Cell biology and clinical implications of adhesion molecules in ...

Clinical & Experimental Metastasis 18: 623–638, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Review

Cell biology and clinical implications of adhesion molecules in colorectal diseases: Colorectal cancers, infections and inflammatory bowel diseases Jörg Haier1,2 & Garth L. Nicolson1 1 The

Institute for Molecular Medicine, Huntington Beach, California, USA; Laboratory, Department of Surgery, Wilhelm-University Münster, Münster, Germany

2 Molecular Biology

Received 29 May 2001; accepted in revised form 11 June 2001

Key words: adhesion molecules, CD44, colorectal carcinoma, Hirschsprung’s disease, selectins, immunoglobulin-like molecules, infection, inflammatory bowel diseases, integrins, prognosis, proteoglycans

Abstract Adhesion molecules are transmembrane proteins that can anchor cytoskeletal proteins on the cytoplasmic side of the cell membrane, while also connecting extracellular structures on the outer surface of the cell membrane. In addition to physical linkages between the extracellular environment and the cytoskeleton, adhesive complexes participate in important signal transduction systems as modulators or receptors. Their functions in cell signaling are probably at least as important as their cytoskeletal and cell attachment properties. Understanding these regulatory functions appears to be of importance in determining of pathological characteristic of numerous diseases. Expression and functional activity of various adhesion molecules have been found in different diseases affecting the colorectum. In this review we summarize recent advantages about the cell biology these diseases and clinical implications.

Introduction Biological roles of cell adhesion molecules Cell adhesion events play important functional roles in different developmental and cellular events, including maintenance of normal tissue structure [1]. The interactions of cells with their environment, such as extracellular matrix (ECM) and other cells, involve cell adhesion events that are mediated by specialized cell surface molecules. These cell adhesion molecules are not only important components of normal tissues, they can also be found as participants in host defense mechanisms, such as leukocyte trafficking during inflammation, and pathological processes, such as formation of tumor metastases [2]. In cancerous processes homotypic cell adhesion between tumor cells at the primary site, heterotypic interactions of malignant cells with leukocytes and platelets during transport in the blood circulation, and cell attachment to the endothelial cells (EC) and ECM in distant organs involve cellular adhesion systems that are represented by a broad spectrum of cell surface molecules from different adhesion molecule families [3, 4]. During adhesion processes cells can form temporary attachments to certain structures, such as during chemotactic cell movements, or they can establish more long-term adhesive interactions, resulting in the formation of intercellular Correspondence to: Dr Jörg Haier, Molecular Biology Laboratory, Department of Surgery, Wilhelm-University Münster, Waldeyerstr. 1, 48149 Münster, Germany. Tel: +49-251-8356326, Fax: +49-251-8358424; E-mail: [email protected]

structures such as desmosomes, adherens junctions and gap and tight junctions. Various tumor cell surface molecules, such as integrins or other adhesion molecules, can directly initiate these interactions or they can act indirectly as receptors for soluble peptides or hormones that modify cellular signaling and thus regulate cell adhesion [5]. The roles of cell adhesion molecules in colorectal diseases will be discussed in this brief review. Cell adhesion molecules can be classified into five main groups: (a) integrins, (b) selectins, (c) immunoglobulin-like, (d) cadherins, and (e) other molecules, such as proteoglycans and glycoconjugates (lectin receptors) [6]. A new group of adhesion molecules has recently been added that includes receptor protein tyrosine phosphatases (RPTP) that can act as homotypic and heterotypic adhesion receptors [7]. Both the extracellular and cytoplasmic domains of RPTPs can interact with other cell adhesion molecules, and they appear to be involved in adhesion regulation and signal transduction. It has not been determined if they play a role in colorectal diseases. Adhesion molecules are usually transmembrane proteins (or cell surface components that can associate with transmembrane proteins) that can form complexes that anchor cytoskeletal proteins, such as actin and actin-binding proteins, on the cytoplasmic side of the cell membrane. At the outer surface they attach to cellular or extracellular structures, and this process is thought to initiate their transmembrane interactions [2, 8]. In addition to physical linkages between the extracellular environment (surrounding cells and ECM) and

624 the cytoskeleton, these adhesive complexes can participate in important signal transduction systems as initiators (receptors) or modulators of cellular signals [5]. Their function(s) in cell signaling are likely to be at least as important as their cytoskeletal and cell attachment properties. An important feature of the intracellular protein complexes induced by the aggregation of adhesion molecules is the subsequent activation of various signal transduction molecules that can trigger further downstream cellular signals [9]. These signaling pathways can regulate gene expression, secretion of enzymes or cytokines, and they can modify cytoskeleton conformational changes, among other properties [1]. Cadherins Cadherins, characterized by their immunoglobin-like structures, belong to a family of transmembrane glycoproteins that are responsible for calcium-dependent intercellular adhesion [10, 11]. This superfamily is divided into more than 10 subclasses, each of which has different immunological characteristics and tissue distributions, and mammalian cells that are organized into solid tissues express one or more member of the cadherin family. Cadherins mediate subclass-specific cell-cell interactions that are involved in selective homotypic cell adhesion properties that are particularly important during tissue development [12]. For example, E-cadherin (epithelial cadherin, L-CAM) is an important regulator of morphogenesis and tissue regeneration in the colonic epithelium that is expressed at the intercellular borders of most epithelial cells. In mucosa of the normal colon E-cadherin immunoreactivity is evenly distributed but distinctly along intercellular borders [13]. The expression and functional activities of E-cadherins are required for colon epithelial cells to remain integrated within the mucosal layer [1]. The overexpression of cadherins can result in tighter cell-cell contacts and their underexpression or inactivation can cause disruption of cell-cell adhesion [14]. For example, an increase in E-cadherin-mediated homotypic cell adhesion can inhibit cell growth, whereas loss of E-cadherin contacts can stimulate cell growth and division [15, 16]. E-cadherins also take part in maintenance of the barrier function of the colonic mucosa through formation of junctional complexes, and they participate functionally in cell polarization in the colon epithelium [17]. In colon adenomas and carcinomas, weaker E-cadherin expression has been observed consistent with its major role in the homotypic adhesion of colorectal epithelial cells [14]. Therefore, suppresion of E-cadherin expression or its function is thought to be involved in the structural alterations seen in these precancerous and cancerous lesions. Decreases in E-cadherins can also enhance the release of colorectal carcinoma cells from the primary tumors [13, 18]. All members of the cadherin family are connected to catenins through their cytosolic domains [18]. Catenins belong to a group of cytoplasmic plaque proteins that connect cell surface molecules with actin filaments. Functionally, E-cadherin is thought to be regulated by its associated cytoplasmic proteins, including α-catenin. Another catenin (β-catenin) has been found to form complexes with the ade-

