Overexpression of Hsp27 in a human melanoma cell line: regulation of E-cadherin, MUC18/MCAM, and plasminogen activator (PA) system

Hsp27 is considered a potential marker for cell differentiation in diverse tissues. Several aspects linked to the differentiation process and to the transition from high to low metastatic potential were analyzed in melanoma cells transfected with Hsp27. E-cadherin plays a central role in cell differentiation, migration, and normal development. Loss of expression or function of E-cadherin has been documented in a variety of human malignancies. We observed by fluorescence-activated cell sorter (FACS) as well as immunofluorescence (IF) analysis a pronounced expression of E-cadherin in Hsp27-transfected A375 melanoma cells compared with control melanoma cells. The expression of the adhesion molecule MUC18/MCAM correlates directly with the metastatic potential of melanoma cells. In contrast to wild-type and neotransfected melanoma cells, in Hsp27-transfected cells the expression of MUC18/MCAM could not be detected by FACS and IF analysis. The plasminogen activator (PA) system plays a central role in mediating extracellular proteolysis and also in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. Hsp27 transfectants revealed elevated messenger ribonucleic acid expression of the urokinase-type PA (uPA) and its inhibitor, PA inhibitor type 1, which might indicate a neutralization effect of the proteolytic activity of uPA. Control cells failed to express both these molecules. The influence of Hsp27 expression on uPA activity and the involvement of E-cadherin could be demonstrated by use of anti-E-cadherin-blocking antibody. Our data provide evidence for an inhibitory-regulatory role of Hsp27 in tumor progression as found in our system.     

MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.     

基质金属蛋白酶MMP-26能有效促进人绒毛膜上皮癌细胞JEG-3浸润能力

胚胎植入过程中,滋养层细胞浸润与肿瘤的迁移过程非常相似,但显著的区别在于前者是受严格调控的有节制的浸润,基质金属蛋白酶(MMPs)的许多成员在其中起重要的作用.MMP-26是近年来发现的MMPs家族的新成员,它在滋养层细胞中的作用所知甚少.利用国际常用的人滋养层细胞模型——人绒毛膜上皮癌细胞系(JEG-3)作为体外实验模型,探讨MMP-26在人滋养层细胞浸润调节中的作用.将含有MMP-26全长cDNA的pCR3.1质粒转染到JEG-3细胞中,获得过量表达MMP-26基因的稳定细胞系JEG-3/MMP-26;细胞浸润分析表明JEG/MMP-26细胞的浸润能力较母本细胞明显增强;RT-PCR和明胶酶谱分析显示JEG-3/MMP-26细胞中MMP-9的表达和分泌水平提高;双荧光免疫细胞化学进一步显示MMP-26和MMP-9蛋白在细胞中有共定位现象.上述结果表明MMP-26能有效促进人滋养层细胞浸润,其作用可能是通过与其他MMP分子(如MMP-9)的协调来实现的.     

Overexpression of Hsp25 in K1735 murine melanoma cells enhances susceptibility to natural killer cytotoxicity

In the present study we used a murine melanoma model to investigate the effect of the 25-kDa heat shock protein (Hsp25) on natural killer (NK) cytotoxicity. The melanoma lines K1735-C123 (low metastatic potential) and K1735-M2 (high metastatic potential) were transfected with hsp25 and a control plasmid. Highly purified interleukin (IL)-2-stimulated DX-5+ NK cells showed enhanced lysis of Hsp25-overexpressing K1735-C123 targets in comparison with controls. In contrast, there was no difference in susceptibility to lysis by purified IL-2-stimulated DX-5+ NK cells between Hsp25-overexpressing and control-transfected K1735-M2 targets. Fluorescence-activated cell sorter analysis revealed that Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic phenotype. Thus, surface localization of Hsp25 does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5+ NK cells in the low-metastatic murine melanoma model investigated.     

