Key words: cadherins, cadherin switch, cancer, cell adhesion, metastasis, tumorigenesis

Summary
Progression to tumor malignancy involves changes in a tumor cell's capabilities to adhere and communicate with neighboring cells and with its extracellular environment. Correlation studies in human cancer specimen and functional experiments with cultured tumor cells and transgenic mouse models have indicated that the loss of the cell adhesion molecule E-cadherin is causally involved in the formation of epithelial cancers (carcinomas). More recently, it has been observed that the function of E-cadherin is replaced or overruled by the expression of mesenchymal cadherins, such as N-cadherin. Although the functional implication of such a ¡§cadherin switch¡¨ remains to be elucidated, recent experimental results demonstrating an interaction of cadherins with tyrosine kinase receptors suggest that changes in cadherin expression may not only modulate tumor cell adhesion but also affect signal transduction and, hence, the malignant phenotype.

Introduction
The majority of human cancers originates from epithelial cells. Normal epithelia is organized by a number of specific intercellular junctions, including tight junctions, adherens-type junctions and desmosomes, which are intimately interconnected with the actin and intermediate filament cytoskeleton. Among the many types of cell-cell adhesion molecules, cadherins play a critical role in establishing adherens-type junctions by mediating Ca⁺⁺-dependent cell-cell adhesion [1-3]. Cadherin-based cell-cell adhesion is critically involved in early embryonic morphogenesis, as exemplified by the early embryonic lethality of mice lacking E-cadherin, a prototype classical cadherin [4, 5]. Cadherin-mediated cell-cell adhesion is accomplished by homophilic protein-protein interactions of extracellular cadherin domains in a zipper-like fashion. The intracellular domain of classical cadherins interacts with various proteins, collectively termed catenins, which assemble the cytoplasmic cell adhesion complex (CCC) that is critical for the formation of extracellular cell-cell adhesion. beta-catenin and gamma-catenin (also called plakoglobin) bind to the same conserved site at E-cadherin¡¦s C-terminus in a mutually exclusive way [6-8], whereas p120^ctn interacts with multiple sites in E-cadherin¡¦s cytoplasmic tail, including its juxtamembrane region [9, 10]. beta-catenin and gamma-catenin bind directly to alpha-catenin, which links the CCC to the actin cytoskeleton. While the dual role of beta-catenin and gamma-catenin in cell adhesion and Wnt-signaling has been extensively studied (see below), the functions of p120^ctn are poorly understood. p120ctn has been implicated both in cell-cell adhesion and in cell migration [11], and recent studies suggest that p120^ctn promotes cell migration by recruiting and activating Rho-family GTPases [12].

E-cadherin and cancer
It has long been known that cell-cell adhesion is dramatically changed during the development of malignant cancer. In particular, in most if not all cancers of epithelial origin, E-cadherin-mediated cell-cell adhesion is lost concomitant with progression towards malignancy, and it has been proposed that the loss of E-cadherin-mediated cell-cell adhesion is a prerequisite for tumor cell invasion and metastasis formation [13]. Multiple mechanisms are found to underly the loss of E-cadherin function during tumorigenesis: mutations or deletions of the E-cadherin gene itself, mutations in the beta-catenin gene, transcriptional repression of the E-cadherin gene, for example by hypermethylation or chromatin rearrangements in the E-cadherin promoter region and, finally, aberrant tyrosine phosphorylation of the components of the CCC (reviewed by reference [14]). Recent reports have highlighted that the DNA binding protein Snail acts as a strong repressor of E-cadherin gene expression in tumor cells, thus inducing tumor malignancy [15-18].

The observation that E-cadherin function is frequently lost in malignant cancers prompted an examination of the functional role of E-cadherin in tumour progression. Using tumour cell lines in culture, several groups demonstrated that re-establishing the functional cadherin complex, for example by forced expression of E-cadherin, resulted in a reversion from an invasive to a benign, epithelial tumour-cell phenotype [13, 19]. Although these experiments clearly demonstrated a critical role for E-cadherin in the suppression of tumour invasion in cultured cells, it remained elusive whether the loss of E-cadherin-mediated cell adhesion is a pre-requisite for tumor progression or whether it is instead a consequence of de-differentiation during tumor progression. We have recently shown that expression of E-cadherin is lost during the transition from well-differentiated adenoma to invasive carcinoma in a transgenic mouse model of pancreatic beta-cell tumorigenesis (RIP1TAG2). To assess whether loss of E-cadherin-mediated cell adhesion is a cause or a consequence of tumor progression in vivo, we crossed RIP1TAG2 mice with transgenic mice that either express wild-type E-cadherin or a dominant negative form of E-cadherin specifically in the pancreatic beta cells [20]. Maintenance of E-cadherin expression during beta-cell tumorigenesis resulted in arrest of tumor development at the adenoma stage. By contrast, expression of a dominant negative E-cadherin induced early invasion and metastasis. These results demonstrate that loss of E-cadherin-mediated cell-cell adhesion is one rate-limiting step in the progression from adenoma to carcinoma in vivo and highlight the role of E-cadherin as a suppressor of tumor invasion.