J. Haier & G.L. Nicolson nomatous polyposis coli (APC) tumor suppressor protein, and E-cadherin and APC bind competitively to the same peptide motif. The β-catenin-APC complex can be phosphorylated, which is thought to be required for initiating the degradation or turnover of this complex. If APC is mutated, as is commonly found in colorectal carcinomas, the complex cannot be formed, resulting in an accumulation of APC and β-catenin in the cytosol. Free cytosolic β-catenin can subsequently act as an potent transcription factor and modify gene expression. The expression levels of β-catenin can also be affected if wildtype APC protein is expressed in APC-mutated cells or if APC-protein is hyperexpressed in unmutated colon carcinoma cells. Furthermore, the linkage of cadherins to α-catenin and then to actin occurs via a specific binding site on the cadherin molecule that is required for cadherin-mediated adhesion. β-catenin is a crucial intermediate for the linkage of α-catenin to the actin filaments [1]. Placental cadherin or P-cadherin is expressed on most epithelial cells and intensely on cells in the placenta [19]. Its cellular distribution is nonrandom, and it is polarized to basal surfaces where it is involved in homotypic cell adhesion and formation of junctional complexes [20]. Integrins The integrins are a family of adhesion molecules consisting of heterodimeric transmembrane proteins containing one αand one β-subunit noncovalently linked in a receptor complex. For the most part integrins mediate adhesion to various ECM components. One of the exceptions is in leukocytes where integrins interact with a specific group of receptors on EC. There are more than 20 different known integrins, and they are grouped according to the type of β-chains present in the complex [21]. To determine the specificity of interactions with their ligands, both subunits are necessary, and integrins are either monospecific, or they can bind to several different ECM components or cell receptors. Integrins are expressed on EC, epithelial cells, platelets, leukocytes, among other cells and their respective tumors [22, 23], but most tissues express a restricted number of integrins. Survival of colonic epithelial cells depends on matrix adhesion, and this is mediated, in part, by interactions between β1 -integrins and ECM components [24]. The expression of integrins on normal colonocytes varies depending on the cellular location within the crypt structure of the mucosa and during organogenesis. For example, in the early phase of embryogenesis colonic epithelium expresses α2 -, α3 -, α5 -, and α8 -integrins, whereas at later stages α2 - α3 -, α6 - and α8 -integrin expression is accentuated at the basal membrane surface. During transition from pseudostratified to simple columnar epithelium a vertical α2 -integrin gradient forms within the primitive crypts. The developing muscur layers show increased expression of α5 -integrins, and the mesenchymal vascular elements show high immunoreactivity for α2 and α6 -integrins during early organogenesis [25]. In normal mature colonocytes, α2 -integrin (collagen receptor) was strongest in crypt cells by immunodetection. Although the results from different studies are not always in agree-

Cell biology and clinical implications of adhesion molecules in colorectal diseases ment, most data show decreases in α2 -integrin expression that may explain the reduced binding of colonocytes to the basement membrane during their movement from the crypts to the mucosal surface where the cells eventually undergo sloughing [26–28]. This differentiation and migration process is accompanied by alterations in the expression and activity of various cell signaling proteins, such as focal adhesion kinase (FAK) and mitogen-activated protein (MAP) [29]. Differences in integrin expression have been found between normal colon mucosa, adenomas and carcinomas. In the normal mucosa two typical patterns of α2 - and α3 integrin expression have been observed: basolateral expression and diffuse cytoplasmic expression [30]. Other integrin subunits, such as those represented by α1 , α3 , αv and β1 , β3 and β4 subunits can be detected throughout the crypt, but they predominate in the superficial enterocytes [31]. In adenomas, monolayered glands showed integrin patterns that differ slightly from that seen in crypt and superficial enterocytes. The complex glands in villous adenomas showed decreased integrin staining and basal polarization [32]. β1 Integrins were usually found to be expressed in adenomas, but the α2 β1 -integrin appeared to be lost in the focal areas of cell contacts [33]. In contrast to adenomas or normal cells, colon carcinomas show weaker integrin staining [34]; however, carcinomas also show considerable heterogeneity in integrin expression [33]. Additionally, the classic fibronectin receptor (α5 β1 -integrin) was frequently found to be expressed in invasive colon carcinomas, whereas the expression of this integrin subunit was usually found to be poor or absent in normal colon epithelium [35, 36]. The β2 -integrins are expressed on mononuclear cells in all layers of the bowel, but they appear predominantly in the mucosa and adventitia. The αL β2 -integrin (also called CD11a) showed the greatest expression in the mucosal layer, whereas the αM β2 -integrin (CD11b) had the highest expression in the adventitial layer. The αX β2 -integrin (CD11c) exhibited the least expression of all α-subunits in β2 -integrins [37]. The β2 -integrin family of adhesion molecules and their ligands, the ICAM molecules, appear to play important roles in intestinal immune responses [37]. In the normal mucosa, the αL β2 -integrin is expressed on some lymphocytes, and the α4 β1 -integrin on most lymphocytes. Lymphocyte- or leukocyte-epithelial cell interactions involving integrin adhesion molecules may be important in immunosurveillance of colon adenocarcinomas, inflammatory bowel disease and celiac disease, where increased levels of proinflammatory cytokines are locally produced within the gut mucosa [38]. Receptor binding and clustering of integrins at the cell surface appear to trigger various signals that can activate signaling cascades or induce interactions with cytoskeleton components [39]. For example, integrins, for the most part, mediate adhesion to the ECM, depending on the type of integrin-binding ECM components expressed. Once cell adhesion occurs, integrin receptors generate regulatory signals inside cells that allow them a certain amount of control over cell anchorage and migration. Integrins important in

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these processes include the β1 - and β3 -integrins, which can be linked to cytoskeletal proteins, such as actin, αactinin and talin. These components are further connected to various other cytoskeleton proteins like vinculin and zyxin [5]. The signaling process also involves phosphorylation and dephosphorylation events, mainly on tyrosine residues [40], and this, in turn, may activate the intracellular signaling cascades. Different protein complexes may be involved in downstream signal transduction after the establishment of cell adhesive interactions, such as focal adhesion kinase (FAK)-paxillin, pp60csrc kinases or protein kinase C [40, 41]. In addition to these ‘downstream’ signaling events, integrin receptor binding can be regulated by ‘upstream’ signal transduction events. These later events include cytoskeletal reorganization [42] and cross signaling from other cell surface receptors, such as the receptors for epidermal growth factor (EGF) or platelet derived growth factor (PDGF) [43]. Thus integrins are functionally and structurally integrated parts of a complex regulatory system that modulates cell adhesion and subsequent secondary cellular events (Figure 1). Selectins Selectins are adhesion molecules that use carbohydrates as receptor ligands. They are important in the interactions of tumor cells with leukocytes/lymphocytes (involving Lselectin), platelets (involving P-selectin) and EC (involving E-selectin). Increased expression of selectins or their increased activities depend on cell activation, such as in EC or leukocytes/lymphocytes by interleukins, tumor necrosis factor (TNF) or toxins or in platelets by thrombin, histamine, O2 -radicals or other procoagulatory substances. Selectins have similar structures containing an N-terminal lectin-domain, EGF-like domains, different numbers of complement-binding domains, a transmembrane and a short intracellular domain. These molecules mediate cell binding to carbohydrates, such as sLea , sLex and the MECA-70 antigens. Although the members of the selectin family are structurally related, they have disparate functions that are dependent on cell type. Selectins appear to play a central role in the targeting and adhesion of circulating tumor cells to EC in various organs. ‘Cross talk’ with other types of adhesion molecules, such as carcinoembryonic antigen (CEA), appears to be involved in the selectin-mediated regulation of cell adhesive interactions. Selectins do not appear to function alone, and the formation of stabilized cell adhesions appears to involve multi-receptor complexes [44]. Various selectins are expressed in different tissues and on different cell types. For example, E-selectin is expressed on the endothelium, but not on colonocytes [45]. Both E- and Pselectins were found to be sporadically expressed in venules [46], whereas E-selectin was shown to be overexpressed on EC in association with various pathological processes in colorectal tissues. For example, there appears to be a relationship between the upregulation of selectin expression on EC and inflammatory reactions found at the invasion front of malignant lesions or in inflammatory bowel diseases. The EC of small vessels adjacent to cancer cell nests, both in

