Transforming growth factor β-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4

Free Radical Biology and Medicine 84 (2015) 54–64

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Original Contribution

Transforming growth factor β-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4 Zi-Miao Liu a, Hong-Yu Tseng a, Hung-Wen Tsai b, Fang-Cheng Su a, Huei-Sheng Huang a,n a b

Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan

art ic l e i nf o

a b s t r a c t

Article history: Received 19 January 2015 Received in revised form 9 March 2015 Accepted 25 March 2015 Available online 1 April 2015

Hepatocellular carcinoma (HCC) is one of the most deadly malignancies worldwide because of its high recurrence rate, high metastatic potential, and resistance to drugs. Elucidation of the mechanisms underlying malignancy in HCC is needed to improve diagnosis, therapy, and prognosis. Previously, we showed that transforming growth factor β-interacting factor (TGIF) antagonizes arsenic trioxide-induced apoptosis of HepG2 cells and is associated with poor prognosis and progression of urothelial carcinoma in patients after radical nephroureterectomy. To determine whether TGIF plays a role in HCC tumorigenesis, we compared the expression of TGIF, its downstream targets, and reactive oxygen species levels between HCC HepG2 cells and the more invasive SK-Hep1 cells. Superoxide production, phosphorylation of c-SrcY416 and AKTS473, and expression of TGIF and NADPH oxidase (Nox) were higher in invasive SK-Hep1 cells than in HepG2 cells. TGIF-overexpressing HepG2 xenograft tumors markedly promoted tumor growth and metastasis to the lungs. Overexpression of TGIF in HepG2 cells increased superoxide production from Nox4, matrix metalloproteinase expression, invadopodia formation, and cellular migration/invasion ability. Conversely, knockdown of TGIF in SK-Hep1 cells attenuated these processes. Using gene knockdown and pharmacological inhibitors, we demonstrate that c-Src/AKT is the upstream signaling that regulates TGIF-induced Nox4 activation and subsequent superoxide production. Taken together, our results implicate TGIF as a potential biomarker for prognosis and target for clinical therapy in patients with advanced HCC. & 2015 Elsevier Inc. All rights reserved.

Keywords: Hepatocellular carcinoma Transforming growth factor factor NADPH oxidase Reactive oxygen species AKT Free radicals

β-interacting

Introduction Hepatocellular carcinoma (HCC) is the sixth most common solid cancer and the third leading cause of cancer-related mortality worldwide, with 782,000 new cases occurring in 2012 [1]. The risk factors for HCC include chronic hepatitis B (HBV) or C (HCV) infection, exposure to aflatoxin B1, tobacco, heavy alcohol consumption, and nonalcoholic fatty liver disease [2]. The early stage of HCC is usually asymptomatic and thus difficult to diagnose. Consequently, many patients are diagnosed in the intermediate or advanced stages and have high levels of intrahepatic metastasis, invasive tumors, and extrahepatic metastasis, which are associated with resistance to chemotherapy and poor prognosis [2]. Therefore, elucidation of the mechanisms that lead to HCC tumor progression is critical to improving therapies and the prognosis of HCC.

n

Corresponding author. Fax: þ886 6 2363956. E-mail address: [email protected] (H.-S. Huang).

http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.028 0891-5849/& 2015 Elsevier Inc. All rights reserved.

Several oncogenic pathways are associated with HCC tumorigenesis, including growth factor pathways, Wnt/β-catenin signaling, Jak/STAT, and PPARγ [3,4]. In mammalian cells, reactive oxygen species (ROS) are the by-products of aerobic respiration, and low levels of ROS play important roles in physiological redox signaling [5]. However, low levels of ROS can also induce oxidative stress to cause genomic instability, DNA damage, and possible cancer progression. Increased oxidative stress is associated with many human metastatic tumors, including HCC [6]. ROS levels are higher in patients with HBV and HCV chronic hepatitis than in healthy controls, as shown by electron paramagnetic resonance analysis [7]. Mice deficient in CuZn superoxide dismutase also have elevated oxidative stress and an increased incidence of nodular hyperplasia or HCC [8]. Tumor cells can produce ROS; however, the exact source of these products is unclear. Endogenous ROS are generated by the mitochondria and by NADPH oxidases (Nox's). The Nox family contains five members (Nox1–5) and two dual oxidases (DUOX1 and 2) [9]. These enzymes are involved in multiple physiological and pathophysiological processes [10].

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Transforming growth factor β-interacting factor (TGIF) interacts with Smad2 to inhibit transcription of TGF-β-responsive genes [11,12] and represses retinoid-regulated gene expression via a specific retinoid response element [13]. TGIF is involved in holoprosencephaly [14], differentiation of preadipocytes [15], hematopoietic stem cell function [16], regulation of placenta vascularization [17], and various types of cancer including leukemia, ovarian cancer, gastric carcinoma, and upper tract urothelial carcinoma [18–21]. The role of TGIF in HCC is unclear. TGIF is regulated by epidermal growth factor (EGF), causing malignant transformation of hepatocytes in mice [22]. We previously demonstrated that TGIF is regulated by the EGFR/PI3K/AKT pathway, antagonizing arsenic trioxide-induced apoptosis of HepG2 cells [23]. In addition, Oncomine analysis (http://www.oncomine.org) demonstrated that TGIF is elevated in HCC compared to normal tissue [24]. Therefore, we hypothesized that TGIF might play a critical role in HCC tumorigenesis. This study aimed to compare the expression of TGIF, its downstream targets, and ROS levels between HCC HepG2 cells and the more invasive SK-Hep1 cells and then to address the roles of TGIF in HCC tumorigenesis.

