E-cadherin, β-catenin, and ZEB1 in malignant progression of cancer
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- Schmalhofer, O., Brabletz, S. & Brabletz, T. Cancer Metastasis Rev (2009) 28: 151. doi:10.1007/s10555-008-9179-y
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The embryonic program ‘epithelial-mesenchymal transition’ (EMT) is activated during tumor invasion in disseminating cancer cells. Characteristic to these cells is a loss of E-cadherin expression, which can be mediated by EMT-inducing transcriptional repressors, e.g. ZEB1. Consequences of a loss of E-cadherin are an impairment of cell-cell adhesion, which allows detachment of cells, and nuclear localization of β-catenin. In addition to an accumulation of cancer stem cells, nuclear β-catenin induces a gene expression pattern favoring tumor invasion, and mounting evidence indicates multiple reciprocal interactions of E-cadherin and β-catenin with EMT-inducing transcriptional repressors to stabilize an invasive mesenchymal phenotype of epithelial tumor cells.
Tumors of epithelial origin develop from benign precursor lesions to invasive carcinomas and metastases. In certain epithelial tumors, e.g. colorectal carcinomas, activation of the embryonic program ‘epithelial-mesenchymal transition’ (EMT) is crucial for the dissemination and invasion of cancer cells . Loss of epithelial differentiation and acquisition of a mesenchymal phenotype allows detachment of cancer cells from the primary tumor mass and dissemination into the surrounding stroma . The most important event of EMT is loss of E-cadherin, which was demonstrated to be a prerequisite for epithelial tumor cell invasion. E-cadherin, encoded by the CDH1 gene, has dual functions in epithelial cells: As cell-cell adhesion molecule and as negative regulator of the canonical WNT signaling cascade, in particular of its central mediator β-catenin. Several transcriptional repressors of the CDH1 gene have been indentified. They include the basic helix-loop-helix (bHLH) family members twist and the E2A gene product E12/47, members of the snail family, snail1 and snail2, and members of the zinc finger homeobox (ZFH) family of repressors, ZEB1 and ZEB2 [3–14]. The purpose of this review is to summarize the knowledge about the molecular interconnections of E-cadherin, β-catenin, and WNT signaling to these transcriptional repressors, in particular to ZEB1. Subsequently, the contribution of this interdependence to induction of EMT, tumor cell invasion, and metastasis will be discussed and exemplified by focusing on the colorectal carcinoma as a model tumor, which is one of the best studied epithelial tumors.
2 Physiology of E-cadherin
2.1 E-cadherin in development and tissue homeostasis
Much of the understanding of E-cadherin and β-catenin function was achieved by experiments with mouse model systems. In mouse embryogenesis zygotic E-cadherin expression starts between the late 4- and the 8-cell stage at E2.0, but is lost gradually in the trophectoderm and parietal endoderm after implantation of the developing embryo into the uterus [20–22]. During gastrulation E-cadherin expression is temporarily suppressed in cells of the epiblast, which loose their epithelial differentiation and acquire a mesenchymal phenotype. These cells delaminate at the primitive streak to populate the intraembryonic mesoderm and the definitive endoderm. Cells of the definite endoderm ultimatively line the gut tube and in these cells loss of CHD1 gene expression is only transient, as they start to reexpress E-cadherin and differentiate into a stratified intestinal epithelium [22–26]. Upregulation of E-cadherin expression is also observed during development of the kidney, brain, and melanocytes [27–31]. These results indicate that E-cadherin expression is dynamically regulated in embryogenesis. E-cadherin is transiently lost in migrating cells, but reexpressed, when cells start to differentiate in epithelial tissues. The physiological importance of E-cadherin for organ development and tissue morphogenesis also becomes evident from different gene targeting experiments of the CDH1 gene in the mouse. Ectopic injection of CDH1+/+ embryonic stem cells gave rise to teratomas, which were composed of differentiated cell types originating from all three germ-layers. In contrast, tumors derived from CDH1−/− embryonic stem cells failed to do so. Of note, reconstitution of E-cadherin expression exclusively caused formation of epithelia . In the developing embryo E-cadherin is a key player during morula compaction and CDH1 null embryos die early during embryogenesis as development of the blastocyst and trophectoderm is impaired [33, 34]. Conditional CDH1 gene targeting approaches revealed severe defects of epidermal as well as mammary and thyroid gland development [33–39]. Loss of E-cadherin expression in mammary epithelial cells was associated with impaired organ function, as alveolar structures in the lactating gland were not properly built up . Furthermore, loss of E-cadherin expression in the skin was demonstrated to impair establishment of apico-basal cell polarity and formation of tight junctions . A transgenic approach, causing overexpression of E-cadherin in intestinal crypts, resulted in reduced proliferation and increased apoptosis in the crypt as well as slower migration of enterocytic cells along the crypt villus axis .