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Figure 1. Functional relationships of adhesion molecules in tumor cell–host organ interactions during metastasis formation

primary and in metastatic lesions, display increased levels of E-selectin expression [47]. In addition, the degree of Eselectin expression was inversely correlated to the distance of the blood vessels from the cancer cell nests. Also, EC adjacent to metastatic lesion expressed more E-selectin than EC adjacent to the primary tumor [47]. In contrast, infiltrating lymphocytes express L-selectin. About 30% of the intraepithelial lymphocytes in the colonic epithelium were found to express L-selectin [48]. Selectins can also be shed from cells. For example, Eselectin has been found in serum samples, but the levels of this selectin are usually quite low in the blood. Under certain pathological conditions, however, serum levels can be considerably higher [49, 50]. A possible source of the soluble forms of E-selectin in blood are leukocytes and to a lesser extent EC, and the baseline serum levels of selectins may reflect normal cellular regeneration processes. Soluble selectins might also be involved as negative regulators of leukocyte trafficking through binding and blockage of selectin receptors [49, 50]. Alternatively, increased levels of E-selectin may be explained as a consequence of increased immunological activity and higher rates of leukocyte consumption. Sialomucins Normal colonic epithelial cells undergo maturation as they traverse the crypt to the lumenal surface, and during this process changes occur in the expression of specific cell surface oligosaccharides that serve as receptors for various carbohydrate-binding adhesion molecules. Such changes

can be seen by the binding of lectins to goblet cell mucins and other glycoconjugates where lectin binding changes as the cells migrate from the crypt and undergo differentiation. For the most part the lectin receptors are cell surface sialomucins. These sialylated mucins appear to play a role in colorectal cell adhesive interactions with both basement membrane ECM and EC-associated ligands. The most important members of this group in the colorectum are various sialo-Lewis or sLe antigens and the CA 19-9 antigen. These antigens were originally identified by their reactions with certain polyclonal or monoclonal antibodies. sLe-antigens are commonly used ligands for selectin-mediated cell adhesion events described above. CA 19-9 and sLex are tumor-associated antigens that are expressed in the entire colorectum, whereas other sialylated carbohydrates, such as sLeb and sLey , have been found only in the distal colon [51]. The affinities and antigenic structures of the sialylated carbohydrates are regulated by the activities of glycosyltransferases and other membrane-bound enzymes, some of which are upregulated in colon carcinomas [52]. This may cause various glycoconjugates to differ in their expression in different cellular compartments, such as local distribution within the crypts and regional distribution between right (ascending colon) and left (rectum) segments of the large bowel [53]. Stepwise modifications in glycoconjugate expression have been found in premalignant and malignant neoplasms [54]. For example, plant lectin leukoagglutinin (L-PHA)reactive oligosaccharides are consistently increased in colon carcinomas compared to normal or benign tissues [55]. Furthermore, expression of 2,6-sialylated sugar chains in-

Cell biology and clinical implications of adhesion molecules in colorectal diseases creased dramatically during malignant transformation from normal colon to benign and malignant colon tumors as demonstrated by the binding of Sambucus nigra agglutinin [56]. Immunoglobin-like superfamily Cell adhesion molecules with an immunoglobin-like (Iglike) structure in their extracellular domain belong to the Ig-like superfamily of adhesion molecules. These adhesion components have wide-ranging functions and participate in a variety of homotypic and heterotypic interactions [57]. Some members of this family, such as ICAM-1, ICAM-2, and VCAM-1, are known to participate in heterotypic cellcell adhesion, whereas others appear to be binding sites and possibly receptors for certain growth factors (PDGF, CSF-1), receptors on T-cells (CD4, CD8), tumor cell antigens (CEA) and a group of molecules that mediate cell-cell-interactions between platelets and EC (CD31, ICAM-1, VCAM-1). This latter Ig-like subgroup takes part in cell-cell-interactions by binding to other adhesion molecules, such as integrins or selectins, that are important in tumor cell interactions during metastasis as well as for cellular immune responses. In colonic tissues ICAM-1 is expressed on the endothelium and tissue mucosa but rarely on mucosal mononuclear cells. Immunohistochemical localization and in-situ hybridization have revealed poor or no expression of ICAM1 on normal colonic epithelium [45]. In colon carcinomas ICAM-1-positive cells were mostly found to be stromal cells, and the most common cell types were identified as macrophages and fibroblasts [58]. After cell activation by soluble factors, such as interleukins, the expression of ICAM-1 becomes strictly polarized in intestinal epithelia [59], whereas ICAM-2 is predominantly found on submucosal endothelium, rarely in colon mucosa and never in ileal mucosa. ICAM-3 can be found on mononuclear cells throughout the bowel wall, and also on the adventitial endothelium [37, 45, 60]. Carcinoembryonic antigen (CEA) is a heavily glycosylated protein that has been used clinically as a tumor marker to detect recurrences of many types of cancers. This Ig-like glycoprotein belongs to a large CEA superfamily of proteins. It is produced in large amounts in essentially all of the colon and in several other types of adenocarcinomas. CEA is expressed intracellularly as well as extracellularly, and it is also secreted in an extracellular form. The intracellular expression of CEA appears to be associated with the degree of atypia in histological sections [61]. EC express CEA on their apical cell surfaces, which may allow CEA-expressing colon carcinoma cells to adhere to EC, in part, via CEACEA interactions [62]. These molecules might also take part in regulating cell binding to ECM components, such as collagens, through interactions with other adhesion molecules [63]. Thus, CEA interactions may facilitate tumor cell extravasation and hematogenous metastasis formation [64]. This group of adhesion molecules might also be involved in morphogenesis during embryonic and fetal development [65].

