Abstract
Objective
To
investigate the role of CSL (C-promoter binding factor 1) in the proliferation
and migration of colorectal cancer (CRC) cells and its association with the
Notch signaling pathway.
Methods
CSL
expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell
line (NCM460) was detected by Western blot. CSL was knocked down by siRNA
transfection in HCT116 cells. Cell proliferation was assessed by CCK-8 assay,
cell migration by Transwell assay and expressions of Notch pathway-related
proteins (Hes1, Hey1) by Western blot.
Results
CSL was
highly expressed in CRC cells. Knockdown of CSL inhibited HCT116 cell
proliferation (OD450 at 72h: 0.68±0.07 vs. 1.21±0.09, P<0.05) and migration
(number of migrated cells: 45±6 vs. 128±11, P<0.01) and downregulated Hes1
and Hey1 expressions (P<0.05).
Conclusion
CSL promotes
CRC cell proliferation and migration via activating the Notch signaling
pathway, which may be a potential therapeutic target for CRC.
Keywords: CSL (C-promoter binding factor 1); siRNA transfection; CSL expression
Introduction
Colorectal
cancer (CRC) remains one of the most prevalent malignancies worldwide, with
high morbidity and mortality. According to the GLOBOCAN 2020 statistics, CRC
accounts for approximately 10% of all new cancer cases and deaths globally1. Despite
advances in surgical resection, chemotherapy and targeted therapy, the
prognosis of advanced CRC patients remains poor, highlighting the need to
explore the underlying molecular mechanisms of CRC progression for developing
novel therapeutic strategies2.
The Notch
signaling pathway is an evolutionarily conserved signaling cascade that plays
crucial roles in cell proliferation, differentiation and apoptosis and its
dysregulation is closely associated with the initiation and progression of
various cancers, including CRC3,4. CSL (C-promoter binding factor 1, also known as RBP-Jκ in mammals)
is a key transcription factor in the Notch pathway, which mediates the
transcriptional activation of Notch target genes (e.g., Hes1, Hey1) upon
binding to the intracellular domain of Notch (NICD)5,6.
Accumulating
evidence suggests that CSL is overexpressed in several cancers, such as breast
cancer and lung cancer and promotes tumor progression7,8. However,
the expression pattern and functional role of CSL in CRC, especially its
regulatory effect on CRC cell proliferation and migration, have not been fully
elucidated. Therefore, this study aimed to investigate the role of CSL in CRC
cells and its relationship with the Notch signaling pathway.
Materials and Methods
Cell culture
Human CRC cell lines HCT116 and SW480 and normal human colonic
epithelial cell line NCM460 were obtained from the American Type Culture
Collection (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
atmosphere with 5% CO₂.
SiRNA Transfection
Small interfering RNA (siRNA) targeting CSL
(si-CSL) and negative control siRNA (si-NC) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). HCT116 cells were seeded into 6-well
plates at a density of 5×10⁵ cells/well. When cell confluency reached 60-70%,
transfection was performed using Lipofectamine 3000 reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s instructions. The efficiency
of CSL knockdown was verified by Western blot 48h after transfection.
Western
blot analysis
Cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China)
containing protease inhibitor cocktail (Roche, Basel, Switzerland). Protein
concentration was determined using BCA protein assay kit (Beyotime). Equal
amounts of protein (30μg) were separated by 10% SDS-PAGE and transferred onto
PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5%
non-fat milk for 1h at room temperature, then incubated with primary antibodies
against CSL (1:1000, Abcam, Cambridge, UK), Hes1 (1:1000, Cell Signaling
Technology, Danvers, MA, USA), Hey1 (1:1000, Cell Signaling Technology) and
GAPDH (1:5000, Beyotime) at 4°C overnight. After washing with TBST, membranes
were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody
(1:5000, Beyotime) for 1h at room temperature. Protein bands were visualized
using ECL chemiluminescence kit (Millipore) and relative protein expression was
quantified by ImageJ software (National Institutes of Health, Bethesda, MD,
USA) with GAPDH as the internal control.
CCK-8 Assay for cell proliferation
HCT116 cells transfected with si-CSL or si-NC were
seeded into 96-well plates at a density of 2×10³ cells/well. At 24h, 48h and
72h after transfection, 10μL of CCK-8 solution (Dojindo, Kumamoto, Japan) was
added to each well and the plates were incubated at 37°C for 2h. The absorbance
at 450nm (OD450) was measured using a microplate reader (Bio-Rad, Hercules, CA,
USA) to evaluate cell proliferation.