Hsp27-induced MMP-9 expression is influenced by the Src tyrosine protein kinase yes

The small heat shock protein hsp27 is associated with aggressive tumor behavior in certain subsets of breast cancer patients. Previously we demonstrated that hsp27 overexpression in breast cancer cells increased in vitro and in vivo invasiveness, suggesting that hsp27 influences the metastatic process. To investigate this role for hsp27, we have utilized MDA-MB-231 breast cancer cells that overexpress hsp27 and cDNA expression array technology. We demonstrate that hsp27 overexpression up-regulates MMP-9 expression and activity and down-regulates Yes expression. Furthermore, our results suggest that Yes may be involved in regulating MMP-9 expression, as well as in vitro invasion. Reconstitution of Yes expression by transfection into hsp27-overexpressing cells decreased MMP-9 expression, and increased in vitro invasiveness, abrogating the phenotype conferred by hsp27 overexpression. Therefore, our results provide a new potential mechanism by which hsp27 affects the metastatic cascade-through regulation of MMP-9 and Yes expression.     

Bcl-2 overexpression in melanoma cells increases tumor progression-associated properties and in vivo tumor growth

In this study, we demonstrated that bcl-2 overexpression in human melanoma cells consistently enhanced the activity of multiple metastasis-related proteinases, in vitro cell invasion, and in vivo tumor growth. In particular, by using the M14 parental cell line, the MN8 control clone, and two bcl-2 overexpressing derivatives, we found that bcl-2 overexpressing cells exposed to hypoxia, when compared to parental cells, expressed higher level of several metalloproteases (MMPs) such as MMP-2, MMP-7, MT1-MMP, and tissue inhibitors of metalloproteases-1 and -2. Moreover, bcl-2 overexpression in melanoma cells enhanced in vitro invasion on matrigel and, in vivo tumor growth. The more aggressive behavior of bcl-2 transfectants tumors is significantly associated to an increase in MMP-2 expression as well as in a more elevated microvessel density as compared to the parental line. Taken together, our data suggest that bcl-2 plays a pivotal role in the regulation of molecules associated with the migratory and invasive phenotype, contributing, in cooperation to hypoxia, to tumor progression.     

Suppression of basement membrane type IV collagen degradation and cell invasion in human melanoma cells expressing an antisense RNA for MMP-1

During progression from benign nevus to vertical growth phase melanoma, melanocytes acquire the ability to invade into the dermis. This process requires rupture of the basal lamina and dissolution of dermal type I collagen. Metastases-derived human melanoma MIM cells have an invasive ability in vitro which is dependent on metalloproteinases. In the present study we analysed the role of type I collagenase (MMP-1) in melanoma invasion using MIM cells in which the constitutive expression of MMP-1 was suppressed by stable transfection with a plasmid vector expressing a 777 bp antisense fragment of MMP-1 genomic DNA. Two clones were isolated in which MMP-1 mRNA expression was blocked by 90–96% with a corresponding loss in protein synthesis. In their morphological appearance and growth rate in vitro these cells were indistinguishable from wild type cells or control cells transfected with the same vector expressing the MMP-1 fragment in the sense orientation. Their mRNA and protein levels for type IV collagenase (MMP-2) were unchanged as assessed by Northern and Western blot analyses and by gelatin zymography. However, when the invasive ability of the cells was measured, we found that in addition to type I collagen, invasion through type IV collagen and a reconstituted, type IV collagen-containing basement membrane (Matrigel) were also significantly inhibited as compared to normal or sense-transfected cells. The results indicate that despite the presence of functional MMP-2, degradation of type IV collagen matrices by the melanoma cells was dependent on expression of MMP-1. © 1997 Elsevier Science B.V. All rights reserved.     

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Expression and Role of Integrins in Invasive Activity of Oncotransformed Fibroblasts Differing in Spontaneous Metastasizing