However, several questions remain to be answered. Tumor invasion is the result of a sequence of multiple cellular events, involving not only changes in cell-cell adhesion but also in cell-matrix adhesion, cell migration, proteolytic degradation of extracellular matrix and so forth. Therefore, it is difficult to envisage that the loss of E-cadherin-mediated cell adhesion per se is sufficient to confer an invasive phenotype to tumor cells. It seems more likely that E-cadherin downregulation results in the activation of specific signaling pathways which, in turn, trigger tumor cell invasion. One of the obvious candidates for activating such signaling pathways is beta-catenin. Besides being a key component of the CCC, beta-catenin plays a key role in Wnt-mediated signal transduction. beta-catenin is usually sequestered in the E-cadherin adherens junction or in tight-junction complexes. Non-sequestered, free beta-catenin is rapidly phosphorylated by glycogen synthase kinase 3beta (GSK-3betaƒw in the adenomatous polyposis coli (APC)/GSK-3beta/axin complex and subsequently degraded by the ubiquitin-proteasome pathway. If the tumor suppressor APC is non-functional, as is the case in many colon-cancer cells, or GSK-3beta activity is blocked by activated Wnt signaling, beta-catenin accumulates at high levels in the cytoplasm. Subsequently, it translocates to the nucleus, where it binds to a member of the TCF/LEF-1 family of transcription factors and modulates expression of TCF/LEF-1-target genes. Target genes of TCF/beta catenin that could be relevant for tumor progression include the proto-oncogene c-Myc and cyclin D1 [21, 22]. Future investigation should focus on the relationship between E-cadherin downregulation and beta-catenin signaling during tumor progression, in particular addressing the issue of whether the loss of E-cadherin results in the activation of the Wnt signaling pathway thus endowing tumor cells with an invasive phenotype.

Not much attention has been devoted to the role of the cytoskeleton upon the loss of E-cadherin-mediated cell-cell adhesion and the induction of tumor malignancy. Cadherin-based adhesion complexes are functionally linked to the dynamics of actin and microtubule cytoskeletal structures [23, 24]. Thus, it can be anticipated that the loss of E-cadherin-mediated cell adhesion leads to dramatic cytoskeletal rearrangements. Cellular factors that connect cadherin function with cytoskeletal organization are likely to play a key role in the structural alterations following the downregulation of cadherin-mediated cell adhesion. Small GTPases of the Rho family are obvious candidates for future investigation, since besides controlling the actin cytoskeleton they are known to modulate cadherin activity [25-27]. Interestingly, among the molecules linking small GTPases with cadherin function is IQGAP1, a protein whose dysregulation has been proposed to correlate with malignancy in gastric cancer [28, 29].

Muir-Torre syndrome
Recent studies have shown that mesenchymal cadherins, in particular N-cadherin, enhance tumor cell motility and migration [30-33], thus showing an opposite effect as compared to E-cadherin. N-cadherin-induced tumor cell invasion can even overcome E-cadherin-mediated cell-cell adhesion [30, 34]. The most intriguing findings in this context come from ex vivo studies documenting de novo expression of the mesenchymal cadherins N-cadherin and cadherin-11 in human tumors which have lost E-cadherin expression [35, 36]. This cadherin conversion recapitulates a well characterized phenomenon occurring during development, when epiblast cells switch from E- to N-cadherin in order to ingress the primitive streak [37, 38]. Based on these observations a novel concept has been formulated that a ¡§cadherin switch¡¨ from epithelial to mesenchymal cadherins supports the transition from a benign to an invasive, malignant tumor phenotype.

