Mutant E-cadherin Breast Cancer Cells Do Not Display Constitutive Wnt Signaling

INTRODUCTION

β-Catenin is a key regulator in the Wnt 3 signal transduction cascade (reviewed in Refs. 1, 2, 3, 4 ). 4 In the absence of Wnt signaling, β-catenin resides in a protein complex including APC, axin/conductin, and GSK-3β (5, 6, 7, 8, 9, 10, 11, 12, 13) . Constitutive phosphorylation of β-catenin by the serine kinase GSK-3β targets β-catenin for ubiquitination byβ -TrCP and subsequent proteosomal degradation (14, 15, 16, 17, 18) . Upon Wnt signaling, the APC destruction complex is inactivated and, as a consequence, β-catenin translocates to the nucleus where it interacts with Tcf/Lef factors and activates target genes (19, 20, 21) . In this bipartite transcription complex,β -catenin provides a potent transactivation domain, whereas Tcf binds DNA in a sequence-specific manner (21) . Recently, the c-Myc, cyclinD1, and Tcf1 genes have been identified as target genes of Tcf4 in colorectal cancer (22, 23, 24) .

The APC tumor suppressor gene was found to be mutated in a majority of colorectal carcinomas (reviewed in Refs. 25 and 26 ). The genetic alterations generally resulted in truncated APC proteins that were no longer able to interact withβ -catenin and axin. Consequently, inactivation of APC leads to nuclear translocation of β-catenin, where it complexes with Tcf4 and inappropriately activates the transcription of target genes (27) . In colorectal cancers with wild-type APC genes (28) , in melanoma (29) , and in some other cancer types (reviewed in Refs. 30 and 31 ), dominant mutations in the β-catenin (CTNNB1) gene have been identified. These mutations affect the GSK-3β phosphorylation sites of β-catenin and were shown to lead to stable Tcf-β-catenin complexes in the nucleus and to constitutive transcriptional activation of target genes (28) . Mutations in the AXIN1 and AXIN2 genes have recently been identified in hepatocellular carcinomas (32) and in mismatch repair-defective colorectal cancers (33) , respectively. These mutations all predicted truncated axin proteins, which would be expected to abolish proper degradation of β-catenin and result in inappropriate activation of Tcf target genes.

E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent cell adhesion between epithelial cells (reviewed in 34 and 35 ). Through its extracellular calcium-binding domains, E-cadherin forms a molecular zipper with other E-cadherin proteins that are located in the adherens junctions between adjacent cells. The intracellular domain of E-cadherin interacts with the actin cytoskeleton via a protein complex containing α-catenin,β -catenin, and γ-catenin (36, 37, 38) . Tyrosine phosphorylation of β-catenin in v-Src- (39) or Ras- (40) transformed cells or in cells stimulated with epidermal growth factor (41) induced release of the E-cadherin-catenin complex from the cytoskeleton and diminished cell adhesion. As a result, the cells had lost their characteristic epithelial growth pattern and had adopted a fibroblast-like cell morphology.

Loss of E-cadherin expression or function has been implicated in tumor invasion and metastasis (reviewed in Ref. 42 ). E-cadherin-mediated cell adhesion seemed to be crucial in the transition from adenoma to carcinoma (43) , and reconstitution of human (44, 45, 46) , canine (47) , murine (48) , or rat (49) cancer cell lines with wild-type E-cadherin cDNA was shown to reverse invasiveness of the cancer cells. Primary tumor specimens of various human cancer types were frequently found to exhibit aberrant E-cadherin protein expression and, consistent with the experimental models, this was often associated with the invasiveness of the tumors (reviewed in Ref. 50 ). Alterations in the E-cadherin (CDH1) tumor suppressor gene have been identified in nearly one-half of lobular breast carcinomas and diffuse-type gastric carcinomas and in a small proportion of gynecological cancers (reviewed in Refs. 51 and 52 ).

The role of E-cadherin as a tumor suppressor protein and the dual role of its binding partner β-catenin in cell adhesion and Wnt signaling could indicate a function for E-cadherin in the Wnt pathway. In the absence of an appropriate Wnt signal, for example, E-cadherin might sequester β-catenin at the cell membrane, thereby preventing the formation of Tcf-β-catenin complexes in the nucleus. Mutation of E-cadherin in cancer cells may disrupt the interaction withβ -catenin, thereby promoting nuclear translocation of β-catenin and inappropriate formation of transcriptionally active Tcf-β-catenin complexes. E-cadherin-null embryonic stem cells had indeed been reported to exhibit Lef-β-catenin-mediated transcriptional activation, which was antagonized by transient expression of wild-type E-cadherin cDNA (53) .

