E. Bruyneel

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.

The intracellular E-cadherin germline mutation V832 M lacks the ability to mediate cell-cell adhesion and to suppress invasion.

E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF–LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF–LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a β-catenin-dependent activation of the WNT pathway.

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.

Effect of temperature on invasion of MO4 mouse fibrosarcoma cells in organ culture

Invasion by MO4 mouse fibrosarcoma cells into fragments of embryonic chick heart or lung in organ culture was studied histologically and ultrastructurally at various temperatures between 12 and 40°C. Invasion was absent for at least 7 days at or below temperatures of 29°C. Invasion was invariably observed at or above 30·5°C. Differences in invasion between 29 and 30·5°C could not be ascribed to differences in growth, migration, or microtubule assembly/disassembly of MO4 cells. Neither could they be explained through differences in the attachment of MO4 cells to the heart fragments. Possible explanations for the absence of invasion at lower temperature are: altered resistance of the extracellular matrix in heart or lung fragments, and deficient expression of fucosylated glycoproteins at the surface of MO4 cells. A population of MO4 cells plated from the parent line and adapted to grow at 28°C (MO4 28 cell line) did not differ in invasiveness from the parent MO4 cells.We conclude that the temperature dependence of invasion in organ culture might indicate as yet unexplored aspects of the mechanisms of tumour invasion.

Effect of inhibitors of glycosylation and carbohydrate processing on invasion of malignant mouse MO4 cells in organ culture.

Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO4 cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasionin vivo. Tunicamycin (1·0μg/ml), 2-deoxy-d-glucose (100mm),β-OH-norvaline (1·0mm), and Monensin (0·1μg/ml) reversibly inhibited the invasion of MO4 cells. At these concentrations the drugs also inhibited the growth of MO4 cells. 1-Deoxynojirimycin (10mm), swainsonine (0·4μg/ml), and Marcellomycin (0·1 μg/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO4 cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO4 cellsin vitro.

Enhancement of tamoxifen-induced E-cadherin function by Ca2+ channel antagonists in human breast cancer MCF7/6 cells.

Despite its intensive use in adjuvant breast cancer therapy for more than 30 years, the exact mechanisms of action of tamoxifen have not yet been fully characterized. Tamoxifen was recently shown to restore the E-cadherin function of human breast cancer MCF7/6 cells and to suppress their invasive phenotype. Because tamoxifen interacts with targets implicated in Ca2+ homeostasis, we explored the possibility that the restoration of E-cadherin function in MCF7/6 cells induced by this drug could be affected by Ca2+ modulators. Two different Ca2+ channel antagonists (verapamil and nifedipine) potentiated the effect of tamoxifen on E-cadherin function, as evaluated with a fast cell aggregation assay. These molecules decreased the tamoxifen concentration needed to restore the E-cadherin function from 10(-6) M to 10(-7) M. When incubated with a Ca2+ channel agonist, Bay K8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methylphenyl)- pyridine-5-carboxylate), the effect of tamoxifen on E-cadherin function was completely abolished. These results demonstrate that the restoration of the E-cadherin function induced by tamoxifen depends, at least in part, on a Ca2+ pathway, and support the evidence of an effect of tamoxifen on Ca(2+)-dependent mechanisms. Our data also suggest that Ca2+ channel modulators could make it possible to decrease the dose of tamoxifen administered to patients without reducing the therapeutic effects.

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.

The Transforming Functions of PI3-kinase-γ Are Linked to Disruption of Intercellular Adhesion and Promotion of Cancer Cell Invasion

The involvement of phosphoinositide 3‐kinases class IA (PI3K‐α and ‐β) in cancer cell proliferation, survival, motility, and invasiveness is now well established. However, the possible contribution of the class IB PI3Kγ in cancer cell transformation remains to be explored. In this study, we have stably transfected the PI3Kγ‐deficient human colon cancer cell line HCT8/S11 with expression vectors encoding either wild‐type PI3Kγ, its plasma membrane targeted form CAAX‐PI3Kγ, or the PI3Kγ lipid and protein kinase‐dead mutant (CAAX‐K832R). We provide evidence that the constitutively active CAAX‐PI3Kγ variant induced collagen type I invasion in HCT8/S11 cells through disruption of cell–cell adhesion, with no apparent impact on cell proliferation and motility. The proinvasive activity of CAAX‐PI3K‐γ was abolished by pharmacological inhibitors targeting PI3‐K activities (wortmannin), Rho‐GTPases, and the Rho‐Rho kinase axis (C3T exoenzyme and Y27632, respectively). Conversely, the wild‐type PI3Kγ and its double mutant CAAX‐K832R were ineffective on cancer cell invasion measured under control or stimulated conditions operated with the proinvasive agents leptin and intestinal trefoil factor. Taken together, our data indicate that PI3Kγ exerts transforming functions via several mechanisms in human colon epithelial cancer cells, including alterations of homotypic cell–cell adhesion and induction of collagen type I invasion through canonical proinvasive pathways.

Methods for Morphological and Biochemical Analysis of Invasion in vitro

ABSTRACT Invasiveness of malignant cells is demonstrated in vitro using three-dimensional shaker cultures of tissue fragments with aggregates of malignant cells. Both tissues are allowed to adhere to each other on a non-adhesive substrate. This method is used to examine the invasiveness of various cell lines with the aim of defining their malignancy. Cellular activities, which are presumed to be involved in invasion are studied : 1) Adhesion of malignant cells to the host tissue; 2) Destruction of the host tissue by invading cells; 3) Phagocytosis of material from the host by the malignant cells.

An anti-invasive concentration of the alkyl-lysophospholipid ET-18-OCH3 enhances the motility of embryonal chick heart cells cultured on solid substrate

Pretreatment of embryonal chick heart fragments with ET-18-OCH3 is known to induce resistance to invasion by several malignant cell lines. Embryonal chick heart fragments or cell suspensions prepared from such fragments were explanted on solid substrate and treated in medium with 10 μg/ml ET-18-OCH3 or with drug-free medium (control) for 48 h. This medium was washed away and replaced by drug-free fresh medium. Twenty-four to 48 h later the fast plasma membrane movements (involved in ruffling, blebbing, fast shape change and fast translocation) were quantified using a simple method based on subtracting two video images taken with an interval of 28 s. The ET-18-OCH3-treated cells showed a higher intensity of fast plasma membrane movements than control cells. Cells around a treated expiant did not show the same radial alignment as in controls, suggesting loss of contact inhibition of movement. Cells from a cell suspension derived from a treated fragment showed faster translocation on solid substrate and faster shape change. We speculate that increased motility of host cells may be involved in resistance to invasion.