Cadherins cell adhesion molecules and cancer
Introduction
Cadherins are calcium-dependent adhesion molecules. Malfunctions in cadherin function and their associated signal transduction pathways are associated with many types of cancer (Gottardi, CJ. et al. 2001). Cancer is often associated with the ability of cells to grow in an anchorage-independent manner (Gottardi, CJ. et al. 2001). These adhesion proteins bind to components outside of the cell as well as those components within the cell (Gilbert, S., 2010). Within the cell, they bind to the actin cytoskeleton. In addition to cell adhesion, cadherins modulate cell signaling through β-catenin Weinberg, R., 2013). β-catenin functions in cell adhesion by binding to sites on the adhesion molecule within the cytoplasm. A physical association between the adhesion molecule and the cytoskeleton results (Weinberg, R., 2013). E-cadherin is a specific type of cadherin that is expressed in epithelial cells and often associates with β-catenin (Weinberg, R., 2013). β-catenin functions in cell signaling through the Wnt-β-catenin cell signaling pathway. The Wnt-β-catenin pathway passes along signals for cell growth and cell division by translocating to the nucleus and binding to various transcription factors (Weinberg, R., 2013). Upregulation of the Wnt signal transduction pathway is involved in tumorigenesis (Gottardi, CJ. et al. 2001). β-catenin accumulation in the cytoplasm leads to an increase in the levels of nuclear β-catenin which drives cell growth through an interaction with lymphocyte enhancer factor/ T cell factor (TCF) (Weinberg, R., 2013). This article is intended to summarize the findings of Cara J. Gottardi, Ellen Wong, and Barry M. Gumbinera in their paper titled: E-Cadherin Suppresses Cellular Transformation by Inhibiting β-Catenin Signaling in an Adhesion-Independent Manner (2001).
The purpose of the Gottardi paper was to assess the impact of E-cadherin binding (or not binding) with β-catenin on tumor suppression and cell growth.
Wnt-β-catenin Signaling Pathway
Cell proliferation is modulated through the Wnt-β-catenin signaling pathway (Weinberg, R., 2013). β-catenin functions in cell signaling by associating, in the nucleus, with transcription factor complexes involved in cell growth and division, specifically, cyclin D1 and Myc (Weinberg, R., 2013). Wnt proteins bind to frizzled membrane receptors, the associated complex functions via Dishevelled to suppress activity of glycogen synthase kinase-3β (GSK-3β). This association inhibits the ability of GSK-3β to phosphorylate β-catenin. As a result, the level of β-catenin in the cytoplasm and nucleus increases. In the nucleus, β-catenin binds to T-cell factor (Tcf) and/or lymphoid enhancer factor (Lef) proteins. The Tcf/Lef-β-catenin complex causes the Groucho repressor protein to fall off (Weinberg, R., 2013). As a result, genes implicated in cell growth (Myc) and division (cyclin D1) are expressed (Weinberg, R., 2013).
Methods
Cell Growth Assays
The researchers assessed the mechanism of E-cadherin to act as a tumor suppressor by evaluating the relationship between cell adhesion and β-catenin cell signaling. The ability of E-cadherin to suppress anchorage-independent growth was evaluated by exposing three different constructed cell lines of SW480 human colon carcinoma cells to soft agar. Wild-type E-cadherin and E-cadherin chimeras were expressed in colon cancer cell lines. Colonies greater than 100 µm in diameter were counted after 14 days. Chimeras lacked adhesive function or β-catenin binding activity respectively.
The constructed cell lines were:
1.) wild-type E-cadherin
2.) E-cadherin–α-catenin fusion product
3.) E-cadherin Δ β-catenin
4.) E-cadherin Δ p120 (control)
5.) IL2R/E-cadherin cytoplasmic chimera construct.
Constructed cell lines were evaluated against a control line. The colony count indicates the relative effectiveness or ineffectiveness of that particular cell construct to inhibit cell growth.
Immunoblot Analysis
The authors used anti-E-cadherin mAb, anti-N-cadherin mAb, anti-c-myc polyclonal antibody, anti-myc epitope mAb, anti-β-catenin, and HRP-conjugated anti-mouse & anti-rabbit secondary antibodies to evaluate the levels of the cadherin constructs, β-catenin and c-myc proteins.