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Another member of the Ig-supergene family that has been shown to mediate homophilic binding is the neural cell adhesion molecule (N-CAM or CD56). The structure of NCAM is similar to the tumor suppressor molecule DCC. NCAMs are apparently only found on neuronal cells where they can act as receptors for RPTP [7, 66]. Several isoforms of N-CAMs have been identified that are expressed preferentially in different tissues and at certain stages of embryonic development [67]. NCAMs are important molecules involved in neural cell migration and proper innervation of developing organs, such as the colon. In addition, N-CAM-positive cells are commonly present in normal intestinal mucosa in inflammatory bowel diseases and in normal colon tissues surrounding colorectal carcinomas [68]. Analysis of mRNA expression demonstrated the presence of NCAM in normal colonic epithelia, and immunohistochemistry showed that NCAM was expressed on the basolateral surface of colonic epithelial cells of the villous tips. In contrast, colon carcinomas showed decreasing expression of NCAM compared to normal tissue, depending on their clinical stage. Later stages were generally found to express the lowest amounts of NCAM [69, 70]. CD44 CD44 is a cell surface adhesion molecule family of different splice variants and is expressed on EC and various tumor cells [71]. Multiple functions have been attributed to the CD44 family of molecules. CD44 plays a role in the production and catabolism of hyaluronate, which is located in the liver and lymph nodes at high concentrations. CD44 mediates cell-cell contacts via glycosaminoglycans, such as hyaluronate on fibroblasts, EC and hematopoietic cells, and in the ECM. Hyaluronate and CD44 have been proposed to be important in predicting cell survival, invasiveness, migration and angiogenesis [71]. For example, ligation of CD95 (APO-1/Fas) cell surface receptors induces apoptosis or cell death in sensitive cells [72]. In human colon carcinoma cells the cross-linking of the apoptosis-associated receptor CD95 with an anti-APO-1 antibody resulted in rapid detachment of the cells from a hyaluronate matrix, suggesting a functional link between the regulation of apoptosis and binding to the hyaluronate receptor CD44. In addition, loss of adhesion of colon cells is usually accompanied by a substantial reduction of this cell surface adhesion molecule after stimulation of the CD95 receptor [72], and increasing amounts of soluble CD44 were found in the supernatant of CD95-triggered cells. Using this mechanism CD44 might contribute to the active disintegration of dying epithelial cells in the early phases of apoptosis. In addition, binding to an immobilized anti-CD44 antibody was found to induce change to a monolayer morphology in colon carcinoma cells, accompanied by FN production and secretion and increased expression of the αv β6 -integrin. These monolayered cells acquired increased resistance to apoptosis mediated by integrin inhibition. Thus CD44-related effects may result in selective advantages for survival of malignant colon cancer cells [73].

628 Other adhesion molecules Lymphocyte homing to normal tissues and recruitment to inflammatory tissue sites are controlled, in part, by the selective expression of chemokines, pro-inflammatory cytokines and mediators and various adhesion proteins and molecules. For example, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a glycoprotein containing immunoglobulin- and mucin-like domains that is selectively expressed in a tissue-selective manner on the EC of high endothelial venules in the gut and gut-associated lymphoid tissues. By interaction with the MAdCAM-1 ligand (α4 β7 integrin) lymphocytes become involved in regulating mucosal immunity, and this and possibly other interactions are involved in allowing lymphocytes to be selectively recruited to intestinal sites [74]. Monoclonal antibodies (MAb) specific for the β7 -integrin or MAdCAM-1 were able to inhibit chronic colonic inflammation in rats, and the MAb blocked the recruitment of lymphocytes to the colitic colon [75]. A molecule structurally and functionally related to the Ig-like superfamily, VCAM-1, is sporadically expressed in venules and constantly in follicular dendritic cells (FDC) in lymphoid follicles in the gut. It can also be found on activated macrophages and EC [76]. This adhesion molecule is important in mediating adhesion of lymphocytes, eosinophils and monocytes to activated EC, and these interactions are mediated specifically by α4 β1 -integrins [76]. Another physiological role for VCAM-1 is its participation in the mediation of lymphocyte adhesion to dendritic cells during normal immune responses, and its expression also appears to be important in a number of developmental pathways [77, 78]. Functional aspects of adhesion molecules Adhesion molecules on colonocytes function in the development and maintenance of tissue structure and normal cellular differentiation and regeneration. Maintaining the proper expression of adhesion molecules is important in colonic mucosa for normal tissue integrity and morphology where these molecules function to maintain normal tissue barriers. In colon cells adhesive properties are vital in determining tissue organization, and adhesion molecules enable the directed cell migrations found during embryonic development, inflammation, immune response, and wound repair. In addition to its functional integrity, the barrier functions of the colonic epithelium against bacterial invasion are strongly dependent on the maintenance of proper adhesion molecule expression [79]. Adhesion molecules also participate in the regulation of gene expression, cell growth, differentiation and programmed cell death [80]. In colonic tissue CEA can directly contribute to colon carcinogenesis by inhibiting colonocyte differentiation [81]. Besides the normal variations in adhesion molecule expression during embryogenesis and cell maturation, changes in their surface expression can also be found during a variety of pathological processes, such as inflammation and malignant transformation. The recruitment of leukocytes from the blood is one of the most important functions of adhesion molecules on

J. Haier & G.L. Nicolson EC [82]. Leukocyte extravasation is regulated by the specific recognition of certain stimuli, such as inflammatory cytokines or bacterial toxins, and the expression of inflammatory adhesion receptors. For example, specific recruitment of leukocytes can be found in acute inflammation, where selective interactions between lymphocytes and high endothelial venules occur in lymphoid tissues [83]. These highly specific and tightly regulated cellular interactions require specific adhesion receptors, but adhesion events cannot be explained solely by cell-specific molecule expression. For example, primary adhesive recognition between circulating leukocytes and EC appears to be mediated mainly by selectin-carbohydrate (sLe) interactions. These very rapid initial adhesion events appear as ‘leukocyte cell rolling’ along the microvascular vessel wall. Leukocyte rolling involves establishment of transient cell adhesions and cell detachment. Under normal physiologic conditions leukocyte extravasation is limited; however, after activation of EC by chemoattractants or soluble activating factors, a variety of activation-dependent adhesion pathways utilizing integrins, ICAM’s or VCAM-1 participate in the stimulation of cell migration, invasion and initiation of immunological functions [84]. The binding of circulating leukocytes to activated EC subsequently results in their activation, leading to further stimulation of cellular responses, such as secretion of chemokines, degradative enzymes, among other molecules [85]. Tumor and metastasis formation During neoplastic transformation epithelial and other cells undergo nuclear modifications that can alter differentiation states and can change their surface structures by differential expression of molecules at the cell surface or by posttranslational modifications, such as glycosylation. Such cell surface structural alterations can involve various adhesive systems, resulting in modified or reduced cellular interactions with components in the extracellular environment. For example, the loss of cell-cell and cell-ECM interactions promotes motility and detachment of tumor cells at the primary tumor site. On the other hand, the establishment of new adhesive interactions is necessary for cell attachment to the endothelium and basement membranes in distant host organs during hematogenous metastasis formation. The sequential model for the development of colorectal cancer metastases involves tumor growth, neovascularisation and invasion at the primary sites, followed by penetration into lymphatics and blood vessels or into body cavities. Malignant cells that loose or undergo reduction in cellular contacts with surrounding cells or ECM can undergo phenotypic change to more motile and unstable cellular anchorage within tissues. Subsequently, malignant cells can be released or detached from the primary tumor and be transported throughout the body via the blood or lymphatic circulations. To survive the circulating tumor cells must adhere to the vessel walls of distant host organs, and they eventually must penetrate the wall or extravasate to avoid blood shear forces and host defense mechanisms. Finally, the metastasizing cells have to survive and grow at the secondary site [2].

