E-Cadherin on Cancer malignancy
The majority of cancers (>90%) are classified as carcinomas originating in the epithelial cells lining the exterior surfaces and internal cavities of the body. These cells exhibit unique morphological characteristics including extensive intercellular complexes, intercellular and cell-cell adhesion to a stable matrix, and distinct apical and basolateral portions of the plasma membrane.
The nature of well-differentiated epithelium allows individual cells to function collectively as a single organized unit Changes in the level of adhesion molecules between cells are important for the development of aggressive carcinomas, especially changes in E-cadherin expression.2 E-cadherin is an adherens junction protein, which through homotypic interactions maintains the barrier function of the epithelium.3 An important hallmark of cancer is the ability of cancer cells to break free from the primary tumor and migrate to distant sites in the body to form new tumors. This metastatic ability generally arises because of the progressive loss of the epithelial characteristics of cancer cells. Currently, E-cadherin is a very active research theme in the field of tumorgenesis.1,2
E-Cadherin Gene and Protein Structure
E-cadherin is a member of the classic cadherin proteins found in epithelial tissue. This protein is encoded by the CDH1 gene. Synonyms of the CDH1 gene include Arc-1, CD324, CDHE, E-cadherin, ECAD, LCAM, Uvomorulin. CDH1 is located on chromosome 16, the long arm of q22.1. This gene is 120 kDa in size, with 16 exons separated by 15 introns. The exons cover an area of up to ~100 kb with base sequences ranging from 115-2245 bp (base pair). Introns range from 120 bp (intron 4) to 65 kb (intron 2). The intron-exon boundary is well preserved compared to other classical cadherins, and in intron 1 a 5′ high-density CpG island was identified that has a role in transcriptional regulation. This island covers the exon 1 to exon 2 area of the human E-cadherin gene, while other exons do not have such features, including the largest exon, exon 16 with 2245 bp (figure 1).2,3
E-cadherin is a type-I transmembrane glycoprotein that is localized to the adherens junction and basolateral membrane of epithelial cells. E-cadherin has several names, including cell adhesion molecule (CAM) 120/80, CD324 antigen, Epithelial cadherin, cadherin 1, cadherin 1 precursor, cadherin type 1, calcium-dependent adhesion protein. E-cadherin is transported from the Golgi complex to the cell surface as a pro-protein and binds to -catenin early in its biosynthesis, whereas P120-catenin and -catenin (which binds to -catenin) only bind after the complex has reached the cell surface. ,4
The structure of E-cadherin consists of 1 extracellular domain, 1 single transmembrane segment and 1 cytoplasmic domain. The extracellular domain consists of 5 repeating extracellular cadherin domains (EC1-5) which bind calcium ions to form rigid linear molecules. The cytoplasmic domain interacts with the actin cytoskeleton through a linking molecule, a protein complex consisting of -, -, and -catenin. – and -catenin share significant homology and bind to a specific domain at the C-terminal E-cadherin. Alpha-catenin links – or -catenin to the actin cytoskeleton (figure 2).1,2
Alpha-catenin is structurally similar to vinculin, a major component of the fibroblast membrane of microfilaments, beta-catenin exhibits homology with Armadillo from Drosophila melanogaster and gamma-catenin is identical to placoglobin, a protein found in desmosomes.
The C-terminal cytoplasmic domain with ~150 residues is highly sequenced, and has been experimentally shown to regulate the intercellular binding function of the E-cadherin extracellular domain through interactions with the cytoskeleton. The juxtamembrane region of the cytoplasmic end of cadherin has been identified as a functionally active area that determines cadherin clustering and adhesion strength; one of the proteins involved in cell clustering and adhesion is p120ctn.2 Binding of the cytoplasmic domain of E-cadherin with -catenin and p120-catenin will stabilize E-cadherin molecules on the cell surface.5
The N-terminal structure of the extracellular domain of E-cadherin has the most adhesive activity. This part of the molecule also has bonding regions for calcium ions located between the chains. Intercellular adhesion is mediated by homophilic interactions of the extracellular domain of E-cadherin via a lateral dimerization process. Parallel dimers can bind to dimers from adjacent cells by forming adhesion points. These findings indicate the presence of bonds between cadherins on the cell surface
E-cadherin is one of the most important molecules in intercellular adhesion in epithelial tissue.2 Adhesion between adjacent cells occurs through homophilic interactions between E-cadherin molecules. The first interaction occurs between adjacent cells through trans-interaction, then the same cell forms a zipper-like structure through lateral association/unification (cis-interaction).
