E-cadherin has been demonstrated to induce growth suppression and decrease the invasiveness of cancer cells and thus has been proposed to be a tumor suppressor gene. The ability of E-cadherin to mediate cell-cell contact and contact inhibition presumably accounts for its antitumor effects, which are attributed to the extracellular domain of the protein. Here we report that blocking the ability of E-cadherin to mediate contact inhibition by either antagonistic antibodies or expression of a mutant form of E-cadherin with the extracellular region deleted does not abrogate growth suppression. Transfection of the E-cadheringene into the human prostate cancer cell line TSU.Pr-1 induced cell-cell contact formation, growth suppression, and redistribution of β-catenin to the cell membrane. Treatment of the E-cadherin transfectant (CAD) with blocking antibodies disrupted cell-cell contact formation but did not influence the growth rate,suggesting that cell-cell interaction is not required for E-cadherin-mediated growth suppression. Similarly, transfection of an E-cadherin construct in which the NH2-terminal(extracellular) region was deleted did not allow cell-cell contact formation but induced growth suppression. In contrast, transfection of an E-cadherin construct in which the COOH-terminal (cytoplasmic) region was deleted did not induce suppression but promoted cell contact formation. In cells expressing E-cadherin lacking the cytoplasmic region, β-catenin was evenly distributed in the cytoplasm. By contrast, in cells expressing E-cadherin lacking the extracellular region, β-catenin was cell membrane associated. Growth suppression was always associated with the localization of β-catenin to the cell membrane. The redistribution of β-catenin from the cytoplasm to the cell membrane initially suggested the involvement of the Wnt signaling pathway in regulating cell growth. However, only small differences inβ-catenin/T-cell factor signaling were detected in control and E-cadherin-expressing cells, suggesting that the Wnt pathway is not involved. Taken together, these findings suggest that E-cadherin-induced growth inhibition may not be solely attributed to contact inhibition but may involve the redistribution of β-catenin from the cytoplasm to the cell membrane, and this redistribution may affect growth pathways independent of T-cell factor.

Cadherins are a family of evolutionarily conserved, surface glycoproteins of Mr120,000–140,000 that facilitate calcium-mediated homophilic binding between cells (1, 2). The E-cadherin molecule consists of a large extracellular domain, a single hydrophobic transmembrane domain, and a cytoplasmic domain. The large extracellular domain is subdivided into five subdomains of homologous repeats 110 amino acids long (EC 1–5). The binding specificity of cadherins is mediated by the first EC domain. The cytoplasmic domain of cadherin binds to the β-catenin protein, which forms complexes withα-catenin (3), actin (4), p120(5), and EGFR2(6), and possibly other proteins.

Cadherins mediate cell-cell adhesion during tissue formation in the embryo and maintain tissue architecture in adult organisms. Thus,cadherins interact dynamically to provide adhesive strength and functional regulation during morphogenesis. In particular, E-cadherin comprises the zona adherens junctions observed between epithelial cells. Cadherin binding is influenced by calcium levels(7), protein kinase C (8), Rac and Rho G proteins (9), epidermal growth factor (10),and v-src kinase (11, 12) and perhaps by other factors as well.

Although the cytoplasmic domain of E-cadherin does not possess any enzymatic activity, it is postulated that E-cadherins may be indirectly involved in signal transduction. β-Catenin is bound primarily to cadherin and forms a complex with α-catenin and actin that stabilizes the cytoskeletal architecture. β-Catenin that is not bound to cadherin can associate with the APC protein and GSK-3β(13). Formation of this complex results in the phosphorylation of serine residues on β-catenin and APC and thereby induces the ubiquitination and proteasome-mediated degradation of the complex (14, 15, 16). If β-catenin or APC protein is mutated such that the degradation does not occur, β-catenin translocates into the nucleus and accumulates (17, 18). In the nucleus,β-catenin forms a heterodimeric transcriptional complex with the TCF or lymphoid enhancer factor and activates gene transcription(19, 20, 21). Besides mutations in β-catenin and/or the APC protein, β-catenin is also stabilized by the Wnt signaling cascade. The Wnt protein in the extracellular matrix binds to its corresponding cell surface receptor, Frizzled (Fz), which activates the disheveled(dsh) protein to inhibit GSK-3β. The inhibition of GSK-3β activity prevents phosphorylation of β-catenin and/or APC protein and stops ubiquitination and degradation. Cadherins may influence Wnt signaling by interacting with and sequestering β-catenin such that the amount of free β-catenin to translocate into the nucleus is reduced(14). The overexpression of cadherins has been shown to antagonize the signaling function of β-catenin.

The loss of cadherin expression or function has been shown to be associated with tumorigenesis and tumor progression(22, 23, 24). Restoration of E-cadherin in cancer cells results in decreased invasiveness (25), growth suppression(26), and terminal differentiation (27). The addition of antibodies that inhibit E-cadherin-mediated cell-cell junctions (26), incubation of the cells in low-calcium media (28), or modulation of the cell density(10) abrogated the growth-inhibitory effects mediated by E-cadherin. Furthermore, the formation of adenomas and perturbations in proliferation were observed in transgenic mice expressing a dominant negative N-cadherin where the extracellular domain was deleted(22, 29). Thus, the ability of E-cadherin to mediate cell-cell contact has been suggested to be the major factor influencing the growth-inhibitory effects. Because of these characteristics,E-cadherin can be considered a tumor suppressor gene.