Taken together, in embryogenesis E-cadherin is important for proper development, organ morphogenesis and tissue formation. Thereby, E-cadherin is a determinant of epithelial cell differentiation. E-cadherin not only is an important mediator of cell-cell adhesion, but also contributes to regulate diverse cellular processes, such as proliferation, migration, apoptosis or maintenance of epithelial cell polarity. This raises the question by which means E-cadherin can influence such a broad variety of responses. E-cadherin must emit downstream signals and the main mediator of these E-cadherin dependent effects appears to be β-catenin. For the involvement of other molecules involved in E-cadherin associated signaling refer to the review of Cavallaro and Christofori . Strikingly, β-catenin was demonstrated to localize to the nucleus, after E-cadherin expression is lost [42–44].
2.2 E-cadherin, β-catenin, and WNT signalling
During embryogenesis canonical WNT signaling, defined by nuclear accumulation of β-catenin, was demonstrated to promote invasive trophoblast differentiation, and activation of the signaling cascade was suggested to contribute to trophoblastic hyperplasia and local invasion . Furthermore, β-catenin is involved in induction of EMT in physiological processes, e.g. gastrulation or heart cushion formation [63–65]. This is supported by the fact that targeted disruption of β-catenin in mice leads to very early embryonic lethality by affecting development at gastrulation . Canonical WNT signaling emerged also as a potent regulator of various additional developmental processes, contributing to hair follicle morphogenesis, thymocyte and neural delevopment, as well as angiogenesis or adipogenesis [67–72]. WNT signaling is also essential for intestinal development. Thereby, β-catenin binds TCF4, a TCF-family member, specific for the intestinal and mammary epithelium . TCF4 expression characterizes the intestinal stem cell compartment and targeted disruption of TCF4 in mice leads to a severe impairment of gut development . The molecular role of β-catenin in the development of these organs and tissues can be deduced by the functions of its defined target genes (see below). Some of these target genes give the competence to act as a stem cell . This is in line with the observed loss of stem cells after targeted deletion of β-catenin in intestine or skin [68, 74]. The function in characterizing the stem cell compartment also indicates the role of nuclear β-catenin in the adult organism. Thereby β-catenin maintains tissue homeostasis, in particular in strong-proliferative, self renewing tissues, like skin or gut.