The adhesion function of E-cadherin plays an important role in epithelial physiology. The complete form of the E-cadherin/catenin complex together with associated actin filaments forms an adherens junction, which fuses 2 plasma membranes with an intercellular gap of only ~25 nmb. This very narrow association stops the movement of individual epithelial cells (known as contact inhibition for motility) and allows organizing these cells into tightly bound layers with functionally distinct apical and basolateral plasma membrane domains. E-cadherins mediating adherens junctions are also important for the formation of tight junctions, an important epithelial structure that forms an apical permeability barrier that blocks the passive diffusion of solutes between the lumen and the interstitial space.
In addition to immobilizing epithelial cells in their respective tissue layers, E-cadherin can also inhibit growth factor (GF) mediated proliferation signaling (ie contact growth inhibition), thereby maintaining integrity and maintaining tissue function. Furthermore, E-cadherin can influence various processes that occur during embryonic development, such as cell sorting and epithelial to mesenchymal transition (EMT), and even maintain embryonic stem cell pluripotency.
E-cadherin expression in embryonic development occurs very early, starting at the two-cell stage. Epithelial differentiation and polarization (basic processes for cell differentiation) occur early on ontogeny in the morula stage, when the embryo condenses and each cell undergoes polarization along the apicosal axis to produce an epithelium-like phenotype. E-cadherin plays an important role in blastomere adhesion and early embryonic capacity to condense. E-cadherin is expressed in the membrane even before morula compaction occurs, is distributed in a non-polar manner and has not shown an adhesive function. The mechanism that makes E-cadherin functional is not known with certainty, but it clearly involves the process of protein phosphorylation.2,3
Another important E-cadherin function under development is controlling mesenchymal epithelial conversion. In contrast to well-differentiated epithelium, mesenchymal cells exhibit an elongated morphology with asymmetric edges, forming only transient adhesions with surrounding cells and the extracellular matrix, also producing various enzymes that can degrade the matrix.1
E-cadherin is mainly expressed on the surface of epithelial cells and is responsible for the formation and maintenance of tissue architecture, maintaining stable intercellular relationships and normal epithelial cell polarity. In addition, E-cadherin is important in regulating the development, integrity and organization of epithelial cells
Intercellular interactions mediated by the E-cadherin/catenin complex play a major role in regulating motility, proliferation, cell differentiation and inhibiting cell proliferation when cell-to-cell contact occurs. The loss of cell-to-cell contact will allow beta-catenin to translocate into the nucleus and stimulate cell proliferation. Reconnected E-cadherin will cause beta-catenin to return to the membrane so that proliferation decreases. This event is known as “contact inhibition”. Loss of inhibitory contact due to E-cadherin mutations, or other causes, is a key characteristic of carcinoma
Therefore, in order to maintain the dynamics of a single layer of epithelial cells, E-cadherin is rapidly removed from the plasma membrane, recycled and returned to the cell surface to then form new connections. The degree of adhesion strength formed can be modulated by regulation of transcription and/or protein degradation through the ubiquitin-proteosome pathway. In addition, junction protein recycling is an alternative mechanism that allows cells to undergo rapid morphology in response to extracellular stimuli, such as low levels of Ca2+ and growth factors such as hepatocyte growth factor (HGF).
