Sj Vermeulen

Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro

The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.

ACTIVATION OF THE E-CADHERIN/CATENIN COMPLEX IN HUMAN MCF-7 BREAST-CANCER CELLS BY ALL-TRANS-RETINOIC ACID

All-trans-retinoic acid (RA), like insulin-like growth factor I (IGF-I) and tamoxifen, inhibit invasion of human MCF-7/6 mammary cancer cells in vitro. For tamoxifen and for IGF-I, activation of the invasion-suppressor function of the E-cadherin/catenin complex was shown to be the most probable mechanism of the anti-invasive action. We did a series of experiments to determine whether the anti-invasive effect of RA also implicated the invasion-suppressor E-cadherin/catenin complex. Human MCF-7/6 mammary and HCT-8/R1 colon cancer cells, both with a dysfunctional E-cadherin/catenin complex, were treated with RA and the function of the complex was evaluated through Ca(2+)-dependent fast aggregation. Fast aggregation of both MCF-7/6 and HCT-8/R1 cells was induced by 1 microM RA. This effect was abolished by antibodies against E-cadherin. RA-induced fast aggregation was not sensitive to cycloheximide, tyrosine kinase inhibitors or antibodies against IGF-I or against the IGF-I receptor. RA did not stimulate IGF-I receptor phosphorylation or alter the E-cadherin/catenin complex, as evidenced by immunoprecipitation. RA up-regulates the function of the invasion-suppressor complex E-cadherin/catenin. Its action mechanism is different from that of IGF-I. RA may act as an anti-invasive agent with unique mechanisms of action.

Regulation of the Invasion Suppressor Function of the Cadherin/Catenin Complex

Invasion is the cause of cancer malignancy. Invasion results from the cross-talk between cancer cells and host cells, building molecular invasion-promoter and invasion-suppressor complexes. The E-cadherin/catenin invasion-suppressor complex is regulated multifactorially, at multiple levels and sometimes in a reversible way. Mutations in the E-cadherin gene combined with loss of the wild type allele, causing irreversible downregulation, has been demonstrated only in a minority of human cancers. Posttranslational and reversible downregulation has been ascribed to tyrosine phosphorylation of beta-catenin. Phosphorylation is also implicated in transmembrane receptor signal transduction through the E-cadherin/catenin complex. E-cadherin interacts with E-cadherin on another cell through a dimeric adhesion zipper, involving the histidine-alanine-valine (HAV) sequence of the first extracellular domains. This is the major extracellular like of the E-cadherin/catenin complex, though not the only one. Intracellularly, the list of proteins that bind to or signal through the complex or through one or more of its elements is steadily growing. Extrinsic factors may influence the complex. At least in vitro, insulin-like growth factor-I, retinoic acid, tangeretin and tamoxifen were shown to upregulate the functions of the E-cadherin/catenin complex including inhibition of invasion.

Subclinical gut inflammation in spondyloarthropathy patients is associated with upregulation of the E-cadherin/catenin complex

OBJECTIVE Previously an upregulation of E-cadherin and its associated molecules α-catenin, β-catenin and plakoglobin has been demonstrated in clinically overt inflammatory bowel disease (IBD). The aim of this study was to investigate the expression of the E-cadherin/catenin complex in subclinically inflamed bowel mucosa from spondyloarthropathy (SpA) patients. METHODS Ileal and colonic biopsy specimens from 19 SpA patients with subclinical inflammatory gut lesions and from seven controls were stained with monoclonal antibodies against E-cadherin, β-catenin and plakoglobin and a polyclonal antibody against α-catenin. E-cadherin mRNA was detected using a riboprobe. Inflammation was histologically classified into acute, chronic active and chronic quiescent forms. RESULTS In acute and chronic active bowel inflammation of SpA patients, upregulation of the E-cadherin/catenin glycoprotein complex could be observed. Chronic lesions in a quiescent state did not show such an upregulation. Furthermore, chronic inflammation was associated with an increase in E-cadherin mRNA. CONCLUSIONS As some of the SpA patients with subclinical gut inflammation develop IBD, upregulation of the E-cadherin/catenin complex in inflamed bowel mucosa from SpA patients may point to early cellular changes in the development of IBD. However, at present it cannot be excluded that increased E-cadherin/catenin complex expression is a bystander phenomenon of active inflammation.

Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay

SummaryMCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993).E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

Activation and inactivation of invasion-suppressor molecules: in vitro analysis

In vitro methods have shown that cancer invasion results from the balance of activation and inactivation of invasion-suppressor and invasion-promoter molecules. Such methods constitute micro-ecosystems that differ from one another mainly by their substrate for invasion, namely components of the basement membrane; collagen type 1 gels; monolayers of different cell types; fragments of different organs. The E-cadherin/catenin complex is an invasion-suppressor complex, the function of which was well documented in experimental and clinical cancer. Loss of E-cadherin resulted in the expression of the invasive phenotype. In a human colon cancer cell line, round cell variants that were E-cadherin-positive, α-catenin negative and invasive reproducibly emerged from epithelioid subclones that were E-cadherin-positive, α-cateninpositive and noninvasive. The E-cadherin/catenin complex was downregulated by synthetic decapeptides that are homologous or identical to the HAV region of the first extracellular domain of E-caderin. Downregulation of the complex at its intracellular side occurs through tyrosine phosphorylation of β-catenin. Upregulation of the function of the complex with inhibition of invasion was demonstrated in variants of the human MCF-7 breast cancer cell family using in vitro methods.