Inverse relation of E-cadherin and autocrine motility factor receptor expression as a prognostic factor in patients with bladder carcinomas

Down-regulation of E-cadherin, an intercellular adhesion molecule, and up-regulation of autocrine motility factor receptor (gp78) expressions have been shown to play a role in tumor cell invasion and metastasis. Monoclonal antibodies against E-cadherin and gp78 were used to stain serial snap-frozen sections of 12 normal bladder and 83 bladder carcinoma specimens (27 noninvasive, 53 invasive, and 3 metastases). In normal urothelium, E-cadherin is expressed while gp78 is not. Positive expression of E-cadherin and negative expression of gp78 were found to be associated with a low risk of clinical progression in the superficial bladder carcinoma patient group. While reduction in E-cadherin concomitantly with an increase in gp78 expression was associated with poor prognosis, 71% of the patients (n = 30) underwent rapid cancer progression, and 32% of the patients died of cancer-related disease at a median of 2 years after initial diagnosis. Thus, it is suggested that reduction of E-cadherin expression associated with an increase in the level of gp78 in bladder cancers may define a high risk group of patients. The dual use of these two antigens may improve early diagnosis of high risk bladder cancer patients and influence treatment decisions.     

Clinical and experimental studies of JPYS in reducing side-effects of chemotherapy in late-stage gastric cancer

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective-tissue-matrix remodelling and the accelerated breakdown associated with tumor development. These MMPs and tissue inhibitor of MMPs (TIMP1) could be expressed by either the cancer or the stromal cells. Expression of mRNAs encoding interstitial collagenase (MMP1), 72-kD type IV collagenase (MMP2) and stromelysin (MMP3), which are probably involved in tumor invasion and metastasis, and of TIMP1 were studied in human mammary pathology by in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells. These data suggest that there is a cooperation between tumor and stromal cells, in particular for the production of 72-kD type IV collagenase, involved in the disruption of basement membranes. A lack of TIMP1 expression from invasive cancer cells would also contribute to matrix destruction.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

Dominant role of E-cadherin in the progression of bladder cancer

PURPOSE: To elucidate factors with a role in the progression of bladder cancer. MATERIALS AND METHODS: One invasive (T24) and two superficial (RT4 and KK47) human bladder cancer cell lines, which express metastasis-related genes, were used. Cells were intravenously inoculated into chick embryos to evaluate metastatic potential to the liver. An orthotopic model with severe combined immunodeficiency mice was also used to investigate both histological appearance and changes in metastasis-related gene expression. Finally, gene expression patterns in a clinical setting were compared between superficial and invasive bladder cancers. RESULTS: In culture condition metastasis-related genes, including matrix metalloproteinases, urokinase-type plasminogen activator, and integrins alpha2 and alpha3 were continually expressed in T24 but only slightly or not at all in RT4 and KK47, respectively. The expression pattern of the metastasis-related genes in vitro reflected the characteristics of the original tumors. Liver metastasis in chick embryos was demonstrated not only with T24 cells, but also with RT4 cells in which enhanced expression of metastasis-related genes was induced. In the orthotopic model, histological appearances were in accordance with the characteristics of the original tumors, although enhanced gene expression was notable with RT4. Expression of E-cadherin by Western blotting was demonstrated only with RT4 under these experimental conditions. Furthermore, predominant E-cadherin mRNA expression was found in superficial and not in invasive human primary bladder cancers; expression of other genes was similar in the two groups. Dominant expression of E-cadherin in superficial tumors was confirmed by immunohistochemical analysis. CONCLUSIONS: These results implicate loss of E-cadherin expression as a critical factor in facilitating the progression of bladder cancer.     

Purification and characterization of two extracellular proteinases from Arthrobacter nicotianae 9458

A community effect was found to occur between heterogeneous tumor cell populations leading to an overall increased tumorigenicity without a clonal dominance of the more tumorigenic clone. In the rat bladder carcinoma cell line NBT-II, this effect appears mediated by the Fibroblast Growth Factor-1 (FGF-1) through either a direct or an indirect signaling pathway. Neovascularization induced by FGF-1 was found not to be responsible for the community effect. The present study shows that the community effect does not involve a direct FGF-1 signaling since tumor cells expressing a dominant-negative FGF receptor mutant were still responding to the highly tumorigenic FGF-1 expressing cells. Tumors arising from inoculates of the FGF-1 producing NBT-II cells mixed with non tumorigenic epithelial MDCK cells contain only the tumorigenic cells indicating that MDCK cells may exerce a helper effect for the growth of the tumor not dependant on their own growth. Therefore the helper function of MDCK cells must be distinguished from a community effect where the contribution of low tumorigenic cells not only provides an in vivo growth advantage to few highly tumorigenic cells but become themselves highly tumorigenic indicating that the community effect may require cell-cell specific cooperativity independent from an helper effect.     

HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis

A significant portion of patients who present with non-muscle invasive "superficial" bladder cancer develop the muscle "invasive" life-threatening form of the disease during subsequent follow-up. In clinical studies, overexpression of the epidermal growth factor receptor (EGFR) and the p21 ras oncogene have been strongly associated with this phenotypic tumor transition. The marked difference in incidence of invasive bladder cancer in Asia compared to the United States has made us hypothesize that, among other factors, dietary influences have an impact on such tumor progression. A significantly higher dietary consumption of soy products exists in Asia and has led to the notion that the isoflavones present in this diet may contribute to a reduction in the number of invasive transitional cell bladder cancers. In this regard, we sought to determine the effect of genistein, a naturally occurring dietary protein tyrosine kinase (PTK) inhibitor, on the growth and motility of human bladder cancer cell lines with diverse EGFR and p21ras expression phenotypes and corresponding invasive behaviors. These effects were compared with those of tyrphostin, a pure synthetic EGFR inhibitor. Our results indicate that both genistein and tyrphostin are effective inhibitors of bladder cancer motility and growth, key factors in the development of muscle invasive disease. In addition, the growth and motility inhibitory effects of genistein and tyrphostin are observed preferentially in cells that overexpress the EGFR. Cells that have a mutated p21ras but do not overexpress the EGFR are less inhibited by these 2 compounds, suggesting that their effect is primarily directed at the EGFR signal transduction pathways proximal to the p21ras gene. Our results would seem to corroborate the notion that a high dietary intake of isoflavones is a likely explanation for the decreased incidence of invasive bladder cancer.     

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP-2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c-ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT-derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin-positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to be regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA-MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.     

Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ

Loss of expression of the intercellular adhesion molecule E-cadherin frequently occurs in invasive lobular breast carcinomas as a result of mutational inactivation. Expression patterns of E-cadherin and the molecules comprising the cytoplasmic complex of adherens junctions, alpha-, beta- and gamma-catenin, were studied in a series of 38 lobular breast carcinomas with known E-cadherin mutation status. The effect of loss of E-cadherin by mutational inactivation (or other mechanisms) on the expression of catenins was investigated. Complete loss of plasma membrane-associated E-cadherin expression was observed in 32 out of 38 invasive lobular carcinomas, for which in 21 cases a mutation was found in the extracellular domain of E-cadherin. In total, 15 frameshift mutations of small deletions or insertions, ranging from 1 to 41 bp, three non-sense mutations, and three splice mutations were identified. Mutations were scattered over the whole coding region and no hot spots could be detected. In all cases, simultaneous loss of E-cadherin and alpha- and beta-catenin expression was found; in 50 per cent of these cases, additional loss of gamma-catenin was observed. In six invasive lobular carcinomas, expression of both E-cadherin and catenins was retained. In none of these carcinomas was an E-cadherin mutation detected. Lobular carcinoma in situ adjacent to invasive lobular carcinoma showed simultaneous loss of E-cadherin and catenins in all the cases studied--remarkably, also, in four cases positive for E-cadherin and catenin expression in the invasive component. These results indicate that simultaneous loss of E-cadherin and alpha-, beta- and gamma-catenin may be an important step in the formation of lobular carcinoma in situ, as a precursor of invasive lobular breast cancer. Events additional to E-cadherin inactivation must be involved in the transition of lobular carcinoma in situ to invasive lobular carcinoma.     

Loss of KAI1 messenger RNA expression in both high-grade and invasive human bladder cancers

The molecular mechanisms responsible for metastasis are not fully understood. Recently, expression of the KAI1 gene on human chromosome 11p11.2 was found to be down-regulated in metastatic prostate cancer cell lines compared with normal human prostate, suggesting that KAI1 may be a metastasis suppressor gene. The aim of this study was to investigate whether there is reduced expression of KAI1 in late-stage bladder cancer. Sixty-six paraffin-embedded bladder tissue sections were analyzed for KAI1 mRNA by in situ hybridization. Nineteen of these were from patients with no histological evidence of bladder cancer, and 47 were from papillary transitional cell carcinomas (TCCs); of these, 16 were highly invasive. KAI1 mRNA was highly expressed in the specimens of normal bladder (11 of 11; 100%), inflammatory bladder (5 of 8; 63%), and noninvasive papillary TCCs of grades 1 and 2 (15 of 24; 63%), compared to grade 3 papillary TCCs (1 of 7; 14%) or invasive TCCs (1 of 16; 6%). The differences in expression between local and invasive disease were statistically significant (P 相似文献   

Glutathione synthesis in the exocrine pancreas

When epithelial cells reach confluency in vitro, a number of energy-requiring activities such as growth and motility are contact-inhibited. We investigated the possible role of the E-cadherin/catenin complex, which acts as an invasion suppressor, in contact inhibition. Three strategies for modulation of the complex were used. Firstly, the cell-cell adhesion and signal transduction functions of E-cadherin were neutralized immunologically in human MCF-7/6 mammary carcinoma cells possessing a complete complex. Secondly, the effect of E-cadherin transfection in E-cadherin negative cell lines was investigated. Thirdly, alpha-catenin deficient variants of the human HCT-8/S11 colon carcinoma cell line were compared with their parent cells. In confluent cultures functional downregulation of the E-cadherin/catenin complex did not alter cell growth nor saturation density. This was shown by cell number counts, protein staining assays, cell cycle analysis, proliferation markers (Ki67 and Proliferating Cell Nuclear Antigen) and apoptosis assays. However, confluent cells with a functionally deficient complex showed positional instability and enhanced succinate dehydrogenase-mediated mitochondrial 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl) tetrazolium bromide (MTT) conversion, as compared to cells with an active complex. Our data indicate that contact inhibition of motility and of mitochondrial enzyme activity, but not of growth is regulated by the E-cadherin/catenin complex in epithelial cells.     

