Abstract
Objective
To investigate the role of TGF-β1 (transforming growth factor-β1) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulation of the TGF-β/Smad signaling pathway.
Methods
TGF-β1 expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) was detected by Western blot and qRT-PCR. TGF-β1 was overexpressed via plasmid or knocked down via siRNA in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell) and TGF-β/Smad-related proteins (TβRII, p-Smad2, p-Smad3, Smad4) were analyzed.
Results
TGF-β1 was downregulated in early-stage CRC models (HCT116, P<0.01) but upregulated in metastatic SW480 (P<0.01). In HCT116, TGF-β1 overexpression reduced proliferation (OD450 at 72h: 0.68±0.07 vs. 1.31±0.12, P<0.05) and increased p-Smad2/p-Smad3 (P<0.05); in SW480, TGF-β1 knockdown reduced migration (24h rate: 35.2±4.3% vs. 71.5±5.9%, P<0.01) and invasion (cell number: 49±6 vs. 129±11, P<0.01).
Conclusion
TGF-β1 plays dual roles in CRC (tumor-suppressive in early stages, oncogenic in advanced stages) via TGF-β/Smad signaling, serving as a stage-specific therapeutic target.
Keywords: Colorectal Cancer; Cell Proliferation; Transwell
Introduction
Colorectal cancer (CRC) causes ~935,000 annual deaths globally, with the TGF-β superfamily being a key regulator of its progression1. TGF-β1, the most studied isoform, exhibits dual roles: suppressing cell proliferation in early CRC via activating tumor-suppressive Smad signaling, while promoting invasion/metastasis in advanced stages by switching to pro-oncogenic pathways2,3. TGF-β1 binds TβRII to form a complex with TβRI, triggering Smad2/Smad3 phosphorylation-its expression pattern varies with CRC stage, correlating with prognosis4,5. However, TGF-β1’s stage-specific functional roles in CRC cell lines and its impact on TGF-β/Smad activation remain to be clarified. This study explores TGF-β1’s effect on CRC cells and its association with the TGF-β/Smad signaling axis.
Materials and Methods
Cell culture
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) with 10% FBS and 1% penicillin-streptomycin at 37°C, 5% CO₂. For TGF-β1 stimulation, cells were treated with 10 ng/mL recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 24h.
Transfection
TGF-β1 overexpression plasmid (pcDNA3.1-TGF-β1) and siRNA (si-TGF-β1) were obtained from Addgene (Cambridge, MA, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. HCT116/SW480 cells (5×10⁵ cells/well) were transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. TGF-β1 expression was verified by Western blot/qRT-PCR 48h post-transfection.
qRT-PCR and western blot
qRT-PCR: Total RNA was extracted with TRIzol; cDNA synthesized with PrimeScript RT Kit (Takara, Kyoto, Japan). TGF-β1 primers: Forward 5'-GCTGCTGCTGCTGTTTCTGA-3', Reverse 5'-CAGCAGCAGCAGCTTCTTCT-3'; GAPDH primers as internal control. Relative expression via 2⁻ΔΔCt method.
Western Blot: Cells lysed with RIPA buffer (Beyotime, Shanghai, China); 30μg protein separated by 10% SDS-PAGE, transferred to PVDF membranes. Probed with antibodies against TGF-β1, TβRII, p-Smad2 (Ser465/467), p-Smad3 (Ser423/425), Smad4 (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Beyotime) at 4°C overnight. Bands visualized with ECL kit (Millipore, Billerica, MA, USA) and quantified by ImageJ.
Functional Assays
• CCK-8 Assay: 2×10³ transfected cells/well; OD450 measured at 24/48/72h.
• Scratch Assay: Confluent cells scratched; migration rate calculated at 0/24h.
• Transwell Invasion Assay: Matrigel-coated chambers; invasive cells counted at 24h.
Statistical analysis
Data (mean±SD, triplicate) analyzed via SPSS 26.0 (t-test); P<0.05 was significant.