Cell biology and clinical implications of adhesion molecules in colorectal diseases The localization of tumor metastases in distant organs is not determined solely by anatomical considerations or blood flow. During hematogenous metastasis formation tumor cells often show selective colonization of various organs independent of the percentage of microcirculation through the organ or whether the organ is the first to be encountered by the blood-borne tumor cell [86]. This multistep process is thought to be determined by the unique properties of the tumor cells and the host organs [87, 88]. Adhesion of metastatic cells to and migration through the microvascular vessel walls of host organs are very important steps in colonization of distant organs [2, 89, 90], and during these steps specific interactions between the tumor cells and endothelial cells, the ECM or surrounding cells are required. Tumor cell surface molecules mediate these interactions, either by direct interactions or as receptors for soluble factors. During the interactions between metastasizing colorectal tumor cells and EC/ECM components adhesion molecules function for the primary cell contact, definitive adhesion stabilization and cell migration. Tumor cells adhere at sites of EC/ECM contacts by sequestering or clustering receptors at zones of adhesive interactions, such as focal adhesions. At this time adhesion has not yet been stabilized, and increasing the shear force in vitro under flow conditions can break the adhesive contacts and release the tumor cells from the EC/ECM. However, if adhesion can be stabilized, tumor cells can resist such hydrodynamic shear forces [89]. Following these initial adhesive interactions, the tumor cells appear to actively change their shape by spreading or flattening to increase contact areas and initiate cell migration through the vessel wall [91, 92]. During adhesion under flow conditions in vitro, colon carcinoma cells showed modest transient interactions with resting human umbilical cord vein endothelial cells (HUVEC) that were comparable with the behavior of leukocytes, such as leukocyte transient adhesion and rolling on IL-1or tumor necrosis factor-activated HUVEC. However, with colorectal carcinoma cells differences in adhesive properties with different ECM components were noted. The initial tumor cell-ECM interactions under flow conditions appeared to depend mainly on biophysical or nonspecific mechanical factors and involved cell surface carbohydrates, whereas specific adhesive interactions (secondary events) were required for further adhesion stabilization. As an example of secondary events, receptor crosslinking by transglutaminase was found to be an important determinant of adhesion stabilization in several cell systems [91]. Moreover, various parameters that describe cell adhesion stabilization under flow conditions with various ECM components correlated with metastatic potential [93]. Recently, we have also shown that adhesive interactions of HT-29 colon carcinoma cells with ECM components under hydrodynamic flow conditions occurred in several steps that were uniquely characteristic, including initial cell-ECM contacts, transient cell adhesion and adhesion stabilization. Using HT-29 cells with different metastatic properties the influence of shear forces that occur under fluid flow conditions demonstrated differences

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in colorectal tumor cell adhesive behaviors in static and dynamic adhesion assays [94]. In parallel with morphological alterations, tumor and host cells undergo functional responses to certain specific adhesive interactions. For example, after stable adhesion cells can release glycosidases and proteases, destroying the integrity of the vascular basement membrane [95]. Matrix proteins, such as type IV collagen, laminin, fibronectin (FN) and vitronectin, are mainly targeted for degradation by different matrix metalloproteinases (MMPs) that have relative substrate specificities as well as nonspecific proteases. Following stable adhesive interactions tumor cells or EC of the host organ can secrete specific MMPs, and these degradative enzymes are then activated by other membrane-bound proteases or by inactivation of their inhibitors and degrade basement membrane ECM at focal sites. Together with the induction of directed cell motility, the porous subendothelial membrane then allows tumor cells to penetrate this structure by active cell locomotion, thereby leaving the circulation and invading the host organ.

Clinical implications Infections The acute host response to gastrointestinal infections caused by invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and subsequent neutrophil transmigration to the luminal side of the crypts occurs. Intestinal EC and epithelial cells play an important role in the recruitment of inflammatory cells to sites of infection through the secretion of chemokines and cell activating autocrine and paracrine factors. These endogenous factors in conjunction with exogenous substances, such as bacterial lipopolysaccarides (LPS), cause massive activation of EC and intestinal immunocompetent cells. A very important characteristic of this activation is an increase in the expression of adhesion molecules on effector cells, such as β2 -integrins, ICAM-1 and selectins [96]. The adhesion of neutrophils to the vascular endothelium and basolateral epithelial membrane within the intestinal wall utilize some common adhesion molecules but are distinct processes during infections. For example, ICAM-1mediated neutrophil adhesion to apical epithelial membranes appears to be similar to adhesion at the epithelial basolateral membrane, but these two adhesion events are dissimilar to adhesion occurring at the vascular endothelium [97]. Increased epithelial ICAM-1 expression in the early time period after infection appears to be the result of direct interactions between invading bacteria and host epithelial cells. Indeed, ICAM-1 was found to localize to areas invaded by bacteria. In contrast, the release of soluble factors by epithelial cells may play only a minor role in mediating increased ICAM-1 expression [98]. Furthermore, ICAM-1 was fond to be expressed on the apical side of polarized intestinal epithelial cells, and its increased expression was accompanied by increased neutrophil adhesion to these cells. Following

630 infection with invasive bacteria, the increased ICAM-1 expression by intestinal epithelial cells may function to trap neutrophils that have transmigrated through the epithelium and maintain the immunocytes in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens [98]. Changes in adhesion molecule expression can influence the pathogenesis of infections of the gut in different ways. As described above, up-regulation of certain adhesion molecules can be found as a part of the inflammatory reaction, and this appears to be a prerequisite for effective immune reactions and host defense. In addition, adhesion molecules on the apical colonocyte surfaces may be important mediators of adhesion of microorganisms to the intestinal wall and subsequent invasion into the mucosal cells or deeper layers of the gut. For example, αv β6 -integrin expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be an essential step in the conversion of asymptomatic enterovirus infection into a clinically apparent disease [99]. Rectal and cervicovaginal mucosa are also common routes of transmission of HIV, although the mechanism(s) of transmission is still unknown. However, expression of αM β2 -integrin was detected in crypts and the surface of rectal mucosa in HIV-1-positive patients, and this could be important in the pathogenesis of HIV-1 [100]. Since this integrin also acts as a receptor for the C3bi complement factor, its expression might facilitate transmission of HIV-1 by enhancing the binding of HIV-antibody complexes in seminal fluid to epithelial cells. Alternatively, since ICAM-1 is a receptor for the αM β2 -integrin, this may promote adhesion between epithelial cells and HIV-1-infected mononuclear cells in seminal fluid [100]. Adhesion molecule expression also appears to be involved in the adhesion to and invasion of eukaryotic parasites through the gut wall. For example, adhesion molecules constitute important elements in schistosomal intestinal granuloma formation. In intestinal granulomas of mice infected with the parasite Schistosoma mansoni the expression of adhesion molecules mediating cell-cell (ICAM-1 and αL β2 -integrin) and cell-matrix (α4 β1 -integrin and syndecan1) interactions were affected. In both the acute and the chronic stages of infection up-regulation of ICAM-1, αL β2 and α4 β1 -integrins were seen in ileal and colonic granulomas, whereas up-regulation of α6 β1 -integrin was not apparent in these intestinal granulomas [101]. Inflammatory bowel diseases Inflammatory bowel diseases are characterized by an intense infiltration of leukocytes, and adhesion molecules expressed on the surface of activated EC are important determinants in this process [102]. Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases, such as ulcerative colitis and Crohn’s disease, correlate with migration of neutrophils across the intestinal epithelium. In vitro modeling of the transepithelial migration has revealed distinct differences from transendothelial migration [103]. Furthermore, enhanced leukocyte binding by intestinal microvascular EC from chronically inflamed tissues in patients with inflamma-

