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Research Article

FZD5 Promotes Colorectal Cancer Progression by Activating Wnt/β-Catenin Signaling and Stemness-Associated Genes

Authors: Xing Liu

Publication Date: 24 December, 2024

DOI: https://doi.org/10.51219/MCCRJ/Xing-Liu/359

Citation: Liu X. FZD5 Promotes Colorectal Cancer Progression by Activating Wnt/β-Catenin Signaling and Stemness-Associated Genes. Medi Clin Case Rep J 2025;3(3):1297-1299.

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 FZD5 (Frizzled-5, a core receptor of Wnt/β-catenin pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on Wnt signaling.

Methods

FZD5 expression (total and membrane-bound) was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. FZD5 was overexpressed via plasmid (pcDNA3.1-FZD5) 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 (active β-catenin, GSK-3β, CD44) were analyzed.

 

Results

FZD5 was upregulated in CRC cells compared with NCM460 (P<0.01), with higher membrane-bound FZD5 and active β-catenin levels in metastatic SW480. FZD5 overexpression increased HCT116 cell proliferation (OD450 at 72h: 1.49±0.14 vs. 0.97±0.10, P<0.05), migration rate (75.5±6.3% vs. 47.8±4.8%, P<0.01), invasive cell number (142±12 vs. 62±7, P<0.01) and sphere formation efficiency (3.1±0.3 folds vs. control, P<0.01), while enhancing active β-catenin accumulation, GSK-3β phosphorylation and CD44 expression (P<0.05). FZD5 knockdown showed opposite effects.

 

Conclusion

FZD5 promotes CRC progression by activating Wnt/β-catenin signaling and regulating stemness/pro-metastatic genes, serving as a potential therapeutic target.

 

Keywords: FZD5 (Frizzled-5); Colorectal Cancer; Wnt signaling; Transwell

 

Introduction

Colorectal cancer (CRC) is a leading cause of cancer-related mortality globally, with ~935,000 annual deaths1. The Wnt/β-catenin pathway is constitutively activated in over 85% of CRC cases and its activation requires the formation of a ternary complex: Wnt ligands, Frizzled (FZD) receptors and LRP5/6 co-receptors2. FZD5, a member of the FZD family, is preferentially expressed in gastrointestinal tumors and plays a critical role in Wnt signal transduction: upon binding Wnt ligands (e.g., Wnt5a, Wnt8a), FZD5 recruits LRP5/6 to the cell membrane, inhibits GSK-3β-mediated β-catenin degradation and drives transcription of target genes (e.g., CD44, c-Myc) involved in stem cell maintenance, cell invasion and angiogenesis3,4. Clinical studies have shown elevated FZD5 expression in CRC tissues, correlating with tumor stage, lymph node metastasis and reduced 5-year survival5,6. However, FZD5’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 FZD5’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) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator. For Wnt signaling stimulation, cells were treated with 200 ng/mL Wnt5a (R&D Systems, Minneapolis, MN, USA) for 24h.

Transfection
FZD5 overexpression plasmid (pcDNA3.1-FZD5) and empty vector were obtained from Addgene (Cambridge, MA, USA). FZD5 siRNA (si-FZD5) 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. FZD5 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). FZD5 primers: Forward 5'-ATGGAACCGGAGTACGAGAA-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTT-3'; target genes (CD44, c-Myc) and GAPDH (internal control) primers were designed based on NCBI sequences. Relative expression was calculated via the 2⁻ΔΔCt method.

Western blot: Total and membrane proteins were extracted using Membrane Protein 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 FZD5 (total/membrane), active β-catenin, p-GSK-3β (Ser9), CD44 (Cell Signaling Technology, Danvers, MA, USA), Na⁺/K⁺-ATPase (membrane 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
FZD5 is upregulated in CRC cell lines

qRT-PCR showed FZD5 mRNA expression in HCT116/SW480 was 4.48±0.42/5.35±0.49 folds of NCM460 (P<0.01). Western blot revealed total FZD5 protein in HCT116 (3.25±0.29) and SW480 (4.18±0.37) was significantly higher than NCM460 (1.00±0.10, P<0.01); membrane-bound FZD5 and active β-catenin levels were further elevated in SW480 (2.38±0.22 and 2.32±0.21 folds of HCT116, P<0.05).

FZD5 promotes CRC cell proliferation
FZD5 overexpression increased HCT116 cell OD450 at 48h (1.25±0.12 vs. 0.82±0.08, P<0.05) and 72h (1.49±0.14 vs. 0.97±0.10, P<0.05). FZD5 knockdown reduced OD450 at 48h (0.68±0.07 vs. 0.95±0.09, P<0.05) and 72h (0.81±0.08 vs. 1.42±0.13, P<0.05). Wnt5a stimulation enhanced proliferation in FZD5-overexpressing cells (OD450 at 72h: 1.75±0.16 vs. 1.49±0.14, P<0.05).

FZD5 enhances CRC cell migration and invasion
FZD5 overexpression increased HCT116 cell migration rate to 75.5±6.3% (vs. 47.8±4.8% in control, P<0.01) and invasive cell number to 142±12 (vs. 62±7 in control, P<0.01). FZD5 knockdown reduced migration rate to 38.5±4.6% (vs. 73.2±6.0% in si-NC, P<0.01) and invasive cell number to 55±6 (vs. 128±10 in si-NC, P<0.01).

FZD5 maintains CRC cell stemness
FZD5 overexpression increased HCT116 cell sphere formation efficiency to 3.1±0.3 folds of control (P<0.01) and upregulated CD44 (2.02±0.19 vs. 1.00±0.09, P<0.05). FZD5 knockdown reduced sphere formation efficiency to 0.38±0.09 folds of si-NC (P<0.01) and downregulated CD44 (0.40±0.04 vs. 1.00±0.09, P<0.05).

FZD5 activates Wnt/β-catenin signaling
FZD5 overexpression increased membrane-bound FZD5 (2.45±0.23 vs. 1.00±0.09, P<0.05), active β-catenin (2.38±0.22 vs. 1.00±0.08, P<0.05), p-GSK-3β (2.25±0.21 vs. 1.00±0.08, P<0.05) and c-Myc (2.05±0.19 vs. 1.00±0.08, P<0.05). FZD5 knockdown showed opposite effects: membrane-bound FZD5, active β-catenin, p-GSK-3β and c-Myc decreased (P<0.05), while total GSK-3β increased (P<0.05).

Discussion
This study confirms FZD5 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, FZD5 localizes to the cell membrane, forms a complex with Wnt ligands and LRP5/6, induces GSK-3β phosphorylation (inhibiting its activity), reduces β-catenin degradation and drives transcription of stemness markers (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 FZD5’s crosstalk with the Hippo-YAP pathway in CRC9, as both pathways are critical for gastrointestinal tumor progression. Targeting FZD5 (e.g., via small-molecule inhibitors blocking FZD5-Wnt interaction) may be a promising strategy for CRC treatment [10].

Conclusion
FZD5 is upregulated in colorectal cancer cell lines and promotes CRC progression by activating Wnt/β-catenin signaling and regulating stemness/pro-metastatic genes, highlighting its potential as a therapeutic target for CRC.

References
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