Abstract
Epithelial-mesenchymal transition (EMT) is involved in the characteristics of malignancy, such as invasion, metastasis, and chemoresistance. In biliary tract cancer (BTC), EMT is induced by transforming growth factor-beta 1 (TGF-β1). The EMT is reversible; therefore, it is conceivable that it could be related to some epigenetic changes. We focused on histone deacetylase (HDAC) inhibitors as regulators of TGF-β1 signaling, and investigated their effect on EMT and chemoresistance. We employed four BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, TFK-1, and gemcitabine-resistant TFK-1) and used vorinostat as the HDAC inhibitor. The relative mRNA expression of an epithelial marker (CDH1) and mesenchymal markers (CDH2, vimentin, SNAI1) were measured by qRT-PCR to evaluate factors associated with EMT. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the chemoresistance of each cell line. In addition, NOD/SCID mice were used to evaluate the effect of vorinostat in vivo. In the parent MzChA-1 and TFK-1 cell lines, TGF-β1 induced EMT and chemoresistance; while vorinostat inhibited the EMT and chemoresistance induced by TGF-β1. In gemcitabine-resistant cell lines that highly expressed TGF-β1, vorinostat inhibited EMT and attenuated chemoresistance. We showed that vorinostat inhibits nuclear translocation of SMAD4 which is a signaling factor of TGF-β1, and this is one of the mechanisms by which vorinostat regulates EMT. We also showed that vorinostat attenuates the binding affinity of SMAD4 to the CDH1-related transcription factors SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. Furthermore, combination therapy with vorinostat and gemcitabine improved survival time in the mice xenografted with gemcitabine resistant MzChA-1 cells. In conclusion, vorinostat regulated TGF-β1-induced EMT and chemoresistance through inhibition of SMAD4 nuclear translocation.
Highlights
Biliary tract cancer (BTC), which has an increasing incidence worldwide, has a poor prognosis because it is difficult to diagnose in the early stages
We examined SMAD4, the key signaling factor of TGF-β1 for epithelial-mesenchymal transition (EMT), and found it was directly stimulated by TGF-β1 signaling, with translocation into the nucleus where it upregulates EMT-related transcription factors such as SNAI1, SNAI2, ZEB1, ZEB2, and TWIST [17,18,19,20,21]
The results showed that the expression of class I histone deacetylase (HDAC) (HDAC-1, HDAC-2, HDAC-3, and HDAC-8) were higher in MzChA-1_GR cells than parent MzChA-1 cells (Table 1)
Summary
Biliary tract cancer (BTC), which has an increasing incidence worldwide, has a poor prognosis because it is difficult to diagnose in the early stages. The 5-year survival rate is less than 30% [1,2,3]. Many patients have unresectable BTC due to local disease spread and/or metastases. Gemcitabine (GEM)-based chemotherapy is often used; the antitumor effect is insufficient, and the median survival time is limited to approximately 10 months [4,5]. To improve the prognosis of BTC patients, the molecular biology of the disease has been studied, including the inflammatory cytokines, epithelial-mesenchymal transition (EMT), DNArepair system, cell signaling, and apoptosis [6,7,8,9]. We previously showed that EMT was observed at the invasion front and in regional lymph node metastases with inflammatory cytokine expression in BTC [10]. Transforming growth factor-beta 1 (TGF-β1) induces EMT and chemoresistance, and chemoresistant BTC cells produce IL-6 and TGF-β1 [10]; we considered that these cytokines could play an important role in promoting EMT in BTC
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