In recent years it became clear that E-cadherin and the associated catenins, in particular β-catenin, not only are static components of adherens junctions, but in addition act as important mediators of downstream signaling cascades. In this respect, E-cadherin contributes to maintain epithelial differentiation in two ways. First, by establishing cell-cell adhesion and apico-basal polarization, and second by acting as negative regulator of active WNT signaling. By recruiting β-catenin to adherens junctions, nuclear localization of β-catenin and thus β-catenin activated target gene expression is inhibited. Still, loss of E-cadherin is not necessarily linked to nuclear localization of β-catenin and transcriptional activation of responsive target genes. It appears that in addition to loss of E-cadherin expression, also components of the destruction complex (see above) have to be impaired to allow β-catenin to localize to the nucleus and exert its transcriptional activity [76, 77]. Of utmost importance is the interaction of GSK3β, APC, and axin with β-catenin [78, 79]. These molecules are negative regulators of the WNT pathway and only if these molecules are functionally assembled in the destruction complex, the amount of nuclear β-catenin can be efficiently regulated. Compiling all data, it is becoming evident that the interconnection of E-cadherin, β-catenin, and WNT signaling not only controls single events, but contributes to control the complex morphogenetic process of EMT. This regulation needs spatial and temporal coordination of multiple events like proliferation, migration, cell-cell adhesion, and differentiation. Loss of E-cadherin expression or loss of function of either APC, axin or GSK3β leads to a reduced degradation and subsequent overexpression of free cytoplasmic β-catenin, which can exert its nuclear function without control (see below). Not surprisingly, E-cadherin and β-catenin have been described to contribute to EMT also in tumorigenesis .
3 E-cadherin in cancer
Sustained CHD1 gene expression contributes to retain an epithelial phenotype and E-cadherin was demonstrated to act as an inhibitor of invasion in numerous tumor cell lines and in in vivo tumor models. In the Rip-Tag mouse model of beta-cell tumors of the pancreas, E-cadherin loss is a causal prerequisite for progression from adenoma to invasive carcinomas, and in human an inverse correlation between expression of E-cadherin and survival of tumor patients was demonstrated [80, 81]. For aggressive carcinomas deregulation of E-cadherin and impairment of adherens junction mediated cell-cell adhesion by different means were reported [80, 82–86]. These different possibilities include genetic alterations, epigenetic inactivation, and transcriptional silencing of the CHD1 gene.
3.1 Genetic and epigenetic inactivation of E-cadherin
Genomic alterations of the CDH1 gene causing loss-of-function of E-cadherin, have been identified in a variety of tumors. In breast cancer, somatic E-cadherin mutations were reported exclusively for sporadic lobular subtype, however not for breast cancers of other histopathological subtypes . Furthermore, somatic mutations of the CHD1 gene were identified in endometrial and ovarial carcinomas, as well as in primary gastric cancers and gastric cancer cell lines [88–90]. In addition, germline mutations of the CHD1 gene were discovered in patients, predisposed to suffer from diffuse gastric cancer [91, 92].
Another means to loose E-cadherin expression in tumor cells is CpG-island hypermethylation of the CDH1 gene promoter [93, 94]. Aberrant methylation of 5′CpG islands in the promoter region of CDH1 was reported for different types of mammary carcinomas and increased with malignant progression [95, 96]. Other reports showed methylation in thyroid, hepatocellular, and prostate cancer, as well as in laryngeal cancer and colorectal cancer [97, 98].
However, although these mechanisms have been described in numerous studies, the most frequent event appears to be another mechanism of transcriptional inhibition of E-cadherin: silencing of CHD1 gene expression by specific transcription factors. Several specific transcriptional repressors, interacting with E-boxes located in the proximal CDH1 gene promoter, have been identified so far (see below).
Taken together, loss of functional E-cadherin, and thus of adherens junction-mediated cell-cell contacts, enables the first step of metastasis: local invasion and dissemination of cancer cells from the main tumor mass.
