Cell adhesion in tumor invasion and metastasis: loss of the glue is not enough.

Tumor cells often show a decrease in cell-cell and/or cell-matrix adhesion. An increasing body of evidence indicates that this reduction in cell adhesion correlates with tumor invasion and metastasis. Two main groups of adhesion molecules, cadherins and CAMs, have been implicated in tumor malignancy. However, the specific role that these proteins play in the context of tumor progression remains to be elucidated. In this review, we discuss recent data pointing to a causal relationship between the loss of cell adhesion molecules and tumor progression. In addition, the direct involvement of these molecules in specific signal transduction pathways will be considered, with particular emphasis on the alterations of such pathways in transformed cells. Finally, we review recent observations on the molecular mechanisms underlying metastatic dissemination. In many cases, spreading of tumor cells from the primary site to distant organs has been characterized as an active process involving the loss of cell-cell adhesion and gain of invasive properties. On the other hand, various examples of metastases exhibiting a relatively benign (i.e. not invasive) phenotype have been reported. Together with our recent results on a mouse tumor model, these findings indicate that 'passive' metastatic dissemination can occur, in particular as a consequence of impaired cell-matrix adhesion and of tumor tissue disaggregation.     

The role of adhesion molecules and chemokine receptor CXCR4 (CD184) in small cell lung cancer

Small-cell lung cancer (SCLC) is a particularly aggressive form of lung cancer. Responsible for this highly malignant phenotype is an early and widespread metastasis with a high propensity of SCLC cells for bone marrow involvement and the ability to develop resistance against chemotherapeutic agents. Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and adhesion molecules. There is growing evidence that the chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 (CD184) regulate migration and metastasis of a variety of cancers including SCLC. SCLC cells express high levels of functional CXCR4 receptors. Engagement of CXCR4 by CXCL12 leads to an upregulation of integrin-mediated adhesion in SCLC and other tumor cells. Activation of CXCR4 chemokine receptors and integrins on SCLC cells promotes adhesion to accessory cells (such as stromal cells) and extracellular matrix molecules within the tumor microenvironment. These adhesive interactions result in an increased resistance of SCLC cells to chemotherapy. As such, inhibitors of the CXCR4/CXCL12 axis and/or integrin activation may increase the chemosensitivity of SCLC cells and lead to new therapeutic avenues for patients with SCLC.     

RIFLE: a novel ring zinc finger-leucine-rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.     

Changing neighbours,changing behaviour: cell adhesion molecule-mediated signalling during tumour progression

Changes in cell-cell and cell-matrix adhesion accompany the transition from benign tumours to invasive, malignant cancer and the subsequent metastatic dissemination of tumour cells. This review discusses a possible role of cell adhesion molecules not only in redirecting a tumour cell's adhesive capabilities but also in modulating intracellular signalling, and with it, tumour malignancy.     

The impact of adhesion on cellular invasion processes in cancer and development

In this paper we consider a simple continuous model to describe cell invasion, incorporating the effects of both cell-cell adhesion and cell-matrix adhesion, along with cell growth and proteolysis by cells of the surrounding extracellular matrix (ECM). We demonstrate that the model is capable of supporting both noninvasive and invasive tumour growth according to the relative strength of cell-cell to cell-matrix adhesion. Specifically, for sufficiently strong cell-matrix adhesion and/or sufficiently weak cell-cell adhesion, degradation of the surrounding ECM accompanied by cell-matrix adhesion pulls the cells into the surrounding ECM. We investigate the criticality of matrix heterogeneity on shaping invasion, demonstrating that a highly heterogeneous ECM can result in a “fingering” of the invasive front, echoing observations in real-life invasion processes ranging from malignant tumour growth to neural crest migration during embryonic development.     

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.     

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.     