Based on the published evidence, the goal of this study was to investigate the ability of E-cadherin to induce growth suppression in the human prostatic cell line TSU.Pr-1. Stable clones of TSU.Pr-1 expressing E-cadherin (CADs) were generated and analyzed for growth suppression. As demonstrated previously, the expression of E-cadherin resulted in an inhibition of growth as compared with the clones transfected with the control vector (Neo). However, the addition of blocking antibodies against E-cadherin inhibited cell-cell interactions but did not affect the growth of CAD, which suggested that the two functions of E-cadherin, the ability to form cell-cell interactions and growth suppression, may be mediated by different domains of the molecule. To further investigate this hypothesis,truncation mutants deleting either the extracellular (ΔN) or cytoplasmic (ΔC) portions of E-cadherin were constructed,transfected, and analyzed for growth-inhibitory activity. Cells expressing only the cytoplasmic portion of E-cadherin lost cell-cell contact formation, but the growth-inhibitory activity was preserved. In contrast, cells expressing only the extracellular portion of E-cadherin retained the ability to form cell-cell interactions, but the growth-inhibitory activity was abolished. Moreover, the cellular distribution of β-catenin correlated with the ability of E-cadherin to induce growth suppression. β-catenin was primarily cytoplasmic in the Neo and ΔC cells, whereas in CAD and ΔN, β-catenin was located at the cell membrane with E-cadherin. These findings indicate that E-cadherin promotes growth suppression in TSU.Pr-1 and its ability to induce growth suppression may not be solely attributed to contact inhibition but may involve the redistribution of β-catenin from the cytoplasm to the cell membrane. However, TCF/β-catenin-mediated transcription was not significantly affected by E-cadherin constructs that did inhibit cell growth. These results suggest that E-cadherin effects on growth are not mediated entirely through modulation of TCF/β-catenin-induced gene expression.

Cells and Treatment.

The mouse E-cadherin cDNA, a generous gift from Dr. M. Takeichi(Kyoto University, Kyoto, Japan; Ref. 30), was subcloned into the eukaryotic expression vector pIRES by partial digestion with EcoRI. The extracellular and cytoplasmic truncated E-cadherin were generated with PCR with the following primers and subcloned into pIRES.

The NH2- and COOH-terminal deletion mutants of E-cadherin were generated by PCR cloning. The forward primer,5′-CCCCCGCGGCCGCCTAGTCGTCCTCGCCACCG-3′, and the reverse primer,5′-CCCCGATATCATGAAGGCGGGAATCGTGG-3′, were used to amplify the transmembrane and cytoplasmic fragment, which was subsequently cloned to pIRES to generate ΔN. To amplify the extracellular and transmembrane fragment, the forward primer,5′-CCCCGATATCATGGGAGCCCGGTGCCGCAG-3′, and reverse primer,5′-CCCCCGCGGCCGCCTAGATGAAGTTTCCAATTTC-3′, were used, and the fragment was cloned to the pIRES.

The constructs were transfected into TSU.Pr-1, a generous gift from Dr. Tony Passaniti (University of Maryland, Baltimore, MD)according to the LipofectAMINE protocol (Life Technologies Inc.,Rockville, MD) and selected for stable transfectants with G418 (500μg/ml) in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin streptomycin (Biofluids, Rockville, MD).

The construction of ΔNC involved the use of the forward primer GAAGGGACGGTCAACAACA and reverse primer TAGCGCTTCAGAACCACTC to amplify a 465-bp fragment from the mouse E-cadherin gene by PCR. The PCR fragment was subcloned into pcDNA3.1/CT-TOPO-GFP vector (Invitrogen, Carlsbad,CA) according to protocol provided by the manufacturer. The construct was transiently transfected into cells with Fugene 6 (Roche,Indianapolis, IN) and incubated for 3 days. The cells were then harvested and analyzed for cell proliferation. The expression of the construct was assessed by visualization of the GFP fusion protein via fluorescence microscopy and immunoblot analysis.

All of the constructs were confirmed by sequencing.

Neutralization of E-cadherin-mediated cell contact by anti-uvomorulin(cadherin) antibodies (DECMA; Sigma, St. Louis, MO) was conducted as follows. Cells were harvested and incubated with various concentrations of neutralizing antibodies while in suspension for 15 min at room temperature. The treated cells were then plated into either 96-well or 24-well microtiter plates and incubated overnight before MTT cell viability analysis or microscopic observation, respectively. Cell morphology was visualized with an inverted microscope fitted with phase-contrast filters at either ×100 or ×200 magnification. Images were captured with the Axiovision software (Carl Zeiss Inc., Thornwood,NY).

MTT Cell Viability and BrdUrd Incorporation Assay.

Cells were trypsinized and plated (1 × 105 cells/well) into 96-well microtiter plates in either the absence or presence of DECMA antibody. The tetrazolium dye MTT (Sigma) was used to assess cell viability as described by Mossman(31) with modifications. Solubilization of the formazin crystals was done in 10% SDS and 0.01 N HCl for 18–24 h. Absorbance was measured at 595 nm with a multiwell plate reader (Bio-Rad,Hercules, CA). Fold increase was determined by the following ratio:(absorbance of sample):(average absorbance at day 0).

The BrdUrd incorporation assay was conducted according to the procedure provided by the manufacturer (Oncogene Research Products, Cambridge,MA), with the following modifications. The cells were initially growth arrested with nocodazole (Sigma) for 18 h, harvested, and plated in microtiter well plates with BrdUrd. At various time points after release from nocodazole-induced growth arrest, the cells were fixed and analyzed for the amount of BrdUrd incorporation.

Data points and error bars represent the average and SD of triplicate samples. Each experiment has been repeated at least twice.

Immunoblot and Immunoprecipitation.

Cells were seeded in 100-mm2 tissue culture dishes and grown to 80% confluence. After washing the cells twice with cold PBS, the cells were lysed, and the lysates were collected according to the protocol from Transduction Laboratories (Lexington,KY). Total protein concentration was measured with the BCA protein assay (Pierce, Rockford, IL). Ten μg of total protein were loaded into each well of 10% SDS-polyacrylamide gel and separated via electrophoresis. The proteins were transferred to PVDF membranes and probed with the indicated antibodies at concentrations recommended by the manufacturer (E-cadherin, α-catenin, β-catenin, and p120;Transduction Laboratories). Antibody complexes were detected by using the secondary antibody goat antimouse immunoglobulin conjugated with alkaline phosphatase (Santa Cruz Biotechnology, Santa Cruz, CA) and the enhanced chemifluorescence kit (Amersham Pharmacia Biotech, Uppsala,Sweden). Secondary antibody was not used for phosphotyrosine analysis because the alkaline phosphatase enzyme was conjugated to the antiphosphotyrosine antibody (RC-20-AP; Transduction Laboratories). The PVDF membranes were scanned with the Storm scanner (Molecular Dynamics,Sunnyvale, CA). Experiments were repeated at least three times.