3.2 E-cadherin and EMT
Cancer cells disseminating from the main tumor mass frequently are characterized by hallmarks of EMT . EMT is a developmental process fundamental to various steps of embryogenesis and has been implicated e.g. during embryo implantation into the uterus, gastrulation, palatal fusion, heart cushion formation, development of the peripheral nervous system, and branching morphogenesis of the mammary gland [65, 99–103]. Thereby, cells loose their epithelial differentiation and acquire a mesenchymal, spindle-shaped phenotype, characterized by loss of cell-cell adhesion, notably disruption of the cadherin–catenin complex, and loss of apico-basal polarization, as well as acquisition of migratory and invasive capabilities [104, 105]. Epithelial markers include cytokeratins 8 and 18, as well as E-cadherin localized at the plasma membrane. In contrast, vimentin expression, membraneous N-cadherin localization, and cytoplasmic relocation or loss of E-cadherin are indicative of a mesenchymal phenotype . Inducers of EMT, such as hepatocyte growth factor, tumour necrosis factor α (TNFa), and transforming growth factor (TGF)β are produced by infiltrating cells or the tumor cells themselves, and trigger expression of a variety of transcriptional repressors. These transcriptional repressors are the intracellular mediators of EMT and were originally identified as inhibitors of E-cadherin expression, contributing to malignant progression of numerous human tumors by repression of CDH1 . These factors include the basic helix-loop-helix (bHLH) family members twist and the E2A gene product E12/47, members of the snail family, snail1 and snail2, and members of the zinc finger homeobox family of repressors, ZEB1 and ZEB2 [3–14]. However, although their involvement in tumorigenesis is just unravelling, it goes far beyond transcriptional inhibition of E-cadherin expression. The importance of these proteins can be deduced from a steadily growing number of identified target genes, e.g. components of the basement mebrane, epithelial cell polarity factors or apoptosis pathways. In addition, most recently two studies reported induction of stem cell phenotypes in breast cancer by EMT [108, 109]. A comprehensive overview on the field was published most recently by Peinado et al. . Whereas in the first years after the notion that EMT is involved in tumorigenesis, the EMT inducers snail1 and snail2 were in the limelight of attention, more recently another player entered center stage: ZEB1.
3.3 ZEB1 in cancer
The transcription factor ZEB1, encoded by the TCF8 gene, is the vertebrate homologue of the ZFH gene family of zinc finger/homeodomain proteins. Much insight on physiological functions was gained by experiments with model organisms. In Drosophila melanogaster, ZEB1 regulates cell fate determination, as well as development of the central nervous system and of mesodermally derived tissues [111, 112]. Involvement in mammalian development was suggested by a conventional gene targeting approach of the TCF8 gene in mouse. Heterozygous animals were vial and fertile, whereas homozygous mutants developed to term and died after birth. Their phenotype was characterized by a severe T-cell deficiency of the thymus, as well as craniofacial, cartilage, and skeletal defects. Mutation of ZEB1 caused a mesenchymal-epithelial-like transition characterized by ectopic expression of E-cadherin in a variety of tissues in these mice, such as nasal and palate mesenchyme or the developing cartilage, as well as embryonic fibroblasts, which clearly displayed an abnormal epithelial-like morphology [113–115].
ZEB1 interacts with the regulatory regions of responsive target genes, by binding to the sequence motif CAGGTG/A, related to the consensus E-box sequence CANNTG [116–119]. ZEB1-dependent repression of genes involved in the development of hematopoietic cells, as well as of extracellular matrix (ECM) components was reported [116, 117, 120–124]. Although the research focused on the ability of ZEB1 to repress target gene transcription, increasing evidence demonstrates that ZEB1 may also act as transcriptional activator, thereby interacting with acetyltransferases p300/pCAF and activated SMADs. This appears to target genes such as smooth muscle actin and myosin or components or the VitD signaling pathway, which were demonstrated to contribute to mesenchymal differentiation [125–130].
Numerous signaling pathways were demonstrated to regulate TCF8 gene expression. TGFβ and TNFα are important mediators of EMT and were shown to be activators of TCF8 gene expression in various cancer cell lines [131–136]. Also IGF1 signaling was suggested to induce EMT in cancer and demonstrated to activate expression of ZEB1 [137–140]. Accumulating evidences also indicates a role of epidermal growth factor receptor (EGFR) signaling in cancer cell metastasis, in particular through activation of the TCF8 gene and inhibition of E-cadherin expression [141–145]. Furthermore estrogen and progesterone dependent activation of the TCF8 gene was reported [126, 146]. A recent report demonstrated a COX2-dependent inhibition of E-cadherin expression. The COX2 metabolite PGE2 induced expression of snail1 and ZEB1 . Repressors of TCF8 are a certain isoform of ZEB1 itself and the Rb-E2F1 cell cycle repressor complex [148, 149].