If E-cadherin is indeed a participant of the Wnt pathway, genetically mutant E-cadherin cancer cells should have constitutive Tcf-mediated transactivation, similar to mutant APC, β-catenin, or axin cancer cells (27 , 28 , 32 , 33) . To test this hypothesis, we performed a mutational analysis of E-cadherin in 26 human breast cancer cell lines and identified 8 cell lines with genetic alterations in E-cadherin. The involvement of E-cadherin in the Wnt signaling pathway was evaluated through determination of transcriptional activation of a Tcf-responsive Luciferase reporter gene in 15 cell lines, including 6 E-cadherin mutant cell lines and 4 cell lines with transcriptional silencing of the E-cadherin gene.

MATERIALS AND METHODS

Cancer Cell Lines.

The breast cancer cell lines used in this study are listed in Table 1 ⇓ . Cell lines EVSA-T, MPE600, SK-BR-5, and the SUM-series were kind gifts of Dr. N. DeVleeschouwer (Institut Jules Bordet, Brussels, Belgium), Dr. H. S. Smith (California Pacific Medical Center, San Francisco, CA), Dr. E. Stockert (Sloan-Kettering Institute for Cancer Research, New York, NY), and Dr. S. Ethier (University of Michigan, Ann Arbor, MI), respectively. Cell line OCUB-F was obtained from Riken Gene Bank. All other cell lines, including colorectal cancer cell line SW480, were obtained from American Type Culture Collection. Cell lines were grown according the suppliers’ recommendations.

PCR.

Genomic DNA was extracted using the Qiagen DNeasy kit. Exonic sequences of E-cadherin (GenBank accession no. Z13009) were amplified using primers designed to anneal to bordering intron sequences (54) . Amplification of genomic DNA (20 ng) was performed by 2 min at 94°C and then by 35 cycles of 30 s at 94°C, 1 min at the appropriate annealing temperature, and 1 min at 72°C, with a final extension of 5 min at 72°C. PCR was performed in 10 mm Tris (pH 9.0) 50 mm KCl, 0.1% Triton X-100, 1.5 mm MgCl₂, 200 μm of each deoxynucleotide triphosphate, and 0.75 units Taq Polymerase (Promega) in a final volume of 15 μl. Exons 4 and 5, as well as exons 8 and 9, were concurrently amplified. For exons 12–16, 0.5 m Betaine and 1% DMSO were included in the reactions.

Microsatellite markers D16S421, D16S496, D16S2621, and D16S2624 were used to PCR-amplify polymorpic loci from the genome of the breast cancer cell lines. PCR was performed essentially as described above, except that the reactions were radioactively labeled with [α-³²P]-dATP, and 5 ng template DNA was used.

Reverse transcription-PCR.

RNA was extracted using the Qiagen RNeasy kit. First-strand synthesis was performed using Ready-to-go-you-prime-first-strand-beads (Pharmacia). E-cadherin transcripts were then amplified by PCR, using E-cadherin-specific primers that annealed to sequences ∼100 bases upstream of the first and downstream of the last codon.

Cycle Sequencing.

E-cadherin amplification products were incubated with 10 units Exonuclease I and 2 units shrimp alkaline phosphatase (United States Biochemical Corp.) for 15 min at 37°C, and the enzymes were then inactivated for 15 min at 80°C. One-tenth of the reaction was sequenced by 60 cycles of 30 s at 94°C, 30 s at the appropriate annealing temperature, and 1 min at 72°C using conditions recommended by the manufacturer (Thermo Sequenase Cycle Sequencing kit; Pharmacia).

Immunohistochemistry.

Cells were grown on collagen-coated coverslips and fixed overnight with acetone. Cells were incubated with either anti-E-cadherin (TL clone #36) or anti-β-catenin (TL clone #14) antibodies (Transduction Labs) or with isotype-matched mouse negative control antibodies (IgG1 and IgG2a; DAKO) and subsequently with fluorescein-conjugated rabbit antimouse secondary antibodies (DAKO). Cells were mounted with Vectashield containing 1 μg/ml 4′,6-diamidino-2-phenylindole.