结肠腺瘤息肉蛋白突变经PI3K/AKT 信号通路影响犬肾细胞MDCK的细胞粘附

结肠腺瘤息肉蛋白（APC）是一个肿瘤抑制因子,它不仅参与Wnt信号通路的传导,而且对细胞粘附、细胞骨架的组织和迁移等都有影响.APC突变发生于大多数结肠癌中.为了探讨APC突变对细胞粘附的影响及机制,本研究利用细胞粘附实验分析了MDCK-APC-N1和对照MDCK-GFP稳定表达细胞株系的细胞粘附情况.实验结果显示,在MDCK细胞中过表达APC-N1导致细胞-细胞间的粘附减少,细胞-基质间的粘附增加.荧光定量PCR和Western印迹实验表明,在MDCK-APC+N1细胞中,E-cadherin表达水平降低,CD29、P-FAK (Y397)、β-catenin和 P-AKT (T308)表达水平升高. 在MDCK-APC-N1细胞中,敲减β-catenin导致E-cadherin表达量升高,而CD29表达没有明显变化.进一步利用PI3K抑制剂LY294002处理MDCK-APC-N1细胞,结果发现,E-cadherin表达量明显升高,CD29表达量明显降低.这些结果揭示,APC-N1可活化 PI3K/AKT 信号通路,进而改变粘附蛋白E-cadherin和CD29影响细胞粘附.     

CD44 in Cancer Progression: Adhesion, Migration and Growth Regulation

It is well established that the large array of functions that a tumour cell has to fulfil to settle as a metastasis in a distant organ requires cooperative activities between the tumour and the surrounding tissue and that several classes of molecules are involved, such as cell-cell and cell-matrix adhesion molecules and matrix degrading enzymes, to name only a few. Furthermore, metastasis formation requires concerted activities between tumour cells and surrounding cells as well as matrix elements and possibly concerted activities between individual molecules of the tumour cell itself. Adhesion molecules have originally been thought to be essential for the formation of multicellular organisms and to tether cells to the extracellular matrix or to neighbouring cells. CD44 transmembrane glycoproteins belong to the families of adhesion molecules and have originally been described to mediate lymphocyte homing to peripheral lymphoid tissues. It was soon recognized that the molecules, under selective conditions, may suffice to initiate metastatic spread of tumour cells. The question remained as to how a single adhesion molecule can fulfil that task. This review outlines that adhesion is by no means a passive task. Rather, ligand binding, as exemplified for CD44 and other similar adhesion molecules, initiates a cascade of events that can be started by adherence to the extracellular matrix. This leads to activation of the molecule itself, binding to additional ligands, such as growth factors and matrix degrading enzymes, complex formation with additional transmembrane molecules and association with cytoskeletal elements and signal transducing molecules. Thus, through the interplay of CD44 with its ligands and associating molecules CD44 modulates adhesiveness, motility, matrix degradation, proliferation and cell survival, features that together may well allow a tumour cell to proceed through all steps of the metastatic cascade.     

Cell-cell and cell-matrix adhesion molecules in human heart and lung transplants

The interaction of immune cells with endothelial and target cells and extracellular matrix in human organ transplants is regulated by a number of receptor-ligand molecules. The molecules mediating intercellular adhesion and activation are classified as integrin, immunoglobulin and selectin families. In the present study the patterns of their cellular expression in human heart and lung transplants are described in normal state and during transplant rejection. The results reveal an organ specific regulation of the different adhesion molecules during transplant rejection. Specific differences were noted in the endothelial expression of vascular ligand molecules in the vascular segments of heart and lung transplants, especially in the lung capillaries. Cell type specific patterns of intercellular and cell-matrix adhesion molecules as their ligands were found in different states of graft rejection. Intravascular and interstitial differences in the expression patterns of leukocyte adhesion receptors support a concept of their stepwise function during graft infiltration. The implications for the organ specific appearance of inflammatory reactions in human heart and lung transplants as for immunosuppressive therapy are discussed.     