J. Haier & G.L. Nicolson tory bowel disease has been recently shown to be an acquired defect, since it is not found in the uninvolved intestinal segments from the same individuals [104]. Moreover, genetic predisposition may be important in this dysregulation, because ICAM-1 expression in tissue specimens from inflammatory bowel diseases was shown to vary among individuals [105, 106]. Adhesion of circulating cells to the vascular endothelium occurs in the early phases of inflammation and is mediated by specific cell adhesion molecules. The expression of various types of adhesion molecules can be increased or decreased in inflamed regions of ulcerative colitis and Crohn’s disease, but their exact pathogenic role in inflammatory bowel diseases remains uncertain. For example, β2 -integrin molecules and their ligands (ICAM-1 and ICAM-2) likely play an important role in the inflammatory responses seen in these diseases. Compared with normal tissues, the immunohistochemical expressions of β2 -integrins were, in general, increased in the colon mucosa and submucosa in Crohn’s disease [107]. For example, αx β2 -integrins expression in the mucosa was significantly greater in Crohn’s disease than in ulcerative colitis. In the muscular and adventitial layers the expression of αx β2 -integrins in Crohn’s disease was similar to normal tissues but increased compared with ulcerative colitis [107], and the expression of αx β2 -ligands (ICAM-1 and ICAM-2) was increased in Crohn’s disease colon and ileum compared with normal tissues [107, 108]. ICAM-1 has been found expressed on the endothelium, but in specimens from inflamed bowel it was also detected on mucosal mononuclear cells. Colon ICAM-1, -2, and -3 expression in Crohn’s disease was similar to that observed in ulcerative colitis, but in Crohn’s disease and ulcerative colitis the expression of E-selectin was also found to be dramatically up-regulated compared to control specimens [109, 110]. The expression of these adhesion receptors in Crohn’s diseases and ulcerative colitis is consistent with a constitutive activation of immune responses in these diseases. The up-regulation of E-selectin and ICAM-1 may play an important role in mediating the inflammatory process in inflammatory bowel diseases [111]. For example, Eselectin can regulate the accumulation of neutrophils in the early stages of the inflammatory process and might be associated with at least the active phase of ulcerative colitis. In contrast, ICAM-1 immunoreactivity in lymphoplasmatic infiltrates can be caused by chronic immune stimulation, which is potentially responsible for the persistence of inflammatory hyperreactivity [110]. Differences in the expression of adhesion molecules in Crohn’s disease suggest that the colonic mucosa in Crohn’s disease contained two types of macrophage/dendritic cells of the same lineage that express intercellular adhesion molecules and major histocompatibility complex (MHC) class-II antigens, consistent with immunological activation of these cells [112]. MAdCAM-1 is expressed in a tissue-selective manner. In normal tissues, MAdCAM-1 is constitutively expressed on endothelium of venules of the intestinal lamina propria. Interestingly, the proportion of venular endothelium within the lamina propria that expresses MAdCAM-1 is increased

Cell biology and clinical implications of adhesion molecules in colorectal diseases at inflammatory foci associated with ulcerative colitis and Crohn’s disease compared with normal tissues. Moreover, MAdCAM-1 is not detected in the majority of normal or inflamed extra-intestinal tissues, including those with mucosal surfaces, such as in chronic gastritis specimens [74]. Marked increases in ICAM-1, α4 β1 - and αl β2 -integrin, VCAM-1 and P- and E-selectin have been observed in specimens from inflammatory bowel diseases [46, 46, 113]. Greater numbers of CD31-expressing blood vessels were also found in inflamed specimens [114]. In contrast, loss of normal membraneous E-cadherin and α-catenin staining was detected at the mucosal edges around epithelial ulcerations in active ulcerative colitis and in 50% of cases with active Crohn’s disease [115]. These changes in expression of E-cadherin and α-catenin correlated significantly with the clinical activity of these diseases [115]. Unlike E-cadherin, P-cadherin was found to be dramatically up-regulated in both Crohn’s disease and ulcerative colitis and especially in dysplastic ulcerative tissues [116]. The observed differences in the expression of cadherins between Crohn’s disease and ulcerative colitis may reflect differences in inflammatory cell infiltrates or in the histopathological differences between the two diseases [109]. Changes in the expression of different adhesion molecules have also been found in relation to acute or chronic stages of inflammatory bowel diseases. For example, the serum levels of soluble adhesion molecules, such as ICAM1, E-selectin and VCAM-1, correlated significantly with the clinical stages of inflammatory bowel diseases [117]. In another study, lymphocyte adhesiveness and aggregation was found to be the most powerful predictive value for disease activity assessed by the Mayo Clinic score for ulcerative colitis, and Harvey-Bradshaw score assessment for Crohn’s disease [118, 119]. However, it remains unclear whether these changes are prerequisites (acquired or genetic predisposition) or the result of these diseases. According to the hypothesis that neutrophils contribute significantly to the epithelial dysfunction that characterizes colitis, several approaches have been undertaken to specifically inhibit these interactions. For example, antibodies directed against adhesion molecules may represent a novel approach to the treatment of intestinal inflammatory disorders. For example, antibodies specific for β7 -integrin subunits or MAdCAM-1 were able to block recruitment of lymphocytes to the inflamed mucosa in a mouse model [120]. This resulted in a significant reduction in the severity of colonic inflammation. Therefore, the adhesive interactions mediated by α4 β7 -integrin and MAdCAM appear to be involved in leukocyte recruitment to the gut in chronic inflammatory diseases, and this could be a relevant new therapeutic target in patients with inflammatory bowel disease [120]. This was confirmed in a recent study where leukocyte adhesion was abrogated by immunoneutralization of VCAM-1 or MAdCAM-1 but not by treatment with an anti-ICAM-1 antibody [121]. However, chronic administration of anti-VCAM-1 antibody, but not anti-ICAM-1 or anti-MAdCAM-1, resulted in significant attenuation of colitis in terms of disease activity index, colon length, ratio

