Background
Lymphoid enhancer factor 1 (LEF-1) is a member of the high mobility group box family that has important roles in organogenesis and colon cancer progression [
1,
2]. LEF-1 has no transcriptional activation potential by itself, but it can act as an architectural transcription factor in the assembly of multiprotein enhancer complexes, together with other lymphoid-specific proteins. For example, LEF-1, combined with ALY and other enhancer binding proteins, regulates transcription in the T cell receptor alpha enhancer [
3,
4]. In addition, LEF-1/TCF proteins have been shown to interact with β-catenin and then transfer it into the cell nucleus for transducing Wnt signals to regulate the downstream genes [
5,
6].
LEF-1 belongs to the multiple promoter gene family, which is usually aberrantly and differentially active in disease. Multiple promoter genes are often characterized by the furthest 5- promoter producing a full-length polypeptide with activities that differ significantly from those encoded by a second downstream promoter that is located inside the gene [
7]. The full-length LEF-1 (LEF-1-FL) isoform, containing the β-catenin binding domain, can promote cell proliferation and inhibit differentiation through Wnt signaling [
8]. This LEF-1 function may be abolished by the expression of a truncated LEF-1 protein that lacks the β-catenin binding domain. This shorter LEF-1 transcript (LEF-1-ΔL) is thought to be an inhibitory isoform [
9,
10], owing to its ability to interact with cofactors that also bind LEF-1-FL, as well as compete for DNA binding sites, and thus has been termed “growth suppressing”.
Both LEF-1 isoforms, however, can stimulate cell differentiation and regulate cellular bio-functions [
11,
12]. The balance between the full and truncated forms may play an important role in cell development. Interestingly, in many colon cancer cell lines, only the LEF-1-FL protein is expressed strongly, and the expression of the short isoform is inhibited significantly [
9,
13]. Therefore, it is essential to understand the molecular functions of these two LEF-1 isoforms and identify whether LEF-1 could be a new therapeutic target in colon cancer.
To determine whether the LEF-1 gene is involved in the development of colon cancers, we searched the expression state of the LEF-1 gene in colon carcinomas and cell lines. Full-length LEF-1 is almost always expressed in colon cancers in situ, and is displaced gradually by the short isoform in the tissues farther from the carcinomas. To further explore the role of LEF-1 in colon carcinogenesis, we expressed the LEF-1 genes stably into the colon cell lines SW480 and HT29. We demonstrated that overexpression of LEF-1-ΔL significantly inhibited the growth of SW480 and HT29 cells and increased their apoptosis in vitro. In vivo, the colon cells expressing LEF-1-ΔL slowed the growth of colon tumors, and neo-angiogenesis was decreased notably. In brief, we present evidence that LEF-1 might play an important role in colon carcinogenesis by acting as a regulator. Overexpression of LEF-1-ΔL might be a promising candidate for treatment of colon cancer by sensitizing it to chemotherapeutic drugs.
Methods
Patient samples
Tumor samples and adjacent normal tissues were acquired during surgery from 22 untreated cancer patients. The study was approved by the Ethical Committee of the Medical Faculty of Medicine of Xi'an Jiaotong University. Informed consent was obtained from all subjects. Every patient sample was divided into three parts, namely tumor tissues, adjacent tissues (the distance from tumors was greater than 2 cm but less than 5 cm) and normal tissues. All samples were immediately frozen by liquid nitrogen and stored at -80°C before protein extraction.
Plasmids
To construct pCDNA3.1-His-LEF-1-ΔL and pCDNA3.1-His-LEF-1-FL, the short and long transcripts of LEF-1 were cloned by PCR from a human lymph node cDNA library. After sequencing, the correct clones were cut from pMD-18 T by HindIII and BamH I, and then inserted into pCDNA3.1/V5-His (Invitrogen), to generate target plasmids. LEF-1 variants and the tag did not fuse together and were expressed independently.
