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Research Article

CSNK1A1 Inhibits Colorectal Cancer Progression by Suppressing Canonical Wnt/ β-Catenin Signaling via β-Catenin Phosphorylation

Authors: Xing Liu

Publication Date: 17 December, 2024

DOI: https://doi.org/10.51219/MCCRJ/Xing-Liu/357

Citation: Liu X. CSNK1A1 Inhibits Colorectal Cancer Progression by Suppressing Canonical Wnt/β-Catenin Signaling via β-Catenin Phosphorylation. Medi Clin Case Rep J 2025;3(3):1291-1293.

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 CSNK1A1 (casein kinase 1 alpha 1, a key regulator of canonical Wnt/β-catenin pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on Wnt signaling.

 

Methods

CSNK1A1 expression was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. CSNK1A1 was overexpressed via plasmid (pcDNA3.1-CSNK1A1) 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, p-β-catenin Ser45, AXIN1, c-Myc) were analyzed.

 

Results

CSNK1A1 was downregulated in CRC cells compared with NCM460 (P<0.01), with lower expression in metastatic SW480. CSNK1A1 overexpression decreased HCT116 cell proliferation (OD450 at 72h: 0.72±0.07 vs. 1.02±0.10, P<0.05), migration rate (40.2±4.7% vs. 49.5±5.0%, P<0.01), invasive cell number (55±6 vs. 68±7, P<0.01) and sphere formation efficiency (0.40±0.04 folds vs. control, P<0.01), while enhancing β-catenin Ser45 phosphorylation (promoting degradation), increasing AXIN1 stability and downregulating c-Myc (P<0.05). CSNK1A1 knockdown showed opposite effects.

 

Conclusion

CSNK1A1 functions as a tumor suppressor in CRC by inhibiting canonical Wnt/β-catenin signaling via β-catenin phosphorylation, serving as a potential therapeutic target for restoring pathway homeostasis.

 

Keywords: CSNK1A1 (casein kinase 1 alpha 1); Transwell; Wnt signaling

 

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 sequential phosphorylation of β-catenin-first by CSNK1A1 (Ser45), then by GSK-3β (Thr41/Ser37/Ser33)-to trigger ubiquitination and degradation2,3. CSNK1A1, a member of the casein kinase 1 family, is a critical upstream kinase in this process: it not only initiates β-catenin phosphorylation but also stabilizes the AXIN1-containing "destruction complex" by phosphorylating AXIN1, further enhancing β-catenin degradation4,5. Clinical studies have shown that CSNK1A1 is frequently downregulated or mutated in CRC tissues, correlating with tumor stage, lymph node metastasis and reduced 5-year survival6,7. However, CSNK1A1’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 CSNK1A1’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
CSNK1A1 overexpression plasmid (pcDNA3.1-CSNK1A1) and empty vector were obtained from Addgene (Cambridge, MA, USA). CSNK1A1 siRNA (si-CSNK1A1) 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. CSNK1A1 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). CSNK1A1 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 CSNK1A1, β-catenin (total/nuclear), p-β-catenin (Ser45), AXIN1, 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
CSNK1A1 is downregulated in CRC cell lines
qRT-PCR showed CSNK1A1 mRNA expression in HCT116/SW480 was 0.45±0.04/0.32±0.03 folds of NCM460 (P<0.01). Western blot revealed CSNK1A1 protein in HCT116 (0.42±0.04) and SW480 (0.28±0.03) was significantly lower than NCM460 (1.00±0.10, P<0.01); nuclear β-catenin levels were inversely elevated in SW480 (2.95±0.27 folds of HCT116, P<0.05), while p-β-catenin (Ser45) was reduced (0.35±0.04 folds of HCT116, P<0.05).

CSNK1A1 inhibits CRC cell proliferation
CSNK1A1 overexpression decreased HCT116 cell OD450 at 48h (0.85±0.08 vs. 1.05±0.09, P<0.05) and 72h (0.72±0.07 vs. 1.02±0.10, P<0.05). CSNK1A1 knockdown increased OD450 at 48h (1.28±0.12 vs. 1.05±0.09, P<0.05) and 72h (1.52±0.14 vs. 1.02±0.10, P<0.05). Wnt3a stimulation partially reversed CSNK1A1-induced proliferation inhibition (P<0.05).

CSNK1A1 Reduces CRC cell migration and invasion
CSNK1A1 overexpression decreased HCT116 cell migration rate to 40.2±4.7% (vs. 49.5±5.0% in control, P<0.01) and invasive cell number to 55±6 (vs. 68±7 in control, P<0.01). CSNK1A1 knockdown increased migration rate to 65.8±6.2% (vs. 49.5±5.0% in si-NC, P<0.01) and invasive cell number to 92±8 (vs. 68±7 in si-NC, P<0.01).

CSNK1A1 suppresses CRC cell stemness
CSNK1A1 overexpression decreased HCT116 cell sphere formation efficiency to 0.40±0.04 folds of control (P<0.01) and downregulated CD44 (0.45±0.04 vs. 1.00±0.09, P<0.05). CSNK1A1 knockdown increased sphere formation efficiency to 2.3±0.2 folds of si-NC (P<0.01) and upregulated CD44 (2.15±0.20 vs. 1.00±0.09, P<0.05).

CSNK1A1 inactivates canonical Wnt/β-catenin signaling
CSNK1A1 overexpression increased p-β-catenin (Ser45) (2.65±0.25 vs. 1.00±0.09, P<0.05) and AXIN1 stability (1.85±0.17 vs. 1.00±0.08, P<0.05), while reducing nuclear β-catenin (0.48±0.04 vs. 1.00±0.09, P<0.05) and c-Myc (0.52±0.05 vs. 1.00±0.08, P<0.05). CSNK1A1 knockdown showed opposite effects: p-β-catenin (Ser45) and AXIN1 decreased (P<0.05), while nuclear β-catenin and c-Myc increased (P<0.05), indicating inhibited β-catenin degradation.

Discussion
This study confirms CSNK1A1 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 cancer8,9. Mechanistically, CSNK1A1 initiates β-catenin phosphorylation at Ser45, a prerequisite for subsequent GSK-3β-mediated phosphorylation and degradation; it also stabilizes AXIN1 to reinforce the destruction complex, thereby suppressing nuclear translocation of β-catenin and transcription of pro-oncogenic genes (e.g., c-Myc)5. Limitations include lack of in vivo validation; future studies should explore CSNK1A1’s interaction with Wnt co-receptors (e.g., LRP6) in CRC10, as CSNK1A1 also phosphorylates LRP6 to modulate Wnt pathway activation. Restoring CSNK1A1 activity (e.g., via small-molecule activators or kinase agonists) may be a promising strategy for CRC treatment.

Conclusion
CSNK1A1 is downregulated in colorectal cancer cell lines and inhibits CRC progression by suppressing canonical Wnt/β-catenin signaling via β-catenin phosphorylation and AXIN1 stabilization, highlighting its potential as a therapeutic target for restoring pathway homeostasis in CRC.

References
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