Western Blotting.

Cell lysates were prepared by resuspension of cells in Laemni sample buffer and subsequent boiling for 5 min. Proteins were separated by electrophoresis in a 3% low-melting point agarose gel and transferred to Hybond-P membranes (Amersham) by overnight capillary blotting. Blots were incubated with anti-APC antibody FE9 (Oncogene Research Products) and then by horseradish peroxidase-conjugated rabbit antimouse secondary antibodies (DAKO). Reactions were visualized through enhanced chemiluminescence (ECL; Amersham).

Reporter Gene Assays.

Tcf reporter constructs pTOPGLOW and pFOPGLOW were generated by insertion of SalI-fragments from the pTOP/FOPFLASH reporter constructs that contain a multimerized Tcf-binding motif (27) into the SalI-linearized p19TATA-luc vector that contains a minimal E1b TATA box upstream of the Firefly Luciferase cDNA.

For transient transfections, cells were grown in RPMI 1640 containing 10% FCS, to 50–80% confluency in six-well plates. Cells were transfected with 1 μg of pTOPGLOW or pFOPGLOW using Fugene-6 (Boehringer Mannheim) in the presence of 10% serum. Transfection efficiencies were determined by cotransfection of 100 ng of pRL-TK reporter construct (Promega) that contained the Renilla Luciferase cDNA under control of the herpes simplex virus thymidine kinase promotor. Cells were harvested 24 h after transfection, washed with PBS, and resuspended in Passive Lysis buffer (Promega). Activities of Firefly and Renilla luciferases were measured sequentially from a single sample using the Dual-Luciferase Reporter Assay System (Promega) on a Lumat LB9507 luminometer. Tcf-mediated gene transcription was defined by the ratio of pTOPGLOW:pFOPGLOW luciferase activities, where the luciferase activity of the internal control reporter pRL-TK was used to correct for differences in transfection efficiency.

RESULTS

E-cadherin Mutational Analysis.

Twenty-six human breast cancer cell lines (Table 1) ⇓ were analyzed for allelic loss of chromosome 16q22 by PCR using microsatellite markers D16S421, D16S496, D16S2621, and D16S2624, which were located in the chromosomal region encompassing the E-cadherin gene. Analysis of genomic DNA from 25 unrelated, randomly selected individuals revealed heterozygosity ratios of 0.36 for marker D16S421, 0.40 for D16S496, 0.88 for D16S2621, and 0.76 for D16S2624. Allelic loss at 16q22 was presumed when a cell line had a single allele size at each of the four loci with a P = 0.01 (i.e., the probability that a heterozygous sample had a single allele size at each locus). Fifteen of 26 (58%) breast cancer cell lines were considered to have loss of heterozygosity at 16q22 (Table 1) ⇓ . None of the 25 DNAs from the unrelated control individuals were homozygous at all four loci, validating the statistical approach.

The coding sequence of E-cadherin was analyzed for genetic alterations in 24 breast cancer cell lines by PCR and by direct cycle sequencing. Homozygous deletions of E-cadherin gene sequences were identified in four breast cancer cell lines (Table 1) ⇓ . Cell line MDA-MB-134VI had a deletion of exon 6 of the E-cadherin gene, cell line MPE600 had a deletion of exon 9, and cell line OCUB-F had a deletion of exon 2. Cell line SK-BR-3 had a homozygous deletion of exons 2 through 12, but had retained exon 1 and exons 13–16 (Fig. 1A) ⇓ . All homozygous deletions were confirmed by duplex PCR using an unrelated primer pair [DPC2′, 500-bp fragment; (55)] . Exons 2–16 of E-cadherin were successfully amplified from the genome of the remaining 20 breast cancer cell lines. Amplification products of exon 1 generally contained fragments of various lengths, likely attributable to primer annealing at homologous sequences in the human genome. Exon 1 was excluded from additional analysis.