Fusion Protein Assay
The ability of β-catenin from SW480 cells to bind to cadherins and/or T-cell factor was assessed using a fusion protein. If E-cadherin inhibits β-catenin/TCF signaling and observations of anchorage-indepedent growth decrease, the authors ask what the relationship is between carcinoma cells (SW480) and β-catenin. Affinity precipitations were performed and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE separates proteins based on molecular size rather than shape or charge. SDS denatures the protein complex and saturates the macromolecule with a negative net charge. Higher molecular weight complexes will have a slower mobility in the gel than lower molecular weight complexes. The fusion protein assay functions by comparing the protein of interest to situations where the protein of interest is accompanied by potential binding partners and glutathione S-transferase (GST) according to:
1.) Protein of interest
2.) Protein of interest + binding partner(s)
3.) Protein of interest + binding partner(s) + GST
A decrease in mobility in lanes 2 and 3 indicates that a binding event has taken place. GST is used to determine whether the gt-labelled protein has bound the target protein. If binding has occurred via the gt-labelled protein, an even greater decrease in mobility will be observed. Cadherin-GST fusion protein and TCF-GST fusion protein were assesed for their respective abilities to bind β-catenin.
Results
E-Cadherin Suppresses Growth in the SW480 Cell Line
The SW480 cell line used in the experiments expressed very low levels of E-cadherin and contained a mutation to allow for upregulation of the β-catenin/wnt signaling pathway.
The wild-type E-cadherin construct was found to inhibit growth of cells on the soft agar plates. When the α-fusion and Δ β-catenin constructs were used, these constructs allowed for growth in the anchorage-independent assay. The IL2R construct showed inhibited cell growth. The authors found that the IL2R construct inhibits cell growth to the greatest degree without participating in any cell adhesion functions. The IL2R construct exhibited the strongest inhibition of cell growth despite the fact that the construct was not expressed as well as the wild-type E-cadherin or the E-cadherin Δ β-catenin construct. The authors claim that the IL2R construct was expressed about as well as the E-cadherin-α-catenin fusion construct.
These results indicate that the β-catenin binding region contains the information necessary for cell growth inhibition.
E-Cadherin's Suppression Activity is Influenced by a Reduction of the β-Catenin Signaling
In order to determine the impact of β-catenin/TCF on the observed E-cadherin growth inhibition, experiments to "rescue" the pathway were conducted. E-cadherin plasmids were transfected and resulted in a decreased number of colonies. When cotransfected with an active form of TCF, inhibition of colonies was reversed.
Thus, the rescue of colony formation when TCF is added indicates the reductive role that the β-catenin/TCF pathway plays in E-cadherin growth suppression.
E-Cadherin Growth Inhibition is Mediated by Cytoplasmic Domain with Minimal Reduction of Nuclear or Cytosolic β-catenin Levels
Experiments were conducted to determine whether growth inhibition was a result of decreased nuclear β-catenin levels. The quantitative assay to detect the amount of bound versus unbound β-catenin showed minimal depletion of β-catenin levels in both the nuclear and cytosolic regions.
Discussion and Significance
The results of the papers provide new evidence regarding cell adhesion and impact of β-catenin signaling. Using the anchorage-independent experiments, they found that E-cadherin suppresses cell growth and is mediated through a β-catenin/TCF-dependent signaling and adhesion-independent manner. SW480 cells contain very low levels of E-cadherin which makes the role of β-catenin in cell growth/inhibition of cell growth of particular interest. The cadherin constructs that exhibit adhesive activity (from the anchorage-independent study) but do not bind β-catenin, failed to exhibit growth inhibition.
When the cadherin constructs were expressed in SW480 cells, cell growth was inhibited. Once active TCF was added, the cell inhibition was reversed and the cells grew. This indicated that E-cadherin growth suppression was facilitated through the inhibition of β-catenin/TCF genes.
Additionally, E-cadherin was found to inhibit β-catenin signaling with minimal changes in cytosolic/nuclear levels of β-catenin.
References
Gottardi C, Wong E, Gumbiner B. E-Cadherin Suppresses Cellular Transformation by Inhibiting β-Catenin Signaling in an Adhesion-independent Manner. 2001. J Cell Biol. 153(5). 1049-1060.
Weinberg, Robert. The Biology of Cancer 2nd Edition. New York, NY: Garland Science, 2013. Print.
Gilbert, Scott. Developmental Biology 9th Edition. Sunderland, MA: Sinauer Associates, 2010. Print.