The activation of lymphoid enhancer factor (LEF)/T-cell factor (TCF)-mediated transcription by sustained expression of β-catenin and the loss of transforming growth factor β (TGF-β) signaling are essential steps in carcinogenesis, particularly for cancers of the colon, breast, and liver. The oncogene c-myc is a common target of both of these signaling pathways and a key regulator of cell cycle progression. Here we have identified a novel LEF/TCF-responsive element in the promoter of the human c-myc gene. β-Catenin activated the transcriptional activity of the c-myc promoter by binding to this element in various cell lines. When TCF-4 was bound to this element, TGF-β dissociated β-catenin and repressed the transcriptional activity of the c-myc promoter. However, TGF-β could not dissociate β-catenin and could not repress c-myc transcription when LEF-1 was bound to the element instead of TCF-4. These findings suggest that enhanced expression of LEF-1, which occurs frequently in colon cancer, may make cells refractory to the down-regulation of c-myc and the subsequent growth arrest induced by TGF-β.

TGF-β3 inhibits the growth of divergent types of cell, and a loss of response to the growth-inhibitory activity of TGF-β has been implicated in carcinogenesis (1, 2, 3). Some types of cancer cell acquire resistance to TGF-β through inactivation of the genes encoding TGF-β receptors and Smads (1, 2, 3). However, many cancer cells lose the growth-inhibitory response to TGF-β in the presence of intact TGF-β receptors and Smads. Among 465 samples of breast cancer cells examined in vivo, 30 (6.6%) lacked phosphorylation of Smad2, suggesting the loss of TGF-β receptor activity; 9 (2%) lacked expression of Smad4, but the rest (>90%) retained normal TGF-β/Smad signaling (4). By contrast, essentially all breast cancer cell lines are resistant to the growth-inhibitory activity of TGF-β. Therefore, it is of great interest to determine how cancer cells lose responsiveness to TGF-β selectively in growth-inhibitory pathways in the presence of normal expression of TGF-β receptors and Smads.

Several oncogene products, such as SV40 large T antigen, adenovirus E1A, Evi-1, activated Ras, and c-Myc have been shown to make cells resistant to growth inhibition by TGF-β. Among these, T antigen (5), E1A (6), Evi-1 (7), Tax (8), c-Ski (9), and activated Ras (10, 11) block Smad function in general either by directly binding to Smads in nuclei (6, 7, 8, 9) or by blocking the translocation of Smads to the nucleus (10) and stabilizing the Smad-binding corepressor, TGIF (11). By contrast, c-myc is a target of the TGF-β/Smad signaling pathway, and TGF-β cannot down-regulate the transcription of c-myc in many cancer cells (12, 13, 14). Stable expression of c-Myc blocks the induction of cyclin-dependent kinase inhibitors and growth inhibition by TGF-β but does not generally affect Smad functions (15). Therefore, it is crucial to determine how the transcription of c-myc becomes resistant to TGF-β to develop an understanding of how cancer cells become refractory to the growth-inhibitory activity of TGF-β. Recently, we and others have identified a TIE/E2F element in the c-myc promoter that is responsive to serum-induced activation and TGF-β-induced inactivation of c-myc transcription. TGF-β/Smad signaling converges on the TIE/E2F element, on which activating signals by serum growth factors function (15, 16).

To assess how cancer cells lose the growth-inhibitory response to TGF-β without affecting the TGF-β/Smad pathway, we have examined the possibility that TGF-β is unable to block transcription of c-myc that is activated by an abnormal carcinogenic signaling. Wnt/β-catenin signaling is frequently activated in colon cancer, hepatocellular carcinoma, and breast cancer (17, 18, 19, 20), and β-catenin activates the c-myc promoter in association with the LEF/TCF family of transcription factors (21). Because TGF-β signaling and/or Smad4 by itself enhance the transcriptional activity of the Xenopus homeobox gene twin (Xtwin) cooperatively with β-catenin and LEF-1 (22, 23), we considered that TGF-β might enhance transcription of c-myc in the presence of Wnt/β-catenin signaling.