631

of colon weight to length, and myeloperoxidase activity in mice [121]. In an animal model for experimental colitis it was shown that blockage of β2 -integrins or other integrins can reduce inflammation and restore barrier function of the colonic epithelium [122]. In addition, in a mouse model of experimental colitis, a specific antisense oligonucleotide that reduces ICAM-1 expression was therapeutically effective [123]. Reductions in ICAM-1 immunostaining and infiltrating leukocytes were observed in the colons of treated animals. The ICAM-1 oligonucleotide also diminished the clinical severity of colitis in mice with established disease without severe side effects [123]. In a small placebocontrolled study these effects were confirmed in 20 patients with Crohn’s disease [124]. At the end of the one-month treatment period 47% (7 of 15) of antisense-treated and 20% (1 of 5) of the placebo-treated patients were in remission (Crohn’s Disease Activity Index < 150). At the end of a six-month follow-up 5 of these 7 responders were still in remission [124]. These results suggest that reduced expression or blocked function of adhesion molecules could be an alternative treatment of inflammatory bowel diseases, but these therapeutic applications are still preliminary and under development [125]. Recent research on inflammatory bowel diseases indicates that chronic infections may underlie a significant proportion of these diseases [126] Chronic infections may be responsible for establishing inflammatory responses and changes seen in the colon epithelium in these diseases. Thus prevention of at least some of these cases could rely on preventing attachment of chronic bacteria and viruses to the colonic epithelium as well as direct treatment of the infections with antibiotics and antivirals [127]. Colorectal carcinoma The detachment of colorectal tumor cells from the primary site, the homotypic interactions between tumor cells and with host cells during transport in the circulation, and the cellular interactions with the endothelium and ECM in distant organs are important for the formation of secondary colorectal and other tumors. The multiple surface molecules that mediate these adhesive interactions include: lectins, glycosyl transferases, integrin- and non-integrin adhesion molecules, glycolipids, among others. The detachment of tumor cells from the primary carcinoma is characterized by the loss of cell–cell adhesion, and as expected the cadherincatenin systems appear to play an essential role in this process in colorectal cells. In poorly differentiated carcinomas, loss of epithelial cell contacts is frequently observed, allowing the cells to break away from the primary tumor, invade surrounding tissue and be released into the lymphatics and blood circulation. E-cadherin appears to be an important adhesion component in the process and its loss or inactivation can enhance detachment and invasion. Adhesion of circulating tumor cells to organ endothelial cells and subendothelial ECM are mediated by several adhesion systems that may or may not involve the same endothelial cell surface receptors used by leukocytes. For example, the initial contacts between carcinoma cells and

632 the microvascular endothelium seem to be related to expression of selectins, such as sialyl-Lewisx (sLex ) and other carbohydrates, intercellular adhesion molecules (ICAM) and possibly annexins [6]. However, integrin-mediated interactions with the subendothelial ECM may be among the more important determinants for organ-specific metastasis of colorectal carcinomas. Changes in the expression of different adhesive systems during malignant transformation and the involvement of adhesion molecules in the formation of distant metastases have stimulated intensive investigations on their prognostic and/or predictive values for assessing colorectal carcinoma patients (Table 1). Like other carcinomas, such as those of the breast, bladder and lung, the expressiond of E-cadherin and αcatenin are down-regulated in poorly differentiated colon cancer cells [128, 129]. In analyzing the expression of the E-cadherin receptor in colorectal carcinomas, it has been shown that this receptor may serve as an independent prognostic marker in Dukes’ stage B colon cancers and can identify patients with poor prognosis and designate them for adjuvant therapy after curative surgical treatment [130]. The frequency of reduced E-cadherin expression was generally higher in tumors with aggressive histopathologic characteristics, lymph node involvement and distant metastases, and the loss or heterogeneous expression of E-cadherin in colorectal cancers correlated closely with advanced clinical stage, including tumor penetration, tumor differentiation, lymph node involvement and distant metastases [131]. It was also significantly associated with an increased incidence of tumor recurrence after apparently curative resection, reduced overall survival rate and reduced disease-free patient survival rate. For example, in a multivariate analysis low level of expression of E-cadherin was identified as an independent factor that is significantly associated with metastases or recurrent disease in 22 Dukes’ stage B colon carcinomas [132]. However, in another study on 90 moderately differentiated Dukes’ B carcinomas this marker did not predict tumor recurrence [133]. Using in-situ hybridization and multivariate analysis, a low level of expression of Ecadherin was found to be an independent prognostic factor that was significantly associated with metastasis or recurrent disease in N0 -colon carcinomas [132, 134]. The expression of E-cadherin-binding proteins (α- and β-catenin) was frequently reduced (about 80%) in primary colorectal carcinomas [135, 136]. Normal mucosa, as well as colon adenomas, showed strong membranous α-catenin expression. In the normal colon, catenin expression was observed in the crypt and surface epithelium; these cells showed reactivity both at the membrane and in the cytosol [137]. The down-regulation of β-catenin expression appears to be associated with malignant transformation, and colorectal cancer cells may have impaired E-cadherin-mediated cell adhesiveness because of the down-regulation of catenin expression [135]. Reduced α-catenin expression in colorectal cancer has been associated with poor differentiation, increased frequency of distant metastases and reduced prognosis [138, 139]. In E-cadherin and β-catenin coexpression experiments about 80% of the colon carcinomas showed similar expression of both pro-

J. Haier & G.L. Nicolson teins, whereas the remaining tumors showed strong positive staining for E-cadherin and reduced expression of β-catenin [136]. There is some evidence in the literature on the increased expression of alternatively spliced variants of the CD44 family of cell adhesion molecules and their association with colorectal tumor metastasis. In colorectal carcinogenesis, expression of CD44 exon v5 seems to be an early tumor marker since it is detectable on small dysplastic polyps but not on normal colon epithelium [140]. The loss of expression of the CD44-v6 isoform seems to be associated with poor prognosis in colorectal cancer and the development of tumor metastases [141]. However, the correlation of expression of CD44 variants with tumor progression and prognosis, such as tumor recurrence and survival, are controversial [142, 143]. For example, CD44-v6 immunoreactivity was detected in 100% of adenomas, and in more than 90% of colorectal carcinomas, but expression was mostly weak in about only one-third of liver metastases. Expression of none of the CD44 variant epitopes was found to be positively correlated with tumor progression or with colorectal tumor metastasis to the liver, results which are inconsistent with a role for CD44 variants as indicators of colonic cancer progression [144]. Using multivariate analysis the expression of another CD44 exon, CD44v8-10, has emerged as an independent prognostic indicator for lymph node and hematogenous metastasis and overall survival [145, 146]. On the basis of a lack of consensus, further examination of CD44 variants and their role in colorectal carcinoma progression and metastasis seems warranted. Not all of the changes found in colorectal tissues are found on tumor cells. The degree of expression or E-selectin was found to be inversely correlated to the distance of the blood vessels from the cancer cells nests. Furthermore, EC adjacent to metastatic lesions expressed E-selectin more extensively than those adjacent to the primary tumor foci [47]. Serum E-selectin levels were also significantly elevated in the metastatic group of patients compared with a non-metastatic patient group [147]. Distinct integrin expression in certain patterns has been found in normal colorectal tissues, primary tumors and their metastases [26]. When compared to their primary tumors, colorectal carcinoma liver metastases showed roughly similar patterns of integrin expression. In various studies, the expression of integrins in normal tissue was determined and compared with different stages of colorectal carcinomas [30, 148]. Generally, a high variability of integrin expression seemed to be related to the degree of differentiation of the original tumor [149]. Correlations have also been found between integrin expression and tumor prognosis and clinical stage. When 19 colorectal carcinomas, 8 adenomas, and 8 normal colon tissues were compared for their integrin expression, malignant transformation of colon cells was found to be associated with infiltrative growth and characterized by diminished or lost expression of α6 -, β1 -, and β4 -integrin subunits [31]. In 96 patients with colorectal carcinomas loss of α2 - or α3 -integrin expression was significantly related to impaired tumor differentiation, more

