Galectin-3 favours tumour metastasis via the activation of β-catenin signalling in hepatocellular carcinoma

Abstract

Background: High probability of metastasis limited the long-term survival of patients with hepatocellular carcinoma (HCC). Our previous study revealed that Galectin-3 was closely associated with poor prognosis in HCC patients.

Methods: The effects of Galectin-3 on tumour metastasis were investigated in vitro and in vivo, and the underlying biological and molecular mechanisms involved in this process were evaluated.

Results: Galectin-3 showed a close correlation with vascular invasion and poor survival in a large-scale study in HCC patients from multiple sets. Galectin-3 was significantly involved in diverse metastasis-related processes in HCC cells, such as angiogenesis and epithelial-to-mesenchymal transition (EMT). Mechanistically, Galectin-3 activated the PI3K-Akt-GSK-3β-β-catenin signalling cascade; the β-catenin/TCF4 transcriptional complex directly targeted IGFBP3 and vimentin to regulate angiogenesis and EMT, respectively. In animal models, Galectin-3 enhanced the tumorigenesis and metastasis of HCC cells via β-catenin signalling. Moreover, molecular deletion of Galectin-3-β-catenin signalling synergistically improved the antitumour effect of sorafenib.

Conclusions: The Galectin-3-β-catenin-IGFBP3/vimentin signalling cascade was determined as a central mechanism controlling HCC metastasis, providing possible biomarkers for predicating vascular metastasis and sorafenib resistance, as well as potential therapeutic targets for the treatment of HCC patients.

Fig. 1. A high level of Galectin-3 expression is significantly associated with vascular invasion and poor survival in HCC patients.

a−c IHC staining shows the expression level of Galectin-3 in HCC tissues and paired adjacent normal tissues, as well as in HCC tissues with or without vascular invasion in the training and testing sets. Scale bars, 100 μm. d The percentage of Galectin-3 IHC staining grade in patients with or without vascular invasion in the training and testing sets. e Tissue IF staining shows the co-expression pattern of Galectin-3 (red) and CD34 (green) in the HCC tumour microenvironment. DAPI, blue. Figure panel pairs representative images taken with different zooming options; Scale bars, 100 μm. f Spearman’s correlation analysis of the association between Galectin-3 and CD34 in the training (left), testing (middle), and vascular invasion (right) sets. g, h Survival analyses between “Galectin-3 high” and “Galectin-3 low” groups for the overall survival (g) and progression-free survival (h) based on IHC staining in the training (left), testing (middle), and vascular invasion (right) sets. N normal, Ca cancer, No without vascular invasion, Yes with vascular invasion. The results represent three independent experiments. **P < 0.01; ***P < 0.001.

Fig. 2. Galectin-3 promotes the angiogenesis and EMT of HCC cells in vitro.

a GO analysis was performed according to the HCC mRNA sequencing data from TCGA to identify Galectin-3-associated GO terms; the top 20 terms are shown. b, c GSEA based on the HCC mRNA sequencing data from TCGA as performed to analyse the correlation between Galectin-3 and angiogenesis (b) and EMT (c). d Western blotting shows the expression of Galectin-3 in HCC cell lines, including Bel-7402, HepG2, Hep3B, SK-Hep1 and Hun7. e qPCR shows the mRNA expression of LGALS3 in Hep3B and SK-Hep1 cells transfected with siControl, siLGALS3#1, siLGALS3#2 or siLGALS3#3. f Western blotting shows the expression of Galectin-3 in Hep3B-vector, Hep3B-Galectin-3, SK-Hep1-vector, SK-Hep1-Galectin-3, HepG2-shControl, HepG2-shGalectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. g–j Tube formation assay (g), Transwell migration assay (h), IF assay for phalloidin (i), and cell adhesion assay (j) in Hep3B-vector, Hep3B-Galectin-3, SK-Hep1-vector, SK-Hep1-Galectin-3, HepG2-shControl, HepG2-shGalectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Scale bars in g, h, and i are 200 μm, 200 μm, and 20 μm, respectively. k PCR array shows the expression of EMT-related genes in Hep3B-vector, Hep3B-Galectin-3, SK-Hep1-vector, SK-Hep1-Galectin-3, HepG2-shControl, HepG2-shGalectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. l Western blotting shows the expression of ECAD, NCAD, vimentin and MMP1 in Hep3B-vector, Hep3B-Galectin-3, SK-Hep1-vector, SK-Hep1-Galectin-3, HepG2-shControl, HepG2-shGalectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Ctrl control, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Activation of the PI3K-Akt-GSK-3β-β-catenin signalling cascade is essential for Galectin-3-induced angiogenesis and EMT of HCC cells.