The IMMUNOcatcher kit (Cytosignal Research Products, Irvine, CA) was used for the immunoprecipitation experiments. Lysates from cells were generated with the Mild Lysis solution, and the batch was precleared with the appropriate preimmune serum according to the protocol provided by the manufacturer. E-cadherin and β-catenin were immunoprecipitated with 1 μg of monoclonal antibodies. Rabbit antiserum againstβ-catenin (5 μg; Santa Cruz Biotechnology) was also used in some of the immunoprecipitation reactions. Precipitated proteins were then analyzed by immunoblotting as described earlier.

Indirect Immunofluorescence.

Indirect immunofluorescence staining was performed according to the procedure described by the manufacturer of the antibody (Transduction Laboratories). Briefly, cells were grown to subconfluence on glass-bottomed microwell dishes (MakTek, Ashland, MA), washed twice with PBS with calcium and magnesium, and fixed with a paraformaldehyde solution (2% paraformaldehyde and 0.1% Triton X-100 in PBS). The fixed cells were washed twice with PBS and blocked with 2% BSA and mouse normal immunoglobulin (Santa Cruz Biotechnology). Monoclonal antibody against E-cadherin (rat anti-uvomorulin; 6 μg/ml;Sigma) was diluted in blocking solution and incubated with cells for 1 h at room temperature. The cells were then washed three times for 5 min with PBS. Donkey antirat immunoglobulin conjugated with 20 μg/ml Alexa 488 (Molecular Probes, Eugene, OR) and mouse anti-β-catenin conjugated to 10 μg/ml TRITC (Transduction Laboratories) were mixed in blocking solution, added to the cells, and incubated for 1 h at room temperature. After washing the cells three times with PBS for 5 min, digitized images of stained cells were captured with a confocal microscope (Axiovert 100 software;Carl Zeiss Inc.) under ×400 magnification. The magnification for Fig. 5 B was increased 3-fold. Images were processed with Adobe Photoshop software (Adobe, San Jose, CA).

TOP/FOP FLASH Reporter Assay.

Reporter assay for TCF was performed as described in Morin et al.(18). Cells (2 × 106) were cotransfected with 1 μg of luciferase reporter plasmid DNA and 0.5 μg of vector DNA containing β-gal gene by using Fugene 6 (Roche). After 24 h of incubation, cells were lysed in 1× lysis buffer (Promega luciferase kit; Promega, Madison,WI). Cell lysate (2–10 μl) was assayed for luciferase activity andβ-gal activity.

E-cadherin expression in the TSU.Pr-1 cell line is silenced by hypermethylation (32), and, as a consequence, the cells do not form intercellular contacts. To investigate the ability of E-cadherin to induce contact-dependent growth suppression, the mouse E-cadherin gene was transfected into TSU.Pr-1 cells, and stable transfectants were selected. As seen in Fig. 1,A, the E-cadherin protein was detected in the lysate of a selected transfectant (CAD) but not in the clone transfected with the control vector (Neo). Interestingly, the constitutive expression of E-cadherin caused a slight increase inβ-catenin and a decrease in p120 levels. α-Catenin levels were not affected by E-cadherin expression. In addition, contacts were observed in CAD cells but not in Neo cells (Fig. 1,B). Moreover, the rate of proliferation of CAD was reduced as compared with Neo cells, as seen in Fig. 1 C. The reduction in proliferation with E-cadherin expression was observed regardless of the cell density on seeding. These findings demonstrate that the expression of E-cadherin resulted in the formation of cell-cell contacts and a decrease in proliferation. These results are consistent with findings in other systems and cell types (26, 28).

Numerous studies have shown that E-cadherin is localized to the membrane and binds to β-catenin via its cytoplasmic tail. Confocal immunofluorescence microscopy analyses were conducted to determine whether E-cadherin in CAD cells was localizing to the membrane of the cell, and whether β-catenin colocalized with E-cadherin in CAD cells. Cells were fixed and double stained with antibodies against E-cadherin and β-catenin. As seen in Fig. 2,A, E-cadherin was detected at the contacts between CAD cells, whereas E-cadherin staining was not observed in the Neo cells. β-Catenin staining was observed throughout the cytoplasm of Neo cells but colocalized at the contacts with E-cadherin in CAD cells. Coimmunoprecipitation experiments confirmed the association of E-cadherin with β-catenin in CAD cells. As seen in Fig. 2 B, antibodies to E-cadherin immunoprecipitated E-cadherin from the lysates of two CAD clones (CAD1 and CAD2). Furthermore, β-catenin was coimmunoprecipitated with E-cadherin in both CAD clones. β-Catenin was detected in the lysate of Neo cells and was not associated with E-cadherin. E-cadherin in CAD cells localizes to the membrane of the cells and binds to β-catenin.

We wondered whether the inhibition of E-cadherin function at the cell surface would reverse the E-cadherin-induced growth suppression. Several studies have demonstrated that treatment with blocking antibodies to E-cadherin can inhibit its function. Treatment of CAD cells with DECMA, a rat polyclonal antibody against E-cadherin,resulted in the disruption of cell-cell interaction (Fig. 3,A). However, the DECMA antibodies did not affect the growth rate of CAD cells (Fig. 3,B). Table 1 also demonstrates the inability of DECMA antibodies to neutralize the growth suppression mediated by E-cadherin in three TSU.Pr-1 clones expressing E-cadherin. These results suggested that the ability to form contacts between cells was unrelated to the growth-suppressive activity of E-cadherin.

