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

Human O. viverrini-associated ICC cell lines, HuCCA-1 and KKU-M213, derived from cells of Thai CCA patients, were generously provided by Professor Stitaya Sirisinha (Mahidol University, Bangkok, Thailand) and Professor Banchob Sripa (Khon Kaen University, Khon Kaen, Thailand) respectively. Cells were maintained at 37 °C in HAM’s F-12 media (GIBCO, Grand Island, NY) consisting of 15 mM HEPES, 14 mM NaHCO₃, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) under a humidified 5% CO₂ atmosphere.

For MTT assay, ICC cells (3 × 10³) were plated onto a 96-well plate in 10% FBS media and cultured for 24 h, then treated with 5 ng/mL h-TGF-β1 (R&D Systems, Minneapolis, MN), or 10 μM SB431542 (TβRI kinase inhibitor) (Cayman, Ann Arbor, MI), or 1 μM U0126 (MEK1/2 inhibitor) (Tocris, Bristol, UK), or a combination of h-TGF-β1 and each inhibitor in 10% FBS media for up to 72 h. Control cells were treated with vehicle (4 µM HCl, 0.001% bovine serum albumin (BSA) and 0.025% dimethyl sulfoxide (DMSO)). At indicated times, the numbers of viable cells were determined by replacing media with 100 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (AppliChem, Darmstadt, Germany) in 10% FBS media. Cells were incubated for 4 h, then supernatant was removed and insoluble formazan dye was dissolved in 200 µL of DMSO. A 540 nm was measured using a Multiskan EX Microplate Reader (Thermo Labsystems, Finland).

In the clonogenic assay, 200 ICC cells were plated onto a 24-well plate in 10% FBS media, then treated with TGF-β in the presence and absence of inhibitor. After 7 days, cells were fixed with 4% paraformaldehyde and stained with 0.5% (w/v) crystal violet in 25% methanol. Colony forming ability was determined by measuring the area of crystal violet stain using an Image J program (NIH, Bethesda, MD).

Confluent cells in a 24-well plate were incubated for 24 h with 0.1% FBS media containing h-TGF-β1 with and without SB431542. Control cells were treated with vehicle. Wounds were created by scratching with a 1-mL-pipet tip and wells were washed with 0.1% FBS media, then 0.1% FBS media containing h-TGF-β1 with and without inhibitor were added. Cells were incubated for 24 h to allow migration into wound area. Images under phase contrast microscope (40 × magnification) were recorded and analyzed using an Image J program at 0 h (immediately after wound creation) and at 24 h.

In vitro invasion and migration abilities of ICC cells were determined using Matrigel-coated and -uncoated Transwells (Corning Inc., Corning, NY) respectively. In the in vitro invasion assay, the upper surface of a Transwell polycarbonate membrane (6.5-mm diameter) was coated with 30 μg of Matrigel (BD Biosciences, Bedford, MA). Then a 200 µL aliquot of 10⁵ ICC cells (pre-incubated with h-TGF-β1, SB431542, U0126, or a combination of h-TGF-β1 with each inhibitor in 0.1% FBS media for 24 h, trypsinized and re-suspended in the same media) was added to the upper chamber, while the lower chamber was filled with the same media. Following incubation at 37 °C for 6 or 12 h, non-invaded cells in the upper chamber were removed and invaded cells attached underneath the membrane were stained with 5% crystal violet in 25% methanol. The numbers of invaded cells were counted in five random fields under a light microscope (100 × magnification).