The amplified E-cadherin sequences were analyzed for alterations by cycle sequencing, using the intronic PCR primers or outwardly directed exonic primers. Alterations in the E-cadherin gene sequence were identified in four breast cancer cell lines (Table 1) ⇓ . Cell line CAMA-1 had an agGTT→aaGTT alteration in the splice acceptor site of exon 12 (Fig. 1B) ⇓ , cell line EVSA-T had an ACTgtaa→ACTaa alteration in the splice donor site of exon 5, and cell line SK-BR-5 had an agATC→acATC alteration in the splice acceptor site of exon 5. Cell line SUM44PE had deleted a thymidine residue at codon 423, exon 9 (ATTTGT→ATTGT). All sequence alterations were confirmed by sequencing of an independently amplified template. The mutational analysis data are summarized in Table 1 ⇓ .

Previously described silent polymorphisms were identified in breast cancer cell lines MDA-MB-134VI, MDA-MB-175, MDA-MB-330, MDA-MB-361, SK-BR-5, SUM102, SUM229, SUM1315, and UACC812 at codon 692, exon 13 (GCC→GCT; frequency 0.25) and in cell lines MDA-MB-468, OCUB-F, and UACC812 at codon 751, exon 14 (AAC→AAT; frequency 0.09). Cell line BT20 had a silent polymorphism at codon 451, exon 10 (ATT→ATC; frequency 0.03), and SUM44PE at codon 115, exon 3 (ACG→ACA; frequency 0.03).

E-cadherin Expression Analysis.

E-cadherin protein expression was analyzed by immunohistochemistry using antibody TL#36 directed against a COOH-terminal epitope (residues 735–883) of E-cadherin. E-cadherin was found to be expressed mainly at the cell membrane in cell lines BT20, DU4475, MCF-7, MDA-MB-361, MDA-MB-468, and T47D, that had a wild-type E-cadherin gene sequence (Fig. 2 ⇓ and Table 1 ⇓ ).

E-cadherin mutant cell lines CAMA-1, EVSA-T, and MPE600, expressed the protein at the cell membrane and weak diffuse in the cytoplasm (Fig. 2 ⇓ and Table 1 ⇓ ). Protein expression was in concordance with sequence analysis of E-cadherin transcripts, which revealed in-frame deletions in all three cell lines. CAMA-1 had deleted exon 11 and the first base of exon 12, where the mutated base in the splice site apparently formed a new acceptor site with the first base of exon 12. EVSA-T had deleted the last 42 bases of exon 5, where the first two bases of this deletion apparently served as a new donor splice site. Cell line MPE600 had skipped exon 9.

The COOH-terminal antibody TL#36 did not detect E-cadherin protein expression in the E-cadherin mutant cell lines MDA-MB-134VI, OCUB-F, SK-BR-3, SK-BR-5, and SUM44PE (Fig. 2 ⇓ and Table 1 ⇓ ). Truncations of the E-cadherin protein in these mutant cell lines were confirmed by shifts in the reading frame of E-cadherin transcripts from OCUB-F and SUM44PE. OCUB-F had deleted exon 2, resulting in a stop codon immediately after the deletion, and the 423delT alteration in SUM44PE predicted the addition of seven new amino acids and then a stop codon.

Cell lines BT549, Hs578T, MDA-MB-231, and MDA-MB-435S did not have detectable E-cadherin protein expression (Fig. 2 ⇓ and Table 1 ⇓ ). The latter three cell lines had been reported to have silenced E-cadherin gene expression through methylation of CpG islands in the promotor region (69) . Methylation-associated silencing in BT549 was inferred from its fibroblast-like growth pattern, which was typical for all cell lines with silenced E-cadherin genes. 5

β-Catenin protein expression patterns were similar to those of E-cadherin in most cell lines; that is, the three E-cadherin mutants with in-frame deletions and the E-cadherin wild-type cell lines expressed both proteins in a comparable pattern, whereas the five mutants with truncated E-cadherin proteins did not have detectable expression of either protein (Fig. 2 ⇓ and Table 1 ⇓ ). The four cell lines that had methylation-associated silencing of E-cadherin seemed to have normal β-catenin expression as judged by immunofluorescence microscopy. Furthermore, cell line DU4475 hadβ -catenin protein expression at the cell membrane in all cells and nuclear β-catenin expression in about one-half of the cells. Reactions with isotype-matched control antibodies were negative for all cell lines. Representative examples of the immunohistochemical analysis are shown in Fig. 2 ⇓ .

Tcf-β-catenin Reporter Gene Assays.