Truncation mutants of E-cadherin were generated to further investigate whether the ability of E-cadherin to form cell-cell contacts could be separated from growth suppression. Through PCR subcloning, the first 540 amino acids of the extracellular domain were removed, and the resulting gene (or mutant) was referred to as ΔN (Fig. 4,A). A second truncation mutant was generated by removing the last 73 amino acids, which include the β-catenin binding domain of E-cadherin, and is referred to asΔC. Immunoblot analyses of E-cadherin expression detected a major band of approximately Mr 37,000 and Mr 115,000 in the lysates of ΔN andΔC, respectively (Fig. 4,B). As expected, the deletion of the extracellular domain (as seen in ΔN; Fig. 4,C) resulted in a loss of cell-cell contact formation. In contrast, the deletion of the β-catenin-binding domain of E-cadherin (ΔC) did not affect the formation of contacts between cells (Fig. 4 B). This is in agreement with a report by Brieher et al.(33), in which Chinese hamster ovary cells expressing the C-cadherin gene lacking the cytoplasmic tail retained homotypic binding capacity, although the adhesive strength was weaker than that of cells expressing the full-length C-cadherin gene.

Staining of the transfectants with antibodies against E-cadherin demonstrated that both truncated E-cadherin proteins localized to the membrane (Fig. 5,A, top left panel). Indeed, despite the loss of cell-cell contact formation with the deletion of the extracellular domain, β-catenin was detected at the cell membrane in ΔN cells as shown by the intense staining in the top right panel of Fig. 5,A. WithΔC, in contrast, cell-cell contact formation was retained, butβ-catenin was distributed throughout the cytoplasm. Visualization ofΔN stained with two different fluorochromes at a higher magnification clearly demonstrates the colocalization of E-cadherin with β-catenin. Double staining of ΔN with E-cadherin (Fig. 5,B, green) andβ-catenin (red) revealed that they colocalized to the cell membrane (yellow). Coimmunoprecipitation studies were conducted on both deletion mutants, and the results confirm the immunofluorescence analysis. The full-length and extracellular truncated E-cadherin were detected in immunoprecipitate complex withβ-catenin from CAD and ΔN lysates, respectively. In contrast, the cytoplasmic truncated E-cadherin was not coimmunoprecipitated together with β-catenin (Fig. 5 C). Taken together, these results show that both deletion mutants localized to the membrane of the cells,and the extracellular truncated E-cadherin bound to and redistributedβ-catenin to the membrane like the full-length E-cadherin, despite the inability to form cell-cell contacts. In contrast, the cytoplasmic truncated E-cadherin could not bind to and redistributeβ-catenin but retained the ability to form cell-cell contacts like the full-length E-cadherin.

To investigate whether the ability to form cell-cell contacts between cells is involved in the growth suppression induced by E-cadherin, the cells were incubated for 4 days and analyzed for cell proliferation. The full-length E-cadherin transfectant showed a 50% reduction in cell proliferation (Fig. 6,A). The expression of the extracellular truncated E-cadherin resulted in an inhibition of cell proliferation similar in magnitude to CAD. Thus,cell contact formation is not required for the growth-inhibitory effects of E-cadherin. However, cell proliferation was not affected by the expression of the cytoplasmic truncated E-cadherin. Because the rate of cell growth is influenced by cell proliferation rate and cell loss through apoptosis, it was possible that E-cadherin could be either decreasing cell proliferation or increasing apoptosis. Cells synchronized in G2-M-phase arrest with nocodazole, a reversible microtubule inhibitor, were analyzed for cell proliferation with BrdUrd incorporation at several time periods after release of the cell cycle block. Sixteen h after release, a significant increase in BrdUrd incorporation was observed with Neo and ΔC cells but not with CAD and ΔN cells (Fig. 6 B). These results indicated that the expression of either the full-length or extracellular truncated Ecadherin inhibits DNA synthesis and hence inhibits cell proliferation. Furthermore, there was no appreciable difference in spontaneous apoptosis as measured through trypan blue exclusion between the cell lines (data not shown). Together with the earlier colocalization results, these findings suggest that the redistribution of β-catenin to the cell membrane results in a decrease in cell proliferation.

To be certain that the redistribution of β-catenin by ΔN was required to suppress growth of TSU.Pr-1 cells, an additional truncation mutant was generated that lacked both the extracellular domain and theβ-catenin-binding site of E-cadherin (ΔNC). The ΔNC mutant construct was transiently transfected into TSU.Pr-1 cells and then analyzed for cell proliferation. As shown previously, the expression of the extracellular truncated E-cadherin (ΔN) inhibited proliferation(Fig. 6 C). The deletion of the β-catenin-binding site of the extracellular truncated E-cadherin abolished the growth-inhibitory activity. These findings further support the hypothesis that theβ-catenin-binding site is necessary for the growth-suppressive activity.

Recent reports have demonstrated that uncomplexed β-catenin can translocate to the nucleus and form a complex with TCF/lymphoid enhancer factor as a component of the activated Wnt signaling pathway(19, 20, 34). Furthermore, the introduction of E-cadherin has been shown to redistribute β-catenin and interfere with the Wnt signaling pathway (35, 36). Cyclin D, which is involved in cell cycle regulation, may be a target of the Wnt pathway(37). Based on the data presented, it was conceivable that the transfection of E-cadherin resulted in the redistribution ofβ-catenin and that the growth suppression was due to inhibition of Wnt signaling. If this idea is correct, cells with forms of E-cadherin that keep β-catenin at the membrane should have defective TCF-mediated gene transcription.