ICC cells (80% confluent) were treated with h-TGF-β1 in 0.1% FBS media with or without SB431542 for 6 and 24 h. RNA was extracted using Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare, Munchen, Germany). Total RNA (1 µg) was reverse transcribed using a random hexamer primer and Improm-II™ reverse transcriptase (Promega BioSciences, Madison, WI). QRT-PCR mixture (20-µL) reaction contained 1 × FastStart Universal SYBR Green Master cocktail (Roche Diagnostics, Mannheim, Germany) and 6 pmol of each specific primer pair (5′-CAGGTGGACCAGCTAACCAA-3′/5′-TGCCAGAGACGCATTGTCA-3′, 5′-CACGACGTCTTCCAGTACCGAGA-3′/5′-CATAGGTCACGTAGCCCACTTGGT-3′, and 5′-GTAACCCGTTGAACCCCATT-3′/5′-CCATCCAATCGGTAGTAGCG-3′ for vimentin and MMP-9 mRNA and 18S rRNA (internal control), respectively). Thermocycling was performed in Stratagene Mx 3000P instrument (Agilent Technologies, Santa Clara, CA) as follows: 95 °C for 10 min; and 40 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min. Relative mRNA levels of treated compared to untreated control cells were determined using 2^−ΔΔCt method [26].

Steady-state mRNA levels of TGF-β, activin and nodal were quantified by qRT-PCR as described above using RNA extracted from 80% confluent cells cultured in 10% FBS media. Specific primer pair for TGF-β, activin and nodal was 5′-AACCCACCCGAAATCTATGAC-3′/5′-GCTGAGGTATCGCCAGGAAT-3′, 5′-GGAGCTCAGACAGCTCTTACC-3′/5′-GCAAATTCTCTTTCTGGTCCCC-3′, and 5′-AGACATCATCCGCAGCCTACA-3′/5′-GTCCATCTGAAACCGCTCTAAG-3′, respectively [27, 28].

ICC cells (80% confluent) were treated with h-TGF-β1 with or without SB431542 or U0126 in 0.1% FBS media. At indicated times, cells were lysed with a solution of 150 mM Tris–HCl pH 7.4, containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 0.1% SDS 1% sodium deoxycholate, 1% Nonidet P-40, 1X protease inhibitor cocktail (Roche Diagnostics), 50 mM NaF, 2 mM Na₃VO₄, 40 mM β-glycerophosphate, and 1 mM dithiothreitol, then subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and incubated with primary antibodies against phospho-Smad2, phospho-ERK1/2, total-ERK1/2 (Cell Signaling Technology, Danvers, MA), vimentin, Slug, β-actin and GAPDH (loading control) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horse-radish peroxidase-conjugated anti-IgG secondary antibodies (Santa Cruz Biotechnology). Chemiluminescent signals of immunoreactive proteins were visualized using Luminata™ Forte Western HRP substrate (Millipore, Billerica, MA) and recorded by a G-Box Chemi XL system (Syngene, Cambridge, UK).

Amounts of MMP-2 and MMP-9 in conditioned media were determined by gelatin zymography and uPA amount by plasminogen-gelatin zymography. Cells (10⁵) were treated with h-TGF-β1 with and without SB431542 or U0126 in 0.1% FBS media. At indicated times, 20 μL aliquots of conditioned media were mixed with non-reducing SDS-PAGE loading buffer and subjected to 7.5% SDS-PAGE containing 1 mg/mL gelatin for detection of MMP-2 and -9, or 10% SDS-PAGE containing 1 mg/mL gelatin plus 10 μg/mL plasminogen for uPA detection. After washing with 2.5% Triton X-100, gels were incubated for 18 h in a solution containing 50 mM Tris–HCl pH 7.5, 10 mM CaCl₂, 1 μM ZnCl₂ and 1% Triton X-100 for gelatinase assay or 100 mM Tris–HCl pH 7.8, 150 mM NaCl and 1% Triton X-100 for uPA assay. Gels were stained with Coomassie blue dye and clear band of 67 and 82kDa in gelatin gel corresponds to MMP-2 and -9 respectively, and of 43 kDa in plasminogen-gelatin gel to that of uPA.

ICC cells (60% confluent) were deposited on coverslips and treated for 24 h with h-TGF-β1 with and without SB431542 or U0126 in 0.1% FBS media, then incubated with phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 2% sucrose, and permeabilized with 0.25% Triton X-100 followed by incubation with 2% BSA in PBS. Cells then were treated with mouse anti-E-cadherin antibody (Santa Cruz Biotechnology), followed by Alexa Fluor^® 488-conjugated anti-mouse IgG secondary antibody (Molecular Probes^®, Eugene, OR). Cell nuclei were stained with DAPI (Molecular Probes^®). Coverslips were mounted with Prolong^® gold antifade reagent (Molecular Probes^®) and examined under a confocal microscope (Olympus FV10; Olympus Corp., Tokyo, Japan).

