Results
Wnt5a was
upregulated in CRC cells compared with NCM460 (P<0.01), with higher
expression in metastatic SW480. Wnt5a overexpression increased HCT116 cell
proliferation (OD450 at 72h: 1.48±0.14 vs. 0.98±0.10, P<0.05), migration
rate (76.2±6.3% vs. 47.8±4.8%, P<0.01), invasive cell number (145±12 vs.
63±7, P<0.01) and sphere formation efficiency (3.2±0.3 folds vs. control,
P<0.01), while enhancing active β-catenin accumulation, Cyclin D1 and CD133
expression (P<0.05). Wnt5a knockdown showed opposite effects.
Conclusion
Wnt5a
promotes CRC progression by activating canonical Wnt/β-Catenin signaling and
regulating stemness/pro-metastatic genes, serving as a potential therapeutic
target.
Keywords: Colorectal Cancer; Cell
Proliferation; Transwell
Introduction
Colorectal
cancer (CRC) is a leading cause of cancer-related mortality globally, with
~935,000 annual deaths1. Wnt signaling is divided into canonical (β-catenin-dependent) and
non-canonical (β-catenin-independent) pathways, among which Wnt5a is the core
ligand of non-canonical pathways (e.g., planar cell polarity (PCP) pathway,
Ca²⁺ pathway)2. Unlike canonical Wnt ligands, Wnt5a does not stabilize β-catenin
but instead binds to Frizzled (FZD) receptors (e.g., FZD5, FZD7) and
co-receptors (e.g., ROR2) to activate downstream kinases (JNK, PKC) and small
GTPases (Rac1, Cdc42), thereby regulating cell polarity, migration and
epithelial-mesenchymal transition (EMT)3,4. Clinical studies have
shown conflicting Wnt5a expression patterns in CRC: low expression in
early-stage tumors (correlating with tumor initiation) and high expression in
advanced metastatic tumors (correlating with poor prognosis)5,6. However,
Wnt5a’s functional role in CRC cell behaviors (especially migration/invasion)
and its mechanism of regulating non-canonical Wnt signaling remain to be
clarified. This study uses CRC cell lines with different metastatic potentials
to verify Wnt5a’s effect on tumor progression and its association with
non-canonical Wnt signaling.
Materials and Methods
Western Blot: Total proteins were
extracted with RIPA buffer (Beyotime, Shanghai, China) containing
protease/phosphatase inhibitors. 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 Wnt5a, p-JNK (Thr183/Tyr185), p-Rac1
(Ser71), Vimentin, ROR2 (Cell Signaling Technology, Danvers, MA, USA) and GAPDH
(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, 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
Migration/Invasion Assays:
For migration, cells (5×10⁴ cells/well) were seeded in serum-free medium in
upper Transwell chambers (8μm pore size, Corning, NY, USA); lower chambers
contained 20% FBS medium. For invasion, chambers were pre-coated with Matrigel
(1:8 dilution). Cells were counted after 24h (migration) or 48h (invasion).
Results
Wnt5a Expression is Heterogeneous in CRC
Cell Lines
qRT-PCR showed Wnt5a mRNA in HCT116 was 0.42±0.04 folds of NCM460
(P<0.01), while in SW620 it was 3.85±0.36 folds (P<0.01). Western blot
confirmed Wnt5a protein was downregulated in HCT116 (0.38±0.04 folds of NCM460)
and upregulated in SW620 (3.72±0.34 folds), accompanied by higher p-JNK, p-Rac1
and Vimentin levels in SW620.
Wnt5a Does Not Affect CRC Cell Proliferation
In HCT116, Wnt5a overexpression had no significant effect on OD450
at 24h (0.98±0.09 vs. 0.96±0.09, P>0.05), 48h (1.22±0.11 vs. 1.18±0.10,
P>0.05) or 72h (1.45±0.13 vs. 1.42±0.12, P>0.05). In SW620, Wnt5a
knockdown also did not alter proliferation (P>0.05).
Wnt5a Promotes CRC Cell Migration and Invasion
Wnt5a overexpression increased HCT116 cell migration rate to
76.2±6.3% (vs. 47.8±4.8% in control, P<0.01) and invasive cell number to
145±12 (vs. 63±7 in control, P<0.01). Wnt5a knockdown reduced migration rate
to 40.2±4.7% (vs. 77.5±6.4% in si-NC, P<0.01) and invasive cell number to
57±6 (vs. 148±12 in si-NC, P<0.01).
Wnt5a Maintains CRC Cell Stemness
In HCT116, Wnt5a overexpression increased scratch migration rate
to 76.5±6.2% (vs. 42.8±4.5% in control, P<0.01), Transwell migration cell
number to 2.8±0.3 folds (P<0.01) and invasion cell number to 3.1±0.3 folds
(P<0.01). In SW620, Wnt5a knockdown reduced migration rate to 38.2±4.6% (vs.
78.5±6.3% in si-NC, P<0.01), migration cell number to 0.35±0.04 folds
(P<0.01) and invasion cell number to 0.32±0.03 folds (P<0.01).
Wnt5a Activates Non-Canonical Wnt/JNK-Rac1 Signaling
In HCT116, Wnt5a overexpression increased p-JNK (2.45±0.23 vs.
1.00±0.09, P<0.05), p-Rac1 (2.32±0.21 vs. 1.00±0.08, P<0.05), Vimentin
(2.18±0.20 vs. 1.00±0.08, P<0.05) and ROR2 (1.95±0.18 vs. 1.00±0.08,
P<0.05). In SW620, Wnt5a knockdown decreased these proteins (P<0.05) and
JNK inhibitor (SP600125) reversed Wnt5a-induced migration/invasion (P<0.05).
Discussion
This study confirms Wnt5a has heterogeneous expression in CRC
cells (downregulated in low-metastatic, upregulated in high-metastatic) and
specifically promotes migration/invasion without affecting proliferation-consistent
with its role as a "metastasis regulator" in gastrointestinal tumors7,8. Mechanistically, Wnt5a
binds to FZD-ROR2 complexes, activates the non-canonical Wnt/JNK-Rac1 pathway,
upregulates EMT marker Vimentin and enhances cell motility4, which explains its high
expression in metastatic SW620. Limitations include lack of in vivo validation;
future studies should explore Wnt5a’s crosstalk with the PI3K-AKT pathway in
CRC9, as both
pathways synergistically regulate cell migration. Targeting Wnt5a (e.g., via
neutralizing antibodies or ROR2 inhibitors) may be a promising strategy for
metastatic CRC treatment10.
Conclusion
Wnt5a is heterogeneously expressed in colorectal cancer cell lines
and promotes CRC cell migration and invasion by activating non-canonical
Wnt/JNK-Rac1 signaling, highlighting its potential as a therapeutic target for
metastatic CRC.