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Constitutive Transcriptional Activation by a β-Catenin-Tcf Complex in APC−/− Colon Carcinoma

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Science  21 Mar 1997:
Vol. 275, Issue 5307, pp. 1784-1787
DOI: 10.1126/science.275.5307.1784

Abstract

The adenomatous polyposis coli (APC) tumor suppressor protein binds to β-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with β-catenin. Nuclei of APC−/− colon carcinoma cells were found to contain a stable β-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed β-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

The product of the APC tumor suppressor gene has been observed to interact with β-catenin and has thus been proposed to regulate cellular signaling events (1). β-Catenin, originally identified on the basis of its association with cadherin adhesion molecules, is now widely recognized as an essential element of the Wingless-Wnt signaling cascade (2). In the absence of Wnt signals, APC simultaneously interacts with the serine kinase glycogen synthase kinase (GSK)-3β and with β-catenin. Phosphorylation of APC by GSK-3β regulates the interaction of APC with β-catenin, which in turn may regulate the signaling function of β-catenin (3). Wnt signaling appears to antagonize GSK-3β activity. Upon Wnt signaling, β-catenin is stabilized and exists primarily as a cytoplasmic monomer (4). Colon carcinoma cells with mutant APC contain large amounts of monomeric, cytoplasmic β-catenin. Reintroduction of wild-type APC removes this cytoplasmic pool and reduces the overall amount of β-catenin (5). Recent evidence indicates that monomeric β-catenin can transduce Wnt signals by associating with T cell factor (Tcf) and lymphoid enhancer factor (Lef) transcription factors (6). We hypothesized that APC may regulate the formation of transcriptionally competent β-catenin-Tcf complexes. If so, loss of APC function would result in uncontrolled transcriptional activation of Tcf target genes, which might contribute to colon tumorigenesis.

There are four known members of the Tcf and Lef family in mammals: the lymphoid-specific factors Tcf-1 and Lef-1 (7, 8) and the less well characterized Tcf-3 and Tcf-4 (9). We performed a qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay for expression of the four Tcf-Lef genes on 43 colon tumor cell lines. Although most colon cell lines expressed more than one of the genes, only hTcf-4 mRNA was expressed in essentially all lines (10).

We then screened a human fetal cDNA library and retrieved clones encoding full-length hTcf-4 (11) (Fig. 1). The predicted sequence of hTcf-4 was most similar to that of hTcf-1. Alternative splicing yielded two COOH-termini that were conserved between hTcf-1 and hTcf-4. The NH2-terminus, which mediates binding to β-catenin in hTcf-1, mouse Lef-1, and Xenopus TCF-3 (6), was also conserved in hTcf-4. Northern (RNA) blot analysis of selected colon carcinoma cell lines (12) revealed extensive expression of hTcf-4 (Fig. 2A). As evidenced by in situ hybridization (Fig. 2, B and C) (13) and Northern blotting (Fig. 2A), hTcf-4 mRNA was readily detectable in normal colonic epithelium, whereas hTcf-1 and hLef-1 were not detectable.

Fig. 1.

Sequence comparison of hTcf-4 and hTcf-1. Two alternative splice forms of hTcf-4 were identified, each encoding a different COOH-terminus. One form (hTcf-4E) was homologous to hTcf-1E (7); the other form (hTcf-4B) was homologous to hTcf-1B. The highly conserved NH2-terminal interaction domain and the HMG-box DNA-binding region are boxed. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. The nucleotide sequence has been deposited in the European Molecular Biology Laboratory database (accession number Y11306). Symbols: ∣, sequence identity;:, sequence similarity.

Fig. 2.

Analysis of hTcf-4 expression. (A) Northern blot analysis of hTcf-4, hTcf-1, and hLef-1 expression in Jurkat T cells (lane 1), colonic mucosa (lane 2), and colon carcinoma cell lines DLD-1 (lane 3), HCT116 (lane 4), SW480 (lane 5), SW620 (lane 6), and HT29 (lane 7). Lane 2 contains 5 μg of total RNA; all other lanes contain 15 μg of total RNA. The positions of 18S and 28S ribosomal RNAs are shown. EtBr, ethidium bromide stain. (B) In situ hybridization of healthy human colon tissue to an hTcf-4 probe. (C) In situ hybridization to a negative control probe (a fragment of the Escherichia coli neomycin resistance gene). Magnifications, ×166.

To investigate whether hTcf-4 functionally interacts with β-catenin, we used two sets of reporter constructs in a β-catenin-Tcf reporter gene assay (7, 14). One set contained three copies of the optimal Tcf motif CCTTTGATC, or three copies of the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFLASH and pFOPFLASH, respectively). The second set contained three copies of the optimal motif, or three copies of the mutant motif, upstream of a minimal herpes virus thymidine kinase promoter driving chloramphenicol acetyltransferase (CAT) expression (pTOPCAT and pFOPCAT, respectively). Epitope-tagged hTcf-4 and a deletion mutant lacking the NH2-terminal 30 amino acids (ΔNhTcf-4) were cloned into the expression vector pCDNA. Transient transfections were performed in a murine B cell line (IIA1.6) that does not express any of the Tcf genes (6).