Moreover, a role of ZEB1 was implicated in the tumorigenesis of renal clear cell carcinomas (RCC). In RCC, expression of ZEB1 was shown to be regulated by HIF1 and, typically, in biopsies of renal clear cell carcinomas (RCC) the expression patterns of HIF1 and E-cadherin were mutually exclusive . Aberrant ZEB1 expression was also detected in tumors of uterine origin, in particular in myometrium-derived malignant leiomyosarcoma and in the stroma of low grade (type I) endometrioid adenocarcinomas. No epithelial ZEB1 expression was observed in normal endometrium and low-grade endometrial cancers. However, in more aggressive type II cancers of endometroid origin, such as FIGO grade 3 endometrioid adenocarcinomas, uterine papillary serous carcinomas, and in carcinosarcoidal tumors of the malignant mixed Mullerian tumor type, ZEB1 is aberrantly expressed in tumor cells of epithelial origin. The high expression level of ZEB1 correlated with loss of E-cadherin expression in surgical specimens and increased migratory and invasive potential of endometroid cancer cells [152, 153]. ZEB1 seems also to be involved in malignant progression of breast cancer, Screening of breast cancer cell lines revealed an inverse correlation of TCF8 and CHD1 gene transcript levels and cell lines with high ZEB1 expression displayed a spindle-like, fibroblastoid phenotype [146, 154–156]. Another regulator of ZEB1 in breast cancer appears to be CCN6. Low CCN6 expression caused an upregulation of snail1 and ZEB1 and was associated with decreased expression of E-cadherin and axillary lymph node metastasis in breast cancer [157–159]. A correlation of ZEB1 expression and loss of E-cadherin was demonstrated in tumor cell lines of lung adenocarcinomas of the non small cell lung cancer (NSCLC) subtype . Another study suggested that E-cadherin repression is regulated in a COX2-dependent manner in NSCLC . Thereby ZEB1 is induced by the COX2 metabolite prostaglandin E2 (PGE2). The inverse pattern of E-cadherin and ZEB1 expression was also demonstrated in human lung adenocarcinomas in vivo, suggesting that PGE2 acts as a paracrine or autocrine modulator of ZEB1 expression.
In summary, ZEB1 contributes to malignant progression of various epithelial tumors, Thereby, ZEB1 is a crucial mediator of EMT, exerting its effects on induction of EMT by inhibiting expression of E-cadherin and miRNAs, which induce an epithelial phenotype [135, 144, 161]. This raises the question of interconnections of ZEB1, E-cadherin and β-catenin with WNT signaling and EMT in cancerogenesis. This point will adressed using the colorectal carcinoma as model tumor in the next section.
4 Compilation: colorectal cancer as a model tumor
β-catenin has been imlpicated in cancer stemness and among the genes activated by β-catenin during colorectal carcinogenesis are factors associated with proliferation, such as cyclin D1 or c-myc, and genes associated with tumor cell survival and a tumor stem cell phenotype, such as survivin and MDR [176–181]. These traits are necessary to give rise to a primary tumor during early stages of tumorigenesis, but insufficient to promote invasion and metastasis. Among the genes targeted by nuclear β-catenin and selectively expressed at the tumor-host interface are many of crucial importance to tumor invasion, such as L1CAM, CD44, TNC, VEGF, PLAUR, PLAU, MMP7, MMP14, LAMC2 and JUN. To invade the host stroma, tumor cells must loose epithelial differentiation and acquire a mesenchymal, motile phenotype. The β-catenin-dependent transcription program being activated in budding tumor cells also comprises mesenchymal markers, e.g. fibronectin, as well as repressors of epithelial differentiation, such as CDX1 and ENC1 [19, 165].
