E/N-cadherin switch mediates cancer progression via TGF-β-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma - PubMed (original) (raw)
E/N-cadherin switch mediates cancer progression via TGF-β-induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma
K Araki et al. Br J Cancer. 2011.
Abstract
Background: Epithelial-to-mesenchymal transition (EMT) is a fundamental process governing not only morphogenesis in multicellular organisms, but also cancer progression. During EMT, epithelial cadherin (E-cadherin) is downregulated while neural cadherin (N-cadherin) is upregulated, referred to as 'cadherin switch'. This study aimed to investigate whether cadherin switch promotes cancer progression in cholangiocarcinoma (CC).
Methods: CC cell lines were examined for migration, invasion, and morphological changes with typical EMT-induced model using recombinant TGF-β1. The changes in E-cadherin and N-cadherin expression were investigated during EMT. We also examined E-cadherin and N-cadherin expression in resected specimens from extrahepatic CC patients (n=38), and the associations with clinicopathological factors and survival rates.
Results: TGF-β1 treatment activated cell migration, invasion, and fibroblastic morphological changes, especially in extrahepatic CC HuCCT-1 cells. These changes occurred with E-cadherin downregulation and N-cadherin upregulation, that is, cadherin switch. Patients with low E-cadherin expression had a significantly lower survival rate than patients with high E-cadherin expression (P=0.0059). Patients with decreasing E-cadherin and increasing N-cadherin expression had a significantly lower survival rate than patients with increasing E-cadherin and decreasing N-cadherin expression (P=0.017).
Conclusion: Cadherin switch promotes cancer progression via TGF-β-induced EMT in extrahepatic CC, suggesting a target for elucidating the mechanisms of invasion and metastasis in extrahepatic CC.
Figures
Figure 1
TGF-_β_1 signalling molecule expression and biological activity of rTGF-_β_1 in CC cells. (A) Expression of TGF-β molecules in CC cells. Total cell extracts were separated by SDS–PAGE using 12% gels, and probed with polyclonal antibodies against TGF-_β_1, phospho-Smad2, and Smad4 to detect their expression. (B) Representative examples of wounding experiments in HuCCT-1 and TFK-1 cells cultured with or without rTGF-_β_1 (5 ng ml−1). HuCCT-1 and TFK-1 cells were wounded (time 0) and maintained for 24 h in conditioned medium with or without rTGF-_β_1 (5 ng ml−1). The arrows point to the edges of the wounds. After 24 h, the wound healing is faster in rTGF-_β_1-treated cells than in untreated cells in both cell lines. (C) Migration assays. The mean cell counts (±s.e.m.; _n_=10) of cells that migrated through the pores to the lower surface are shown. HuCCT-1 cells with or without rTGF-_β_1 treatment were tested for migration using a modified Boyden chamber. *P<0.05 vs control cells. (D) Invasion assays. The mean cell counts (±s.e.m.; _n_=10) of HuCCT-1 cells that invaded through the pores to the lower surface are shown. *P<0.05 vs control cells. (E and F) Proliferation assays. The mean optical densities (±s.e.m.; _n_=3) of the CC cells are shown. HuCCT-1 and TFK-1 cells were cultured on 96-wells with or without rTGF-_β_1 (5 ng ml−1). The reagent was injected after 0, 24, 48, 72 or 96 h of culture and the cells were incubated for a further 2 h. The optical densities were detected using a microplate reader. *P<0.05 vs 0 h.
Figure 2
Immunocytochemical analysis of changes in actin filaments in HuCCT-1 and TFK-1 cells in response to rTGF-_β_1 treatment. HuCCT-1 and TFK-1 cells were incubated with rTGF-_β_1 (5 ng ml−1) for 24 h. Actin filaments were stained with Alexa Fluor-conjugated phalloidin (1:50; red).
Figure 3
Basal and activation statuses of phospho-Smad2, Smad4, E-cadherin, and N-cadherin in HuCCT-1 cells treated with TGF-β. HuCCT-1 cells were stimulated with rTGF-_β_1 (5 ng ml−1), and the cells were extracted after 1, 3, 6, 12, 24, 48 or 72 h. The levels of phosphorylated and total proteins were detected by western blotting analysis. C indicates control samples. (A) Time-dependent changes in the expression levels of phospho-Smad2, Smad4, and _β_-actin in response to rTGF-_β_1 stimulation. Densitometric analyses of the data are shown under the western blotting bands. *P<0.05 vs control cells. (B) Time-dependent changes in the expression levels of E-, N-cadherin, and _β_-actin in response to rTGF-_β_1 stimulation. Data represent the means±s.e.m. of triplicate analyses.
Figure 4
Immunohistochemical staining of E-cadherin and N-cadherin in primary EHCC samples. (A) High E-cadherin expression in a primary EHCC. (B) Reduced E-cadherin expression in a primary EHCC. (C) High N-cadherin expression in a primary EHCC. (D) Low N-cadherin expression in a primary EHCC.
Figure 5
Relationships between postoperative survival and E-cadherin and N-cadherin expression. (A) The cancer-specific survival rates at 5 years after surgery were 53.4% for the high E-cadherin expression group and 0% for the low E-cadherin expression group. Kaplan–Meier curves are shown (_P_=0.0059, Log-rank test). (B) The cancer-specific survival rates at 5 years after surgery were 0% for the N-cadherin-positive group and 36.3% for the N-cadherin-negative group. Kaplan–Meier curves are shown (_P_=0.8025, Log-rank test). (C) Number of patients in the following four subgroups: E-cadherin high expression/N-cadherin-positive; E-cadherin high expression/N-cadherin-negative; E-cadherin low expression/N-cadherin-positive; and E-cadherin low expression/N-cadherin-negative. (D) Kaplan–Meier curves of the E-cadherin high expression/N-cadherin-negative and E-cadherin low expression/N-cadherin-positive subgroups are shown (_P_=0.017, Log-rank test). The cancer-specific survival rates at 5 years after surgery were 50.9% for the E-cadherin high expression/N-cadherin-negative group and 0% for the E-cadherin low expression/N-cadherin-positive group.
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