Therefore, the β-catenin/TCF reporter assay was performed to compare the transcriptional activity of β-catenin in the various cells. Theβ-catenin/TCF reporter assay requires the use of the TOP FLASH reporter construct that consists of four consensus TCF-binding sites placed upstream of the luciferase reporter gene. The FOP FLASH reporter construct contained mutations in the TCF-binding sites and served as a negative control. As shown in Fig. 7,very low levels of β-catenin transactivating activity were detected in the Neo cells. Furthermore, differences in β-catenin transcriptional activity among Neo, CAD, ΔN, and ΔC were insignificant. These findings indicated that β-catenin/TCF transcription was very low in all of the cells, and the expression of E-cadherin did not affect this activity in a significant way. In addition, transient transfection of the dominant positive β-catenin mutant (S37F; Ref. 38) could not reverse the growth-inhibitory effects of E-cadherin (data not shown). Thus, the growth-inhibitory effects of β-catenin redistribution are not clearly related to alteration in the Wnt-TCF signal transduction pathway. Taken together, although the expression of E-cadherin results in growth suppression and β-catenin redistribution to the cell membrane,inhibition of the Wnt signaling pathway by reducing the availableβ-catenin does not seem to be the mechanism by which E-cadherin induces growth suppression in these cells. These results suggest thatβ-catenin influences signaling through other growth pathways in a fashion that is independent of its ability to promote TCF-induced transcription. Alternatively, E-cadherin may be affecting another pathway that is unrelated to the redistribution of β-catenin.

Several studies have shown that E-cadherin, in association withβ-catenin, can associate with either receptor tyrosine kinases(10, 11, 12) or receptor tyrosine phosphatases(39). In particular, activation of the EGFR has been shown to be inhibited by E-cadherin-mediated cell-cell adhesion(10). Furthermore, Zolfaghari and Djakiew(40) have demonstrated the expression of the EGFR on the surface of TSU.Pr-1. Hence it is possible that the formation of the E-cadherin/β-catenin complex could prevent EGFR signaling that would lead to suppression of growth. However, immunoblot analysis of cell lysates of Neo, CAD, ΔN, and ΔC revealed that these cells do not express any detectable levels of EGFR (data not shown). Immunoprecipitation analysis with antibodies to the EGFR also confirmed the absence of the EGFR in the cells (data not shown). The reason for the discrepancy between our results and those of Zolfaghari and Djakiew(40) is not clear. In addition, immunoblot analysis with antiphosphotyrosine antibodies did not detect any significant differences in levels of specific tyrosine-phosphorylated substrates as a consequence of E-cadherin expression (data not shown). The data indicated that the growth-inhibitory activity of E-cadherin in TSU.Pr-1 cells does not involve the association of EGFR with E-cadherin and the modulation of protein tyrosine phosphorylation. This implies that the activation or suppression of tyrosine kinases or tyrosines phosphatases is not a central feature of cadherin-mediated growth suppression.

Several studies have shown that the Src tyrosine kinase substrate p120 is also associated with the juxtamembrane region of cadherin independent of β-catenin (5, 41). Altered levels of p120 have been observed in various cancer cells (42, 43, 44). Based on these findings, it is possible that the expression of E-cadherin alters the level of p120 in TSU.Pr-1 cells, which could lead to growth suppression. However, as seen in Fig. 8,a, no significant differences in the level of p120 (or the Mr 100,000 isoform) were observed with the expression of E-cadherin and both E-cadherin mutants. Furthermore,van Hegel et al.(45) have shown that p120 localizes to the nucleus in cadherin-negative cancer cell lines, and the expression of E-cadherin results in the redistribution of p120 to the cell membrane. Although p120 was distributed throughout cytoplasm and at the cell membrane in NEO and CAD cells (data not shown), it is possible that the ability of E-cadherin to complex with p120 could be associated with growth suppression. If this were the case, then the deletion of either the NH2- or COOH-terminal of E-cadherin should result in the dissociation of p120. To investigate this, total lysates from CAD, ΔN, and ΔC were precipitated with normal mouse immunoglobulin (control immunoglobulin) and anti-E-cadherin antibodies, and the precipitates were analyzed for the association with p120. The full-length and truncated E-cadherin were precipitated by anti-E-cadherin. The Mr 100,000 isoform of p120 coimmunoprecipitated with the full-length E-cadherin and both truncated mutants of E-cadherin (Fig. 8, A and B); however, β-catenin coimmunoprecipitated with the full-length E-cadherin and the NH2-terminal truncated E-cadherin (Fig. 5). Thus,these findings indicate that the level of p120 does not change with E-cadherin expression, and truncation of either the ectodomain or theβ-catenin-binding site does not affect the ability of p120 to associate with E-cadherin. Thus, the ability of E-cadherin to inhibit the growth of TSU.Pr-1 cells does not correlate with its ability to bind p120.

Cadherins, which are cell adhesion proteins, have been shown to be down-regulated during tumor progression (46). Cadherin expression is considered a prognostic marker in prostate cancer. In particular, the ability of E-cadherin to mediate cell contact has been associated with contact-inhibition and suppression of cell invasion. In this paper, the prostate carcinoma cell line TSU.Pr-1 was transfected with the mouse E-cadherin gene, which shares high homology(1) and functions in various human cancer models(47, 48) and cell contact formation and growth suppression were induced. Furthermore, β-catenin relocalized from the cytoplasm to the cell membrane in association with E-cadherin expression. In agreement, studies have shown that the transfection of E-cadherin into mouse mammary carcinoma cells (26) and colon carcinoma cells (25, 48) resulted in an increase in cell-cell adhesion and growth suppression with the redistribution of β-catenin. However, the growth-inhibitory effects of E-cadherin as reported by St. Croix et al.(26) were only observed in three-dimensional cultures and not seen in monolayer cultures. E-cadherin-induced growth suppression, in contrast, was observed in TSU.Pr-1 cells when grown as either subconfluent or confluent monolayer cultures, as seen by Efstathiou et al.(48) and Miyaki et al.(25) in colon cancer cells. The direct association of both effects of E-cadherin was confirmed in breast cancer cells; when E-cadherin-transfected cells were treated with neutralizing anti-E-cadherin antibodies, cell-cell aggregation and growth suppression were reversed (26). However, in our studies with subconfluent monolayer cultures of E-cadherin-transfected TSU.Pr-1 prostate cancer cells, neutralizing antibodies to E-cadherin prevented cell-cell adhesion but did not affect growth inhibition. It is possible that the conflicting results could be explained by the difference in either culture conditions or cell types. Nevertheless,our data with blocking antibodies suggest that the ability of E-cadherin to facilitate cell-cell adhesion may not be entirely responsible for growth suppression.