ZEB1 expression has been associated with tumor cell migration, invasion, and metastasis, in particular in colorectal carcinomas [140, 144, 152, 188, 198]. Epithelial tissues are surrounded by a basement membrane, which induces epithelial differentiation and basal-apical polarization. ZEB1 interferes with these traits of an epithelial phenotype by inhibiting expression of basement membrane components, of E-cadherin and desmosomal proteins, as well as intracellular effectors of cell polarity [155, 156, 188, 198]. Overcoming the basement membrane is a prerequisite for epithelial tumor cells to invade the stroma and subsequently to disseminate and metastasize . In this context, ZEB1-mediated loss of the basement membrane contributes to tumor progression and disturbance of tissue architecture in a dual fashion: by loosing a mechanical barrier and by loss of inductive signals, and thus subsequent loss of epithelial polarity. ZEB1 also inhibits basal-apical differentiation by directly targeting cell polarity factors, such as AP1M2, PATJ, and CRB3 [156, 198, 200, 201]. These data also demonstrated a direct role of ZEB1 in metastasis by suppression of LGL2. Indeed, LGL2 is classified as tumor suppressor, as loss of its Drosophila homologue lgl caused metastatic tumors . ZEB1 also inhibits epithelial differentiation by transcriptional inhibition of the microRNA (miR)-200 family. miR-200 family members were demonstrated to be expressed in epithelial tissues and negatively correlated with expression of TCF8 [203, 204]. When transfected into tumor cell lines of colorectal origin, a mesenchymal to epithelial differentiation was induced by miR-200 family members, including upregulation of E-cadherin expression. The miR-200 family inhibited migration and invasion of undifferentiated cancer cell lines. Intriguingly, ZEB1 is also a direct target of the miR-200 family and ZEB1 was demonstrated to directly suppress transcription of two members of the miR-200 family, miR-141 and miR-200c . As both miRNAs affect the expression of molecules acting in a proinvasive manner, such as cofilin, the leptin receptor, TGFβ2 and, in particular, ZEB1, this suggests an EMT-enhancing feed forward loop in invading cancer cells [131, 132, 135, 161, 205]. Supporting this model, knockdown of ZEB1 in colorectal cancer cells abolished liver metastasis in a nude mice xenograft approach . This is in line with the observation that expression of ZEB1 and selective loss of basement membrane in invasive tumor regions of colorectal carcinomas are a strong predictor of poor patient survival and metachronous distant metastasis .
An enormous knowledge about the invasion and metastasis of epithelial cancer cells has been gained in the last 20 years. This process is best understood in colorectal cancers. In colorectal carcinomas disseminating cancer cells undergo EMT at the tumor-host interface, that is they loose their epithelial differentiation and acquire a mesenchymal phenotype, allowing detachment of cancer cells from the primary tumor mass. The most important hallmark of EMT is loss of E-cadherin, which is mediated by different transcriptional repressors, e.g. ZEB1 and snail1. E-cadherin has a dual function in epithelial cells as cell-cell adhesion molecule and as negative regulator of the canonical WNT pathway, in particular its central mediator β-catenin. After loss of E-cadherin expression, β-catenin localizes to the nucleus and activates a target gene expression program, linking EMT to WNT signaling. However, WNT signaling is also linked to EMT, by direct activation of snail2 or by indirect activation of ZEB1 via other WNT target genes, e.g. COX2 or IGF1. Taken together, this suggests a feed forward loop of dedifferentiation in invading cancer cells in colorectal carcinomas. This loop involves E-cadherin, β-catenin and ZEB1 and interference with these crosstalks might offer novel therapeutic opportunities in the future.
This work was supported by grants to T.B. from the EU MCSC contract no. 037297, the DFG (no. BR 1399/4-3) and the Deutsche Krebshilfe (no. 106958).