The pTOPFLASH reporter was strongly transcribed upon cotransfection with the combination of β-catenin and hTcf-4 plasmids, but not with the individual plasmids or with the combination of β-catenin and ΔNhTcf-4 plasmids. No enhanced transcription was detected in cells transfected with the negative control pFOPFLASH (Fig. 3A). These results show that interaction of the NH2-terminus of hTcf-4 with β-catenin results in transcriptional activation.

Fig. 3.

Transactivational properties of β-catenin-hTcf-4. All reporter assays were performed as duplicate transfections. For each condition, both values are shown. (A) Reporter gene assays in IIA1.6 B cells. Cells were transfected by electroporation with 1 μg of luciferase reporter plasmid, 5 μg of β-catenin expression plasmid, and 3 μg of hTcf-4 expression plasmids. Empty pCDNA was added to a total of 10 μg of plasmid DNA. (B) Reporter gene assays in SW480 colon carcinoma cells. Cells were transfected with 0.3 μg of the indicated luciferase reporter gene, 0.7 μg of pCATCONTROL as internal control, the indicated amounts of pCMVNeo-APC, and empty pCDNA to a total of 2.5 μg of plasmid DNA. Control CAT values (pCATCONTROL) are given in the right panel.

In three APC−/− carcinoma cell lines, SW480, SW620, and DLD-1 (15), the transcriptional activity of the pTOPFLASH reporter was 5 to 20 times that of pFOPFLASH. Cotransfection of SW480 cells with the reporter gene and an APC expression vector abrogated the transcriptional activity in a dose-dependent manner (Fig. 3B). In contrast, APC had no effect on a cotransfected internal control (pCATCONTROL) or on the basal transcription of pFOPFLASH (Fig. 3B). The use of pTOPCAT and pFOPCAT instead of pTOPFLASH and pFOPFLASH led to comparable observations. The constitutive transcriptional activity of Tcf reporter genes in APC−/− colon carcinoma cells was in stark contrast to the inactivity of these genes in noncolonic cell lines, including IIA1.6 B cells (Fig. 3A); the C57MG breast carcinoma cell line; the Jurkat and BW5147 T cell lines; the Daudi and NS1 B cell lines; the K562 erythromyeloid cell line; the HeLa cervical carcinoma line; the HepG2 hepatoma cell line; 3T3, 3T6, and Rat-1 fibroblasts; and the kidney-derived SV40-transformed COS cell line (7, 16).

To investigate whether a functional β-catenin-hTcf-4 complex exists constitutively in APC−/− cells, we used HT29-APC1 colon carcinoma cells (17), in which APC is controlled by a metallothionein promoter. Induction by Zn2+ restores wild-type amounts of APC and leads to apoptosis (17). HT29-Gal cells that carry a Zn2+-inducible LacZ gene were used as a control. The only Tcf family member expressed in HT29 is hTcf-4 (Fig. 2A). In nuclear extracts from uninduced HT29-derived transfectants, we readily detected hTcf-4 by gel retardation (Fig. 4) (18). An additional band of slightly slower mobility was also observed. The addition of a β-catenin antibody resulted in the specific retardation of the latter band, indicating that it represented a β-catenin-hTcf-4 complex (Fig. 4) (17). After Zn2+ induction for 20 hours, the amount of β-catenin-hTcf-4 complex was reduced by five-sixths in HT29-APC1 cells, whereas no marked change was observed in HT29-Gal cells (Fig. 4). The overall amount of cellular β-catenin does not change during this induction period in HT29-APC1 cells (17).

Fig. 4.

Constitutive presence of β-catenin-hTcf-4 complexes in APC−/− cells. Gel retardation assays were performed on nuclear extracts from the indicated cell lines before and after a 20-hour exposure to Zn2+. Samples in lanes 1, 4, 7, and 10 were incubated under standard conditions. Anti-β-catenin (0.25 μg) was added to the samples in lanes 2, 5, 8, and 11. A control antibody (human CD4, 0.25 μg) was added to the samples in lanes 3, 6, 9, and 12. NS, nonspecific band also observed with mutant (nonbinding) probe (lane Mt).

On the basis of these data, we propose the following model. In normal colonic epithelium, hTcf-4 is the only expressed member of the Tcf family. The interaction of β-catenin with hTcf-4 is regulated by APC. When appropriate extracellular signals are delivered to an epithelial cell, β-catenin accumulates in a form that is not complexed with GSK-3β-APC and that enables its nuclear transport and association with hTcf-4. The high mobility group (HMG) domain of hTcf-4 binds in a sequence-specific fashion to the regulatory sequences of specific target genes; β-catenin supplies a transactivation domain. Thus, transcriptional activation of target genes occurs only when hTcf-4 is associated with β-catenin. The hTcf-4 target genes remain to be identified. However, the link with APC and β-catenin suggests that these genes may participate in the generation and turnover of epithelial cells. Upon the loss of wild-type APC, monomeric β-catenin accumulates in the absence of extracellular stimuli, leading to uncontrolled transcription of the hTcf-4 target genes. The apparent de novo expression of other members of the Tcf family in some colon carcinoma cell lines might lead to a further deregulation of Tcf target gene expression by the same mechanism. The control of β-catenin-Tcf signaling is likely to be an important part of the gatekeeper function of APC (19), and its disruption may be an early step in malignant transformation.

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