a IF staining shows the colocalisation of β-catenin (green) and Galectin-3 (red) in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Scale bars, 20 μm. b Western blotting shows the expression of phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-GSK-3β and GSK-3β in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. c, d Hep3B-Galectin-3 cells were treated with DMSO, PF294002 (20 μmol/L), AZD5363 (10 μmol/L) or AR-A014418 (20 μmol/L). Western blotting shows the expression of Galectin-3, phospho-PI3K, PI3K, phospho-Akt, Akt, phospho-GSK-3β and GSK-3β (c). Western blotting of the expression of β-catenin in the whole cell, cytoplasm and nucleus (d). e, f Statistical diagrams of the tube formation assay, Transwell migration assay and cell adhesion assay. g, h Proteome angiogenesis array shows angiogenesis-related soluble proteins (g), and PCR array and western blotting show EMT-related genes/proteins (h) in Hep3B-shControl and Hep3B-shβ-catenin cells based on Galectin-3 overexpression. i Analysis of the IGFBP3 and VIM promoter identified a TCF4-binding site. Moreover, CHIP was performed using IgG and TCF4 antibodies, followed by qPCR in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. j IF staining shows the colocalisation of β-catenin (green) and IGFBP3 (red) (left) or vimentin (red) (right) in Hep3B-vector, Hep3B-Galectin-3, Huh7-shControl and Huh7-shGalectin-3 cells. Scale bars, 20 μm. k Western blotting shows the expression of β-catenin, IGFBP3 and vimentin in Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl and Hep3B-Galectin-3-shβ-catenin cells (upper) and Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells (bottom). Ctrl control, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. Galectin-3 facilitates tumorigenesis and lung metastasis of HCC cells in vivo via β-catenin signalling.

a, b Representative images and photo flux of tumour growth of Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl and Hep3B-Galectin-3-shβ-catenin cells in BALB/c nude mice at day 7, 14 and 21. c, d Representative images and photo flux of tumour growth of Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells in BALB/c nude mice at day 7, 14 and 21. e, f IHC staining shows the expression of CD34, IGFBP3 and vimentin in xenografts. Scale bars, 100 μm. g, h Representative images and photo flux of lung metastasis of Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl and Hep3B-Galectin-3-shβ-catenin cells in BALB/c nude mice at day 14, 21 and 28. i, j Representative images and photo flux of lung metastasis of Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin3-β-catenin cells in BALB/c nude mice at day 14, 21 and 28. k, l H&E staining shows micro-metastatic pulmonary nodules of Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl, Hep3B-Galectin-3-shβ-catenin, Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells in BALB/c nude mice. Scale bars, 100 μm. Ctrl control, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Molecular deletion of Galectin-3-β-catenin signalling synergistically improves the antitumour effect of sorafenib in vitro and in vivo.

a–f After cell adhesion, DMSO or sorafenib (20 μmol/mL) were administered every two days for the colony formation assay, (a, b), CCK8 assay (c, d) and apoptosis assay (e, f) in Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl, Hep3B-Galectin-3-shβ-catenin, Huh7-shControl, Huh7-shGalectin-3, Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells. Scale bars in a and b, 5 mm. g, h Representative images and photo flux of tumour growth of Hep3B-vector, Hep3B-Galectin-3, Hep3B-Galectin-3-shControl, Hep3B-Galectin-3-shβ-catenin, Huh7-shControl, Huh7-shGalectin-3 Huh7-shGalectin-3-vector and Huh7-shGalectin-3-β-catenin cells in BALB/c nude mice treated with DMSO or sorafenib at day 12, 19, and 26. i Mice were sacrificed at day 27. The tumour weights were measured. Ctrl control, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6. High-level Galectin-3-β-catenin-IGFBP3/vimentin axis is correlated with vascular invasion-mediated sorafenib resistance and poor prognosis in large-scale clinical samples.

a–d Sorafenib-sensitive patients without vascular invasion and sorafenib-resistant patients with vascular invasion were selected from the training set. a IHC staining shows the expression levels of Galectin-3, β-catenin, IGFBP3 and vimentin. Scale bars, 50 μm. b–d Tissue IF staining shows the co-expression pattern of Galectin-3 (red) and β-catenin (green) (b); Galectin-3 (red), IGFBP3 (green) and CD34 (magenta) (c); and Galectin-3 (red), vimentin (green) and CD326 (magenta) (d). DAPI, blue. Figure panel pairs the representative images taken with different zooming options; Scale bars, 100 μm. e–h Spearman’s correlation analysis for the relationship between Galectin-3 and nuclear β-catenin (e); Galectin-3 and IGFBP3 (f); Galectin-3 and vimentin (g); nuclear β-catenin and IGFBP3 (h) and nuclear β-catenin and vimentin (i) in the training set according to IHC staining. j–o Analysis of the differences between the high and low expression of nuclear β-catenin (j, k), IGFBP3 (l, m) and vimentin (n, o) for the OS and PFS based on IHC staining in the training set. p, q Kaplan–Meier analysis of the OS (p) and PFS (q) in the training set containing 278 HCC patients according to both Galectin-3 and nuclear β-catenin expression (left), nuclear β-catenin and IGFBP3 expression (middle) and nuclear β-catenin and vimentin expression (right), as assessed by IHC staining. No without vascular invasion, Yes with vascular invasion. β-cat β-catenin, G3 Galectin-3. The results represent three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.