The primers used for cloning were as follows:
LEF-1-ΔL F: 5’- GGAAAGCATCCAGATGGAGGC-3’;
LEF-1-ΔL R: 5’- AATGAGCTTCGTTTTCCACCATG -3’;
LEF-1-FL F: 5’- CACAGCGGAGCGGAGATTACA -3’;
LEF-1-FL R: 5’- AATGAGCTTCGTTTTCCACCATG -3’;
Cell culture and transfection
The human colon cell lines SW480 and HT29 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mML glutamine (GIBCO/BRL). SW480 and HT29 cells were stably transfected using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Stable cell lines, including SW480-pCDNA3.1/V5-His, SW480-LEF-ΔL, HT29-pCDNA3.1/V5- His, HT29- LEF-1-ΔL and HT29-LEF-1-FL, were selected in the presence of 800 μg/ml G-418 (MERCK, Germany) for SW480 cells and 600 μg/ml for HT29 cells, and were maintained in RPMI1640 containing 10% FBS and 400 μg/ml of G-418.
MTT assay
Cells of the co-culture were collected on days 1-5 and were then seeded in 96-well plates (4 × 103 cells per well) with 100 μl of the medium. An equal volume of fresh medium containing 20% MTT (5 mg/ml) was added. Cells were incubated further at 37°C for 4 h, followed by the addition of 150 μl of dimethyl sulfoxide (Sigma) to each well and mixing by shaking at room temperature for 10 min. The absorbance was measured at 490 nm. Each experiment was repeated at least three times, and the data were analyzed with the Student’s t-test, with P < 0.05 being considered statistically significant.
Cell cycle analysis
Cells (1 × 106) were collected and washed with PBS, then fixed by incubating in 75% alcohol for 30 min at room temperature. After washing with cold PBS three times, cell were resuspended in 1 ml of PBS containing 40 μg propidium iodide (PI, Sigma) and 100 μg RNase A (Sigma) and incubated at 37°C for 30 min. Samples were analyzed for DNA contents, using a FACScalibur™ (BD Immunocytometry Systems, San Jose, CA). Each experiment was repeated at least three times.
Apoptosis
Apoptotic cells were detected using the AnnexinV-FITC Apoptosis Detection KIT I (Pharmingen, San Diego, CA), according to the manufacturer’s instructions.
Caspase activity assays
Caspase-3 fluorogenic substrate, Ac-DEVD-AFC, came from BD Biosciences (San Jose, CA, USA). Caspase activity in cell lysates was determined according to the manufacturer’s instructions, using an Aminco-Bow-man series-2 spectrofluorometer (440/500-nm excitation/emission), and expressed as a fold increase of caspase-3 over the control.
The plate colony-forming assay process mainly refers to the work of Franken NA et al. [
14]. Briefly, cells were plated in 35-mm plates at a density of 500 cells per well in the complete medium. Cells were cultured at 37°C in 5% CO
2 for 14 days. After fixation using methanol for 10 min, the cells were stained with Giemsa stain for 15 min and colonies (with more than 50 cells) were photographed and counted by Image Pro Plus 6.0 software. Each experiment was repeated at least three times, and data were analyzed with one-way ANOVA analysis and LSD-
t test.
Migration assay
Chemotaxis experiments were performed in polycarbonate transwell inserts (5 μm pore diameter, Corning Costar Corp.). Soluble SDF-1 (Peprotech) was added in the lower chamber at a concentration of 100 ng/ml. Cells (2 × 105) were seeded in the upper compartment and were cultured at 37°C for 18 h. Migrated cells in the lower chamber were photographed and counted under a microscope.
ECM adhesion assay
Wells of a 96-well plate with l0 g/L BSA and 5 mg/L matrigel at 4°C overnight. Some wells were left uncoated as negative controls. The coated plates were incubated at 37°C in a CO2 incubator for 45-60 min on the next day. Cells were counted and diluted to 4 × 105/ml and 50 μl of the cell suspension was added in each well. After incubation in a CO2 incubator at 37°C for 1 h, non-adherent cells were removed by washing with PBS. The number of adherent cells was counted with the MTT assay.
Western blotting
Whole-cell extracts were prepared by lysing cells with the RIPA buffer (50 mM Tris–HCl, pH7.9, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40, and 0.1 mM PMSF). Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and were electroblotted onto polyvinylidene difluoride membranes. Membranes were probed using mouse-anti-human LEF-1 (1 C3.1D10, Chemicon International, Temecula, CA), monoclonal anti-β-actin (AC-74, Sigma) at appropriate dilutions, followed by incubation with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG antibody (Sigma). In some experiments, mouse-anti-His tag (R94025, Invitrogen) was used. Blots were developed using an enhanced chemiluminescence system (Roche, Basel, Switzerland).