Four closely related lines of RSV-transformed Syrian hamster fibroblasts differing drastically in their spontaneous metastatic capacity were investigated for the surface expression of integrins, in vitro invasion, and production of MMP-2 collagenase. The highly metastasizing HET-SR-2SC-LNM cells differ from the lowly metastasizing parental HET-SR cells in a high level of the surface expression of the collagen-specific alpha1beta1, alpha2beta1, and alphavbeta3 integrins, a high invasive activity, and an increased production of MMP-2. The same properties are characteristic for the actively metastasizing cells of the independent HET-SR-1 line. The lowly metastasizing fibroblasts that are derived from HET-SR-2SC-LNM retain a high level of the expression of the alpha1beta1 and alpha2beta1 integrins, but, unlike the parental line, they exhibit a decreased expression of the alphavbeta3 integrin, invasion in Matrigel, and MMP-2 production. Substrate stimulation of the signal function of the collagen-specific integrins increases the production of MMP-2 by the metastatically active fibroblasts. Inhibition of the signal activity of the integrins by RGD-containing pentapeptide or by genistein reduces markedly in vitro invasion in Matrigel and MMP-2 production. The role of specific properties of the extracellular matrix surrounding tumor cells and of specific surface integrins expressed in these cells in developing of the malignant phenotype is discussed.     

Hsp25-induced radioresistance is associated with reduction of death by apoptosis: involvement of Bcl2 and the cell cycle

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.     

DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

Oct-3/4 promotes migration and invasion of glioblastoma cells

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct-3/4 expression, a self-renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct-3/4 in glioblastoma cell invasiveness. The present study established Oct-3/4-overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct-3/4 expressing-glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct-3/4 overexpression resulted in upregulated FAK and c-Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct-3/4 expressing-glioblastoma cells and associated with membrane ruffles during cell migration. Oct-3/4 expressing-cells exhibited increased MMP-13 mRNA expression and MMP-13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct-3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP-13 expression and altering integrin signaling. Therefore, Oct-3/4 might contribute to tumor promoting activity in glioblastomas.     

siRNA联合沉默MMP-9和FAK基因对小鼠黑色素瘤高转移细胞B16F10体外侵袭和迁移的影响

目的： 观察双基因联合干扰MMP-9和FAK对小鼠黑色素瘤高转移细胞B16F10体外侵袭、迁移能力的影响。方法：分别构建pGV102-MMP9-siRNA,pGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,实验分为空白对照组、Anti-MMP-9组,Anti-FAK组、Anti-MMP-9 &FAK组、阴性对照组。经G418筛选GFP+克隆,流式细胞仪分析阳性率,激光共聚焦观察转染后细胞形态,半定量RT-PCR检测各组B16F10细胞MMP-9和FAK基因的mRNA转录水平,Transwell侵袭、迁移实验测定各组B16F10细胞体外侵袭、迁移能力。结果： 经G418筛选,3个转染组阳性率分别为92.41±1.64%,95.72±0.21%,91.52±0.11%,且转染后细胞形态良好;与空白对照组相比,3个转染组的MMP-9,FAK mRNA转录水平下降明显（P<0.01）,迁移、侵袭能力明显降低（P<0.01）,但Anti-MMP-9 &FAK组细胞侵袭迁移能力显著低于Anti-MMP-9 组和Anti-FAK组（P<0.01）。结论： 相比单独沉默MMP-9 或FAK,联合沉默MMP-9 和FAK可明显降低小鼠黑色素瘤B16F10细胞体外迁移、侵袭能力。     

Pancreatic cancer cells invasiveness is mainly affected by interleukin-1beta not by transforming growth factor-beta1

BACKGROUND: We investigated in vitro whether IL-1beta and TGF-beta1 affect pancreatic cancer cell growth, adhesion to the extracellular matrix and Matrigel invasion. MATERIALS AND METHODS: Adhesion to fibronectin, laminin and type I collagen, and Matrigel invasion after stimulation with saline, IL-1beta and TGF-beta1 were evaluated using three primary and three metastatic pancreatic cancer cell lines. RESULTS: Extracellular matrix adhesion of control cells varied independently of the metastatic characteristics of the studied cell lines, whereas Matrigel invasion of control cells was partly correlated with the in vivo metastatic potential. IL-1beta did not influence extracellular matrix adhesion, whereas it significantly enhanced the invasiveness of three of the six cell lines. TGF-beta1 affected the adhesion of one cell line, and exerted contrasting effects on Matrigel invasion of different cell lines. CONCLUSIONS: IL-1beta enhances the invasive capacity of pancreatic cancer cells, whereas TGF-beta1 has paradoxical effects on pancreatic cancer cells; this makes it difficult to interfere with TGF-beta1 signaling in pancreatic cancer treatment.     

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.     

Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.     

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.