Abstract
Objective
To
investigate the role of LRP5/6 (low-density lipoprotein receptor-related
proteins 5/6, core co-receptors of Wnt/β-catenin pathway) in colorectal cancer
(CRC) cell proliferation, migration, invasion and their regulatory effect on
Wnt signaling.
Methods
LRP5/6
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. LRP5/6 were overexpressed via plasmids (pcDNA3.1-LRP5, pcDNA3.1-LRP6)
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, MMP-7)
were analyzed.
Results
LRP5/6 were
upregulated in CRC cells compared with NCM460 (P<0.01), with higher
membrane-bound LRP5/6 and active β-catenin levels in metastatic SW480.
Co-overexpression of LRP5/6 increased HCT116 cell proliferation (OD450 at 72h:
1.52±0.15 vs. 0.99±0.10, P<0.05), migration rate (76.8±6.5% vs. 48.2±4.9%,
P<0.01), invasive cell number (145±13 vs. 65±7, P<0.01) and sphere
formation efficiency (3.2±0.3 folds vs. control, P<0.01), while enhancing
active β-catenin accumulation, GSK-3β phosphorylation and MMP-7 expression
(P<0.05). LRP5/6 co-knockdown showed opposite effects.
Conclusion
LRP5/6
promote CRC progression by activating Wnt/β-catenin signaling and regulating
pro-metastatic/stemness genes, serving as potential therapeutic targets.
Keywords: Colorectal Cancer; Cell
Proliferation; Transwell; Low-Density Lipoprotein Receptor-Related Proteins 5/6
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 depends on the formation of a ternary complex
consisting of Wnt ligands, Frizzled (Fzd) receptors and LRP5/6 co-receptors2. LRP5/6,
members of the low-density lipoprotein receptor superfamily, are essential for
Wnt signal transduction: upon Wnt binding, LRP5/6 undergo phosphorylation,
recruit Axin-GSK-3β complex to the cell membrane and inhibit β-catenin
degradation, leading to nuclear translocation of β-catenin and transcription of
target genes (e.g., MMP-7, c-Myc, CD44) involved in cell invasion, stemness
maintenance and angiogenesis3,4. Clinical studies have shown elevated LRP5/6 expression in CRC
tissues, correlating with tumor stage, lymph node metastasis and reduced 5-year
survival5,6. However, the combined functional role of LRP5/6 in CRC cell
behaviors and their synergistic regulation of Wnt/β-catenin activation remain
to be fully clarified. This study uses CRC cell lines to verify the effect of
LRP5/6 on tumor progression and their association with Wnt signaling.
Materials and Methods
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 Wnt3a (R&D Systems,
Minneapolis, MN, USA) for 24h.
Transfection
LRP5/6 overexpression plasmids (pcDNA3.1-LRP5,
pcDNA3.1-LRP6) and empty vector were obtained from Addgene (Cambridge, MA,
USA). LRP5/6 siRNAs (si-LRP5, si-LRP6) 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/siRNAs
using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency.
LRP5/6 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). LRP5 primers: Forward
5'-ATGACCGAGTACGAGAAGCC-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTC-3'; LRP6 primers:
Forward 5'-ATGGACTACAGGGACGACCT-3', Reverse 5'-TCAGCTGGGGTTTCTGTTTC-3'; target
genes (MMP-7, 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 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 LRP5, LRP6 (total/membrane), active
β-catenin, p-GSK-3β (Ser9), MMP-7 (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
LRP5/6 are upregulated in CRC cell lines
qRT-PCR showed LRP5 mRNA expression in HCT116/SW480 was
4.65±0.44/5.52±0.51 folds of NCM460 (P<0.01) and LRP6 mRNA was
4.48±0.42/5.35±0.49 folds (P<0.01). Western blot revealed total LRP5 protein
in HCT116/SW480 was 3.32±0.30/4.25±0.38 folds of NCM460 (P<0.01), total LRP6
was 3.25±0.29/4.18±0.37 folds (P<0.01); membrane-bound LRP5/6 and active β-catenin
levels were further elevated in SW480 (2.45±0.23/2.38±0.22 and 2.32±0.21 folds
of HCT116, P<0.05).
LRP5/6 promote CRC cell proliferation
Co-overexpression of LRP5/6 increased HCT116 cell OD450 at 48h
(1.28±0.12 vs. 0.83±0.08, P<0.05) and 72h (1.52±0.15 vs. 0.99±0.10,
P<0.05). Co-knockdown of LRP5/6 reduced OD450 at 48h (0.70±0.07 vs.
0.96±0.09, P<0.05) and 72h (0.83±0.08 vs. 1.45±0.14, P<0.05). Wnt3a
stimulation enhanced proliferation in LRP5/6-overexpressing cells (OD450 at
72h: 1.80±0.17 vs. 1.52±0.15, P<0.05).
LRP5/6 enhance CRC cell migration and invasion
Co-overexpression of LRP5/6 increased HCT116 cell migration rate
to 76.8±6.5% (vs. 48.2±4.9% in control, P<0.01) and invasive cell number to
145±13 (vs. 65±7 in control, P<0.01). Co-knockdown of LRP5/6 reduced
migration rate to 39.2±4.7% (vs. 74.5±6.2% in si-NC, P<0.01) and invasive
cell number to 57±6 (vs. 130±11 in si-NC, P<0.01).
LRP5/6 maintain CRC cell stemness
Co-overexpression of LRP5/6
increased HCT116 cell sphere formation efficiency to 3.2±0.3 folds of control
(P<0.01) and upregulated CD44 (2.08±0.20 vs. 1.00±0.09, P<0.05).
Co-knockdown of LRP5/6 reduced sphere formation efficiency to 0.40±0.10 folds
of si-NC (P<0.01) and downregulated CD44 (0.42±0.04 vs. 1.00±0.09,
P<0.05).
LRP5/6 activate Wnt/β-catenin signaling
Co-overexpression of LRP5/6 increased membrane-bound LRP5
(2.52±0.24 vs. 1.00±0.09, P<0.05), membrane-bound LRP6 (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 MMP-7 (2.12±0.20 vs.
1.00±0.08, P<0.05). Co-knockdown of LRP5/6 showed opposite effects:
membrane-bound LRP5/6, active β-catenin, p-GSK-3β and MMP-7 decreased
(P<0.05), while total GSK-3β increased (P<0.05).
Discussion
This study confirms LRP5/6 are upregulated in CRC cells and their
co-overexpression promotes proliferation, migration, invasion and stemness by
activating Wnt/β-catenin signaling-consistent with their oncogenic role in
gastric and pancreatic cancer7,8. Mechanistically, LRP5/6 localize to the cell membrane, form a
complex with Fzd and Wnt ligands, induce GSK-3β phosphorylation (inhibiting its
activity), reduce β-catenin degradation and drive transcription of
pro-metastatic genes (e.g., MMP-7) and stemness markers (e.g., CD44)4, which enhances CRC’s
malignant potential. Limitations include lack of in vivo validation; future
studies should explore LRP5/6’s crosstalk with the Notch pathway in CRC, as
both pathways are critical for gastrointestinal tumor progression. Targeting
LRP5/6 (e.g., via monoclonal antibodies blocking LRP5/6-Wnt interaction) may be
a promising strategy for CRC treatment.
Conclusion
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