The tumor formation method was based on Hu et al. [
15]. In detail, cells (5 × 10
6) were injected subcutaneously into nude mice. Eighteen days after the initial inoculation, tumor growth was monitored every 3 days by measuring the tumor length (L) and width (W) with a sliding caliper. Tumor size was calculated as L × W
2 × 0.51. Thirty days after the initial inoculation, tumors were excised and weighed. All animal experiments were approved by and performed in accordance with guidelines from the Animal Experiment Administration Committee of the Medicine of Xi'an Jiaotong University, to comply with international humanitarian standards.
Histology and immunohistochemistry
Tissues were fixed in 4% paraformaldehyde, embedded in OCT, sectioned in 10 μm thicknesses, and stained with hematoxylin and eosin by standard methods. Immunohistochemistry was performed by standard procedures, with rat antimouse CD31 (1:200 dilution; Chemicon International) or rabbit antimouse HIF1α antibody (1:200 dilution; Chemicon International) as the primary antibody. Secondary antibodies included horseradish peroxidase–conjugated goat antirat IgG or antirabbit IgG (Boster BioTec, Wuhan, China). Samples were developed using a standard DAB reagent and were observed under a microscope. Microvessels were counted by different technicians to evaluate density, based on “hot fields,” which showed the most concentrated vessel regions. For quantification, pictures were captured and then pixels were counted by Image-Pro Plus 6.0 software (MediaCybernetics Inc., Bethesda, MD).
Statistics
Images were imported into Image Pro Plus 6.0 software, and pixels for each color were analyzed to quantitatively represent positively stained cells. Statistical analysis was performed with the SPSS 12.0 program. Results are expressed as mean ± SD. Comparisons between groups were undertaken using an unpaired Student's t-test. P < 0.05 was considered statistically significant.
Discussion
LEF-1 is one of the DNA-binding transcription factors that functions in the Wnt signaling pathway, and it acts by recruiting β-catenin to Wnt target genes for regulation. The first exon of the LEF-1 gene encodes the domain that is necessary and sufficient to bind to β-catenin, and a second promoter for transcription is located downstream of this exon. The intronic promoter produces a shorter isoform without the β-catenin binding domain, but retains the DNA binding domain8. LEF-1 interacts with transcription co-repressors through a domain in the central portion of the protein. Therefore, shorter polypeptides should function as constitutive transcription repressors or competitive inhibitors of Wnt signaling by binding to Wnt response elements in target genes, thereby disallowing β-catenin access and constitutively inhibiting transcription by recruiting a repressor. Since Wnt signaling directs many important processes during development, and constitutive Wnt signaling is tightly linked to the formation of cancers, the relative expression patterns of activating and repressing LEF isoforms could be important for the Wnt signal throughput to target genes in both normal and abnormal settings.
To understand LEF-1 function in colon cancer, we employed two colon cell lines, SW480 and HT29, to construct stable cell lines expressing each of two LEF-1 isoforms. The full-length form of LEF-1, TCF1 and TCF4 can be detected in the SW480 cell line. But both forms of LEF-1 could not be found in the HT29 cell line, and the only TCF family member expressed in HT29 was TCF4. Okamura et al. [
19] and Reya et al. [
16] reported that LEF-1 was involved in the regulation of T cell proliferation and apoptosis in pro-B cells. Our data also showed that modulation of LEF-1 resulted in significant changes in colon cell proliferation. As we had hypothesized, LEF-1-ΔL significantly inhibited the proliferation of SW480 and HT29
in vitro by blocking the cell cycles at the G
0/1 phase. Consistent with this, the expression of cyclin D1 and c-Myc, two down-stream target genes of Wnt signaling that can induce S phase entry in many cancer cell lines [
20,
21], was also decreased under the regulation of the short form of LEF-1 (data not shown). Overexpression of LEF-1-ΔL also induced up-regulation of the Rb protein and down-regulation of cyclin E and D3 (data not shown). This is unusual, because it has been generally accepted that the activity of Rb protein is regulated by phosphorylation catalyzed by cyclin-dependent kinase complexes. Up-regulation of the Rb protein level might neutralize the effect of increased cyclin E and D3 in cell cycle progression, which led to the inhibition of cell proliferation in colon cells.
