Abstract
Objective
To
investigate the role of Notch4 in colorectal cancer (CRC) cell proliferation,
migration, stemness maintenance and its regulatory mechanism in the Notch
signaling pathway.
Methods
Notch4
expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell
line (NCM460) was detected by Western blot and qRT-PCR. Notch4 was
overexpressed via plasmid transfection or knocked down via siRNA in HCT116
cells. Cell proliferation was assessed by CCK-8 assay, migration by scratch
wound healing assay, stemness by sphere formation assay and expressions of
Notch pathway-related proteins (NICD4, Hes1, Hey1) by Western blot.
Results
Notch4 was
highly expressed in CRC cells (P<0.01). Notch4 overexpression increased
HCT116 cell proliferation (OD450 at 72h: 1.32±0.12 vs. 0.89±0.09, P<0.05),
migration rate (24h: 71.2±5.8% vs. 42.5±4.3%, P<0.01), sphere formation
ability (sphere number: 38±4 vs. 15±3, P<0.01) and upregulated NICD4, Hes1,
Hey1 expressions (P<0.05). Notch4 knockdown showed opposite effects.
Conclusion
Notch4
promotes CRC cell malignant behaviors and stemness via activating the Notch
signaling pathway, serving as a potential therapeutic target for CRC.
Keywords: Colorectal Cancer; Cell
Proliferation; Transwell
Introduction
Colorectal
cancer (CRC) remains a major global health burden, with approximately 1.9
million new cases and 935,000 deaths annually1. The progression of CRC
is driven by multiple molecular abnormalities, among which the dysregulation of
the Notch signaling pathway plays a critical role2,3. The Notch family
consists of four transmembrane receptors (Notch1-4) and five ligands and their
interaction mediates cell fate decisions including proliferation,
differentiation and stemness maintenance4. While Notch1 and Notch2
have been extensively studied in CRC, the functional role of Notch4 in CRC
remains largely unclear.
Notch4 is
uniquely associated with vascular development and has been implicated in the
progression of multiple cancers, such as breast cancer and glioblastoma5,6. In
gastrointestinal malignancies, Notch4 overexpression has been reported in
gastric cancer, where it promotes cell invasion and metastasis7. However, the
expression pattern of Notch4 in CRC and its impact on CRC cell biological
behaviors (especially stemness, a key factor in chemotherapy resistance and
recurrence) have not been fully elucidated. This study aimed to explore the
function of Notch4 in CRC cells and its regulatory effect on the Notch
signaling pathway.
Materials and Methods
Human CRC cell lines HCT116 and SW480 and normal human colonic
epithelial cell line NCM460 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, Gibco) and 1% penicillin-streptomycin (Gibco)
at 37°C in a humidified incubator with 5% CO₂.
Plasmid transfection and SiRNA knockdown
Notch4 overexpression plasmid (pcDNA3.1-Notch4)
and empty vector (pcDNA3.1) were obtained from Addgene (Cambridge, MA, USA).
SiRNA targeting Notch4 (si-Notch4) and negative control siRNA (si-NC) were
purchased from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells were
seeded into 6-well plates (5×10⁵ cells/well) and transfected with plasmids or
siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) when confluency
reached 60-70%. Notch4 expression was verified by Western blot and qRT-PCR 48h
post-transfection.
qRT-PCR
and western blot analysis
Total RNA extraction, cDNA synthesis and qRT-PCR were performed as
previously described [8]. Notch4 primers: Forward 5'-GCTGCTGCTGCTGTTTCTGA-3',
Reverse 5'-CAGCAGCAGCAGCTTCTTCT-3'; GAPDH primers: Forward
5'-GAAGGTGAAGGTCGGAGTC-3', Reverse 5'-GAAGATGGTGATGGGATTTC-3'.
For Western blot, cells were lysed with RIPA buffer (Beyotime,
Shanghai, China) containing protease inhibitors. Protein (30μg) was separated
by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA)
and incubated with primary antibodies against Notch4 (1:1000, Abcam, Cambridge,
UK), NICD4 (1:1000, Cell Signaling Technology, Danvers, MA, USA), Hes1 (1:1000,
Cell Signaling Technology), Hey1 (1:1000, Cell Signaling Technology) and GAPDH
(1:5000, Beyotime) at 4°C overnight. After incubation with HRP-conjugated
secondary antibody (1:5000, Beyotime), bands were visualized with ECL kit
(Millipore) and quantified by ImageJ.
CCK-8 assay
Transfected HCT116 cells (2×10³ cells/well)
were seeded into 96-well plates. At 24h, 48h, 72h, 10μL CCK-8 solution
(Dojindo, Kumamoto, Japan) was added and absorbance at 450nm was measured using
a microplate reader (Bio-Rad, Hercules, CA, USA).
Scratch wound healing assay
Transfected HCT116 cells were seeded into
6-well plates to confluency. A scratch was made with a 200μL pipette tip. Wound
width was measured at 0h and 24h and migration rate was calculated as (wound
width at 0h - wound width at 24h)/wound width at 0h × 100%.