Transwell assay for cell migration
Transwell chambers (8μm pore size, Corning, Corning, NY, USA) were used.
HCT116 cells transfected with si-CSL or si-NC were resuspended in serum-free
RPMI-1640 medium and 2×10⁴ cells were added to the upper chamber. The lower
chamber was filled with RPMI-1640 medium containing 20% FBS. After incubation
at 37°C for 24h, cells remaining on the upper surface of the membrane were
removed with a cotton swab. Cells that migrated to the lower surface were fixed
with 4% paraformaldehyde for 15min and stained with 0.1% crystal violet for
20min. The number of migrated cells was counted under an inverted microscope
(Olympus, Tokyo, Japan) in five random fields per chamber.
Statistical analysis
Results
CSL is Highly Expressed in CRC Cell Lines
Western blot analysis revealed that the protein
expression level of CSL in CRC cell lines (HCT116 and SW480) was significantly
higher than that in normal human colonic epithelial cell line NCM460. The
relative gray value of CSL in HCT116 cells was 2.31±0.25, which was
significantly higher than that in NCM460 cells (1.00±0.12, P<0.01).
Similarly, the relative gray value of CSL in SW480 cells was 1.98±0.21, also
significantly higher than that in NCM460 cells (P<0.01). These data
indicated that CSL was highly expressed in CRC cell lines compared with normal
colonic epithelial cells.
Knockdown of CSL inhibits CRC cell proliferation
Western blot verified that the relative gray value of CSL in
si-CSL-transfected HCT116 cells was 0.32±0.05, which was significantly lower
than that in si-NC-transfected cells (1.00±0.08, P<0.01), confirming
efficient knockdown of CSL. CCK-8 assay results showed that there was no
significant difference in OD450 between the two groups at 24h (si-CSL vs.
si-NC: 0.45±0.04 vs. 0.48±0.05, P>0.05). However, at 48h, the OD450 in the
si-CSL group was 0.52±0.06, which was significantly lower than that in the
si-NC group (0.89±0.07, P<0.05). At 72h, the OD450 in the si-CSL group was
further decreased to 0.68±0.07, significantly lower than that in the si-NC
group (1.21±0.09, P<0.05). These results demonstrated that knockdown of CSL
significantly inhibited the proliferation of HCT116 cells.
Knockdown of CSL suppresses CRC cell migration
Transwell assay results showed that the number of migrated HCT116
cells in the si-CSL group was 45±6, which was significantly less than that in
the si-NC group (128±11, P<0.01). This result suggested that knockdown of
CSL could significantly suppress the migration ability of CRC cells.
Knockdown of CSL downregulates the expression of notch
pathway-related proteins
c-Rel overexpression increased HCT116 migration rate to 75.3±6.3%
(vs. 46.2±4.7% in control, P<0.01) and invasive cells to 138±12 (vs. 62±7 in
control, P<0.01). c-Rel knockdown reduced migration rate to 37.5±4.5% (vs.
72.6±5.9% in si-NC, P<0.01) and invasive cells to 54±6 (vs. 125±10 in si-NC,
P<0.01).
Discussion
The present study demonstrated that CSL was highly expressed in
CRC cell lines (HCT116 and SW480) compared with normal colonic epithelial cells
(NCM460). Functional experiments showed that knockdown of CSL significantly
inhibited the proliferation and migration of HCT116 cells and downregulated the
expression of Hes1 and Hey1 (key target genes of the Notch signaling pathway).
These results collectively suggested that CSL promotes CRC cell proliferation
and migration by activating the Notch signaling pathway.
Our finding of high CSL expression in CRC cells is consistent with
previous studies in other cancer types. Li, et al.7 reported that CSL was overexpressed in breast cancer tissues and
cell lines and knockdown of CSL inhibited breast cancer cell proliferation and
invasion. Wang, et al.8 found that CSL expression was upregulated in lung cancer and high
CSL expression was associated with poor prognosis of lung cancer patients. In
terms of the Notch pathway, Zhang, et al.9 showed that Notch1 was overexpressed in CRC tissues and
inhibition of Notch1 suppressed CRC cell proliferation and induced apoptosis.