ICC cells (2.5 × 10⁵) in 10% FBS media were cultured in a 6-well plate for 24 h, then transfected with 15 nM siRNA targeting mRNA of MMP-9 (siMMP-9), Smad2/3 (siSmad2/3), Slug (siSlug) (Santa Cruz Biotechnology) or 15 nM AllStars siRNA Negative Control (siNeg) (Qiagen, Valencia, CA) using Lipofectamin^® RNAiMAX reagent (Invitrogen, Carlsbad, CA) following manufacturer’s protocol. After 24 or 48 h, cells were treated with or without h-TGF-β1 in 0.1% FBS media for another 24 h. Invasive ability of treated cells then was determined using the Transwell assay as described above.

TGF-β1 is involved in invasion and metastasis of a number of cancers including CCA [22, 23]. This property was tested for an ICC cell line, KKU-M213, derived from tissue of a Thai ICC patient. Cell migration was evaluated using wound healing assay following treatment of KKU-M213 cells with 5 ng/mL h-TGF-β1 for 24 h. This resulted in a threefold increase in cell migration compared to untreated control, and this effect was abolished upon treatment with 10 µM TβRI inhibitor, SB431542 (Fig. 1a). Using a Transwell assay, h-TGF-β1 also significantly induced in vitro invasive ability of KKU-M213 cells, which also was inhibited by 10 µM SB431542 (Fig. 1b).

TGF-β enhances tumor invasion and metastasis through promoting EMT, a process mediated by a group of transcription factors, such as Slug and Snail, via the canonical signaling Smad pathway [29, 30]. The existence of this TGF-β signaling pathway in KKU-M213 cells was demonstrated by Western blot detection of phospho-Smad2, which was induced within the first 30 min of treatment with 5 ng/mL h-TGF-β1 and was maintained for up to 24 h; phosphorylation was almost completely inhibited by 10 µM SB431542 (Fig. 2a).

EMT induced by h-TGF-β1 was demonstrated by determining the increase in levels of Slug, E-cadherin, vimentin and matrix degrading enzymes. Slug level was raised 1.5–2.5 folds following 2–6 h of exposure to 5 ng/mL h-TGF-β1, which was maintained for up to 24 h (Fig. 2a). However, E-cadherin level remained unchanged during 24 h of h-TGF-β1 treatment, but was translocated from plasma membrane to cytosol (Fig. 2b). Vimentin mRNA level was induced 6 ± 2 folds by h-TGF-β1 (Fig. 2a). Zymography was employed to demonstrate that h-TGF-β1 elevated MMP-9 secretion into conditioned media, being apparent (sixfold increase) after 24 h and increased (14 folds) after another 24 h, but this phenomenon was nearly completely abolished by SB431542 (Fig. 2c). The cells also secreted small quantities of uPA and MMP-2, both of which were not affected significantly by exposure to h-TGF-β1 (Fig. 2c). Silencing MMP-9 gene expression by siMMP-9 reduced TGF-β1-induced MMP-9 secretion by approximately 60% (Fig. 3a). This silencing effect significantly inhibits both basal (by 50 ± 10%) and h-TGF-β1-activated invasiveness (by 48 ± 5%) (Fig. 3b).