The expression of a mutant N-cadherin gene, which is missing the extracellular domain, in transgenic mice resulted in an increase in cell proliferation and apoptosis that led to adenomas (22, 29). In contrast, our findings indicate that the deletion of the extracellular domain of E-cadherin does not increase cell proliferation and apoptosis. The conflicting findings could be explained by the fact that the mutant E-cadherin in our system does not compete with endogenous E-cadherin in TSU.Pr-1 cells. The mutant N-cadherin in transgenic mice acts as a dominant negative mutation and competes with endogenous E-cadherin, which leads to an increase in cell proliferation as reported by Hermiston and Gordon (29).

To further investigate whether different regions of E-cadherin may be involved in either cell-cell adhesion or growth suppression, mutants comprised of either the extracellular or cytoplasmic region of E-cadherin were expressed in TSU.Pr-1 cells. Deletion of the extracellular region of E-cadherin resulted in the loss of cell-cell adhesion but persistence of growth suppression. This is in accordance with Zhu and Watt (27), who demonstrated that the expression of a dominant negative E-cadherin mutant inhibited proliferation of human epidermal keratinocytes. The dissociation of cell-cell adhesion and growth suppression effects of E-cadherin was further demonstrated in cells expressing E-cadherin with the cytoplasmic region truncated; growth suppression, but not cell-cell adhesion, was abrogated. These findings indicate that the extracellular region of the E-cadherin is involved in cell-cell adhesion but not growth suppression, whereas the cytoplasmic region is necessary for growth suppression.

Recent studies have shown that the transcriptional activity ofβ-catenin/TCF in the Wnt pathway is involved in transformation and growth proliferation. Some of the target genes that are activated by the Wnt pathway are involved in growth regulation such as c-myc(49) and cyclin D (36). Moreover,E-cadherin can influence the availability of β-catenin to interact with TCF to activate the Wnt pathway (35, 36, 50, 51). Because the expression of either the full-length E-cadherin or cytoplasmic region of E-cadherin resulted in the binding and redistribution of β-catenin, it was conceivable that the expression of E-cadherin would inhibit the Wnt pathway by reducing the availability of free β-catenin. However, in TSU.Pr-1 cells, this was not the case. The expression of either the full-length E-cadherin or the cytoplasmic region of E-cadherin did not influence β-catenin/TCF transcriptional activity. The expression of the dominant positiveβ-catenin mutant could not reverse the growth-inhibitory activity of E-cadherin, which further undermines the idea that inhibition of the Wnt pathway is the mechanism for E-cadherin-induced growth suppression. Another possible mechanism by which E-cadherin could be suppressing growth could be through the modulation of various receptor tyrosine kinases, such as the EGFR (10), EphA2 receptor tyrosine kinase (52), or phosphatases (39). However,immunoblot analysis with antibodies directed to phosphotyrosine residues of lysates from TSU.Pr-1 cells either expressing or not expressing E-cadherin failed to detect any significant differences in tyrosine-phosphorylated proteins. This finding suggests that the growth-suppressive effects of E-cadherin are not related to alterations in tyrosine kinase or phosphatase activity. Alternatively, expression of E-cadherin could redistribute other proteins such as p120, which binds to the E-cadherin (5, 41). van Hegel et al.(45) have shown that the expression of exogenous E-cadherin or the up-regulation of E-cadherin expression resulted in the redistribution of p120 from the nucleus to the cell membrane. Several studies have detected changes in the level or the redistribution of p120 in colon cancer (42), adenomatous polyps of the colon (44), bladder cancer(53), and breast cancer (54). Our results,however, did not show any significant differences in the level or association of p120 as a consequence of the expression of the full-length or truncated E-cadherin in TSU.Pr-1 cells. Thus, althoughβ-catenin is redistributed to the cell membrane with E-cadherin expression, our data seem to rule out the inhibition of the Wnt pathway, alterations in EGFR signaling, and altered p120 nuclear translocation as mechanisms of E-cadherin-induced growth inhibition in TSU.Pr-1 cells.

In summary, we report that the adhesiveness and growth-inhibitory effects of E-cadherin are separable. Adhesiveness is controlled by the extracellular region, and growth suppression is controlled by the cytoplasmic region. Furthermore, although the cytoplasmic region of E-cadherin redistributed β-catenin to the membrane,E-cadherininduced growth suppression is unrelated toβ-catenin/TCF transcriptional activity. Indeed, from results with a dominant active form of β-catenin, it appears that the growth-suppressive effects of E-cadherin are either entirely independent of β-catenin or involve some function of β-catenin other than transcription.

Fig. 1.

Stable expression of E-cadherin results in cell-cell contact and growth suppression. A, immunoblot analysis of various adhesion proteins in Neo and CAD lysates. Cell lysates were separated through a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was then assembled into a Multiscreen apparatus and blotted with various monoclonal antibodies against E-cadherin, α-catenin, β-catenin, and p120. Numberson the top of the blot refer to the monoclonal antibody concentration (μg/ml). Numbers on the left of the blot refer to the molecular weight in thousands. B, cell-cell contact formation observed with E-cadherin expression. Stable clones of TSU.Pr-1 transfected with control vector (Neo) or E-cadherin expression vector(CAD) were plated on tissue culture dishes and incubated in media as described in “Materials and Methods.” Magnification,×100. C, growth suppression associated with E-cadherin expression. Cell proliferation was assessed via MTT proliferation assay.

Fig. 1.

Stable expression of E-cadherin results in cell-cell contact and growth suppression. A, immunoblot analysis of various adhesion proteins in Neo and CAD lysates. Cell lysates were separated through a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was then assembled into a Multiscreen apparatus and blotted with various monoclonal antibodies against E-cadherin, α-catenin, β-catenin, and p120. Numberson the top of the blot refer to the monoclonal antibody concentration (μg/ml). Numbers on the left of the blot refer to the molecular weight in thousands. B, cell-cell contact formation observed with E-cadherin expression. Stable clones of TSU.Pr-1 transfected with control vector (Neo) or E-cadherin expression vector(CAD) were plated on tissue culture dishes and incubated in media as described in “Materials and Methods.” Magnification,×100. C, growth suppression associated with E-cadherin expression. Cell proliferation was assessed via MTT proliferation assay.