In the present study using TCF-4, which is expressed in normal colorectal tissue, TGF-β dissociated β-catenin from TCF-4 and repressed transcriptional activity of the c-myc promoter. However, TGF-β could not dissociate β-catenin from LEF-1, which is expressed frequently in colon cancer and metastatic melanoma (24, 25). As a result, TGF-β could not repress transcription of c-myc in the presence of LEF-1.

Cells.

Clone 4-2 of Mv1Lu R mutant (R4-2) was provided by J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY). COS7, HepG2, and NIH3T3 cells were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and 10 μg/ml gentamicin (Sigma).

DNA Constructs.

A luciferase reporter construct containing −2329/+510 of the human c-myc promoter (pHx-luc) and the pGL3-c-myc promoter (−40/+16)-Lux have been described previously (16). Deletion constructs (−1616/+16 and −1415/+16) of the c-myc promoter were amplified using PCR and subcloned into the SacI/HindIII sites of pGL3-basic (Promega, Madison, WI). Other deletion constructs (−1616/−1416, −1567/−1525, and −1528/−1416 with or without mutations in TCF binding consensus sequence) of the c-myc promoter were also amplified using PCR and subcloned into the SacI/BglII sites of the pGL3-c-myc promoter (−40/+16)-Lux. Luciferase reporters containing three copies of TBE3 or three copies of mTBE3 were constructed by subcloning of (TCTTTGATCAGA) × 3 or (TCTTTGGCCAGA) × 3 into the SacI/BglII sites of the pGL3-c-myc promoter (−40/+16)-Lux. The underlined letters mean substituted nucleotides. To obtain expression constructs for β-catenin, HA-TCF-4, HA-TCF-4 (d1-30; HA-TCF-4 lacking β-catenin binding domain), and HA-LEF-1, each cDNA was amplified by PCR and subcloned into a pCAGGS vector using a XhoI linker. The original cDNAs for β-catenin, TCF-4, and LEF-1 were kindly provided by S. Ishihara (Kyoto University, Kyoto, Japan), B. Vogelstein (Howard Hughes Medical Institute and Johns Hopkins Oncology Center, Baltimore, MD), and K. Jones (The Salk Institute for Biological Studies, La Jolla, CA), respectively. All sequences were confirmed by sequencing. pcdef3-ALK-5(TD)HA and pcDNA3-FLAG-Smad3 have been described previously (26).

DNA Transfection and Luciferase Assay.

Cells were transfected using FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s recommendations. Cells were lysed 24 h after transfection and analyzed by the dual luciferase reporter assay system (Promega) using a luminometer (AutoLumat LB953; EG & G Berthold, Bad Wildbad, Germany). Luciferase activities were normalized to the activity of sea pansy luciferase of cotransfected pRL-TK (Promega).

DNAP.

DNAP was performed as described previously (16). Briefly, cell lysates prepared in 1% NP40, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1.5% aprotinin, and 20 mm Tris-HCl (pH 7.5) were precleared with streptavidin-agarose beads (Sigma), and incubated with 30 nmol of biotinylated double-stranded oligonucleotides in the presence of 10 μg of poly(dI-dC) (Roche Diagnostics) at 4°C for 1 h. DNA-bound proteins were mixed with streptavidin-agarose (Sigma) for 1 h with end-over-end rotation, washed three times with the cell lysis buffer, and analyzed by SDS-PAGE and Western blotting. The sequences of the upper strands of the probe DNA were three repeats of the following sequences: TCTTTGATCAGA for TBE3; and TCTTTGGCCAGA for mTBE3.

Western Blotting.

Cells were solubilized as described above. Aliquots of total cell lysates or precipitates obtained by DNAP were separated by SDS-PAGE. Proteins were electrotransferred to polyvinylidene difluoride membranes (ProBlott; Applied Biosystems, Foster City, CA) and subjected to Western blotting. Anti-β-catenin (Clone 14; Transduction Laboratories, Lexington, KY), anti-HA (3F10; Roche Diagnostics), or anti-FLAG (M2; Sigma) antibodies were used as the primary antibodies for Western blotting. Antibodies that reacted were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ).