Until recently, cadherin was thought to be involved only in homophilic interactions, but now E-cadherin has also been shown to be a ligand for two integrins, alphaEbeta7 and alpha2beta1. The first interaction serves to maintain intraepithelial lymphocytes in the mucosal tissue, while the second contributes to the organization of the epithelial layers
The Role of E-cadherin in Cancer Cells
a. E-cadherin expression in cancer
The progressive accumulation of somatic mutations in a number of different genes characterizes the process of tumorigenesis (tumor formation). Many genes are involved in the process of tumorigenesis and are components of one of the many signal transduction pathways of molecular networks. It is now clear that epithelial malignancy in some aspects can be explained by changes in the adhesion properties of neoplastic cells
In stark contrast to their parent epithelial cells, carcinoma cells usually do not show contact inhibition of either their motility or growth. The most prominent feature shown by E-cadherin is that it promotes contact inhibition so that E-cadherin is a strong molecular barrier that must be overcome if tumor cells are to proliferate, detach, and spread. Loss of function of E-cadherin is often a marker of molecular changes that occur during tumor progression. This is due to a variety of mechanisms, including transcriptional repression, epigenetic silencing, inactivating mutations, endocytosis and fragment separation by proteolysis.
In addition to its role in normal cells, this gene can play a major role in the transformation of malignant cells, especially in tumor development and progression. Emphasis of E-cadherin expression is considered ap as one of the main molecular events responsible for the occurrence of intercellular adhesion dysfunction. Most tumors have an abnormal cellular architecture, and hence, loss of tissue integrity can lead to local invasion. Thus, loss of function of E-cadherin as a tumor suppressor protein correlates with increased tumor invasiveness and metastasis, so E-cadherin is often referred to as a suppressor of invasion gene.2 Ultimately, reduced or absent E-cadherin expression is an indication of outcome. poor clinical condition in several malignancies such as esophageal tumors, lung tumors, cervical tumors and Willm’s tumors.1,5
The metastatic ability of cancer cells is also thought to arise due to the progressive loss of epithelial characteristics of cancer cells by adopting more mesenchymal phenotypes in a process called EMT. Downregulation of E-cadherin is a standard feature in the development of EMT and loss of E-cadherin function in cancer plays an important role in the transition to a malignant phenotype.
E-cadherin-mediated loss of epithelial cell-to-cell binding is a hallmark of the transformation of benign lesions to invasive metastatic cancer. However, there is evidence that E-cadherin also plays a role in signal transduction pathways including wnt signaling, along with other important molecules involved, such as beta-catenin.
Expression of E-cadherin can suppress Wnt/b-catenin signaling by binding to -catenin at the site of cell-to-cell contact. In the absence of Wnt ligand binding to the Frizzled receptor, -catenin will be phosphorylated by CK1 kinase and GSK-3B, which in turn undergoes ubiquitination and proteasomal degradation.
However, once Wnt signaling is initiated due to lack or absence of E-cadherin expression, phosphorylation of -catenin will be inhibited, leading to accumulation of free -catenin in the cytoplasm and subsequently translocated into the nucleus to form an active complex of transcription factor -catenin/TCF/ LEF (T cell factor-lymphoid enhancer factor) and ultimately results in the expression of Wnt target genes. One of the target genes of this complex is encoding the c-MYC protein, which explains why constitutive activation of the Wnt pathway can lead to malignancy (figure 4).
b. E-cadherin Fragment Cleavage in cancer
Strong intercellular binding is a major barrier to cancer cell mobility, and the loss of cell-cell adhesion by E-cadherin is a fundamental change that occurs during the development of invasive cancer. Proteolytic processes and the release of membrane protein fragments (called shedding ectodomains) are common pathways by which cells modify the functional properties of membrane proteins. It is now known that proteolysis of E-cadherin fragments can promote tumor growth, survival and motility suggesting that fragmented E-cadherin fragments can convert these tumor suppressors into oncogenic factors.