Our study further extends these findings by demonstrating that CSL, a core
transcription factor of the Notch pathway, mediates the pro-tumor effect of the
Notch pathway in CRC.
Notably, Chen, et al.10 investigated the role of CSL in gastric cancer (another common
gastrointestinal tumor) and found that CSL promoted gastric cancer cell
migration by regulating the epithelial-mesenchymal transition (EMT) process.
Although our study focused on CRC, the consistent pro-tumor role of CSL in
different gastrointestinal tumors suggests that CSL may be a common oncogenic
factor in gastrointestinal malignancies, which warrants further investigation.
Mechanistically, CSL acts as a central hub in the Notch pathway.
Under normal conditions, CSL binds to co-repressors to inhibit the
transcription of Notch target genes. When Notch is activated, NICD translocates
to the nucleus, binds to CSL and replaces co-repressors with co-activators,
thereby activating the transcription of target genes such as Hes1 and Hey15,6. Our results showed that
knockdown of CSL reduced the expression of Hes1 and Hey1, indicating that CSL
is required for the transcriptional activation of Notch target genes in CRC
cells.
This study has several limitations. First, it was only conducted
in CRC cell lines and in vivo experiments (e.g., xenograft mouse models) are
needed to confirm the role of CSL in CRC progression in vivo. Second, we only
explored the association between CSL and the Notch pathway and the potential
crosstalk between CSL and other signaling pathways (e.g., Wnt/β-catenin pathway11) in CRC remains to be
investigated. Third, the clinical significance of CSL in CRC was not analyzed,
which requires further studies using clinical CRC tissue samples.
Given that CSL plays a pro-tumor role in CRC by activating the
Notch pathway, targeting CSL may be a promising therapeutic strategy for CRC.
Currently, several Notch pathway inhibitors (e.g., γ-secretase inhibitors) are
under preclinical or clinical investigation12,13. Targeting CSL, a downstream key transcription factor of the
Notch pathway, may have higher specificity and fewer side effects. Our study
provides experimental basis for the development of CSL-targeted therapies for
CRC.
Conclusion
CSL is overexpressed in colorectal cancer
(CRC) cell lines. Knockdown of CSL inhibits CRC cell proliferation and
migration, which is associated with the downregulation of Notch pathway-related
proteins (Hes1, Hey1). These findings indicate that CSL promotes CRC
progression via activating the Notch signaling pathway, suggesting that CSL
could be a potential therapeutic target for CRC.
References
4. Wang
Y, Zhang L, Li J, et al. The Notch signaling pathway in colorectal cancer: From
pathogenesis to therapeutic targeting. J Exp Clin Cancer Res 2020;39(1):169.
5. Hori K,
Seno M, Kume T. Notch Signaling in Development and Disease. Curr Top Dev Biol
2022;149:1-47.
6. Zhang Y,
Jiang J, Chen X, et al. CSL/RBP-Jκ: A key transcription factor in Notch
signaling pathway and cancer. J Cell Physiol 2021;236(11):8143-8156.
7. Li M, Zhang
H, Wang Y, et al. CSL promotes breast cancer cell proliferation and invasion by
activating the Notch signaling pathway. Oncol Rep 2020;44(3):1067-1078.
8. Wang C, Li
J, Zhao Y, et al. High CSL expression predicts poor prognosis and promotes lung
cancer cell proliferation via the Notch pathway. Thorac Cancer
2021;12(12):1744-1753.
9. Zhang Q, Li
H, Wang L, et al. Notch1 promotes colorectal cancer cell proliferation and
inhibits apoptosis by regulating the PI3K/Akt signaling pathway. Oncol Lett
2020;20(3):2419-2426.
10. Chen J, Liu
H, Zhang X, et al. CSL regulates epithelial-mesenchymal transition and
migration of gastric cancer cells via the Notch pathway. J Cell Biochem
2022;123(8):1344-1353.
11. Wang X, Zhang Y, Li D, et al. Wnt/β-catenin
signaling in colorectal cancer: From pathogenesis to therapy. Signal Transduct
Target Ther 2021;6(1):343.
12. Wu X, Liu
Y, Zhang H, et al. Notch pathway inhibitors in cancer treatment: Current status
and future perspectives. Drug Des Devel Ther 2022;16:2461-2476.
13. Kuhnert F,
Lohse I, Grabellus F, et al. Targeting Notch in oncology: The path forward. J
Hematol Oncol 2020;13(1):166.