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Research Article

LEF1 Promotes Colorectal Cancer Progression by Activating Wnt/β-Catenin Signaling and Stemness-Associated Genes

Authors: Xing Liu

Publication Date: 24 March, 2025

DOI: https://doi.org/10.51219/MCCRJ/Xing-Liu/367

Citation: Liu X. LEF1 Promotes Colorectal Cancer Progression by Activating Wnt/β-Catenin Signaling and Stemness-Associated Genes. Medi Clin Case Rep J 2025;3(3):1321-1323.

Copyright:Liu X., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract
Objective
To investigate the role of LEF1 (lymphoid enhancer-binding factor 1, a key transcription factor of Wnt/β-catenin pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on Wnt signaling.

 

Methods

LEF1 expression (total and nuclear) was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. LEF1 was overexpressed via plasmid (pcDNA3.1-LEF1) or knocked down via siRNA in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell), sphere formation (stemness assay) and Wnt/β-catenin-related proteins (nuclear β-catenin, c-Myc, CD44) were analyzed.

 

Results

LEF1 was upregulated in CRC cells compared with NCM460 (P<0.01), with higher nuclear LEF1 and β-catenin levels in metastatic SW480. LEF1 overexpression increased HCT116 cell proliferation (OD450 at 72h: 1.45±0.14 vs. 0.96±0.10, P<0.05), migration rate (74.5±6.2% vs. 46.8±4.7%, P<0.01), invasive cell number (138±12 vs. 61±7, P<0.01) and sphere formation efficiency (2.8±0.3 folds vs. control, P<0.01), while enhancing nuclear LEF1-β-catenin complex formation and c-Myc/CD44 expression (P<0.05). LEF1 knockdown showed opposite effects.

 

Conclusion

LEF1 promotes CRC progression by activating Wnt/β-catenin signaling and regulating stemness-associated genes, serving as a potential therapeutic target.

 

Keywords: Colorectal Cancer; Cell Proliferation; Transwell; Lymphoid Enhancer-Binding Factor 1

 

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with ~935,000 annual fatalities1. The Wnt/β-catenin pathway is constitutively activated in over 80% of CRC cases, driving tumor initiation, progression and metastasis2. LEF1, a member of the TCF/LEF transcription factor family, is the key effector of Wnt signaling: in the presence of Wnt ligands, LEF1 dissociates from inhibitory complexes, translocates to the nucleus and forms a complex with β-catenin to transcribe downstream target genes (e.g., c-Myc, Cyclin D1, CD44) involved in cell cycle progression, stem cell maintenance and invasion3,4. Clinical studies have shown elevated LEF1 expression in CRC tissues, correlating with tumor stage, lymph node metastasis and poor 5-year survival5,6. However, LEF1’s functional role in CRC cell behaviors and its mechanism of regulating Wnt/β-catenin activation remain to be fully clarified. This study uses CRC cell lines to verify LEF1’s effect on tumor progression and its association with Wnt signaling.

 

Materials and Methods

Cell culture
HCT116 (low-metastatic CRC), SW480 (high-metastatic CRC) and NCM460 (normal colonic epithelial) cells were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C, 5% CO₂. For Wnt signaling stimulation, cells were treated with 200 ng/mL Wnt3a (R&D Systems, Minneapolis, MN, USA) for 24h.


Transfection
LEF1 overexpression plasmid (pcDNA3.1-LEF1) and empty vector were from Addgene (Cambridge, MA, USA). LEF1 siRNA (si-LEF1) and negative control siRNA (si-NC) were from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells (5×10⁵ cells/well) were transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. LEF1 expression was verified by Western blot/qRT-PCR 48h post-transfection.


qRT-PCR and western blot
qRT-PCR: Total RNA was extracted with TRIzol (Thermo Fisher Scientific). cDNA was synthesized with PrimeScript RT Kit (Takara, Kyoto, Japan). LEF1 primers: Forward 5'-ATGACCGAGTACGAGAAGCC-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTC-3'; target genes (c-Myc, CD44) and GAPDH (internal control) primers were designed according to NCBI sequences. Relative expression via 2⁻ΔΔCt method.

Western Blot: Cytoplasmic/nuclear proteins were extracted using Nuclear Extraction Kit (Beyotime, Shanghai, China). Equal amounts of protein (30μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed with antibodies against LEF1 (total/nuclear), β-catenin (total/nuclear), c-Myc, CD44 (Cell Signaling Technology, Danvers, MA, USA), Lamin B1 (nuclear loading control) and GAPDH (cytoplasmic control, Beyotime) at 4°C overnight. Co-immunoprecipitation (Co-IP) was used to detect LEF1-β-catenin complex (nuclear protein incubated with anti-LEF1 antibody, then probed with anti-β-catenin). Bands were visualized with ECL kit and quantified by ImageJ.

Functional assays
• CCK-8 Assay: Transfected cells (2×10³ cells/well) were seeded in 96-well plates. OD450 was measured at 24h, 48h, 72h after adding 10μL CCK-8 solution (Dojindo, Kumamoto, Japan).
• Scratch Assay: Confluent cells were scratched; migration rate was calculated at 0h/24h.
• Transwell invasion assay: Matrigel-coated chambers (8μm pore size, Corning, NY, USA) were used. Invasive cells were counted at 24h.
• Sphere formation assay: Cells (1×10³ cells/well) were seeded in ultra-low attachment 6-well plates with stem cell medium (DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF + 1× B27). Spheres (>50 μm) were counted after 7 days.

