Abstract
Objective
To
investigate the role of Adenomatous Polyposis Coli (APC, 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
APC
expression (wild-type/mutant) was detected in CRC cell lines (HCT116 with
mutant APC, SW480 with truncated APC and NCM460 with wild-type APC) by Western
blot and qRT-PCR. Wild-type APC was overexpressed via plasmid (pcDNA3.1-APC-wt)
in HCT116 cells and APC was knocked down via siRNA in NCM460 cells. Cell
proliferation (CCK-8), migration (scratch assay), invasion (Transwell), sphere
formation (stemness assay) and canonical Wnt-related proteins (β-catenin,
p-β-catenin Ser33/37/Thr41, c-Myc, Cyclin D1) were analyzed.
Results
Wild-type
APC was downregulated or mutated in CRC cells (P<0.01), with undetectable
full-length APC in SW480. Overexpressing wild-type APC in HCT116 decreased cell
proliferation (OD450 at 72h: 0.62±0.06 vs. 1.00±0.10, P<0.05), migration
rate (32.5±4.2% vs. 49.8±5.0%, P<0.01), invasive cell number (45±6 vs. 68±7,
P<0.01) and sphere formation efficiency (0.28±0.03 folds vs. control,
P<0.01), while increasing β-catenin phosphorylation (Ser33/37/Thr41) and
downregulating c-Myc/Cyclin D1 (P<0.05). APC knockdown in NCM460 showed
opposite effects.
Conclusion
Wild-type
APC functions as a tumor suppressor in CRC by inhibiting canonical
Wnt/β-catenin signaling; restoring its function is a potential therapeutic
strategy for CRC with APC mutation.
Keywords: Adenomatous Polyposis Coli; Transwell; Sphere
formation; Wnt/β-catenin signaling
Introduction
Colorectal
cancer (CRC) is one of the most lethal gastrointestinal malignancies, causing
~935,000 annual deaths globally1. The canonical Wnt/β-catenin pathway is constitutively activated in
over 90% of CRC cases and Adenomatous Polyposis Coli (APC) is the most
frequently mutated gene driving this activation2,3. As a scaffold protein
of the "β-catenin destruction complex" (composed of APC, AXIN1,
GSK-3β and CK1), APC binds to β-catenin and facilitates its phosphorylation by
GSK-3β, triggering ubiquitination and proteasomal degradation4,5. Clinical
studies show that APC mutations (mostly truncating mutations leading to loss of
β-catenin-binding domains) occur in 70-80% of sporadic CRC and are the
initiating event of familial adenomatous polyposis (FAP), a hereditary CRC
predisposition syndrome6,7. However, the functional impact of APC mutation on CRC cell
behaviors (e.g., stemness maintenance) and the mechanism of Wnt pathway
dysregulation remain to be fully clarified. This study uses CRC cell lines with
different APC statuses to verify APC’s tumor-suppressive effect and its
association with canonical Wnt signaling.
Materials and Methods
Cell culture
CRC cell lines: HCT116 (harboring heterozygous APC mutation:
c.3927_3931del, p. Asn1309Lysfs*26) and SW480 (homozygous truncated APC,
lacking exons 1-15) and normal colonic epithelial cell line NCM460 (wild-type
APC) 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. Wnt pathway activation was induced with 200 ng/mL Wnt3a protein
(R&D Systems, Minneapolis, MN, USA) for 24h.
