Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
Sritananuwat et al. Cancer Cell Int
Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells
Phaijit Sritananuwat 0 2
Natthaporn Sueangoen 0 1
Parichut Thummarati 0
Kittiya Islam 0
Tuangporn Suthiphongchai 0
0 Department of Biochemistry, Faculty of Science, Mahidol University , Bangkok 10400 , Thailand
1 Present Address: Research Center, Faculty of Medicine Ramathibodi Hospital, Mahidol University , Bangkok , Thailand
2 Present Address: Faculty of Pharmaceutical Sciences, Ubon Ratchathani University , Ubon Ratchathani , Thailand
Background: Transforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells. Methods: TGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/ phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells. Results: h-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine's ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knockdown suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions. Conclusions: Inhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch.
Cholangiocarcinoma; ERK1/2; Invasion; Slug; Smad2/3; TGF-β1
Background
Cholangiocarcinoma (CCA), a bile duct malignancy,
is one of the most severe forms of cancer with a 5-year
survival rate < 5% [
1
]. Although intrahepatic CCA (ICC)
is not frequently found worldwide, its incidence has
increased during the past decades [
2
]. Incidence of ICC
is high in Asia [
3
], particularly in northeast Thailand (96
per 100,000 in males, about 100 times higher than that in
USA and Europe) [
4
].
Risk factors of ICC include hepatolithiasis, primary
sclerosis cholangitis, cirrhosis, and liver fluke
(Opisthorchis viverrini or Clonorchis sinensis) infection, depending
on geographical location [
4
]. Interestingly, the majority of
these risk factors are those associated with inflammation.
In Southeast Asia, including northeast Thailand, O.
viverrini infection is the major risk factor for CCA [
5
]. In a
hamster model, this parasite damages bile duct epithelia,
initiates inflammation, enhances peribiliary fibrosis, and
increases transforming growth factor (TGF)-β, IL-1β and
TNF-α levels [
6, 7
]. In addition, exposure of O.
viverriniinfected hamsters to N-nitrosodimethylamine promotes
liver fibrosis and cholangiocarcinogenesis [8].
TGF-β regulates a diversity of cellular functions, such
as embryogenesis, cell proliferation, inflammation, and
fibrogenesis [
9
]. During liver injury, TGF-β expression is
up-regulated and promotes liver fibrosis [
10
]. However
in carcinogenesis, TGF-β initially functions as a tumor
suppressor in the early stages of the disease, but acts as
a tumor promoter at the later stages [
11
]. This change in
TGF-β from being a tumor suppressor to a tumor
promoter is known as ‘TGF-β paradox’ switch.
In the early stages of cancer development, TGF-β
inhibition of cell proliferation and induction of apoptosis
occur via a variety of mechanisms, viz., enhancing levels
of pro-apoptotic Bcl (Bim) or cyclin-dependent kinase
inhibit (...truncated)