TGF-β1 signaling-mediated lymphangiogenesis in gastric cancer

Abstract

Background

Recent studies have shown that TGF-β1 may have an important role in gastric cancer progression and metastasis. However, the role of TGF-β1 in lymph node metastasis and lymphangiogenesis, one of the most important steps of gastric cancer dissemination, is largely unknown. The goal of this study was to investigate the role of TGF-β1 signaling in gastric cancer and the molecular mechanisms involved in lymphangiogenesis.

Methods

Two gastric cell line models, MKN45 and KATOIII, were selected for this study. The potential role of TGF-β1 signaling in in vitro lymphangiogenesis was investigated. The expression of TGF-β1 pathway molecules and VEGF-C, a representative prolymphangiogenic factor, was examined by RT-PCR, western blot, or ELISA in response to the TGF-β1 and TGF-β1 receptor I inhibitor treatment. To elucidate whether Smad3 binds to the specific DNA sequences in the VEGFC promoter, we performed an electrophoretic mobility shift assay (EMSA). The cell line-specific effects of the Smad-dependent and Smad-independent pathways on lymphangiogenesis were also examined. Tube formation of lymphatic endothelial cells was assayed on a matrigel to evaluate TGF-β1-activated tumor cell-stimulated lymphangiogenesis.

Results

Two gastric cell line models, MKN45 and KATOIII, showed functional regulation of TGF-β1 signaling pathway. Conditioned media of both cells were able to induce the TGF-β1 signaling pathway in a cell line which only expresses TGF-β receptor II. TGF-β1 induced activation of Smad2/3 and Smad pathway-modulated VEGF-C expression. Phosphorylated and activated Smad3 in the nucleus bound to the promoter of VEGFC in KATO III cells. Of note, in MKN45 cells, the Smad-independent AKT pathway was also activated in response to TGF-β1 and induced VEGF-C expression. Inhibition of TGF-β1 signaling down-regulated the expression of VEGF-C and blocked tube formation of lymphatic endothelial cells in vitro.

Conclusion

TGF-β1 signaling may promote in vitro lymphangiogenesis through VEGF-C production in gastric cancer cells. Cell line-specific Smad-dependent and -independent pathways were able to induce the expression of VEGF-C and enhance tube formation of lymphatic endothelial cells.