EMSA.

EMSA was performed as described previously (27). Briefly, the sticky ends of the SacI/BglII sites flanking the three tandem repeats of TBE3 or mTBE3 were labeled with [α-32P]dATP using Klenow fragments of DNA polymerase. Binding reactions containing 2 × 104 dpm of probe DNA and 3 μg of COS7 lysates were performed for 20 min at 37°C in 18 μl of binding buffer. The protein-DNA complexes were then resolved in 5% polyacrylamide gels at 4°C.

TGF-β Signaling Represses c-myc Transcription Induced by β-Catenin and TCF-4.

β-Catenin up-regulated transcriptional activity of pHx-luc as reported previously (21). Exogenously expressed TCF-4 did not greatly affect this activity, but NH2-terminally truncated TCF-4 (d1-30) blocked β-catenin-induced transcriptional activation, which suggested that β-catenin up-regulated c-myc transcription through binding to endogenous transcription factors of the LEF/TCF family (Fig. 1,A). To assess the effects of TGF-β signaling on the c-myc transcription that was up-regulated by β-catenin and TCF-4, the active form of the TGF-β type I receptor (ALK-5TD) and/or Smad3 were coexpressed with β-catenin and TCF-4. Both ALK-5TD and Smad3 repressed β-catenin-induced transcription of c-myc in a dose-dependent manner, and coexpression of ALK-5TD and Smad3 resulted in further suppression (Fig. 1 B).

Identification of a β-Catenin/TCF-responsive Element in the c-myc Promoter.

To identify the β-catenin/TCF-responsive element(s) in the c-myc promoter, we made several 5′-deletion constructs of the c-myc promoter and found that the DNA fragment between nucleotides −1616 and −1415 contained major responsive element(s) to both β-catenin and TGF-β in R4-2 cells (Fig. 2,A). Because this region was different from the β-catenin/LEF-1-responsive elements reported previously, we used two other cell lines to examine the cell specificity of the significance of this region. In all three cell lines, R4-2, HepG2, and NIH3T3, the responsiveness of the c-myc promoter to both β-catenin and TGF-β was lost when the −1616/−1415 fragment of the c-myc promoter was deleted (Fig. 2, B and C).

To study this responsive element further, we examined three smaller sections of the DNA sequence. Both fragments −1616/−1567 and −1528/−1416 responded to β-catenin (Fig. 3,A). The former fragment did not contain an obvious TCF binding consensus sequence, but the latter had a unique LEF/TCF binding sequence, which we named TBE3 (Fig. 3 B), following the previously given names TBE1 and TBE2.

We focused on the −1528/−1416 DNA fragment and generated sequences with or without two base substitutions in TBE3. The −1528/−1416 fragment was sufficient to respond to β-catenin, and its activity was completely lost by a mutation in TBE3 (Fig. 3,C). DNA fragments consisting of three tandem repeats of TBE3 with or without the mutation shown in Fig. 3,B were then examined for response to β-catenin and TGF-β. TBE3 was found to be sufficient to respond to β-catenin. Moreover, this element maintained a responsiveness to TGF-β signaling in R4-2 (Fig. 3 D), HepG2, and NIH3T3 cells (data not shown), indicating that both β-catenin/TCF and TGF-β signaling converge on TBE3.

TGF-β Cannot Repress c-myc Transcription Induced by β-Catenin and LEF-1.

We examined further the effects of TGF-β signaling on the c-myc transcription induced by β-catenin and LEF-1. This experiment seemed especially interesting because LEF-1 is selectively expressed in most colon cancers. As we expected, TGF-β-signaling could not repress c-myc transcription induced by β-catenin and LEF-1 (Fig. 4,A). Overexpression of LEF-1 reversed the pHx-luc and 3× TBE3-Lux activities repressed by TGF-β signaling in a dose-dependent manner, whereas TCF-4 showed no such activity (Fig. 4 B; data not shown).

Smad3 Dissociates β-Catenin from TCF-4 but Forms a Ternary Complex with LEF-1 and β-Catenin.

