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Research Article

AXIN1 Inhibits Colorectal Cancer Progression by Suppressing Canonical Wnt/β- Catenin Signaling and Pro-Oncogenic Genes

Authors: Xing Liu

Publication Date: 09 December, 2024

DOI: https://doi.org/10.51219/MCCRJ/Xing-Liu/353

Citation: Liu X. AXIN1 Inhibits Colorectal Cancer Progression by Suppressing Canonical Wnt/β-Catenin Signaling and Pro- Oncogenic Genes. Medi Clin Case Rep J 2025;3(3):1280-1282.

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 AXIN1 (a key negative regulator of canonical Wnt/β-catenin pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on Wnt signaling.

 

Methods

AXIN1 expression was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. AXIN1 was overexpressed via plasmid (pcDNA3.1-AXIN1) or knocked down via siRNA in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell), sphere formation (stemness assay) and canonical Wnt-related proteins (β-catenin, GSK-3β, c-Myc) were analyzed.

 

Results

AXIN1 was downregulated in CRC cells compared with NCM460 (P<0.01), with lower expression in metastatic SW480. AXIN1 overexpression decreased HCT116 cell proliferation (OD450 at 72h: 0.68±0.06 vs. 0.99±0.10, P<0.05), migration rate (38.5±4.6% vs. 48.2±4.9%, P<0.01), invasive cell number (52±6 vs. 65±7, P<0.01) and sphere formation efficiency (0.35±0.04 folds vs. control, P<0.01), while reducing β-catenin accumulation, enhancing GSK-3β activity and downregulating c-Myc (P<0.05). AXIN1 knockdown showed opposite effects.

 

Conclusion

AXIN1 functions as a tumor suppressor in CRC by inhibiting canonical Wnt/β-catenin signaling, serving as a potential therapeutic target for restoring pathway homeostasis.

 

Keywords: Colorectal Cancer; Western Blot; Cell Proliferation; Canonical Wnt-related Proteins

 

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related mortality globally, with ~935,000 annual deaths1. The canonical Wnt/β-catenin pathway is constitutively activated in over 85% of CRC cases and its activity is tightly regulated by the "destruction complex" composed of AXIN1, APC, GSK-3β and CK12. AXIN1 (Axin-1) acts as a scaffold protein in this complex, facilitating GSK-3β-mediated phosphorylation and degradation of β-catenin-thus preventing nuclear translocation of β-catenin and transcription of pro-oncogenic target genes (e.g., c-Myc, Cyclin D1)3,4. Clinical studies have shown that AXIN1 is frequently downregulated or mutated in CRC tissues, correlating with tumor stage, lymph node metastasis and reduced 5-year survival5,6. However, AXIN1’s functional role in CRC cell behaviors (especially pathway suppression) and its mechanism of regulating Wnt/β-catenin homeostasis remain to be fully clarified. This study uses CRC cell lines to verify AXIN1’s tumor-suppressive effect and its association with canonical 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) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. For Wnt pathway activation, cells were treated with 200 ng/mL Wnt3a protein (R&D Systems, Minneapolis, MN, USA) for 24h.

Transfection
AXIN1 overexpression plasmid (pcDNA3.1-AXIN1) and empty vector were obtained from Addgene (Cambridge, MA, USA). AXIN1 siRNA (si-AXIN1) and negative control siRNA (si-NC) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells (5×10⁵ cells/well) were seeded in 6-well plates and transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. AXIN1 expression was verified by Western blot and qRT-PCR 48h post-transfection.


qRT-PCR and Western Blot
qRT-PCR: Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using PrimeScript RT Kit (Takara, Kyoto, Japan). AXIN1 primers: Forward 5'-ATGGAACCGGAGTACGAGAA-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTT-3'; target genes (c-Myc, Cyclin D1) and GAPDH (internal control) primers were designed based on NCBI sequences. Relative expression was calculated via the 2⁻ΔΔCt method.