Sphere formation assay
Transfected HCT116 cells (1×10³ cells/well)
were seeded into ultra-low attachment 6-well plates (Corning, Corning, NY, USA)
in serum-free DMEM/F12 medium (Gibco) supplemented with EGF (20ng/mL), bFGF
(20ng/mL) and B27 (1%, Gibco). After 7 days of culture, spheres with diameter
>50μm were counted under an inverted microscope (Olympus, Tokyo, Japan).
Statistical analysis
Data were presented as mean ± SD (triplicate
experiments). 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
Notch4 is Overexpressed in CRC Cell Lines
qRT-PCR showed Notch4 mRNA expression in HCT116 and SW480 cells
was 3.87±0.35 and 3.12±0.29 folds of NCM460 cells (P<0.01). Western blot
revealed Notch4 protein relative gray values in HCT116 (2.95±0.26) and SW480
(2.43±0.22) were significantly higher than NCM460 (1.00±0.11, P<0.01),
indicating Notch4 overexpression in CRC cells.
Notch4 Regulates CRC Cell Proliferation
Notch4 overexpression increased HCT116 cell OD450 at 48h
(1.05±0.09 vs. 0.72±0.06, P<0.05) and 72h (1.32±0.12 vs. 0.89±0.09,
P<0.05). Notch4 knockdown reduced OD450 at 48h (0.51±0.07 vs. 0.88±0.08,
P<0.05) and 72h (0.63±0.06 vs. 1.25±0.10, P<0.05), demonstrating Notch4
promotes CRC cell proliferation.
Notch4 Enhances CRC Cell Migration
Notch4 overexpression increased HCT116 cell migration rate at 24h
(71.2±5.8% vs. 42.5±4.3%, P<0.01). Notch4 knockdown decreased migration rate
(28.6±3.9% vs. 69.3±5.5%, P<0.01), indicating Notch4 enhances CRC cell
migration.
Notch4 maintains CRC cell stemness
Notch4 overexpression
increased HCT116 cell sphere number (38±4 vs. 15±3, P<0.01). Notch4
knockdown reduced sphere number (8±2 vs. 36±4, P<0.01), suggesting Notch4
maintains CRC cell stemness.
Notch4 Activates the Notch Signaling Pathway
Notch4 overexpression upregulated NICD4, Hes1, Hey1 protein
relative gray values (2.78±0.25, 2.51±0.23, 2.37±0.21 vs. 1.00±0.09,
P<0.05). Notch4 knockdown downregulated these proteins (0.35±0.04,
0.32±0.03, 0.29±0.03 vs. 1.00±0.08, P<0.05), confirming Notch4 activates the
Notch signaling pathway.
Discussion
This study demonstrated Notch4 overexpression in CRC cell lines
and Notch4 regulates CRC cell proliferation, migration, stemness via activating
the Notch signaling pathway. These findings highlight Notch4 as a key oncogenic
factor in CRC.
Notch4's overexpression in CRC aligns with its role in other
cancers. For instance, Notch4 overexpression in breast cancer promotes cell
proliferation and stemness5 and in glioblastoma, it enhances invasion and chemotherapy
resistance6. In
gastric cancer, Notch4 activates the Notch pathway to drive tumor progression7, which is consistent with
our observation in CRC, suggesting Notch4 may play a conserved oncogenic role
in gastrointestinal malignancies.
Mechanistically, Notch4 activation involves cleavage to release
NICD4, which translocates to the nucleus and forms a complex with CSL to
activate target genes (Hes1, Hey1)4,8. Our results showed Notch4 overexpression upregulates NICD4,
Hes1, Hey1, while knockdown has the opposite effect, confirming Notch4-mediated
activation of the Notch pathway in CRC. This is supported by Wang, et al.9, who reported Notch4/NICD4
signaling promotes pancreatic cancer cell stemness by upregulating Hes1.
Notably, CRC stem cells (CSCs) are responsible for tumor
initiation, recurrence and chemotherapy resistance10. Our sphere formation assay showed Notch4 regulates CRC cell
stemness, which is consistent with Li, et al.11, who found Notch4 maintains CSC properties in colorectal adenoma.
This suggests Notch4 may contribute to CRC recurrence by preserving CSCs,
providing a new target for overcoming therapy resistance.
This study has limitations. First, it was conducted in CRC cell
lines; in vivo studies (xenograft models) are needed to validate Notch4's role.
Second, we only explored the Notch pathway; crosstalk with other pathways
(e.g., Wnt/β-catenin12) requires investigation. Third, the clinical significance of
Notch4 in CRC needs analysis with patient tissues.
Targeting Notch4 may be a promising CRC therapy. Current Notch
inhibitors (γ-secretase inhibitors) have off-target effects13, while Notch4-specific
inhibitors could improve specificity. Our study provides evidence for
developing Notch4-targeted therapies for CRC.
Conclusion
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