Tumor cell adhesion to the extracellular matrix and signal transduction mechanisms implicated in tumor cell motility,invasion and metastasis

Summary The relationship between tumor cell adhesion to the extracellular matrix (ECM) and invasion and metastasis formation is one of the most intensively studied topics in cancer biology within the last 10–15 years. The aberrant molecular relationships between malignant tumor cells and their surrounding ECM have been implicated at virtually every stage of the metastatic process; ranging from steps that involve the local invasion of tumor cells away from the primary tumor to those that are involved in mediating extravasation through microvessel-associated basement membranes at the site(s) of metastasis formation. The complexity of tumor metastasis has required that a reductionist approach be taken in order to identify and relate specific molecular mechanisms involved in tumor cell adhesion to various aspects of tumor metastasis. The intensive research efforts into cell adhesion and tumor cell biology have generated many significant new concepts towards our understanding of the molecular aspects of tumor cell adhesion and metastasis. Our purpose in this article is to briefly summarize the relationship of ECM-stimulated tumor cell adhesion to the processes of tumor cell motility and invasion. This is followed by a discussion of certain aspects of signal transduction pathways that may impact on cell motility, with an emphasis on the relationship between phosphatidylinositol hydrolysis and actin polymerization, as well as certain GTP-binding protein-(G-protein) mediated events that could influence cytoskeletal organization and cell motility. Our emphasis is based on increasing evidence that implicates members of the signal transduction G-proteins in the motility and invasion of many normal and transformed cells.     

Over-expression of cyclin D1 induces glioma invasion by increasing matrix metalloproteinase activity and cell motility.

In order to define the role of cyclin D1 in the progression of malignant glioma, cells over-expressing cyclin D1 were constructed (a-1 cells). They exhibited significantly increased invasiveness as compared with mock-transfected cells. Since cellular invasion is thought to depend on extracellular-matrix degradation, we determined whether cyclin-D1 expression modifies the activity of matrix metalloproteinases (MMPs). Increased gelatinolytic activity of latent type MMP-2 (proMMP-2) and active MMP-2 was observed in a-1 cells. Moreover, cyclin-D1 expression was associated with increased activation of proMMP-9 through MMP-3. Wound assays showed an increase of cell motility in a-1 cells. Cyclin-D1 expression was found to be associated with up-regulation of Rac1, which modulates the formation of ruffling membranes and cell motility. Our results show that cyclin D1 may modulate invasive ability by increasing MMP activity and cell motility, and suggests a novel function of cyclin D1 in the progression of malignant gliomas.     

Tumor invasion and host extracellular matrix

In this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.     

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion.

Meningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells.     

Modulation of neuroblastoma cell differentiation by the extracellular matrix.

The effect of a complex in vitro synthesized extracellular matrix (ECM) and its components on growth and phenotypical differentiation of a human neuroblastoma (NB) cell line (HTLA230) was investigated. Rat smooth-muscle-cell (R22CIF)-derived ECM composed of collagen, glycoproteins, and glycosaminoglycans (GAGs) promoted spontaneous neurite outgrowth of HTLA230 cells but did not alter their growth kinetic or cloning efficiency as compared with cells seeded onto gelatin-coated dishes. The matrix significantly enhanced, quantitatively and qualitatively, the responsiveness of HTLA230 cells to retinoic acid (RA), and a substantially reduced growth rate was observed in the presence of RA with cells grown on the ECM. Biochemical modification of the composition of the R22CIF-matrix by trypsin digestion and/or high-salt extraction (4 M guanidinium) demonstrated that the ratio of chondroitin sulfate to hyaluronic acid (HA) present in the ECM determines the capacity of the matrix to promote NB differentiation. A human fibroblast (T-1)-derived ECM, which has a biochemical composition of the GAG component similar to that of the trypsinized R22CIF-matrix, but which has a high amount of glycoproteins, confirmed these results. Nerve-growth-factor (NGF)-induced differentiation in a variant HTLA 230 cell line was inhibited when cells were grown on an ECM with a low ratio of chondroitin sulfate/HA. The composition of the ECM thus modulates the responsiveness to various differentiation-inducing agents and alters the phenotype of NB cells.     

Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors.

The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.     

Tumor invasion and extracellular matrix degradative enzymes: regulation of activity by organ factors.