Not only Smad but ERK1/2 has been reported to act as downstream effectors contributing to TGF-β-induced EMT [31]. The notion that in ICC cells h-TGF-β1 also acts via MEK/ERK pathway was tested by investigating the effects of MEK1/2 inhibitor U0126 on h-TGF-β1-induced invasiveness of KKU-M213 cells. Exposure to 5 ng/mL h-TGF-β1 for 24 h resulted in 2- to 6-fold increase in ERK1/2 phosphorylation, which was inhibited by 10 µM SB431542 (Fig. 4a and Additional file 1: Figure 1a). After 24 h in the presence of 1 µM U0126, h-TGF-β1-induced vimentin expression was reduced 23 ± 9% (Fig. 4b and Additional file 1: Figure 1b) and MMP-9 secretion almost 75% (Fig. 4c), but not that of Smad2/3 phosphorylation or Slug expression (Fig. 4d). In addition, U0126 attenuated TGF-β ability to reduce E-cadherin membrane localization (Fig. 4e) and to induce invasion by approximately 70% (Fig. 4f). These results demonstrate that TGF-β1-induced KKU-M213 cell invasiveness through induction of EMT and MMP-9 secretion was, at least in part, through activation of ERK1/2 pathway. The effects of TGF-β and U0126 on invasion were also confirmed in another ICC cell line, HuCCA-1, which showed 2.5-fold increase in cell invasiveness upon TGF β stimulation. This effect could be inhibited (to some extent) by co-treatment with U0126 (Additional file 2: Figure 2). Interestingly in the absence of h-TGF-β1, invasiveness of both cell lines was also attenuated by U0126, indicating that ERK1/2 pathway was involved also in ICC cell basal invasiveness.

Smad phosphorylation and Slug production are well-known downstream regulators of TGF-β1-induced progression of many types of cancers [29, 30]. Thus, we examined their roles in ERK1/2 activation and invasiveness of h-TGF-β1-induced KKU-M213 cell by knocking down Smad2/3 and Slug expression levels by means of siRNA. Knocking down Smad2/3 in KKU-M213 cells reduced h-TGF-β1 ability to induce ERK phosphorylation 35 ± 9%, but not in the case with Slug knock down cells (Fig. 5a and Additional file 3: Figure 3a). Smad2/3 silencing also suppressed Slug (30 ± 10%) and vimentin (50 ± 10%) expression levels (Fig. 5a and Additional file 3: Figure 3c and d), cell invasion (45 ± 8%) (Fig. 5b), cell migration (47 ± 4%) (Fig. 5c) and MMP-9 secretion (60 ± 15%) (Fig. 5d). Similarly, Slug silencing inhibited h-TGF-β1-induced KKU-M213 cell migration (40 ± 6%), cell invasion (39 ± 3%), vimentin expression (75 ± 15%) and Smad phosphorylation (26 ± 5%) (Fig. 5a–c and Additional file 3: Figure  3b and d) but only marginally affected MMP-9 secretion (Fig. 5d).

In general, TGF-β1 exhibits anti-proliferative and apoptotic activities during the early stages of tumorigenesis [12]. However, in the later stages of cancer progression, transformed cells are able to escape from this TGF-β1 growth inhibitory effect [13]. In order to determine if this latter phenomenon also applies in ICC, KKU-M213 cells were cultured in 10% FBS media and permitted to proliferate (4- to 5 folds) over a period of 72 h (Fig. 6a). Surprisingly, the proliferation is significantly reduced 25 ± 4% upon h-TGF-β1 (5 ng/mL) treatment (Fig. 6a). U0126 (1 µM) also possessed similar anti-proliferative effect and h-TGF-β1 in combination with inhibitor does not significantly enhance the effect of either one alone (Fig. 6a).

Colony formation assay was performed to investigate the long-term effect of h-TGF-β1 and U0126 on proliferation of the two ICC cell lines, KKU-M213 and HuCCA-1. The ability of cells to proliferate was quantitated from determination of colony area. KKU-M213 cells were sensitive to TGF-β anti-proliferative activity (74 ± 1% reduction in colony area after 7 days of TGF-β-treatment compared to control), while blocking ERK activation with U0126 alone only slightly reduced colony area (13 ± 1% compared to control), but interestingly, when combined the colony area was further diminished (84 ± 1% compared to control) (Fig. 6b). Similarly with HuCCA-1 cells, TGF-β produced 14 ± 5% reduction in colony area, U0126 treatment alone 36 ± 5% reduction and the combination of TGF-β and U0126 54 ± 2% reduction (Additional file 4: Figure 4). These results indicate that blocking ERK1/2 activation assisted in TGF-β anti-proliferative function in ICC cells.