Cleavage of E-cadherin by -secretase occurs on the extracellular surface of the plasma membrane catalyzed by several proteases, including matrix metalloproteinases (MMP-2, MMP-7, MMP-9, and MT1-MMP), A-disintegrin-and-metalloproteinase ( ADAM10 and ADAM15), plasmin. This cleavage transforms the characteristic 120 kDa mature E-cadherin into an 80 kDa extracellular N-terminal phagment and a 38-kDa intracellular C-terminal fragment. Ectodomain fragments [termed soluble E-cadherin (sE-cad)] are released from the plasma membrane and diffuse into the extracellular environment and even the bloodstream to act as paracrine/autocrine signaling molecules. In contrast, the intracellular C-terminal fragment (E-cad/CTF1) remains bound to the plasma membrane until cleavage occurs at the intracellular surface via -secretase (Presenilin-1/2).
This enzyme breaks the adherens junction and releases a 33-kDa intracellular fragment (E-cad/CTF2) into the cytosol where it plays a role in intracellular signaling. This fragment can also undergo further proteolysis by caspase-3 to produce a 29-kDa fragment (E-cad/CTF3) whose function is unknown (Figure 5).
sE-cad increases motility and invasion
Separation of the ectodomain fragment of E-cadherin was first found in media from breast carcinoma cells. In several subsequent studies, investigators observed a significant increase in sE-cad in the serum of cancer patients from various tumor types and reported that a significant increase in sE-cad was associated with invasive disease course and/or poor prognosis. The first function related to sE-cad is impaired intercellular adhesion based on the observation that in vitro cell therapy with sE-cad decreases cell aggregation and increases migration and invasion. The sE-cad mechanism for these changes involves at least 4 steps: (Figure 5)
1. The cleavage of E-cadherin degrades the whole molecule, so that the reserves of E-cadherin that are competent for the adhesion process on the plasma membrane will be reduced.
2. Because sE-cad has the ability to form homophilic bonds, the full-length E-cadherin homodimer between adjacent cells can be blocked.
3. sE-cad has chemotactic properties and can be trapped in the extracellular matrix and acts as an anchor/anchor of intact E-cadherin molecules from migrating cells.
4. sE-cad causes upregulation (upregulation) of major MMPs (MMP-2, MMP-9, and MT1-MMP) which degrades the basement membrane resulting in tumor invasion into the stroma. Then, because MMP-9 is also known to cleave E-cadherin, protease induction by sE-cad also gives positive feedback to continuously produce sE-cad.
sE-cad induces EGFR-dependent growth and survival signaling
It is known that E-cadherin regulates cell signaling processes. Activation of the receptor tyrosine kinase (RTK) occurs via receptor dimerization and autophosphorylation, and clustering of E-cadherin molecules after intercellular homophilic interactions can trigger ligand-independent dimerization and EGFR activation. Since the extracellular domain of E-cadherin can interact with EGFR, cleavage and separation of this domain into the extracellular environment can convert this molecule into a soluble growth factor.
This new function of sE-cad was first demonstrated in breast cancer cells. The EGFR (ErbB) family of RTK consists of 4 members: EGFR (HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These receptors can be both homodimeric and heterodimeric in response to ligand binding. Najy and colleagues, observed that sE-cad can bind to HER2 and HER3, stabilize their interactions, and activate signals to promote proliferation and migration (Figure 6). Because HER2 overexpression is common in breast cancer and is associated with a poor prognosis, proteolytic release of sE-cad may stimulate this pathway to promote aggressive growth and tumor metastasis.
The signaling process by sE-cad can also influence apoptosis through ligand-independent EGFR activation. Studies by David et al., collectively demonstrated that sE-cad acts as an anti-apoptotic factor, which means that sE-cad may play an important role in the survival and initiation of preneoplastic conditions in epithelial tissues.
The relationship between sE-cad and EGFR also suggests a reciprocal relationship in which EGFR activation promotes MMP and ADAM-dependent sE-cad. This suggests a feedback mechanism by which EGFR activation generates sE-cad, which then further activates EGFR signaling to continuously promote oncogenic proliferation and invasion (Figure 6).