Degradation of the extracellular matrix (ECM) during cellular invasion is important to the formation of tumor metastasis. Several types of ECM degradative enzymes have been implicated in metastatic invasion by malignant cells. These enzymes are regulated by various factors which vary qualitatively and quantitatively between organs. Tissue and plasma activators of zymogens, enzyme inhibitors, and cell surface enzyme receptors directly control localized enzyme activities, and the expression of enzymes is modulated by various cytokines, growth factors and ECM components. Thus, the interaction of tumor cells with an organ environment affects the metastatic behavior of the cells. This organ factor regulation is illustrated using human colon carcinoma cells implanted in different organ sites of nude mice. The cells growing in the cecal wall produced high levels of ECM degradative enzymes and metastasized to lymph nodes and liver. In contrast, enzyme production was repressed in colon carcinoma tumors growing in the subcutis and no metastases were formed.     

Inhibition of cyclooxygenase-2 decreases breast cancer cell motility, invasion and matrix metalloproteinase expression

Background  

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the formation of prostaglandins. The inducible isoform of COX (COX-2) is highly expressed in aggressive metastatic breast cancers and may play a critical role in cancer progression (i.e. growth and metastasis). However, the exact mechanism(s) for COX-2-enhanced metastasis has yet to be clearly defined. It is well established that one of the direct results of COX-2 action is increased prostaglandin production, especially prostaglandin E₂ (PGE₂). Here, we correlate the inhibition of COX-2 activity with decreased breast cancer cell proliferation, migration, invasion and matrix metalloproteinase (MMP) expression.     

Colonial morphology of tumour cells and susceptibility to cytolysis by tumour necrosis factor. The role of cellular fibronectin deposition in the extracellular matrix

The tumour cell lines U937A and L929 form large, loosely packed colonies in vitro and can be killed by the cytokine tumour necrosis factor (TNF). In contrast, their TNF-resistant mutants U937A/R and L929/R form tightly packed colonies. Since cells which form loose colonies have increased metastatic potential it is important to understand the factors governing colonial morphology. To this end, we have compared the extracellular matrices (ECMs) of the 'loose' lines, U937A and L929 with their 'tight' mutants. By immunofluorescence, a polyvalent anti-U937A serum revealed a fibrillar network in the ECMs of the 'loose' lines which was absent in the 'tight'. On Western blotting of ECMs the antiserum detected an additional 300 kDa protein in the 'loose' lines which was subsequently shown to be cellular fibronectin. The four lines secreted comparable amounts of fibronectin and this was qualitatively indistinguishable between 'loose' and 'tight' cells by peptide mapping or lectin binding. It is concluded that the differences in colonial morphology are due to the 'tight' mutants' inability to incorporate fibronectin into the ECM.     

Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer.

There is increasing evidence that urokinase secreted by tumor cells can be bound to a cell surface receptor retaining its full potential to activate plasminogen and subsequently cleave basement membrane constituents. This study was undertaken to discriminate between soluble and cell surface bound urokinase as a potential mediator of in vitro invasion by cultured colon cancer. Extracellular matrix invasion by a colon cancer cell line GEO, characterized as being a poor secretor of urokinase and having few receptors (less than 10(4) receptors/cell) was not augmented when these cells were made to secrete up to 8 times as much urokinase, in response to an exogenous urokinase gene driven by the Rous sarcoma virus long terminal repeat promoter. The majority of the plasminogen activator (greater than 95%) appeared in the culture medium, this reflecting the low numbers of binding sites displayed by GEO cells. In contrast, the cell line HCT 116 equipped with 10 times as many binding sites, (greater than 10(5)/cell), the majority of which are occupied with endogenous ligand, was an efficient invader of the extracellular matrix. Inhibition of urokinase binding to the cell surface receptors using an antibody to the A chain of the plasminogen activator reduced invasion by 65%. The cell line RKO is equipped with 3 x 10(5) receptors/cell, 15% of which are tagged with endogenous urokinase. Pretreatment of these cells with a concentration range of urokinase known to result in the majority of these binding sites being charged with the plasminogen activator led to a dose dependent increase in extracellular matrix invasion. Together, these data suggest that for cultured colon cancer, at least, invasion is a function of the amount of cell surface receptor bound urokinase.     