Transcriptional activation mediated by Tcf-β-catenin protein complexes was determined in 15 breast cancer cell lines, including 6 cell lines that had genetic alterations of E-cadherin and 4 cell lines that had methylation-associated silencing of the gene (Fig. 3 ⇓ and Table 1 ⇓ ). Cells were transiently transfected with either the pTOPGLOW or pFOPGLOW reporter constructs, which contained multimerized wild-type or mutant Tcf-binding motifs upstream of the Firefly Luciferase cDNA driven by a minimal E1b TATA box together with the pRL-TK internal control reporter construct that contains the Renilla Luciferase cDNA driven by the HSV-TK promotor. Tcf-mediated gene transcription was defined by the ratio of pTOPGLOW:pFOPGLOW luciferase activity after 24 h, each corrected for luciferase activities of the pRL-TK reporter, where no transcriptional activation equals 1.

Cell line DU4475, which had a wild-type E-cadherin gene sequence, showed a 300-fold increase in transcriptional activity of the pTOPGLOW reporter as compared with the negative control pFOPGLOW. None of the other 14 breast cancer cell lines had enhanced transcription of the TOPGLOW reporter (Table 1 ⇓ and Fig. 3A ⇓ ). As a control, the APC mutant colorectal cancer cell line SW480 had a 600-fold enhanced transcriptional activity of pTOPGLOW as compared with pFOPGLOW (27) . All reporter gene assays were performed at least twice in duplicate transfections.

Possible aberrations of APC proteins in cell line DU4475 were addressed by Western blot analysis. Expression of truncated APC proteins (M_r150,000–200,000) but not wild-type APC was detected with monoclonal antibody FE9, which is directed against an NH₂-terminal epitope of APC (Fig. 3B) ⇓ .

DISCUSSION

E-cadherin Alterations in Breast Cancer.

Mutational analysis of the E-cadherin gene in 26 human breast cancer cell lines revealed genetic alterations in 8 cell lines. Although four of these mutant cell lines had been described previously (70) , our characterization revealed some novel aspects. Whereas the E-cadherin gene sequence of cell line CAMA-1 was reported to be wild-type (70) , we identified a point-mutation in the splice acceptor site of exon 12 (Fig. 1B) ⇓ . Our analysis of E-cadherin transcripts in CAMA-1, as well as in MPE600, revealed in-frame deletions in both cell lines. Immunohistochemistry using an antibody directed against a COOH-terminal epitope of E-cadherin revealed abundant expression of the mutant proteins (Fig. 2) ⇓ , excluding the amplification of aberrant transcripts. The E9 antibody that was used by Hiraguri et al. (70) may have failed to detect protein expression in CAMA-1 and MPE600 because it is directed against an extracellular epitope of E-cadherin that might have been affected in the mutant cell lines.

Three of 8 E-cadherin mutant breast cancer cell lines had in-frame deletions of gene sequences, whereas mutational analyses of primary breast carcinomas (54 , 71) had identified in-frame deletions in only 2 of 27 mutant tumors. This discrepancy is not readily explained by differences in experimental procedures (PCR versus SSCP) but possibly reflects biases in either the cell line collection or in the sampling of primary tumor specimens. In this respect, it should be noted that in-frame deletions were the predominant E-cadherin gene alterations identified in primary gastric carcinomas (51 , 52) .

Alterations in the E-cadherin gene had been identified in nearly one-half of lobular breast carcinomas (54 , 71) , a histological subtype that represents 10–20% of primary breast carcinomas. The relatively high percentage of E-cadherin mutants in our cell line collection (8 of 26; 31%) may be attributable to the homozygous deletion of gene sequences in one-half of the mutant cell lines. Homozygous deletions would not, however, be detected in primary tumor specimens because of the inevitable presence of non-neoplastic cells in these samples. Four breast cancer cell lines had no detectable E-cadherin protein expression because of methylation-associated transcriptional silencing. Together with the 8 genetic E-cadherin mutants, 12 of 26 (46%) breast cancer cell lines had aberrant E-cadherin protein expression, a frequency that is in concordance with ample immunohistochemical analyses of primary breast carcinomas (50) .

Interestingly, of four E-cadherin mutant breast cancer cell lines with known histological subtype, only one was of lobular origin and three were of ductal origin (Table 1) ⇓ . Also, all four cell lines with silenced E-cadherin genes were of ductal origin. This is in contrast to genetic analyses on primary breast carcinomas, where mutant E-cadherin genes were identified exclusively in cancers of lobular histology (54 , 71) . The reason for this discrepancy is presently unclear, but it may again reflect biases in these tumor collections.