Western Blot: Total and 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 primary antibodies against AXIN1, β-catenin (total/nuclear), GSK-3β (total/p-Ser9), c-Myc (Cell Signaling Technology, Danvers, MA, USA), Lamin B1 (nuclear loading control) and GAPDH (total protein control, Beyotime) at 4°C overnight. 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 and 72h after adding 10μL CCK-8 solution (Dojindo, Kumamoto, Japan).
• Scratch Assay: Confluent cells were scratched with a 200μL pipette tip. Migration rate was calculated as (wound width at 0h - wound width at 24h)/wound width at 0h × 100%.
• Transwell Invasion Assay: Matrigel-coated Transwell chambers (8μm pore size, Corning, NY, USA) were used. Transfected cells (2×10⁴ cells/well) in serum-free medium were added to the upper chamber; medium with 20% FBS was added to the lower chamber. 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 ± standard deviation (SD, n=3). Statistical analysis was performed using SPSS 26.0 software (IBM, Armonk, NY, USA) with independent samples t-test. P<0.05 was considered statistically significant.

Results
AXIN1 is downregulated in CRC cell lines
qRT-PCR showed AXIN1 mRNA expression in HCT116/SW480 was 0.42±0.04/0.28±0.03 folds of NCM460 (P<0.01). Western blot revealed AXIN1 protein in HCT116 (0.38±0.04) and SW480 (0.25±0.03) was significantly lower than NCM460 (1.00±0.10, P<0.01); nuclear β-catenin levels were inversely elevated in SW480 (2.85±0.26 folds of HCT116, P<0.05).

AXIN1 inhibits CRC cell proliferation
AXIN1 overexpression decreased HCT116 cell OD450 at 48h (0.82±0.08 vs. 1.02±0.09, P<0.05) and 72h (0.68±0.06 vs. 0.99±0.10, P<0.05). AXIN1 knockdown increased OD450 at 48h (1.25±0.12 vs. 1.02±0.09, P<0.05) and 72h (1.48±0.14 vs. 0.99±0.10, P<0.05). Wnt3a stimulation partially reversed AXIN1-induced proliferation inhibition (P<0.05).

AXIN1 reduces CRC cell migration and invasion
AXIN1 overexpression decreased HCT116 cell migration rate to 38.5±4.6% (vs. 48.2±4.9% in control, P<0.01) and invasive cell number to 52±6 (vs. 65±7 in control, P<0.01). AXIN1 knockdown increased migration rate to 62.8±6.0% (vs. 48.2±4.9% in si-NC, P<0.01) and invasive cell number to 88±8 (vs. 65±7 in si-NC, P<0.01).

AXIN1 suppresses CRC cell stemness
AXIN1 overexpression decreased HCT116 cell sphere formation efficiency to 0.35±0.04 folds of control (P<0.01) and downregulated CD44 (0.42±0.04 vs. 1.00±0.09, P<0.05). AXIN1 knockdown increased sphere formation efficiency to 2.1±0.2 folds of si-NC (P<0.01) and upregulated CD44 (2.05±0.19 vs. 1.00±0.09, P<0.05).

AXIN1 inactivates canonical Wnt/β-catenin signaling
AXIN1 overexpression reduced nuclear β-catenin (0.45±0.04 vs. 1.00±0.09, P<0.05), c-Myc (0.48±0.04 vs. 1.00±0.08, P<0.05) and p-GSK-3β (Ser9) (0.52±0.05 vs. 1.00±0.08, P<0.05) (indicating enhanced GSK-3β activity). AXIN1 knockdown showed opposite effects: nuclear β-catenin, c-Myc and p-GSK-3β increased (P<0.05), while β-catenin degradation was inhibited.

Discussion
This study confirms AXIN1 is downregulated in CRC cells and its overexpression exerts tumor-suppressive effects by inhibiting proliferation, migration, invasion and stemness-consistent with its role in gastric and pancreatic cancer7,8. Mechanistically, AXIN1 stabilizes the Wnt destruction complex, enhances GSK-3β-mediated β-catenin phosphorylation and degradation and reduces nuclear translocation of β-catenin, thereby suppressing transcription of pro-oncogenic genes (e.g., c-Myc)4. Limitations include lack of in vivo validation; future studies should explore AXIN1’s interaction with APC (another destruction complex component) in CRC9, as concurrent loss of AXIN1 and APC often exacerbates Wnt pathway activation. Restoring AXIN1 expression (e.g., via gene delivery or small-molecule stabilizers) may be a promising strategy for CRC treatment10.

Conclusion
AXIN1 is downregulated in colorectal cancer cell lines and inhibits CRC progression by suppressing canonical Wnt/β-catenin signaling, highlighting its potential as a therapeutic target for restoring pathway homeostasis in CRC.

References
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