E-cadherin cleavage activates Wnt/β-catenin . signaling pathway
The linking component of the cadherin-catenin complex is -catenin, which functions as a major mediator of the Wnt signaling pathway. In the classical Wnt pathway, Wnt ligand binding triggers the accumulation of -catenin, to translocate into the nucleus and bind to the transcription factor T-cell factor/lymphocyte enhancer factor-1 to activate growth-promoting target genes. By forming a complex with -catenin, E-cadherin will effectively bind this protein to the plasma membrane and prevent its transcriptional activity. However, cleavage of E-cadherin destroys this complex and releases -catenin to potentiate the oncogenic Wnt signaling pathway (Fig. 6). This pathway is also directly enhanced by intracellular E-cad/CTF2 fragments. In addition to liberating b-catenin, cleavage of E-cadherin also liberates P120 catenin, which translocates into the nucleus and binds to the Kaiso transcriptional repressor to abolish repression of various target genes of the Wnt/b-catenin pathway. It is also important that the E-cad/CTF2 fragment can remain bound to P120 catenin to enhance its inhibitory effect on Kaiso-mediated transcriptional repression (Fig. 6).
These studies demonstrate a dual mechanism of E-cadherin cleavage that facilitates the signaling of the Wnt/b-catenin pathway. The resulting aberrant activation of the Wnt/b-catenin pathway which functions to support the rapid proliferation of tumor cells and also increases tumor cell survival, as the expression of nuclear E-cad/CTF2 also exhibits suppressive activity against the induction of apoptosis. In addition, E-cadherin cleavage may also promote the acquisition of cancer stem cell phenotypes, because the Wnt/b-catenin signaling pathway is involved in stem cell regeneration. Furthermore, because MMP-7 is a Wnt/b-catenin target that has been shown to cleave full-length E-cadherin, activation of this gene can establish a positive feedback loop for continuous E-cadherin cleavage to ensure continuous oncogenic signaling.
1. E-cadherin is an important tumor-inhibiting gene.
2. The involvement of E-cadherin in the wnt signaling process, indicates that the same molecule can have different functions and that E-cadherin can regulate cellular responses shaped by external signals received by the cell. In this case, E-cadherin can regulate the process of migration, proliferation, apoptosis and cell differentiation.
3. E-cadherin cleavage produces protein fragments that are oncogenic, another mechanism that can increase motility, invasion, survival and tumor growth, also plays a role in signaling the WNT pathway to ensure continuous oncogenic signaling.
4. One important future approach in gene therapy is to develop methods to prevent the decrease in E-cadherin levels in tumors. To mencPreventing the potential for metastasis in almost all types of epithelial tumors can be initiated by targeting this molecule. Even so, this will be difficult because the process of decreasing E-cadherin levels is influenced by many different mechanisms, from large mutations and deletions to repression of gene transcription, as well as stimulation of signal transduction from formation of E-cadherin adhesion complexes.
5. Low expression of E-cadherin in tumor tissue or high levels of its fragments (sEcad and E-cad/CTF2) in the circulation signify invasiveness and severe proliferation of tumor cells which will result in poorly differentiated tumors.
6. The levels of E-chaderin and the levels of its fragments (sEcad and E-cad/CTF2) can be used to determine the severity of the disease and the degree of tumor metastasis.
7. E-cadherin and its fragments can determine the prognosis of the disease and the type of therapy in malignancy.
- David JM, Rajasekaran AK, Dishonorable Discharge: The Oncogenic Roles of Cleaved E-Cadherin Fragments. American Association for Cancer Research. 2012.
- Slaus NP. Tumor suppressor gene E-cadherin and its role in normal and malignant cells. Cancer Cell International.
- Bossche JV, Malissen B. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. The American Society of Hematology. 2012.
- E-Cadherin (Calsium dependent adhesion molecules).
- Alami J, Williams BR, Yeger H. Differential expression of E-cadherin and b catenin in primary and metastatic Wilms’s tumours. BMJ. 2003.
- Tight Junction. https://en.wikipedia.org/wiki/Tight_junction Structure
dr. Suhardi.Mkes.SpA,Dr.dr. Nadirah R. Ridha, MKes., Sp.A(K),Prof. Dr. dr. H. Dasril Daud, Sp.A(K)
BAGIAN ILMU KESEHATAN ANAK
FAKULTAS KEDOKTERAN UNIVERSITAS HASANUDDIN