We examined the effects of TGF-β signaling on the β-catenin/TCF-4 or β-catenin/LEF-1 complex formed on TBE3. When TCF-4 was binding to TBE3 together with β-catenin, activated Smad3 bound to both TCF-4 and to β-catenin and dissociated β-catenin from TCF-4. By contrast, LEF-1 could bind Smad3 together with β-catenin (Fig. 5,A). The ternary complex of LEF-1, β-catenin, and Smad3 was shown to form on TBE3 in an EMSA analysis (Fig. 5 B).

Myc family proteins promote proliferation, growth, and apoptosis; inhibit terminal differentiation; and, when deregulated, are strongly involved in the genesis of many types of cancer (28). Repression of c-myc is an initial critical event that is induced by TGF-β and precedes cell growth arrest (2). Constitutive expression of c-myc makes cells resistant to TGF-β-induced cell growth arrest (16, 29), and the levels of c-myc are not down-regulated by TGF-β in many cancer cells (12, 13, 14). In cancer cells, c-myc regulation may become refractory to TGF-β through chromosomal abnormalities, such as transpositions or amplifications involving the c-myc gene. Amplification of the c-myc gene has been described in ∼15% of all human tumors (30). In other cancers, expression of functional TGF-β receptors or Smads is lost by mutations or hypermethylation in the genes encoding the TGF-β receptors or Smads (1, 3). Frequently, however, many cancer cells lose responsiveness to TGF-β only in growth regulation and retain other TGF-β transcriptional responses (15). Thus far, no known molecular mechanisms can explain how cancer cells lose response to TGF-β in a growth-regulatory gene-specific manner in the absence of a chromosomal abnormality of c-myc.

Here, we have identified a potential mechanism of c-myc resistance to TGF-β, as summarized in Fig. 6. An exchange of the TCF family transcription factor occupying the LEF/TCF-responsive element in the c-myc promoter from TCF-4 to LEF-1 renders the cells resistant to TGF-β-induced repression of c-myc. LEF-1 is highly expressed in most colon cancers (24). Recently, LEF-1 has been shown to be up-regulated in cancer cells through activated Wnt signaling (31). We have found that TBE3-mediated transcriptional activity was highly active in the absence of exogenous stimuli in some colon cancer cell lines and that endogenous LEF-1, TCF-4, and β-catenin bind to the TBE-3 in these cells.4 It will be interesting to examine whether blocking the expression of LEF-1 can recover the reactivity of colon cancer cells to TGF-β-induced repression of c-myc and thus reestablish cell growth arrest.

In the present study, we have identified a significant functional difference between LEF-1 and TCF-4 in relation to TGF-β signaling. Recently, different LEF/TCF isoforms have been shown to vary in their ability to bind nucleosomal templates (32), but the molecular basis underlying the involvement of LEF-1 in carcinogenesis rather than other LEF/TCF family members has not been revealed thus far. The resistance of LEF-1 to TGF-β signaling, demonstrated in this study, may explain why LEF-1 has a specific role in carcinogenesis.

We have identified a new target element of the β-catenin/LEF/TCF complex in the c-myc promoter, which we named TBE3 (Fig. 3 B). This element seemed to be responsible for the β-catenin/LEF/TCF response, at least in the three cell lines that we tested. However, the reason for the difference of the functional responsive elements with the sites reported by He et al. (21) remains to be solved. To address this issue, additional studies should entail in vivo analysis using chromatin immunoprecipitation or in vivo footprinting.

In summary, in the present study we found that two LEF/TCF family members, TCF-4 and LEF-1, interact with β-catenin to produce a similar transcriptional activity on TBE3 in the c-myc promoter. However, these two transcription factors show different responses to TGF-β signaling. This finding provides new insight into the functional differences of LEF/TCF family members and into the role of up-regulated LEF-1 expression in cancer cells.

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.

1

Supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

3

The abbreviations used are: TGF-β, transforming growth factor β; LEF, lymphoid enhancer factor; TCF, T-cell factor; TBE3, third TCF/LEF binding element; mTBE3, mutant TBE3; DNAP, DNA affinity precipitation; EMSA, electrophoretic mobility shift assay.