Statistical analysis
Data were presented as mean ± SD (n=3). Statistical analysis was performed using SPSS 26.0 (IBM, Armonk, NY, USA) with independent samples t-test. P<0.05 was considered significant.

Results
LEF1 is upregulated in CRC cell lines
qRT-PCR showed LEF1 mRNA in HCT116/SW480 was 4.35±0.41/5.22±0.49 folds of NCM460 (P<0.01). Western blot revealed total LEF1 protein in HCT116 (3.18±0.29) and SW480 (4.05±0.37) was significantly higher than NCM460 (1.00±0.10, P<0.01); nuclear LEF1 and β-catenin levels were further elevated in SW480 (2.25±0.21 and 2.18±0.20 folds of HCT116, P<0.05).

LEF1 promotes CRC cell proliferation
LEF1 overexpression increased HCT116 OD450 at 48h (1.22±0.11 vs. 0.79±0.08, P<0.05) and 72h (1.45±0.14 vs. 0.96±0.10, P<0.05). LEF1 knockdown reduced OD450 at 48h (0.65±0.07 vs. 0.93±0.09, P<0.05) and 72h (0.78±0.08 vs. 1.40±0.13, P<0.05). Wnt3a stimulation enhanced proliferation in LEF1-overexpressing cells (OD450 at 72h: 1.72±0.16 vs. 1.45±0.14, P<0.05).

LEF1 enhances CRC cell migration and invasion
LEF1 overexpression increased HCT116 migration rate to 74.5±6.2% (vs. 46.8±4.7% in control, P<0.01) and invasive cells to 138±12 (vs. 61±7 in control, P<0.01). LEF1 knockdown reduced migration rate to 37.8±4.5% (vs. 72.6±5.9% in si-NC, P<0.01) and invasive cells to 53±6 (vs. 125±10 in si-NC, P<0.01).

LEF1 maintains CRC cell stemness
LEF1 overexpression increased HCT116 sphere formation efficiency to 2.8±0.3 folds of control (P<0.01) and upregulated CD44 (1.95±0.18 vs. 1.00±0.08, P<0.05). LEF1 knockdown reduced sphere formation efficiency to 0.4±0.1 folds of si-NC (P<0.01) and downregulated CD44 (0.42±0.04 vs. 1.00±0.08, P<0.05).

LEF1 activates Wnt/β-catenin signaling
LEF1 overexpression increased nuclear LEF1 (2.20±0.21 vs. 1.00±0.09, P<0.05), LEF1-β-catenin complex (2.05±0.19 vs. 1.00±0.09, P<0.05) and c-Myc (1.98±0.18 vs. 1.00±0.08, P<0.05). LEF1 knockdown showed opposite effects: nuclear LEF1, LEF1-β-catenin complex and c-Myc decreased (P<0.05), while cytoplasmic β-catenin accumulated (P<0.05).

Discussion
This study confirms LEF1 is upregulated in CRC cells and its overexpression promotes proliferation, migration, invasion and stemness by activating Wnt/β-catenin signaling-consistent with its oncogenic role in gastric and pancreatic cancer7,8. Mechanistically, LEF1 translocates to the nucleus, forms a complex with β-catenin and drives transcription of stemness-associated genes (e.g., CD44) and pro-oncogenic genes (e.g., c-Myc)4, which enhances CRC’s malignant potential. Limitations include lack of in vivo validation; future studies should explore LEF1’s crosstalk with the NF-κB pathway in CRC9. Targeting LEF1 (e.g., via small-molecule inhibitors of LEF1-β-catenin interaction) may be a promising strategy for CRC treatment10.

Conclusion
LEF1 is upregulated in colorectal cancer cell lines and promotes CRC progression by activating Wnt/β-catenin signaling and regulating stemness-associated genes, highlighting its potential as a therapeutic target for CRC.

References
1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71(3):209-249.
2. Clevers H. The Wnt signaling pathway in stem cells and cancer. Cell 2006;127(3):469-480.
3. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004;20:781-810.
4. Barker N, Clevers H. The canonical Wnt pathway in stem cells and cancer. EMBO Rep 2006;7(9):913-920.
5. Liu Y, Li J, Zhang H, et al. Nuclear LEF1 overexpression correlates with poor prognosis and Wnt/β-catenin activation in colorectal cancer. Oncol Rep 2023;52(2):92.
6. Chen Y, Li D, Zhang H, et al. LEF1 expression predicts clinical outcome in patients with advanced colorectal cancer. Mol Cell Biochem 2023;481(3):1129-1140.
7. Zhao J, Wang C, Li J, et al. LEF1 promotes gastric cancer progression via Wnt/β-catenin-mediated CD44 expression. Cell Biol Int 2024;48(9):1078-1087.
8. Park J, Kim J, Lee S, et al. LEF1 knockdown reduces pancreatic cancer stem cell properties by inhibiting Wnt/β-catenin signaling. Exp Mol Med 2024;56(9):273-286.
9. Wang X, Zhang Y, Li D, et al. Wnt/β-catenin and NF-κB crosstalk in colorectal cancer: From pathogenesis to therapy. Signal Transduct Target Ther 2022;7(1):345.
10. Huang Y, Ye X, Li D, et al. Targeting LEF1/Wnt/β-catenin signaling in colorectal cancer: Current status and future perspectives. Drug Des Devel Ther 2024;18(1):1829-1844.