell-matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.  相似文献   

2.
Previous studies show that aqueous garlic extract and its derivatives (e.g. S-allylcysteine [SAC]) prevent carcinogen-induced breast tumorigenesis. However, investigations testing the effect of SAC on later stages of breast tumorigenesis and/or metastasis have produced mixed results. Here we show that SAC significantly reduced anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose- and time-dependent fashion, and sub-lethal SAC-treatment altered mammary tumor cell adhesion and invasion through components of the extracellular matrix. We provide evidence to suggest increased expression of E-cadherin and reduced MMP-2 expression and activity are partially responsible for inhibition of mammary tumor cell invasion by SAC. Because E-cadherin and MMP-2 are important in cancer metastasis, these results suggest a link between SAC induction of E-cadherin and reduction of MMP2 activity with the inhibition of cell motility and invasion; thus providing evidence that events leading to breast cancer metastasis are repressed by sub-lethal SAC-treatment.  相似文献   

3.
E-cadherin, which has a widely acknowledged role in mediating calcium-dependent cell-cell adhesion between epithelial cells, also functions as a tumor suppressor. The ectodomain of human E-cadherin contains four potential Nglycosylation sites at Asn residues 554, 566, 618, and 633. We investigated the role of E-cadherin N-glycosylation in cell cycle progression by site-directed mutagenesis. We showed previously that all four potential N-glycosylation sites of E-cadherin w ere N-gly cosylated in human breast carcinoma MDA-MB-435 cells. Removal of N-glycan at Asn633 dramatically affected E-cadherin stability. In this study we showed that E-cadherin mutant missing N-glycans at AsnS54, Asn566 and Asn618 failed to induce cell cycle arrest in Gt phase and to suppress cell proliferation in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn554 and Asn566, but not at Asn618, seemed to be indispensable for E-cadherin-mediated suppression of cell cycle progression. Removal of N-glycans at either Asn554 or Asn566 of E-cadherin was accompanied with the activation of the extracellular signal-regulated protein kinase signaling pathway. After treatment with PD98059, an inhibitor of the extracellular signal-regulated protein kinase signaling pathway, wild-type E-cadherin transfected MDA-MB-435 and E-cadherin N-glycosylation-deficient mutant transfected MDA-MB-435 cells had equivalent numbers of cells in G1 phase. These rmdings implied that N-glycosylation might be crucial for E-cadherin-mediated suppression of cell cycle progression.  相似文献   

4.
E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.  相似文献   

5.
The calcium-dependent homophilic cell adhesion molecule E-cadherin typically connects epithelial cells. The extracellular portion of the mature transmembrane protein consists of five homologous domains. The four sequences linking these domains contain the structural amino acid motif DXXD that is thought to be involved in direct calcium binding. In gastric cancer patients mutations affecting this motif between the second and third domain are frequently seen. In order to determine the functional significance of similar sequence alterations with regard to their location, we analyzed single amino acid substitutions changing the DXXD motif to DXXA in each linker region according to a mutation found in gastric cancer (D370A). The cDNA sequences coding for DQND, DVLD and DVND were changed (D257A, D479A, D590A, respectively) and stably expressed in E-cadherin negative MDA-MB-435S mammary carcinoma cells. We found that the D257A and D370A mutations result in abnormal protein localization, changes in the actin cytoskeleton, markedly reduced homophilic cell adhesion, and altered cell morphology. Unexpectedly, the tumor-associated D370A mutation but not the D257A mutation induced increased cell motility. The D479A mutation only had slight functional consequences whereas cells expressing the D590A mutant did not differ from cells expressing the wild-type molecule. Although the putative calcium binding motif DXXD is located at repetitive positions in the extracellular portion of E-cadherin, our results indicate that it has different functions depending on the location. Remarkably, tumor cells select for mutations in the most critical domains resulting both in loss of function (decreased cell adhesion) and in gain of function (increased cell motility). Since multiple DXXD motifs are typically seen in other cadherins, our structure-function study is relevant for this gene family in general.  相似文献   

6.
Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.  相似文献   

7.
Metastatic cells exhibit considerable flexibility in their adhesive interactions with other cells or components of the extracellular matrix. This review will describe the involvement of specific adhesion receptors, extracellular matrix molecules and cell dissociating cytokines in the metastatic cascade. We will particularly focus on disturbance of intercellular adhesion as a prerequisite for the release of invasive cells from carcinomas. We suggest that cell dissociation in these tumours is accomplished by loss of function or expression of the epithelial cell adhesion molecule E-cadherin, and through the activity of cell motility factors such as the scatter factor.  相似文献   

8.
Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the alpha3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the alpha2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-alpha2beta1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-alpha3beta1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.  相似文献   

9.
Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of alpha(2), alpha(3), alpha(4), alpha(5), and beta(1) integrin subunits. Additionally, blocking antibodies to beta(1) integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.  相似文献   

10.
Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.  相似文献   

11.
CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.  相似文献   

12.
6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells   总被引:1,自引:0,他引:1  
Gingerol (Zingiber officinale Roscoe, Zingiberaceae) is one of the most frequently and heavily consumed dietary condiments throughout the world. The oleoresin from rhizomes of ginger contains [6]-gingerol (1-[4′-hydroxy-3′-methoxyphenyl]-5-hydroxy-3-decanone) and its homologs which are pungent ingredients that have been found to possess many interesting pharmacological and physiological activities, such as anti-inflammatory, antihepatotoxic and cardiotonic effects. However, the effects of [6]-gingerol on metastatic processes in breast cancer cells are not currently well known. Therefore, in this study, we examined the effects of [6]-gingerol on adhesion, invasion, motility, activity and the amount of MMP-2 or -9 in the MDA-MB-231 human breast cancer cell line. We cultured MDA-MB-231 cells in the presence of various concentrations of [6]-gingerol (0, 2.5, 5 and 10 μM). [6]-Gingerol had no effect on cell adhesion up to 5 μM, but resulted in a 16% reduction at 10 μM. Treatment of MDA-MB-231 cells with increasing concentrations of [6]-gingerol led to a concentration-dependent decrease in cell migration and motility. The activities of MMP-2 or MMP-9 in MDA-MB-231 cells were decreased by treatment with [6]-gingerol and occurred in a dose-dependent manner. The amount of MMP-2 protein was decreased in a dose-dependent manner, although there was no change in the MMP-9 protein levels following treatment with [6]-gingerol. MMP-2 and MMP-9 mRNA expression were decreased by [6]-gingerol treatment. In conclusion, we have shown that [6]-gingerol inhibits cell adhesion, invasion, motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer cell lines.  相似文献   

13.
The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.  相似文献   

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The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.  相似文献   

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Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-xL cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-xL-clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosis-resistance to cytokines was induced in 435 cells by Bcl-xL-expression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-xL and 435/Neo cells at 1:3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-xL cDNA. These results showed that 435/Bcl-xL cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-xL expression. We conclude that Bcl-xL expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorage-independent growth.  相似文献   

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