4

H. Suzuki and M. Kato, unpublished observations.

Fig. 1.

TGF-β signaling blocks the human c-myc promoter activity that is up-regulated by β-catenin and TCF-4. A, overexpression of β-catenin up-regulates c-myc promoter activity in a TCF-dependent manner. B, a constitutively active form of the TGF-β type I receptor (ALK5TD) and Smad3 repress the c-myc promoter activity induced by β-catenin and TCF-4. A luciferase reporter construct containing the human c-myc promoter (pHx-luc, 0.5 μg/well) was transfected into R4-2 cells, together with expression constructs encoding β-catenin (0.5 μg/well), wild-type TCF-4 (TCF4; 0.5 μg/well), TCF-4 (d1-30; 0.5 μg/well), ALK-5TD (0.03 or 0.1 μg/well), and/or Smad3 (0.2 or 0.6 μg/well), as indicated.

Fig. 1.

TGF-β signaling blocks the human c-myc promoter activity that is up-regulated by β-catenin and TCF-4. A, overexpression of β-catenin up-regulates c-myc promoter activity in a TCF-dependent manner. B, a constitutively active form of the TGF-β type I receptor (ALK5TD) and Smad3 repress the c-myc promoter activity induced by β-catenin and TCF-4. A luciferase reporter construct containing the human c-myc promoter (pHx-luc, 0.5 μg/well) was transfected into R4-2 cells, together with expression constructs encoding β-catenin (0.5 μg/well), wild-type TCF-4 (TCF4; 0.5 μg/well), TCF-4 (d1-30; 0.5 μg/well), ALK-5TD (0.03 or 0.1 μg/well), and/or Smad3 (0.2 or 0.6 μg/well), as indicated.

Close modal
Fig. 2.

A β-catenin- and TGF-β-responsive region is located in the −1616/−1415 fragment of the human c-myc promoter. pHx-luc (−2329/+510), c-myc promoter (−1616/+16)-Lux, c-myc promoter (−1415/+16)-Lux, or c-myc promoter(−40/+16)-Lux was transfected into (A) R4-2, (B) HepG2, or (C) NIH3T3 cells, together with β-catenin (0.5 μg/well), Smad3 (0.2 or 0.6 μg/well), and ALK-5TD (0.1 μg/well), as indicated.

Fig. 2.

A β-catenin- and TGF-β-responsive region is located in the −1616/−1415 fragment of the human c-myc promoter. pHx-luc (−2329/+510), c-myc promoter (−1616/+16)-Lux, c-myc promoter (−1415/+16)-Lux, or c-myc promoter(−40/+16)-Lux was transfected into (A) R4-2, (B) HepG2, or (C) NIH3T3 cells, together with β-catenin (0.5 μg/well), Smad3 (0.2 or 0.6 μg/well), and ALK-5TD (0.1 μg/well), as indicated.

Close modal
Fig. 3.

Identification of the third β-catenin/TCF-responsive element (TBE3) in the human c-myc promoter. A, the c-myc promoter contains at least two β-catenin-responsive elements in the −1616/−1415 region. B, schematic representation of the third consensus LEF/TCF binding element (TBE3) found in the region −1528/−1416 and the mutation introduced in TBE3 (mTBE3). C, 2-base substitutions in TBE3 disable the enhancer activity of the −1528/−1416 DNA fragment in response to β-catenin (0.5 μg/well). D, the 3× TBE3-Lux reporter construct was introduced into R4-2 cells together with β-catenin (0.5 μg/well), Smad3 (0.2 or 0.6 μg/well), and ALK-5TD (0.1 μg/well), as indicated.

Fig. 3.