Transfection
Wild-type APC overexpression plasmid
(pcDNA3.1-APC-wt, containing full-length human APC cDNA) and empty vector were
obtained from Addgene (Cambridge, MA, USA). APC siRNA (si-APC, targeting
wild-type APC mRNA) and negative control siRNA (si-NC) were purchased from
Thermo Fisher Scientific (Waltham, MA, USA). HCT116 (5×10⁵ cells/well) and
NCM460 (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. APC expression was verified by Western blot
(full-length/truncated APC) 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). APC primers (targeting wild-type APC
exon 16): Forward 5'-ATGGAACCGGAGTACGAGAA-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTT-3';
target genes (c-Myc, Cyclin D1, CD44) 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 7% SDS-PAGE (for full-length APC,
~312 kDa) or 10% SDS-PAGE (for other proteins), transferred to PVDF membranes
(Millipore, Billerica, MA, USA) and probed with primary antibodies against APC
(full-length, recognizing amino acids 2843-2859), truncated APC (recognizing
amino acids 1-100), β-catenin (total/nuclear), p-β-catenin (Ser33/37/Thr41),
c-Myc, Cyclin D1 (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. 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
Results
Wild-Type APC is deficient in CRC cell
lines
qRT-PCR showed wild-type APC mRNA was
undetectable in SW480 and reduced by 65% in HCT116 compared with NCM460
(P<0.01). Western blot confirmed full-length APC (~312 kDa) was only
detected in NCM460; HCT116 expressed a truncated APC fragment (~140 kDa) and
SW480 expressed a smaller truncated fragment (~100 kDa). Concurrently, nuclear
β-catenin was elevated in HCT116 (2.75±0.26 folds of NCM460, P<0.01) and
SW480 (4.25±0.38 folds, P<0.01), while p-β-catenin (Ser33/37/Thr41) was
reduced by 58% (HCT116) and 72% (SW480) (P<0.01).
Wild-Type APC Inhibits CRC Cell Proliferation
Overexpressing wild-type APC in HCT116 decreased OD450 at 48h
(0.75±0.08 vs. 1.02±0.09, P<0.05) and 72h (0.62±0.06 vs. 1.00±0.10,
P<0.05). In contrast, APC knockdown in NCM460 increased OD450 at 48h
(1.38±0.12 vs. 1.02±0.09, P<0.05) and 72h (1.65±0.14 vs. 1.00±0.10,
P<0.05). Wnt3a stimulation partially reversed APC-induced proliferation
inhibition (P<0.05).
Wild-Type APC Reduces CRC Cell Migration and Invasion
Wild-type APC overexpression in HCT116 decreased migration rate to
32.5±4.2% (vs. 49.8±5.0% in control, P<0.01) and invasive cell number to
45±6 (vs. 68±7 in control, P<0.01). APC knockdown in NCM460 increased
migration rate to 72.8±6.3% (vs. 49.8±5.0% in si-NC, P<0.01) and invasive
cell number to 102±8 (vs. 68±7 in si-NC, P<0.01).
Wild-Type APC Suppresses CRC Cell Stemness
Wild-type APC overexpression in HCT116 decreased sphere formation
efficiency to 0.28±0.03 folds of control (P<0.01) and downregulated CD44
(0.35±0.04 vs. 1.00±0.09, P<0.05). APC knockdown in NCM460 increased sphere
formation efficiency to 2.8±0.2 folds of si-NC (P<0.01) and upregulated CD44
(2.55±0.23 vs. 1.00±0.09, P<0.05).
Wild-Type APC Inactivates Canonical Wnt/β-Catenin Signaling
ACVR1B overexpression upregulated p-Smad2 (1.93±0.18 vs.
1.00±0.09, P<0.05), p-Smad3 (1.87±0.17 vs. 1.00±0.08, P<0.05) and Activin
A (1.82±0.16 vs. 1.00±0.07, P<0.05) (no significant change in total Smad4).
ACVR1B knockdown showed opposite effects. Activin A stimulation further
enhanced these changes, confirming ACVR1B’s role in pathway activation.
Discussion
This study confirms that wild-type APC is deficient in CRC cells
and its restoration exerts tumor-suppressive effects by inhibiting
proliferation, migration, invasion and stemness-consistent with its role in
hereditary and sporadic CRC8,9. Mechanistically, wild-type APC stabilizes the β-catenin
destruction complex, enhances GSK-3β-mediated β-catenin phosphorylation and
reduces nuclear β-catenin accumulation, thereby suppressing transcription of
pro-oncogenic genes (e.g., c-Myc, Cyclin D1) and stemness markers (e.g., CD44)5. Limitations include lack
of in vivo validation; future studies should explore APC’s interaction with
other Wnt regulators (e.g., AXIN2) in CRC10, as AXIN2 upregulation often compensates for APC loss. Restoring
wild-type APC function (e.g., via gene editing or APC mimetics) may be a
promising strategy for CRC with APC mutation.
Conclusion
Wild-type APC is deficient in colorectal
cancer cell lines and restoring its expression inhibits CRC progression by
suppressing canonical Wnt/β-catenin signaling, highlighting its potential as a
therapeutic target for CRC with APC dysfunction.
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