The Wnt Pathway in Breast Cancer.

Int-1/Wnt-1 was originally identified as an oncogene that had been activated in murine breast carcinomas through mouse mammary tumor virus integrations (72 , 73) . Furthermore, Tcf4, the Tcf/Lef family member that is involved in the inappropriate Wnt pathway activation in colorectal cancer, is specifically expressed in epithelia of the intestine and mammary gland (74) . It was, therefore, perhaps somewhat surprising that we detected Tcf-mediated transcriptional activation in only 1 of 15 breast cancer cell lines. This cell line, DU4475, was found to express truncated APC proteins and had nuclear β-catenin protein expression, both consistent with Wnt pathway activation. These results were confirmed by a recently reported mutational analysis of Wnt pathway members in 24 breast cancer cell lines, where DU4475 was also identified as the only cell line with a mutation in the APC gene (E1577stop; Ref. 75 ). The low mutation frequency of APC in breast cancer cell lines is similar to that reported for primary breast carcinoma specimens (2 of 31; 6%; Ref. 76 ). Our results thus confirm that inactivation of APC is rare in breast cancer, but they also indicate that inappropriate activation of the Wnt pathway through mutation of other members of the signaling cascade is an uncommon event in breast carcinogenesis. Because activation of the Wnt pathway is a prerequisite for evasion of tumor suppressive mechanisms in the intestine, the biology of breast cancers appears, in this respect, to be quite distinct from that of colorectal cancers.

E-cadherin and the Wnt pathway.

None of six genetically mutant E-cadherin breast cancer cell lines, and four cell lines with transcriptionally silenced E-cadherin genes exhibited Tcf-mediated transcriptional activation. These results indicate that mutant E-cadherin tumor suppressor proteins do not constitutively activate the Wnt pathway, and thus do not resemble mutant APC, β-catenin, or axin proteins in colorectal and hepatocellular cancer (27 , 28 , 32 , 33) . Similar results were recently reported for human breast cancer cell lines that had lost E-cadherin protein expression (77) . Direct evidence that E-cadherin is not involved in the Wnt pathway, however, had not been provided because the absence of E-cadherin expression had not been substantiated by mutations in the E-cadherin gene. Notably, E-cadherin expression in these cell lines might have been silenced in association with methylation of CpG islands in the E-cadherin promotor region. This epigenetic mechanism of inactivation is not yet fully understood, but it has been shown to be heterogeneous and changing dynamically in human breast cancers (78) and therefore disputable would lead to constitutive activation of Wnt signaling in the cancer cells. The observed Tcf-β-catenin-signaling activity in E-cadherin-null embryonic stem cells (53) seems in contrast with these and our results and may reflect an inherently reduced efficiency of embryonic stem cells to degrade free β-catenin proteins. Sanson et al. (79) elegantly demonstrated that the function of E-cadherin in cell adhesion does not affect Wnt signaling in Drosophila. It is, however, entirely possible that the E-cadherin function that is abrogated through E-cadherin gene mutations in human cancers is distinct from its function in cell adhesion, and E-cadherin could thus still be involved in the Wnt pathway.

Here, we conclusively excluded E-cadherin as a participant of the Wnt pathway through extensive analysis of natural E-cadherin mutants with in-frame deletions, truncating mutations, or methylation-associated silenced E-cadherin genes. The five mutant breast cancer cell lines with truncated E-cadherin proteins would be expected to be incapable of sequestering β-catenin at the cell membrane, because these mutant proteins have deleted the COOH-terminal β-catenin interaction site. Indeed, none of these five truncation mutants expressed β-catenin at the cell membrane, but, surprisingly, neither did they have detectable β-catenin expression elsewhere in the cell (Fig. 2) ⇓ . Normal activity of the APC destruction complex is likely to prevent any significant increase in levels of freeβ -catenin in these E-cadherin truncation mutants. Release ofβ -catenin from the cell membrane is thus in itself not sufficient to lead to transcriptionally active Tcf-β-catenin complexes in the cell nucleus. The apparently normal expression of β-catenin proteins in the three breast cancer cell lines with in-frame deletions of E-cadherin reinforces that abolishment of β-catenin sequestering does not bear functional significance in the E-cadherin tumor suppressive pathway.