Close modal
Fig. 2.

Expression of E-cadherin results in the redistribution ofβ-catenin from the cytoplasm to membrane. A,immunofluorescence staining of Neo and CAD cell lines for E-cadherin and β-catenin. Neo (top two panels) and CAD(bottom two panels) were stained with monoclonal antibodies against E-cadherin (left two panels) andβ-catenin (right two panels) as described in“Materials and Methods.” Magnification, ×400. B,coimmunoprecipitation of β-catenin with transfected E-cadherin in two CAD clones. Lysates from Neo and two E-cadherin-expressing clones(CAD1 and CAD2) were immunoprecipitated with either normal mouse control immunoglobulin (IP:Control Ig) or monoclonal antibodies against E-cadherin(IP:Anti-E-cadherin). The first three lanes contain total cell lysates. Numbers on the left of the figure refer to the molecular weight in thousands.

Fig. 2.

Expression of E-cadherin results in the redistribution ofβ-catenin from the cytoplasm to membrane. A,immunofluorescence staining of Neo and CAD cell lines for E-cadherin and β-catenin. Neo (top two panels) and CAD(bottom two panels) were stained with monoclonal antibodies against E-cadherin (left two panels) andβ-catenin (right two panels) as described in“Materials and Methods.” Magnification, ×400. B,coimmunoprecipitation of β-catenin with transfected E-cadherin in two CAD clones. Lysates from Neo and two E-cadherin-expressing clones(CAD1 and CAD2) were immunoprecipitated with either normal mouse control immunoglobulin (IP:Control Ig) or monoclonal antibodies against E-cadherin(IP:Anti-E-cadherin). The first three lanes contain total cell lysates. Numbers on the left of the figure refer to the molecular weight in thousands.

Close modal
Fig. 3.

Treatment with neutralizing antibodies against E-cadherin disrupts cell-cell contact but not growth suppression. A, loss of cell-cell contact of CAD cells with antibody treatment. CAD cells were incubated with either control or a rat monoclonal antibody against E-cadherin (DECMA), seeded on tissue culture dishes, and incubated overnight. Magnification, ×200. B, treatment of CAD cells with antibodies against E-cadherin does not affect growth suppression. Neo or CAD cells were incubated with various concentrations of DECMA antibodies for 48 h. Cell viability was assessed by the MTT proliferation assay.

Fig. 3.

Treatment with neutralizing antibodies against E-cadherin disrupts cell-cell contact but not growth suppression. A, loss of cell-cell contact of CAD cells with antibody treatment. CAD cells were incubated with either control or a rat monoclonal antibody against E-cadherin (DECMA), seeded on tissue culture dishes, and incubated overnight. Magnification, ×200. B, treatment of CAD cells with antibodies against E-cadherin does not affect growth suppression. Neo or CAD cells were incubated with various concentrations of DECMA antibodies for 48 h. Cell viability was assessed by the MTT proliferation assay.

Close modal
Fig. 4.

Truncation of the extracellular region results in the loss of cell-cell contact. A, schematic drawing of the extracellular domain (ΔN) and cytoplasmic domain (ΔC) truncation mutations of E-cadherin. B, loss of cell-cell contact with the truncation of the extracellular domain of E-cadherin. Stable transfected cells were seeded on tissue culture dishes and incubated overnight. Magnification, ×100. C, expression of truncated E-cadherin mutants. Proteins from cell lysates were separated through SDS-polyacrylamide gel and blotted with monoclonal antibodies against E-cadherin. A431 served as a positive control for E-cadherin. The arrows on the right point to the full-length E-cadherin (CAD), the cytoplasmic truncated E-cadherin (ΔC), and the extracellular truncated E-cadherin(ΔN), in descending order. Numbers on the left refer to the molecular weight in thousands.

Fig. 4.

Truncation of the extracellular region results in the loss of cell-cell contact. A, schematic drawing of the extracellular domain (ΔN) and cytoplasmic domain (ΔC) truncation mutations of E-cadherin. B, loss of cell-cell contact with the truncation of the extracellular domain of E-cadherin. Stable transfected cells were seeded on tissue culture dishes and incubated overnight. Magnification, ×100. C, expression of truncated E-cadherin mutants. Proteins from cell lysates were separated through SDS-polyacrylamide gel and blotted with monoclonal antibodies against E-cadherin. A431 served as a positive control for E-cadherin. The arrows on the right point to the full-length E-cadherin (CAD), the cytoplasmic truncated E-cadherin (ΔC), and the extracellular truncated E-cadherin(ΔN), in descending order. Numbers on the left refer to the molecular weight in thousands.

Close modal
Fig. 5.

Truncation of the extracellular domain, but not the cytoplasmic domain, retains the ability to redistribute β-catenin. A, immunostaining of ΔN and ΔC cells for E-cadherin and β-catenin. ΔN (top two panels) and ΔC(bottom panels) were stained with monoclonal antibodies against E-cadherin (left two panels) and β-catenin (right two panels) as described in “Materials and Methods.” Magnification, ×400. B, colocalization of E-cadherin and β-catenin in ΔN cells. ΔN cells were stained with E-cadherin (green) and β-catenin (red). The rightpanel is the composite image of E-cadherin and β-catenin images. The yellow color is the result of the colocalization of E-cadherin and β-catenin. Magnification, ×1200. C, coimmunoprecipitation ofβ-catenin with extracellular domain truncation E-cadherin. Lysates from CAD, ΔN, and ΔC cells were immunoprecipitated with antisera against β-catenin and blotted with monoclonal antibodies against E-cadherin and β-catenin. Numbers on the left side refer to the molecular weight in thousands.

Fig. 5.