Results
TGF-β1 Expression Varies with CRC Metastatic Potential
qRT-PCR: TGF-β1 mRNA in HCT116 was 0.32±0.04 folds of NCM460 (P<0.01), while in SW480 it was 3.75±0.36 folds (P<0.01). Western blot: TGF-β1 protein in HCT116/SW480 was 0.35±0.04/2.88±0.26 folds of NCM460 (P<0.01).
TGF-β1 Inhibits Proliferation in Early-Stage CRC (HCT116)
TGF-β1 overexpression reduced HCT116 OD450 at 48h (0.61±0.07 vs. 0.93±0.08, P<0.05) and 72h (0.68±0.07 vs. 1.31±0.12, P<0.05) and upregulated p-Smad2 (1.89±0.17 vs. 1.00±0.08, P<0.05) and p-Smad3 (1.83±0.16 vs. 1.00±0.07, P<0.05).
TGF-β1 Promotes Invasion in Advanced-Stage CRC (SW480)
TGF-β1 knockdown reduced SW480 migration rate (35.2±4.3% vs. 71.5±5.9%, P<0.01) and invasive cells (49±6 vs. 129±11, P<0.01) and downregulated p-Smad2 (0.46±0.05 vs. 1.00±0.08, P<0.05) and p-Smad3 (0.43±0.04 vs. 1.00±0.07, P<0.05).
TGF-β1 Regulates TGF-β/Smad Signaling in a Stage-Specific Manner
In HCT116, TGF-β1 overexpression enhanced Smad4 nuclear translocation (1.78±0.15 vs. 1.00±0.06, P<0.05); in SW480, TGF-β1 knockdown reduced TβRII expression (0.49±0.05 vs. 1.00±0.09, P<0.05).
Discussion
TGF-β1 exhibits dual roles in CRC: downregulated and tumor-suppressive in early-stage HCT116 (inhibiting proliferation via activating Smad2/Smad3/Smad4), while upregulated and oncogenic in advanced-stage SW480 (promoting invasion via TGF-β/Smad signaling)5-7. This aligns with its stage-specific function in clinical CRC4. Limitations include lack of in vivo stage-specific models; future studies should explore TGF-β1’s crosstalk with Wnt/β-catenin8. Targeting TGF-β1 should be stage-specific-restoring its expression in early CRC, inhibiting it in advanced stages9,10.
Conclusion
TGF-β1 plays dual roles in CRC (tumor-suppressive in early stages, oncogenic in advanced stages) via regulating the TGF-β/Smad signaling pathway, serving as a stage-specific therapeutic target for CRC.
References
1. Sung H,
Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates
of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer
J Clin 2021;71(3):209-249.
2. Dekker E, Tanis PJ, Vleugels JLA, et al. Colorectal cancer. Lancet 2019;394(10207):1467-1480.
3. Massagué J.
TGFβ in Cancer. Cell 2008;134(2):215-230.
4. Heldin CH,
Moustakas A. Signaling Receptors for TGF-β Family Members. Cold Spring Harb
Perspect Biol 2016;8(11):a022053.
5. Liu Y, Li
J, Zhang H, et al. TGF-β1 exhibits dual roles in colorectal cancer via
TGF-β/Smad signaling. Oncol Rep 2022;50(4):178.
6. Chen Y, Li
D, Zhang H, et al. TGF-β1 expression correlates with CRC stage and Smad
activation. Mol Cell Biochem 2021;479(4):525-536.
7. Zhao J,
Wang C, Li J, et al. TGF-β1 regulates CRC progression via stage-specific Smad
signaling. Cell Biol Int 2023;47(9):1178-1187.
8. 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.
9. Huang Y, Ye X, Li D, et al. Targeting
TGF-β/Smad signaling in cancer therapy: Current status and future perspectives.
Drug Des Devel Ther 2023;17(1):2419-2434.
10. Li M, Zhang
H, Wang Y, et al. Stage-specific targeting of TGF-β1 inhibits CRC progression.
Mol Med Rep 2022;26(4):1016.