Identification of the third β-catenin/TCF-responsive element (TBE3) in the human c-myc promoter. A, the c-myc promoter contains at least two β-catenin-responsive elements in the −1616/−1415 region. B, schematic representation of the third consensus LEF/TCF binding element (TBE3) found in the region −1528/−1416 and the mutation introduced in TBE3 (mTBE3). C, 2-base substitutions in TBE3 disable the enhancer activity of the −1528/−1416 DNA fragment in response to β-catenin (0.5 μg/well). D, the 3× TBE3-Lux reporter construct was introduced into R4-2 cells together with β-catenin (0.5 μg/well), Smad3 (0.2 or 0.6 μg/well), and ALK-5TD (0.1 μg/well), as indicated.

Close modal
Fig. 4.

TCF-4 and LEF-1 mediate similar enhancer activity with β-catenin of the c-myc promoter but have completely distinct responses to TGF-β signaling. A, pHx-luc activity was repressed by TGF-β signaling in the presence of TCF-4 but was resistant to TGF-β signaling in the presence of LEF-1. B, overexpression of LEF-1 (0.03, 0.1, 0.3, or 1.0 μg/well) but not TCF-4 (0.03, 0.1, 0.3, or 1.0 μg/well) cancels the TGF-β-induced repression of 3× TBE3-Lux activity in R4-2 cells.

Fig. 4.

TCF-4 and LEF-1 mediate similar enhancer activity with β-catenin of the c-myc promoter but have completely distinct responses to TGF-β signaling. A, pHx-luc activity was repressed by TGF-β signaling in the presence of TCF-4 but was resistant to TGF-β signaling in the presence of LEF-1. B, overexpression of LEF-1 (0.03, 0.1, 0.3, or 1.0 μg/well) but not TCF-4 (0.03, 0.1, 0.3, or 1.0 μg/well) cancels the TGF-β-induced repression of 3× TBE3-Lux activity in R4-2 cells.

Close modal
Fig. 5.

LEF-1 forms a ternary complex with β-catenin and Smad3 on TBE3. A, Smad3 dissociates β-catenin from TCF-4 but does not dissociate β-catenin from LEF-1. Three tandem repeats of TBE3 with a biotin label were mixed with total cell lysates from COS7 cells transfected with β-catenin (0.5 μg/well), HA-TCF-4 (0.5 μg/well), HA-LEF-1 (0.5 μg/well), and/or Smad3 (0.2 or 0.6 μg/well with 0.1 μg/well of ALK-5TD), and DNA-bound proteins were precipitated and subjected to Western blotting, as indicated (DNAP). Total cellular extracts were also subjected to Western blotting to monitor expression levels of the transfected proteins (total). B, EMSA showing that LEF-1 forms a ternary complex with β-catenin and Smad3 on TBE3. Anti-β-catenin, anti-FLAG, and anti-HA antibodies were used to detect β-catenin, FLAG-Smad3, and HA-TCF-4, respectively, in the shifted bands.

Fig. 5.

LEF-1 forms a ternary complex with β-catenin and Smad3 on TBE3. A, Smad3 dissociates β-catenin from TCF-4 but does not dissociate β-catenin from LEF-1. Three tandem repeats of TBE3 with a biotin label were mixed with total cell lysates from COS7 cells transfected with β-catenin (0.5 μg/well), HA-TCF-4 (0.5 μg/well), HA-LEF-1 (0.5 μg/well), and/or Smad3 (0.2 or 0.6 μg/well with 0.1 μg/well of ALK-5TD), and DNA-bound proteins were precipitated and subjected to Western blotting, as indicated (DNAP). Total cellular extracts were also subjected to Western blotting to monitor expression levels of the transfected proteins (total). B, EMSA showing that LEF-1 forms a ternary complex with β-catenin and Smad3 on TBE3. Anti-β-catenin, anti-FLAG, and anti-HA antibodies were used to detect β-catenin, FLAG-Smad3, and HA-TCF-4, respectively, in the shifted bands.

Close modal
Fig. 6.

Model showing how the increased expression of LFF-1 may block the normal repression of c-myc expression that is induced by TGF-β.

Fig. 6.

Model showing how the increased expression of LFF-1 may block the normal repression of c-myc expression that is induced by TGF-β.

Close modal

We thank S. Ishihara, B. Vogelstein, and K. Jones for providing samples of β-catenin, TCF-4, and LEF-1 cDNA, respectively.

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