Cell biology and clinical implications of adhesion molecules in colorectal diseases

633

Table 1. Cell adhesion molecules with changes in the expression patterns during the adenoma-carcinoma-progression sequence in colorectal tumors. Family

Name

Function

Normal expression

Expression in colorectal carcinomas

Integrins

VLAs LFAs gp

ECM-cell adhesion, leukocyte-EC adhesion, platelet-EC adhesion, gut homing receptor

Colonocytes, EC, fibroblasts, leukocytes, platelets

α2 -, α6 -, β1 -, β4 -integrins reduced; α5 -integrin hyperexpressed

Immunoglobin gene superfamily

ICAM VCAM PECAM (CD31)

Antigen recognition, leukocyte adhesion and trafficking

EC, fibroblasts, leukocytes

Hyperexpressed in tumor margins, reduced within carcinomas

CEA (CD66)

Homotypic cell adhesion

Colonocytes

Hyperexpressed

Cadherins

Homotypic cell adhesion

Colonocytes

Reduced or lost

Lectins

sLex sLea (CA 19-9) sLex Galectin-3

ECM-cell adhesion, heterotypic EC adhesion

Colonocytes, EC

Hyperexpressed

Selectins

E-, P-, L-selectin

Leukocyte-EC adhesion, platelet-EC adhesion

EC, leukocytes, platelets

Hyperexpressed

Proteoglycan receptors

CD44

Hyaluronate adhesion

Colonocytes, EC

Hyperexpressed, different exons

VLA – very late antigens, LFA – leukocyte function associated antigens; gp – platelet glycoproteins; ICAM – intercellular adhesion molecules; VCAM – vascular cell adhesion molecule; MAdCAM – mucosal addressin cell adhesion molecule; PECAM – platelet endothelial cell adhesion molecule; CEA – carcinoembryonic antigen; ECM – extracellular matrix; EC – endothelial cells.

advanced Dukes’ stage and survival rate [33], while loss of expression of the β1 -subunit was significantly correlated with lymph node metastasis and depth of invasion. A strong correlation was also observed between the expression of the α6 -laminin receptor and the degree of colorectal carcinoma differentiation, invasive and metastatic properties [130]. Colorectal carcinomas with increased metastatic potential and with poor prognosis are characterized by high contents of certain carbohydrate antigens. The levels of these carbohydrate antigens apparently increase during colorectal carcinoma progression from non-metastatic to metastatic tumors [150], suggesting their involvement in the metastatic processes. For example, in retrospective studies the level of tumor-associated sLex antigens was inversely correlated to the post-surgical survival of colon carcinoma patients [151]. Multivariate analysis revealed that the sLex status was an independent predictive factor for colorectal disease recurrence, depth of invasion, and histologic type [152, 153]. The CA 19-9 antigen had been studied for years before it was identified as a monosialosyl Lea blood group antigen. Its contents in adenoma and carcinoma specimens were found to be significantly higher than in the normal surrounding mucosa [154, 155]. Higher tumor stages correlated with higher tissue marker values of CA 19-9 antigen in 115 primary colorectal carcinomas and 64 normal colorectal mucosa specimens, and its content in carcinoma specimens was significantly higher than in the normal mucosa [156].

The concentrations of CEA in tumor specimens and blood show a high degree of correlation with the risk of carcinoma relapse [157–159]. Conversely, there was no correlation between tissue CEA content and tumor differentiation [159]. Immunohistochemical expression confirmed the predictive value of CEA tissue contents in colorectal tumor specimens [160]. In addition, in other studies serum CEA levels determined by radioimmunoassays and CEA tissue contents determined by immunohistochemical staining correlated with poor patient survival, and they appear to have similar prognostic values [157, 161]. For example, using multivariate analysis in a large study on 572 lymph node negative colon cancer patients preoperative CEA serum levels and overall clinical stage predicted patient survival [162]. The overall 5-year survival rate was also predicted in a multivariate analysis of 2330 patients with all stages of colorectal carcinomas [163]. In cancer tissues, the presence or infiltration of inflammatory cells has been suggested to be an independent marker of host surveillance. It has also been assumed that the presence of EC in an activated state may indicate occurring inflammatory reactions [164]. For example, since venules distributed along the invasive margins of colorectal carcinomas expressed E- and P-selectins and ICAM-1 (EC phenotypical features similar to those observed in active inflammatory lesions), these EC might act as immunologically-activated effector cells. EC cells at the invasive margins of tumors

634 appear to express different adhesion molecules compared to EC within tumors. Since the majority of blood vessels within tumors lack immunoreactivity for adhesion molecules (Eand P-selectins and ICAM-1), these EC are thought to be immunologically inactive [164]. The presence of tumor-associated macrophages is thought to be an important factor that may modulate carcinoma progression. This may occur due to the release of a variety of active substances, such as activated oxygen species, free radicals, etc. that can directly modify tumor genomes. The invasive margins seen in colorectal carcinomas appear to be similar to sites of active inflammation, where antigen presenting cells (macrophages) and lymphocytes may interact via ICAM-1/αL β2 -integrin adhesion systems [58]. Alternatively, macrophages themselves may interact directly with tumor cells to cause growth inhibition, death and alterations in gene expression [165, 166]. Hirschsprung’s disease Hirschsprung’s disease is characterized by a segmental loss of inervation in the colonic muscle layers that results in disturbed motility of the colon and impaired fecal transport. Although the pathogenesis of Hirschsprung’s disease is not fully understood, advances in understanding Hirschsprung’s disease involve changes in specific adhesion molecules. The absence of ganglion cells in Hirschsprung’s disease has been attributed to the failure of migration of neural crest cells. As a possible etiological mechanism of this disease, the association of Hirschsprung’s disease with a reduction in or lack of neural cell adhesion molecule (NCAM) expression in the aganglionic portions of the bowel has been described. Loss of NCAM may result in the failure of neurocytes to migrate to aganglionic regions [167]. The expression of the neuron specific L1 molecule, which plays an important role in cell adhesion, neural cell migration and neurite outgrowth, is reduced in the extrinsic nerve fibers in aganglionic colon [168]. Similar reductions in NCAM were demonstrated in colonic muscle specimens from patients with Hirschsprung’s disease [169, 170]. This reduced expression may perturb neural crest migration and prevent adequate neurite outgrowth, resulting in aganglionic segments and abnormal nerve bundles of extrinsic fibers in the colons of these patients [168, 171]. In general, a Hirschsprung’s disease-like regional defect appears to be the result of a generalized abnormality of neural cell adhesion molecule expression and/or function [172]. The expression of ICAM-1 and MHC class II on hypertrophic nerve trunks suggests the presence of an immunologic response in the pathogenesis of Hirschsprung’s disease [173]. These findings support the hypothesis that the aganglionosis may be caused by failure of nerve cell differentiation as a result of microenvironmental changes after the cell migration, such as reduced levels of neurotrophic factors or enhanced immunological activity [174].

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