The echinoderm adhesome

Although the development of sea urchin embryos has been studied extensively and clearly involves both cell adhesion and cell migration, rather little is known about the adhesion receptors and extracellular matrix molecules involved. The completion of the genome of Strongylocentrotus purpuratus allows a comprehensive survey of the complement of cell-cell and cell-matrix adhesion molecules in this organism. Furthermore, the phylogenetic position of echinoderms offers the opportunity to compare the complement of adhesion proteins between protostome and deuterostome invertebrates and between invertebrate and vertebrate deuterostomes. Many aspects of development and cell interactions differ among these different taxa and it is likely that analysis of the spectrum of adhesion receptors and extracellular matrix proteins can open up new insights into which molecules have evolved to suit particular developmental processes. In this paper, we report the results of an initial analysis along these lines. The echinoderm adhesome (complement of adhesion-related genes/proteins) is similar overall to that of other invertebrates although there are significant deuterostome-specific innovations and some interesting features previously thought to be chordate or vertebrate specific.     

Suppression of Mouse Melanoma Metastasis by EA-1, A Monoclonal Antibody Specific for α6 Integrins

The rat monoclonal antibody (mAb) termed EA-1 was originally selected for its capacity to block the adhesion of T lymphocyte progenitors to mouse thymic endothelium. Here we show that the mAb EA-1 recognizes the α6 chain of α6β1 and α6β4 integrins. Both molecules are present at a high level on the luminal and basolateral side of vascular endothelium and α6β1 integrin is expressed on the highly metastatic cell lines B16/129 (melanoma) and KLN-205 (carcinoma). These lung specific tumors bind preferentially to lung frozen sections, and EA-1 blocked this interaction in vitro. Moreover, mAb EA-1 inhibited experimental metastasis to the lung of B16/129 cells injected intravenously. Metastasis in vivo was blocked when the antibody was injected into mice before or simultaneously with the melanoma cells, as well as when melanoma cells were precoated with EA-1 before injection. We suggest that α6 integrins play a dual role in the metastatic process, mediating the adhesion of tumor cells to the luminal surface of the endothelium and the adhesion to laminin in the subendothelial extracellular matrix during extravasation. Despite the fact that α6 integrins are laminin receptors, EA-1 did not interfere with melanoma cell binding to laminin fragments. Our antibody EA-1 may therefore recognize a binding domain on α6 integrins of a novel ligand involved in cell-cell interaction.     

Molecular mechanisms of tumor invasion and metastasis: an integrated view

As tumors progress to increased malignancy, cells within them develop the ability to invade into surrounding normal tissues and through tissue boundaries to form new growths (metastases) at sites distinct from the primary tumor. The molecular mechanisms involved in this process are incompletely understood but those associated with cell-cell and cell-matrix adhesion, with the degradation of extracellular matrix, and with the initiation and maintenance of early growth at the new site are generally accepted to be critical. This article discusses current knowledge of molecular events involved in these various processes. The potential role of adhesion molecules (eg. integrins and cadherins) has undergone a major transition over the last ten years, as it has become apparent that such molecules play a major role in signaling from outside to inside a cell, thereby controlling how a cell is able (or not) to sense and interact with its local environment. Similarly the roles of proteolytic enzymes and their inhibitors (eg. matrix metalloproteinases and TIMPs) have also expanded as it has become apparent that they not only have the abilities to break down the components of the extracellular matrix but also are involved in the release of factors which can affect the growth of the tumor cells positively or negatively. Recent work has highlighted the importance of the later, post-extravasational stages of metastasis, where adhesion and proteolysis are now known to play a role along with other processes such as apoptosis, dormancy, growth factor-receptor interactions and signal transduction. Recent work has also demonstrated that not only the immediate cellular microenvironment, in terms of specific cell-cell and cell-matrix interactions, but also the extended cellular microenvironment, in terms of vascular insufficiency and hypoxia in the primary tumor, can modify cellular gene expression and enhance metastasis. Mechanisms of metastasis appear to involve a complex array of genetic and epigenetic changes many of which appear to be specific both for different types of tumors and for different sites of metastasis. Our improved understanding of the expanded roles of the individual molecules involved has resulted in a mechanistic blurring of the previously described discrete stages of the metastatic process.     

