Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells

Cancer Cell International, Sep 2017

Kittiya Islam, Natthaporn Sueangoen, Parichut Thummarati, Phaijit Sritananuwat, Tuangporn Suthiphongchai

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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)


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Kittiya Islam, Natthaporn Sueangoen, Parichut Thummarati, Phaijit Sritananuwat, Tuangporn Suthiphongchai. Blocking ERK1/2 signaling impairs TGF-β1 tumor promoting function but enhances its tumor suppressing role in intrahepatic cholangiocarcinoma cells, Cancer Cell International,