Of course, these observations raise a number of important questions. For example, E-cadherin and N-cadherin are both classical cadherins and on first sight seem to involve similar mechanisms of cell-cell adhesion. Hence, the basis for their functional differences is not clear. It has been proposed that, unlike E-cadherin, N-cadherin (and, presumably, other mesenchymal cadherins) promotes a dynamic adhesion state in tumor cells, allowing not only the dissociation of single cells from the tumor mass but also their interactions with endothelial and stromal components [30, 35, 36]. Moreover, N-cadherin-expressing breast carcinoma cells were specifically sensitized to FGF-2-induced invasion and upregulation of the proteolytic enzyme MMP-9 [30]. These results have two important implications. First, they imply that matrix-degrading enzymes can mediate the pro-invasive effects of N-cadherin. Second, they underscore a functional interaction between N-cadherin and fibroblast growth factor receptor (FGFR) signaling. In fact, such a crosstalk has been shown in neurons, where it supports neurite outgrowth, an event strictly related to cell migration and invasion [39]. In addition, FGFR signaling has been implicated in N-cadherin-induced motility of breast cancer cells [34]. Finally, work in our laboratory has recently demonstrated a physical association between N-cadherin and FGFR in beta tumor cells of the endocrine pancreas [40], although the functional relevance of this interaction for tumor progression and metastasis remains to be verified. Notably, E-cadherin has also been found to associate with FGFR in tumor cells of the exocrine pancreas, and FGF stimulation enhances cell-cell adhesion in this system [41].

Although the latter result seems to contradict many reports showing that FGFR signaling decreases cell adhesion and triggers tumor cell invasion, it raises the interesting possibility that FGFR can associate with different cadherins in a cell type- or tumor type-specific manner. Verifying this hypothesis in vivo and elucidating the biological significance of the interaction between FGFRs and the cadherin systems in the context of tumor progression represents a stimulating challenge for the future.

Another crucial issue in the context of the cadherin switch is the molecular mechanism(s) regulating the conversion from one cadherin type to another. Interestingly, the cadherin switch in epiblast cells can be recapitulated in vitro by treating the cells with hepatocyte growth factor (HGF) [42]. The latter is known to play a pathogenic role in various tumors by supporting their invasion [43], and it is tempting to speculate that the induction of the cadherin switch may be part of HGF¡¦s tumorigenic capabilities. Islam et al. reported that the suppression of N-cadherin expression in invasive squamous carcinoma cells resulted in the induction of E- and P-cadherin expression and reversion to an epithelial phenotype. In contrast, forced expression of N-cadherin in epithelial-like squamous cells caused downregulation of E- and P-cadherin and acquisition of an invasive phenotype [32]. This implies that the expression of N-cadherin during tumor progression might be necessary and sufficient to induce the loss of E-cadherin-mediated cell-cell adhesion and to support the tumor transition towards malignancy.

Conclusions
The cadherin switch and its functional implication in tumor malignancy is an exciting research area in tumor biology, and it is expected to give some insights into how tumors acquire an invasive and metastatic phenotype. However, several issues need to be addressed before considering the cadherin switch as a crucial step in tumor progression. Thus far, the cadherin switch in vivo has only been described during the development of malignant melanoma and prostate carcinoma [36, 44, 45]. Further studies on other tumor types are required to establish whether a switch in cadherin expression is a common mechanism underlying tumor progression. In vitro observations on various tumor cell lines suggest that this might indeed be the case [35]. In addition, although the classical cadherin family comprises at least 30 members [46], not many attempts have been made to investigate the expression of cadherins other than E- or N-cadherin in different tumor types. Such systematic studies might provide evidence of tumor-specific cadherin repertoires, thus raising the possibility that many more members of the cadherin family are involved in cadherin switches and that the cadherins involved in the switch vary in a tumor-specific manner. Forced expression or genetic ablation of particular cadherin genes during embryonic development or in appropriate mouse models of tumorigenesis or other disease will help in unraveling the functional role of the cadherin switch(es) in physiological and pathological processes.

Extensive investigations on the functional relevance of the cadherin switch in vivo may not only provide additional insights into the molecular mechanisms underlying tumor progression, but may also allow the identification of novel molecular targets for anti-cancer therapy.

Acknowledgments
We apologize to those colleagues whose work we could not cite due to space restrictions. Research in the laboratory of the authors is supported by Boehringer Ingelheim, the Austrian Industrial Research Promotion Fund (FFF) and by the Austrian Science Foundation (FWF).

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