Truncation of the extracellular domain, but not the cytoplasmic domain, retains the ability to redistribute β-catenin. A, immunostaining of ΔN and ΔC cells for E-cadherin and β-catenin. ΔN (top two panels) and ΔC(bottom panels) were stained with monoclonal antibodies against E-cadherin (left two panels) and β-catenin (right two panels) as described in “Materials and Methods.” Magnification, ×400. B, colocalization of E-cadherin and β-catenin in ΔN cells. ΔN cells were stained with E-cadherin (green) and β-catenin (red). The rightpanel is the composite image of E-cadherin and β-catenin images. The yellow color is the result of the colocalization of E-cadherin and β-catenin. Magnification, ×1200. C, coimmunoprecipitation ofβ-catenin with extracellular domain truncation E-cadherin. Lysates from CAD, ΔN, and ΔC cells were immunoprecipitated with antisera against β-catenin and blotted with monoclonal antibodies against E-cadherin and β-catenin. Numbers on the left side refer to the molecular weight in thousands.

Close modal
Fig. 6.

Truncation of the cytoplasmic domain resulted in the loss of growth suppression. A, growth curves of Neo, ΔN,ΔC, and CAD cells for 4 days. Cell viability was assessed by the MTT proliferation assay on days 0, 2, and 4. B, expression of full-length E-cadherin or extracellular domain truncated E-cadherin inhibits DNA synthesis. Cells were released from nocodazole arrest, and DNA synthesis was assessed by the BrdUrd incorporation assay, as indicated in “Materials and Methods.” C, deletion of the β-catenin binding site of ΔN abolishes growth suppression. Neo,ΔN, and transiently transfected ΔNC cells were plated, incubated overnight, and analyzed for proliferation by MTT.

Fig. 6.

Truncation of the cytoplasmic domain resulted in the loss of growth suppression. A, growth curves of Neo, ΔN,ΔC, and CAD cells for 4 days. Cell viability was assessed by the MTT proliferation assay on days 0, 2, and 4. B, expression of full-length E-cadherin or extracellular domain truncated E-cadherin inhibits DNA synthesis. Cells were released from nocodazole arrest, and DNA synthesis was assessed by the BrdUrd incorporation assay, as indicated in “Materials and Methods.” C, deletion of the β-catenin binding site of ΔN abolishes growth suppression. Neo,ΔN, and transiently transfected ΔNC cells were plated, incubated overnight, and analyzed for proliferation by MTT.

Close modal
Fig. 7.

β-Catenin//TCF transcriptional activity was not affected by E-cadherin expression. Neo, CAD, ΔN, and ΔC cells were transiently transfected with the β-catenin/TCF response element-driven luciferase reporter plasmid (TOP; □) or the mutant β-catenin/TCF response element-driven luciferase reporter plasmid (FOP; ). Luciferase activity was measured as an indicator for β-catenin/TCF transcriptional activity. The β-gal expression plasmid was cotransfected to normalize for transfection efficiency.

Fig. 7.

β-Catenin//TCF transcriptional activity was not affected by E-cadherin expression. Neo, CAD, ΔN, and ΔC cells were transiently transfected with the β-catenin/TCF response element-driven luciferase reporter plasmid (TOP; □) or the mutant β-catenin/TCF response element-driven luciferase reporter plasmid (FOP; ). Luciferase activity was measured as an indicator for β-catenin/TCF transcriptional activity. The β-gal expression plasmid was cotransfected to normalize for transfection efficiency.

Close modal
Fig. 8.

Expression of E-cadherin does not affect p120 levels, and truncation of E-cadherin does not affect p120 association. A, total lysates from NEO, CAD, ΔN, and ΔC cells were probed with anti-p120 antibodies. B, detection of p120 in lysates from CAD, ΔN, and ΔC immunoprecipitated for E-cadherin. Total lysates were immunoprecipitated with normal mouse immunoglobulin (Control Ig) and monoclonal anti-E-cadherin (E-cadherin). Membranes were hybridized with monoclonal anti-p120. Bands at Mr38,000 and Mr 53,000 represent the light and heavy immunoglobulin chain. Numbers on the left of the figure indicate the molecular weight in thousands.

Fig. 8.

Expression of E-cadherin does not affect p120 levels, and truncation of E-cadherin does not affect p120 association. A, total lysates from NEO, CAD, ΔN, and ΔC cells were probed with anti-p120 antibodies. B, detection of p120 in lysates from CAD, ΔN, and ΔC immunoprecipitated for E-cadherin. Total lysates were immunoprecipitated with normal mouse immunoglobulin (Control Ig) and monoclonal anti-E-cadherin (E-cadherin). Membranes were hybridized with monoclonal anti-p120. Bands at Mr38,000 and Mr 53,000 represent the light and heavy immunoglobulin chain. Numbers on the left of the figure indicate the molecular weight in thousands.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: EGFR, epidermal growth factor receptor; APC, adenomatous polyposis coli; GSK-3β,glycogen synthase kinase 3β; TCF, T-cell factor; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BrdUrd,bromodeoxyuridine; PVDF, polyvinylidene difluoride; β-gal,β-galactosidase.

Table 1

Neutralizing antibody against E-cadherin does not inhibit the growth suppression induced by E-cadherin in three CAD clones

CellsFold increase after 2 days of incubationa
UntreatedDECMA (15 μg/ml)
Neo 8.6 ± 0.1b 9.3 ± 0.3 
CAD1 4.6 ± 0.8 4.0 ± 0.7 
CAD2 3.7 ± 0.3 3.6 ± 1.0 
CAD3 4.5 ± 0.7 5.3 ± 0.9 
CellsFold increase after 2 days of incubationa
UntreatedDECMA (15 μg/ml)
Neo 8.6 ± 0.1b 9.3 ± 0.3 
CAD1 4.6 ± 0.8 4.0 ± 0.7 
CAD2 3.7 ± 0.3 3.6 ± 1.0 
CAD3 4.5 ± 0.7 5.3 ± 0.9 
a

Fold increase was determined using the MTT viability assay as described in “Materials and Methods.”

b

Average and SD of triplicate samples.

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