Histamine alters endothelial barrier function at cell-cell and cell-matrix sites

To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.     

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells

Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.     

Study of cell-matrix adhesion dynamics using surface plasmon resonance imaging ellipsometry

The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.     

Rap1, a mercenary among the Ras-like GTPases

The small Ras-like GTPase Rap1 is an evolutionary conserved protein that originally gained interest because of its capacity to revert the morphological phenotype of Ras-transformed fibroblasts. Rap1 is regulated by a large number of stimuli that include growth factors and cytokines, but also physical force and osmotic stress. Downstream of Rap1, a plethora of effector molecules has been proposed on the basis of biochemical studies. Here, we present an overview of genetic studies on Rap1 in various model organisms and relate the observed phenotypes to in vitro studies. The picture that emerges is one in which Rap1 is a versatile regulator of morphogenesis, by regulating diverse processes that include establishment of cellular polarity, cell-matrix interactions and cell-cell adhesion. Surprisingly, genetic experiments indicate that in the various model organisms, Rap1 uses distinct effector molecules that impinge upon the actin cytoskeleton and adhesion molecules.     

Muscarinic signaling in carcinoma cells

We previously reported that activation of M(3) muscarinic acetylcholine receptors (mAChR) generates anti-proliferative signals and stimulates cadherin-mediated adhesion in the SCC-9 small cell lung carcinoma (SCLC) cell line. The current study was undertaken to determine the frequency of functional mAChR expression among different SCLC cell lines, and to test the ability of mAChR to generate anti-proliferative signals in different SCLC cell lines. The potential role of Rac1 in SCLC cell-cell adhesion was also investigated. Exposure to the mAChR agonist carbachol induces robust Ca(2+) mobilization (indicated by intracellular fluorescence of the Ca(2+)-binding dye Indo-1) in three SCLC cell lines (SCC-9, SCC-15, and NCI-H146), modest Ca(2+) mobilization in one SCLC cell line (NCI-H209), and no detectable Ca(2+) mobilization in two SCLC cell lines (SCC-18 and NCI-H82). The M(3) mAChR-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits Ca(2+) mobilization in all SCLC cell lines responding to carbachol. Incubation with carbachol for four hours significantly inhibits [3H]thymidine uptake in three of the four SCLC cell lines expressing functional mAChR (SCC-9, SCC-15, and NCI-H146 cells), but does not significantly alter [3H]thymidine uptake in the other SCLC cell lines examined. These results indicate that SCLC cell lines often express functional mAChR which elicit anti-proliferative signals when activated. To investigate the role of Rac1 in SCLC adhesion, SCC-9 cells were transiently transfected with cDNA constructs coding for Rac1, constitutively active Rac1(Val-12), or dominant negative Rac1(Asn-17) tagged to green fluorescent protein (GFP). SCC-9 cells expressing GFP-tagged constitutively active Rac1(Val-12) exhibit increased cell-cell adhesion in comparison to cells expressing GFP-Rac1 or GFP-Rac1(Asn-17). Constitutively active GFP-Rac1(Val-12), but not GFP-Rac1 or GFP-Rac1(Asn-17), accumulates at cell-cell junctions in SCC-9 cells. These results indicate that activated Rac1 increases SCLC cell-cell adhesion, consistent with the possibility that Rac1 activation contributes to increased SCLC cell-cell adhesion induced by mAChR stimulation. These findings indicate that activation of mAChR may play a significant role in regulating the proliferation and adhesion of SCLC cells. The demonstration by other investigators that acetylcholine is expressed by a variety of cells in the airways supports the possibility that acetylcholine may activate mAChR expressed by SCLC cells in primary tumors.     

Vascular endothelial-cadherin regulates cytoskeletal tension, cell spreading, and focal adhesions by stimulating RhoA

Changes in vascular endothelial (VE)-cadherin-mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin-mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.