To our surprise, no increments in the sub G0/G1 population were detected for both SW480-ΔL and HT29-ΔL cells. This would be expected based on the results shown in Figure
3 where an increase in annexin-V positive cells was observed for both these cell lines. We think that there are two main reasons, as follows: (1) As is known, annexin-V, belonging to the protein family of annexins, with anticoagulant properties, has been shown to be a useful tool in detecting early apoptotic cells since it preferentially binds to negatively charged phospholipids such as PS in the presence of Ca2
+ and shows minimal binding to phosphatidylcholine and sphingomyelin. Changes in PS asymmetry, which are analyzed by measuring annexin-V binding to the cell membrane, were detected before morphological changes associated with apoptosis occurred and before membrane integrity was lost. Apoptotic cells become annexin-V positive after nuclear condensation starts, but before the cell becomes permeable to PI. Therefore, as tools of detection for apoptosis, annexin-V staining is more sensitive than DNA flow cytometric analysis. In our results, the cells we detected may be in a stage of early apoptosis, which could be detected by annexin-V staining but not by cell cycle analysis. (2) SW480 and HT29 cells are adherent and should be disaggregated to have a single cell suspension before annexin-V staining. Any procedure that affects the integrity of the plasma membrane will result in cell positivity for annexin-V. The binding of annexin-V to phosphatidylserine may be affected in adherent cells, which are usually detached from plastic dishes by enzymatic treatment, with their membrane integrity unaltered. Therefore, the apoptosis-positive rate of HT29-dL cells detected by annexin-V staining would be higher than that detected by cell cycle analysis.
We further found that LEF-1 could influence the expression of CXCR4 in SW480 and HT29. It has been demonstrated that SDF-1/CXCR4 signaling, one of the most important chemokine receptor–ligand complexes, was considered to play multiple roles in cell migration, proliferation, chemotactic responses, adhesion, secretion of MMPs and angiopoietic factors in the development of many systems and tumor cells through some possible pathways [
22‐
26]. Luo et al. [
27] and others [
28‐
31] have reported that SDF-1/CXCR4 signaling might interact with the Wnt/β-catenin/LEF-1 pathway to regulate the development of the central nervous system. Wang et al. [
32] showed that the abrogation of CXCR4 could influence the pancreatic cancer cell phenotype, including cell proliferation, colony formation and cell invasion. Wnt target genes were also inhibited in CXCR4-knockdown cells. Samara et al. [
33] found that the binding of CXCR4 to CXCL12 lead to an increase in head and neck squamous cell carcinoma (HNSCC) cell adhesion and MMP-9 secretion, suggesting that CXCR4 may be a novel regulator of metastatic processes in HNSCC. Interestingly, our results indicated that inactivation of Wnt signaling via overexpression of the short form of LEF-1 also down-regulated the expression of CXCR4 in colon cancer cell lines, but CXCR4 expression was enhanced by the full form of LEF-1 in HT29. The changes of CXCR4 in colon cell lines might trigger a series of alterations in cell behavior and biological functions, such as migration, adhesion and microvessel formation. According to our data, the regulation of the CXCR4 protein expression might be a new mechanism in regulating colon cell function by the Wnt pathway.
We further analyzed whether reduced tumor vascularization observed in cells expressing the LEF-1-ΔL variant also occurred in tumors of the same size. As shown in Figure
7, tumor vascularization of HT29-LEF-1-ΔL was reduced slightly compared with the controls, but no statistical differences were obtained. According to our results, colon cell lines expressing LEF-1-ΔL grow much slower than those expressing the full-length both
in vitro and
in vivo. As is known, rapid proliferation of tumor cells can cause hypoxia of the internal tumor tissues, which can induce the formation of new blood vessels through high level cytokines such as VEGF. Therefore, the significant difference in microvessels between colon tumors formed by different LEF-1 cell lines may be a result of the difference in the ability for cell proliferation. In the early stage of tumor formation, hypoxia caused by both tumor cells was at a low level, which could not induce statistical differences in the observed microvessels. Therefore, the reduction in tumor vascularization of HT29-LEF-1-ΔL formed gradually during the process of tumor growth, rather than in the initial stages of tumor formation. Though there are many details of tumor formation in colon cell lines with LEF-1 variants to be further understood, the proliferating capability of these cells may be one of the most important inherent initiating agents that can influence tumor growth.
Competing interests
The author(s) declare that they have no competing interests.
Authors’ contributions
S-H W. and Y-C W. performed most of the experiments including cell culture and prepared the manuscript, examination of cell bio-function and FACS analyses. W-J W. and T T. helped in the construction of plasmids. K-J N. supervised the study. All authors read and approved the final manuscript.