Development of anin vitro extracellular matrix assay for studies of brain tumor cell invasion

Summary Invasion of brain by tumor cells is an inherent feature of the malignant phenotype. Assays to quantitate invasiveness should provide a powerful tool to investigate this phenomenon. We have developed a modifiedin vitro assay to measure tumor cell invasion, attachment, and chemotaxis using a barrier of the complex basement membrane Matrigel on gelatin-coated filters. Within 5 hours, 7.8% of U251MGp and 2.6% of SF126 human malignant glioma cells invaded the Matrigel and filter, compared with 0.8% of normal human leptomeningeal cells. The extent of invasion was directly proportional to incubation time and filter pore size and inversely proportional to the Matrigel concentration. Cells from exponentially growing U251MG_p cultures invaded more readily (10.9%) than cells from plateau-phase cultures (2.3%); however, labeling studies with bromodeoxyuridine showed that quiescent cells and rapidly dividing cells were equally capable of invading. This suggests that the mechanisms underlying invasion by malignant glioma cells are distinct from those underlying proliferation and indicates the need for therapy aimed specifically at invasive behavior. In a practical application of this assay to test a potential anti-invasive strategy, monoclonal antibodies to the subunit of an integrin receptor mediating attachment to the extracellular matrix inhibited invasion by U251MG_p cells in a dose-dependent manner. This assay should allow evaluation of the cellular and molecular basis of brain tumor progression and perhaps aid the development of rationally designed drugs that limit tumor invasion. It may also allow prediction of the clinical behavior of neoplasms in individual patients.     

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.     

Relationship between extracellular matrix interactions and degree of differentiation in human colon carcinoma cell lines

Human colon carcinoma cell lines that vary in their degree of differentiation were examined for their ability to interact with extracellular matrix components. For this purpose, established cell lines were classified on the basis of several criteria that relate to degree of differentiation. These criteria include histology of the original tumor, histology of xenografts, in vitro morphology, and carcinoembryonic antigen expression. On this basis, the cell lines used were either moderately well or poorly differentiated. The poorly differentiated cell lines adhered to surfaces coated with laminin or reconstituted basement membrane extract (Matrigel) to a significantly greater extent than the moderately well differentiated lines with the exception of one moderately well differentiated line that was derived from a highly aggressive signet ring cell carcinoma. In addition, the poorly differentiated cell lines exhibited considerable spreading on laminin and Matrigel after adherence that was not evident for the moderately well differentiated lines. The adherence of these cell lines on fibronectin-coated surfaces did not correlate as well with differentiation although, in general, poorly differentiated cell lines adhered better than moderately well differentiated lines. None of the cells that adhered to fibronectin exhibited the extensive spreading seen on laminin. The specificity of tumor cell interactions with extracellular matrix glycoproteins was examined using synthetic peptides which correspond to sequences within these proteins that are recognized by cell surface receptors. The pentapeptide YIGSR-NH2 significantly inhibited the adherence and spreading of the tumor cell lines on laminin, but not on fibronectin. The peptide RGDS, however, did not inhibit tumor cell interactions with laminin although it did inhibit their interactions with fibronectin. Thus, the interactions of colon carcinoma cells with laminin and fibronectin are probably mediated by separate receptors. Taken together, the data demonstrate that cells derived from colon carcinomas exhibit considerable variation in their ability to interact with extracellular matrix components, and that this variability is related to the degree of differentiation of original tumor.     

By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility,invasion and lung colonization

Interstitial fluid flow and associated shear stress are relevant mechanical signals in cartilage and bone (patho)physiology. However, their effects on chondrosarcoma cell motility, invasion and metastasis have yet to be delineated. Using human SW1353, HS.819.T and CH2879 chondrosarcoma cell lines as model systems, we found that fluid shear stress induces the accumulation of cyclic AMP (cAMP) and interleukin-1β (IL-1β), which in turn markedly enhance chondrosarcoma cell motility and invasion via the induction of matrix metalloproteinase-7 (MMP-7). Specifically, shear-induced cAMP and IL-1β activate PI3-K, ERK1/2 and p38 signaling pathways, which lead to the synthesis of MMP-7 via transactivating NF-κB and c-Jun in human chondrosarcoma cells. Importantly, MMP-7 upregulation in response to shear stress exposure has the ability to promote lung colonization of chondrosarcomas in vivo. These findings offer a better understanding of the mechanisms underlying MMP-7 activation in shear-stimulated chondrosarcoma cells, and provide insights on designing new therapeutic strategies to interfere with chondrosarcoma invasion and metastasis.     

