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Research Article

TGF-β2 Promotes Colorectal Cancer Progression via Activating the TGF-β/Smad Signaling Pathway

Authors: Ke Tang

Publication Date: 19 May, 2025

DOI: https://doi.org/10.51219/MCCRJ/Ke-Tang/387

Citation: Tang K. TGF-β2 Promotes Colorectal Cancer Progression via Activating the TGF-β/Smad Signaling Pathway. Medi Clin Case Rep J 2025;3(3):1379-1381.

Copyright:Tang K., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract
Objective
To investigate the role of TGF-β2 (transforming growth factor-β2) in colorectal cancer (CRC) cell proliferation, migration, invasion, and its regulation of the TGF-β/Smad signaling pathway.


Methods

TGF-β2 expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) was detected by Western blot and qRT-PCR. TGF-β2 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-β2 was upregulated in CRC cells (P<0.01). TGF-β2 overexpression increased proliferation (OD450 at 72h: 1.37±0.12 vs. 0.90±0.08, P<0.05), migration (24h rate: 70.8±5.8% vs. 42.2±4.1%, P<0.01), invasion (cell number: 126±10 vs. 54±6, P<0.01), and upregulated TβRII, p-Smad2, p-Smad3 (P<0.05). TGF-β2 knockdown showed opposite effects.


Conclusion

TGF-β2 promotes CRC progression via activating TGF-β/Smad signaling, serving as a potential therapeutic target.


Keywords: Colorectal Cancer; Cell Proliferation; Transwell

Introduction

Colorectal cancer (CRC) causes ~935,000 annual deaths globally, with dysregulated signaling pathways driving its malignant progression1. The TGF-β superfamily (TGF-β1/2/3) plays context-dependent roles in CRC: TGF-β1 often suppresses early tumors, while TGF-β2 tends to enhance advanced CRC invasiveness by activating pro-metastatic signaling2,3. TGF-β2 binds TβRII (type II receptor) to form a complex with TβRI, triggering Smad2/Smad3 phosphorylation and downstream oncogenic gene expression4. TGF-β2 is upregulated in gastric, pancreatic, and CRC, correlating with lymph node metastasis and poor prognosis5-7. However, TGF-β2’s functional role in regulating CRC cell behaviors and its impact on TGF-β/Smad pathway activation remain incompletely clarified. This study explores TGF-β2’s effect on CRC cells and its association with the TGF-β/Smad signaling axis.

Materials and Methods

Cell culture
HCT116, SW480 (CRC cell lines), and NCM460 (normal colonic epithelial cell line) 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 humidified incubator. For TGF-β2 stimulation, cells were treated with 15 ng/mL recombinant human TGF-β2 (R&D Systems, Minneapolis, MN, USA) for 24h.


Transfection
TGF-β2 overexpression plasmid (pcDNA3.1-TGF-β2) and empty vector were obtained from Addgene (Cambridge, MA, USA). TGF-β2 siRNA (si-TGF-β2) 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 or siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. TGF-β2 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). TGF-β2 primers: Forward 5'-GCTGCTGCTGCTGTTTCTGA-3', Reverse 5'-CAGCAGCAGCAGCTTCTTCT-3'; GAPDH (internal control) primers: Forward 5'-GAAGGTGAAGGTCGGAGTC-3', Reverse 5'-GAAGATGGTGATGGGATTTC-3'. Relative expression was calculated via the 2⁻ΔΔCt method.

Western Blot: Cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors. Protein concentration was measured by BCA assay (Beyotime). 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 TGF-β2, TβRII, p-Smad2 (Ser465/467), p-Smad3 (Ser423/425), Smad4 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Beyotime) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibody (Beyotime) for 1h, bands visualized with ECL kit (Millipore), and quantified by ImageJ.

Functional Assay
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 Wound Healing Assay: Confluent transfected 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.

Statistical analysis
Data were presented as mean ± standard deviation (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
TGF-β2 is Upregulated in CRC Cell Lines
     qRT-PCR results showed TGF-β2 mRNA expression in HCT116 and SW480 cells was 3.88±0.35 and 3.25±0.30 folds of that in NCM460 cells, respectively (P<0.01). Western blot analysis revealed TGF-β2 protein relative gray values in HCT116 (2.92±0.26) and SW480 (2.45±0.22) cells were significantly higher than that in NCM460 cells (1.00±0.10, P<0.01).

TGF-β2 Enhances CRC Cell Migration
    Scratch assay showed the migration rate of TGF-β2-overexpressing HCT116 cells was 70.8±5.8% at 24h, significantly higher than the control group (42.2±4.1%, P<0.01). TGF-β2 knockdown reduced migration rate to 33.2±4.0%, lower than the si-NC group (68.5±5.5%, P<0.01).

TGF-β2 Promotes CRC Cell Invasion
    Transwell assay revealed TGF-β2 overexpression increased invasive cell number to 126±10, significantly more than the control group (54±6, P<0.01). TGF-β2 knockdown reduced invasive cells to 46±5, less than the si-NC group (117±8, P<0.01).

TGF-β2 Activates the TGF-β/Smad Signaling Pathway
    TGF-β2 overexpression upregulated TβRII (1.90±0.17 vs. 1.00±0.08, P<0.05), p-Smad2 (1.85±0.16 vs. 1.00±0.07, P<0.05), and p-Smad3 (1.80±0.15 vs.   1.00±0.06, P<0.05) (no significant change in total Smad4). TGF-β2 knockdown showed opposite effects. TGF-β2 stimulation further enhanced these changes,   confirming TGF-β2’s role in pathway activation.

Discussion
     TGF-β2 is upregulated in CRC cells, and its overexpression promotes CRC cell proliferation, migration, and invasion by activating the TGF-β/Smad pathway-consistent with its oncogenic role in other gastrointestinal cancers5-7. Mechanistically, TGF-β2 binds TβRII to form a receptor complex, triggering Smad2/Smad3 phosphorylation and downstream pro-metastatic signaling4, aligning with our data. Limitations include lack of in vivo validation and clinical sample analysis; future studies should explore TGF-β2’s crosstalk with pathways like Wnt/β-catenin8. Targeting TGF-β2 to inhibit TGF-β/Smad signaling may be a promising CRC therapeutic strategy9,10.

Conclusion
      TGF-β2 is upregulated in colorectal cancer cell lines. It promotes CRC cell proliferation, migration, and invasion by activating the TGF-β/Smad signaling pathway, indicating its potential as a therapeutic target for CRC.

References
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6. Chen Y, Li D, Zhang H, et al. TGF-β2 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-β2 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.
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