CD4(+) CD25(+) regulatory T cells promote hepatocellular carcinoma invasion via TGF-β1-induced epithelial–mesenchymal transition

BACKGROUND: CD4(+) CD25(+) regulatory T cells (Tregs), a crucial component of the infiltration of immune cells in tumor microenvironment, are associated with progression and metastasis of hepatocellular carcinoma (HCC). METHODS: The mechanism of Tregs in the invasion and metastasis of HCC was investigated in vivo and in vitro using immunohistochemical analysis, western blot, and quantitative reverse transcription-PCR (qRT-PCR). RESULTS: Analysis of 78 clinical HCC samples indicated that high expression of Tregs was strongly associated with poor cancer-free survival and overall survival of patients. The reduced expression of E-cadherin and enhanced expression of Vimentin and transforming growth factor-beta 1 (TGF-β1) were found in HCC tissue compared with normal liver tissue. The HCC Hepa1-6 cells were treated with the supernatant of Tregs-conditioned medium (Tregs-CM) to investigate the epithelial-mesenchymal transition (EMT) and TGF-β1. Western blot and qRT-PCR also showed that down-regulated E-cadherin and up-regulated Vimentin and TGF-β1 were found in Tregs-CM-treated Hepa1-6 cells. An experiment of tumorigenicity in C57 mice showed larger and heavier tumors in Tregs-CM-treated group than in the control group. Tregs produced higher TGF-β1 compared with Tregs treated with FOXP3 shRNA. TGF-β1 with neutralizing antibodies was used to deplete TGF-β1 in Tregs-CM, which enhanced expression of E-cadherin, reduced expression of Vimentin and TGF-β1, and decreased migratory and invasive capacity of Hepa1-6 cells. CONCLUSION: Tregs could promote the invasion and migration of Hepa1-6 cells, which are possibly maintained by TGF-β1-induced EMT. This study showed that the development of therapeutic strategies against TGF-β1 pathway is valuable in HCC therapy.