Quantitative in vitro assay for tumor cell invasion through extracellular matrix or into protein gels

A new quantitative method for the study of tumor cell invasion in vitro is presented. It is intended to facilitate the study of the mechanisms of invasion using an isolated basement membrane without the involvement of stromal structures or using defined protein gels. Cells are allowed to migrate through the pores of a Nuclepore polycarbonate filter into a protein gel on a nitrocellulose filter, or they may have to penetrate a cell-derived extracellular matrix (ECM) to reach the gel. Experiments with a nonmetastatic mouse lymphoma (Eb) and its two metastatic variants (ESb and ESb-MP) showed that the metastatic lines penetrated a Matrigel (a gel containing the components of a basement membrane) much better than the nonmetastatic cell line, but only the most metastatic line (ESb) was able to penetrate into a native collagen I gel. The presence of an ECM on the polycarbonate filter reduced the number of cells invading a fibrin gel, demonstrating that the dense, fibrillar structure of the cell-derived ECM was a barrier to the tumor cells. The metastatic lines penetrated the ECM to a 4- to 6-fold higher extent than the nonmetastatic cell line. It is concluded that in order to metastasize efficiently, the tumor cells must be able to penetrate many different kinds of barriers.     

Regulation of breast cancer cell motility by T-cell lymphoma invasion and metastasis-inducing protein

Introduction  

T-cell lymphoma invasion and metastasis-inducing protein (Tiam1) is an Ras-related C3 botulinum toxin substrate (Rac)-specific guanine nucleotide exchange factor that was isolated based on its ability to induce a metastatic phenotype. In polarized migrating keratinocytes, Tiam1 is found at the leading edge, where it cooperates with the protease-activated receptor 1 (Par1) complex to establish front-to-rear polarity. Although a positive correlation has been observed between Tiam1 expression and tumor grade in a variety of human malignancies, including breast, its role in breast cancer cells has not yet been examined.     

Induction of matrix metalloproteinase-1 and glioma cell motility by nitric oxide

High grade gliomas invariably recur due in a large part to tumor cells permeating normal brain in an inaccessible, diffuse manner. Previous work demonstrates that the expression of matrix metalloproteinases (MMP) contributes to this characteristic. Not only can MMPs assist a cell in traversing its environment by clearing extracellular matrix molecules, but they can also impact non-traditional downstream signals that affect a cell’s ability to interact and respond to its surroundings. Contributions to the induction of MMP expression and functional significance in glioma are still under investigation. Evidence in other cancer settings indicates that nitric oxide (NO) may play a role in tumor/cell progression that can influence MMP production. Matrix metalloproteinase-1 (MMP-1), also known as interstitial collagenase, and the constitutive nitric oxide synthases (NOS) have been shown to be over-expressed in high grade gliomas. In the current study we investigated the potential involvements of NO with regard to MMP-1 and functional glioma cell movement. With the treatment of the NO donor sodium nitroprusside (SNP), there was significant induction of MMP-1 mRNA, secreted MMP-1 protein and motility of glioma cell lines within 48 h. RNA inhibition of MMP-1 through transient transfection of three MMP-1 specific siRNAs revealed a marked abrogation of the NO-mediated induction of motility. In addition, application of the NOS inhibitor N_ω-Nitro-l-arginine methyl ester (l-NAME) impaired movement of glioma cells. These data provide evidence for a regulatory axis of high grade glioma cell movement from NO through MMP-1, with NOS inhibitor results showing promise for future pharmacologic investigation.     

Autocrine motility factor and the extracellular matrix. I. Coordinate regulation of melanoma cell adhesion,spreading and migration involves focal contact reorganization

Cellular interactions with the extracellular matrix are complex and are involved in numerous biological processes. These interactions may be modulated by cytokines such as tumor cell autocrine motility factor, a secreted molecule that regulates cellular growth and migration by a receptor-mediated pathway. In this report we provide evidence suggesting that high- and low-metastatic K1735 melanoma cells coordinate their attachment, spreading and migratory responses to autocrine motility factor and the extracellular substratum through alterations in focal adhesion plaque architecture that are dependent on both inherent cellular metastatic phenotype and the composition of the supporting matrix. Thus, since activation of the autocrine motility factor receptor has previously been shown to enhance the experimental metastasis of the high- but not the low-metastatic K1735 cells, differences in melanoma cell malignancy in this system may be due in part to a coordinated interplay between cytokine-mediated responses and extracellular matrix-directed regulation of cellular adhesion, spreading and motility. Int. J. Cancer 76:120–128, 1998.© 1998 Wiley-Liss, Inc.