E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1)

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.     

Altered gamma-catenin expression correlates with poor survival in patients with bladder cancer

PURPOSE: We studied the expression of alpha-, beta-, gamma- catenin and E-cadherin in transitional cell carcinoma (TCC) and normal bladder epithelium and correlated these results with pathological and clinical parameters. MATERIALS AND METHODS: We used an avidin-biotin immunoperoxidase technique to examine the cellular localization of alpha-catenin, beta-catenin, gamma-catenin and E-cadherin in 68 TCC and 14 normal bladder biopsies. RESULTS: E-cadherin, alpha-catenin, beta-catenin and gamma-catenin were expressed in a normal membranous pattern in all normal bladder epithelium specimens. Loss of normal surface E-cadherin, alpha-catenin, beta-catenin and gamma-catenin expression was found in 52/68 (76.4%) tumors, 57/68 (83.8%) tumors, 54/68 (79.4%) tumors and 54/68 (79.4%) tumors (p <0.001). There was a significant correlation between the loss of normal membranous expression of catenins and E-cadherin and increased grade (p <0.05). A highly significant correlation was observed between the loss of expression of E-cadherin, alpha-catenin and gamma-catenin, but not beta-catenin, with increased TNM stage (p <0.05). The abnormal expression of gamma-catenin as well as E-cadherin was correlated with poor survival (p <0.05). CONCLUSIONS: E-cadherin-gamma-catenin complex may be a useful prognostic marker in bladder cancer. Work is in progress to establish whether normal membranous catenin expression can be enhanced by gene transfer or biological therapy to induce a less invasive and metastatic phenotype.     

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.     

Expression of transforming growth factors beta-1, beta 2 and beta 3 in human bladder carcinomas

We previously detected elevated transforming growth factor beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. Fibroblasts expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, fibroblasts released only 19% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by fibroblasts with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer.     

Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.     

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.     

The effects of IL-6 on cell adhesion and e-cadherin expression in breast cancer

Interleukin 6 (IL-6) is a pleiotropic inflammatory cytokine and its role in cancer is not yet clear. The effects of IL-6 on four breast cancer cell lines and normal mammary epithelium, cultured from milk were tested. Four different patterns of response to IL-6 were found depending on the differentiation status of the cells. In normal mammary epithelial cultures, the effects of IL-6 were mainly growth inhibitory, whereas in MCF-7, IL-6 had growth inhibitory and anti-adhesive effects. In T-47D and ZR-75-1 the anti-adhesive effects were prominent although the growth inhibitory effects were not. These anti-adhesive effects were associated with epithelioid to fibroblastoid morphological changes and a local decrease in E-cadherin expression. In the highly invasive cell line MDA-MB-231, which does not express E-cadherin, no effects of IL-6 were seen. IL-6 levels in the serum of 60 breast cancer patients were found to be increased in 27% (16/60) compared to 2% (1/50) in a control group. Furthermore, it was found that altered E-cadherin expression was seen in 69% of the primary tumours, although no significant association was found between raised serum IL-6 levels and altered E-cadherin expression. Finally IL-6 serum levels did not effect the survival of breast cancer patients. The authors therefore implicate IL-6 as a possible factor important in breast cancer progression and metastasis formation, although the clinical significance of this cytokine in breast cancer patients could not be established.     

Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinase expression

To assess the role of gelatinases in mouse skin tumor progression and their link to the expression of E-cadherin (E-CD), the cell-cell adhesion protein, we used the highly metastatic squamous HaCa4 cell line and several HaCa4-derived clones obtained by transfection of the mouse E-CD cDNA. Expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein activity were present in E-CD (-) HaCa4 and control clones in culture, but they were strongly diminished in E-CD (+) clones (E24 and E62) at subconfluence. To explore the suppressive effect of the cell-cell contacts mediated by E-CD on MMP-9 expression, we introduced a plasmid encoding mouse E-CD antisense cDNA into the E24 cell clone. The transfectant P1-clones obtained with reduced or absent E-CD expression showed increased levels of MMP-9 gelatinase, motility in vitro, and metastatic potential in vivo. Expression of MMP-9 in the various cell clones was also negatively modulated by cell density, but this effect was much stronger in E-CD (+) cells, despite the fact that all of the cell clones analyzed maintained the expression of P-cadherin and made cell-cell contacts at high cell density. Our results indicate that in this cell system, the E-CD-mediated cell-cell contacts are involved in the down-regulation of MMP-9 expression. Thus, the loss of E-CD triggers a migratory and invasive phenotype in mouse squamous carcinoma cells.