E-cadherin and metastasin (mts-1/S100A4) expression levels are inversely regulated in two tumor cell families.

Metastasin is putatively associated with cytoskeletal proteins and may influence cell motility, although its exact physiological role is not known. Because E-cadherin is an invasion suppressor molecule, and metastasin a metastasis-inducing molecule, we wondered which molecule was playing a dominant role in the balance that finally leads to noninvasiveness or invasiveness. The expression levels of E-cadherin and metastasin were monitored in two mouse tumor cell families and were found to be inversely regulated. Moreover, overexpression obtained via transfection of plasmids coding for either one of these two molecules abrogated expression of the other molecule as investigated via Northern and Western blotting experiments. Invasiveness was accordingly influenced. Expression levels of alpha- and beta-catenins were not influenced by up-regulated metastasin, but their intracellular distribution was disturbed. The present results suggest that metastasin-induced invasiveness of several malignant tumor cells is at least partially caused by down-regulation of E-cadherin.

Molecular cloning of the human p120ctn catenin gene (CTNND1): expression of multiple alternatively spliced isoforms.

Catenins were discovered as proteins that are linked to the cytoplasmic domain of transmembrane cadherins. Among these junctional plaque proteins are several members of the Armadillo gene family: beta-catenin, plakoglobin, and p120ctn. Recently it became clear that some catenins also mediate nuclear signaling. We performed a detailed analysis of the human p120ctn gene (HGMW-approved symbol CTNND1) and its transcripts. The human p120ctn gene comprises 21 exons, potentially encoding up to 32 protein isoforms as products of alternative splicing. Human isoforms, designated 1 to 4, differ from each other by the start codon used. Additional isoforms are derived from combinations with alternatively used exons A (exon 18) and B (20), near the end of the open reading frame, and also with exon C (11) in the middle of the open reading frame. Hence, the longest isoform is of type 1ABC and comprises 968 amino acid residues. The functional consequence of the observed multitude of p120ctn splice variants awaits further study, but tissue-specific expression was obvious. Further, we demonstrate that the exon organization, which is not simply related to the Armadillo repeat structure, is very well conserved between the p120ctn gene and the related ARVCF gene, but not at all between these two genes and the beta-catenin or plakoglobin genes. The present data favor the concept that p120ctn is the prototype of a subfamily of Armadillo proteins, comprising ARVCF, p0071, delta-catenin/NPRAP, and plakophilins 1 and 2, that are more related to each other than to other Armadillo proteins.

Mechanisms of downregulation of transfected E-cadherin cDNA during formation of invasive tumors in syngeneic mice.

Loss of E-cadherin expression has been observed both in experimental tumors and in human cancers and is related to invasiveness and poor differentiation. The E-cadherin-negative mouse mesenchymal tumor cell line MO4 was transfected with several plasmids expressing mouse E-cadherin cDNA. These plasmids differed from each other by the extent of E-cadherin-specific 3' untranslated region (UTR) sequences and by the use of different constitutive promoters. Transfectants were isolated that expressed functional E-cadherin in a homogeneous way. In syngeneic mice, such MO4-Ecad transfectants invariably produced malignant fibrosarcoma-like tumors, which were completely E-cadherin-negative at the protein level. Northern blotting revealed that E-cadherin mRNA expression was downregulated in some but not all MO4-Ecad tumors. Downregulation was caused by mRNA instability triggered by particular 3' UTR sequences. This in vivo downregulation of E-cadherin in malignant MO4-Ecad tumors turned out to be reversible and is likely to be mediated by host factors to be further identified.

Enlarged cell-associated proteoglycans abolish E-cadherin functionality in invasive tumor cells.

Mouse and dog epithelial cell lines, expressing high levels of the Ca(2+)-dependent cell-cell adhesion molecule E-cadherin in vitro, generated invasive and metastatic tumors in athymic mice. From these tumors, neoplastic cell lines were isolated. All ex vivo isolates retained high expression levels of E-cadherin at their surface. Nevertheless, some showed a fusiform morphotype, were defective in Ca(2+)-dependent cell aggregation, and were invasive in vitro, indicating that E-cadherin was not functional. Cell-associated proteoglycans were found to be enlarged in these variants as compared to their counterparts with functional E-cadherin. Treatment of the cells with the drug 4-methylumbelliferyl beta-D-xyloside specifically reduced the amount and size of cell-associated proteoglycans. This same drug induced an epithelial morphotype, increased Ca(2+)- and E-cadherin-dependent cell aggregation, and abrogated invasiveness without influencing E-cadherin expression levels. Our results indicate that enlarged proteoglycans can prevent the homophilic binding of E-cadherin, probably by steric hindrance. This is one more mechanism by which carcinomas may counteract invasion-suppressor genes and acquire malignancy.