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. 2021 Apr 22;18(12):2466-2479.
doi: 10.7150/ijms.54239. eCollection 2021.

Immune checkpoint molecules are regulated by transforming growth factor (TGF)- β 1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma

Ritu Shrestha  1   2 , Kim R Bridle  1   2 , Darrell H G Crawford  1   2 , Aparna Jayachandran  1   2   3
Affiliations

Immune checkpoint molecules are regulated by transforming growth factor (TGF)- β 1-induced epithelial-to-mesenchymal transition in hepatocellular carcinoma

Ritu Shrestha et al. Int J Med Sci. .

Abstract

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-β1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-β1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-β1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-β1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-β1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-β1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-β1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-β1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-β1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-β1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-β1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-β1-mediated EMT and regulation of immune checkpoints in HCC.

Keywords: epithelial-to-mesenchymal transition; hepatocellular carcinoma; immune checkpoint molecules; transforming growth factor-β1.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
EMT induced by TGF-β1 modulates expression of immune checkpoint molecules in HCC cells. (A) qRT-PCR revealed upregulation of PD-L1 and B7-H3 upon treatment with 10 ng/ml of TGF-β1 for 72 h in Hep3B cells. (B) Western blot analysis revealed upregulated expression of PD-L1 and B7-H3 upon treatment with 10 ng/ml of TGF-β1 for 72 h in Hep3B cells. (C) qRT-PCR revealed upregulation of PD-L1 and B7-H3 upon treatment with 10 ng/ml of TGF-β1 for 72 h in PLC/PRF/5 cells. (D) Western blot analysis revealed upregulated expression of PD-L1 and B7-H3 upon treatment with10 ng/ml of TGF-β1 for 72 h in PLC/PRF/5 cells. (n=3, *p<0.05, **p<0.01). GAPDH was used as loading control.
Figure 2
Figure 2
EMT mediated by TGF-β1 in HCC cells is reversible. qRT-PCR revealed upregulation of E-cadherin and Occludin and downregulation of N-cadherin, Vimentin, Snai1 and Fibronectin following EMT reversal assay in (A) Hep3B and (B) PLC/PRF/5 cells (n=3, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001).
Figure 3
Figure 3
TGF-β1 induces reversible EMT in Hep3B cells. (A) Fluorescence microscopy demonstrated lower expression of E-cadherin and higher expression of Vimentin and N-cadherin during EMT whereas higher expression of E-cadherin and lower expression of Vimentin and N-cadherin was observed following MET (scale bar = 200 µm, magnification 20X). (B) Western blot analysis revealed decreased expression of E-cadherin and increased expression of Vimentin and N-cadherin during EMT and upregulation of E-cadherin and downregulation of Vimentin and N-cadherin during reversal or MET (n=3, ***p<0.005, ****p<0.001). GAPDH was used as loading control.
Figure 4
Figure 4
TGF-β1-mediated EMT is reversible in PLC/PRF/5 cells. (A) Fluorescence microscopy revealed decreased expression of E-cadherin and increased expression of Vimentin and N-cadherin during EMT whereas increased expression of E-cadherin and decreased expression of Vimentin and N-cadherin was observed following MET (scale bar = 200 µm, magnification 20X). (B) Western blot analysis revealed downregulation of E-cadherin and Occludin and upregulation of Vimentin and N-cadherin during EMT and upregulation of E-cadherin and downregulation of Vimentin and N-cadherin during MET. (n=3, **p<0.01, ***p<0.005). GAPDH was used as loading control.
Figure 5
Figure 5
TGF-β1-mediated EMT modulates migratory ability of HCC cells. (A) Morphology changes in Hep3B and PLC/PRF/5 cells during EMT and MET (scale bar = 500 µm, magnification 10X). (B) Transwell migration assay revealed increased motility of Hep3B and PLC/PRF/5 cells upon TGF-β1-induced EMT and decreased motility following MET (scale bar = 500 µm, magnification 10X). (C) The wound healing assay demonstrated that the migratory ability is increased upon EMT and decreased following MET in Hep3B and PLC/PRF/5 cells (scale bar = 500 µm, magnification 10X).
Figure 6
Figure 6
Expression of immune checkpoint molecules is reversed following reversal of EMT in Hep3B cells. (A) qRT-PCR, (B) fluorescence microscopy (scale bar = 200 µm, magnification 20X) and (C) western blot analysis demonstrated upregulation of PD-L1 and B7-H3 upon induction of EMT by TGF-β1 and downregulation of PD-L1 and B7-H3 upon reversal of MET. (n=3, * p<0.05, **p<0.01, ***p<0.005, ****p<0.001). GAPDH was used as loading control.
Figure 7
Figure 7
Expression of immune checkpoint molecules is reversed following reversal of EMT in PLC/PRF/5 cells. (A) qRT-PCR, (B) fluorescence microscopy (scale bar = 200 µm, magnification 20X) and (C) western blot analysis demonstrated upregulation of PD-L1 and B7-H3 upon induction of EMT by TGF-β1 and downregulation of PD-L1 and B7-H3 upon reversal of MET. (n=3, *p<0.05, **p<0.01, ****p<0.001). GAPDH was used as loading control.
Figure 8
Figure 8
Knockdown of B7-H3 can reverse TGF-β1-induced EMT in HCC cells. siRNA-mediated silencing of B7-H3 expression in Hep3B cells resulted in reversal of TGF-β1-induced EMT as demonstrated by (A) qRT-PCR and (B) western blot analysis. Knockdown of B7-H3 expression in PLC/PRF/5 cells caused reversal of TGF-β1-induced EMT as demonstrated by (C) qRT-PCR and (D) western blot analysis. GAPDH was used as loading control. (n=3, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001).
Figure 9
Figure 9
Silencing of B7-H3 reduces motility of HCC cells. Transwell migration revealed that number of cells migrating reduced significantly following B7-H3 knockdown in both Hep3B and PLC/PRF/5 cells (scale bar = 500 µm, magnification 10X) (n=3, **p<0.01, ****p<0.001).
Figure 10
Figure 10
Coordinate expression of TGF-β1 and immune checkpoint molecules in HCC patients. Kaplan-Meier survival curves demonstrating the (A) gene expression of TGF-β1/PD-L1 and overall survival, and (B) gene expression of TGF-β1/B7-H3 and overall survival in HCC patient samples. Red curve represents high-risk group, while green curve represents low-risk group . Markers (+) represent censoring samples. X-axis represents the study time in days. Y-axis represents survival probability.
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