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generation was detected by lucigenin chemiluminescence using a high-performance chemiluminescence analyzer (CLA-2100; Tohoku Electronic Industrial Co. Ltd., Rifu, Japan). Cells (2  105) were incubated with 20 μM lucigenin solution in an absolutely dark chamber of the chemiluminescence analyzer. The chemiluminescence counts were continuously recorded at 10-s intervals for 5 min. Each measurement was performed at least in triplicate. The results were analyzed using chemiluminescence Analyzer Data Acquisition software (Tohoku Electronic Industrial Co). Determination of Nox activity Cells (2  105) were gently scraped and centrifuged at 400 g for 10 min at 4 1C and resuspended in 35 μl/dish of ice-cold DMEM after treatment. The cells (2  104) were then added to prewarmed (37 1C) DMEM containing 1 μM NADPH and 20 μM lucigenin to a final 600-μl volume to initiate the reaction followed by immediate measurement of chemiluminescence in a chemiluminescence analyzer (CLA-2100; Tohoku Electronic Industrial Co.). Appropriate blanks and controls were established, and the chemiluminescence was measured continuously at 10-s intervals for 5 min. The activity of Nox was expressed as counts per million cells.

Materials and methods

Transient transfection and reporter assay

Reagents and antibodies

The transfection was performed using Lipofectamine 2000 reagent according to the manufacturer's instructions with a slight modification. HepG2 cells (8  104) were subcultured in a 12-well plate with 2 ml of fresh culture medium per well for 24 h before transfection. Plasmids were mixed with Lipofectamine 2000 reagent in 1 ml of Opti-MEM, and then incubated with cells at room temperature (25 1C) for 30 min. Cells were incubated in the mixture at 37 1C in a humidified atmosphere of air/CO2 (19/1) for 48 h and then lysed for the measurement of luciferase activity as described previously [25]. The luciferase activity was determined and normalized to the amounts of total proteins.

Nicotinamide adenine dinucleotide phosphate (NADPH), wortmannin, dichlorofluorescin diacetate (DCFH-DA), lucigenin, and tiron were from Sigma–Aldrich Co. (St. Louis, MO, USA). Wizard Plus MiniPrep DNA purification system, pGL-3, and luciferase assay system were from Promega (Madison, WI, USA). Lipofectamine 2000 reagent, TRIzol RNA extraction kit, SuperScript III, Dulbecco's modified Eagle's medium (DMEM), and Opti-MEM were from Invitrogen (Grand Island, NY, USA). Antibodies against TGIF, phospho-AKTS473, and AKT were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin and Flag were from Sigma–Aldrich. Antibodies against phospho-c-SrcY416 were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against matrix metalloproteinases 2 and 9 (MMP2 and MMP9) were from Millipore (Billerica, MA, USA). The shRNA (small-hairpin RNA) of candidate genes and a luciferase control (pLKO.1shluc) plasmid construct were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica (Taipei, Taiwan). The shRNAtargeted sequences were as follows: (i) pLKO.1-TGIF-shRNA targets the human TGIF gene sequence 50 -GCAAGAGATGAATTGCATTAT-30 ; (ii) pLKO.1-Nox4-shRNA targets the human Nox4 gene sequence 50 -AGTAACCAGAACAACTCATAT-30 . Cell culture HepG2 and SK-hep1 cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Cells were grown at 37 1C in a humidified atmosphere of air/CO2 (19/1, v/v). ROS detection The nonfluorescent DCFH can be oxidized by ROS to highly fluorescent DCF. HepG2 or SK-hep1 cells in 35-mm dishes were incubated with 100 μM DCFH-DA in DMEM for 30 min at 37 1C and then trypsinized and washed with ice-cold phosphate-buffered saline (PBS) to quantify the fluorescence emitted from the fluorescent DCF using a fluorescence-activated cell sorter (FACScan, Becton–Dickinson, San Jose, CA, USA). In addition, superoxide

Stable TGIF-overexpressing HepG2 transfectants HepG2 cells were transfected with pcTGIF or empty vector using Lipofectamine 2000 reagent. After incubation in normal medium with 100 μg/ml G418 for about 2 weeks, individual clones resistant to G418 were isolated and continuously cultured in 24-, 12-, and 6-well plates. Stable TGIF-overexpressing HepG2 transfectants (TG) were identified by detection of TGIF mRNA and protein expression. Stable TGIF-silencing SK-hep1 transfectants 293 T cells (8  106) were incubated on 10-cm petri dishes coated with poly-L-lysine for 24 h to package lentiviral vectors. TransIT-LT1 reagent (45 μl) in Opti-MEM (750 μl) was mixed with the packaging vector pCMV-ΔR8.91 (6.75 μg), the envelope vector pMD.G (0.75 μg), and the transfer vector pLKO.1-TGIF-shRNA (7.5 μg) or pLKO.1-shluc (7.5 μg). After incubation at room temperature for 20 min, the plasmid-containing mixture was transferred to the 293 T cells for 16 h. After 1% (w/v) BSA-containing medium was added for 2 days, the supernatant of the culture cells was harvested. The media containing lentiviral vectors were centrifuged at 2500 rpm for 10 min, and the viral supernatants were passed through 0.45-μm filters and stored at  80 1C. SKhep1 cells (5  105) were cultured on 6-cm petri dishes with 4 ml medium, followed by infection with the harvested lentiviral supernatants (1 ml) and Polybrene (8 μg/ml) for 24 h. The infected cells were incubated in 2 μg/ml puromycin-containing medium for selection of stable TGIF-silencing SK-hep1 transfectants.

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Fold changee of F supeeroxide prod duction

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HepG2 12.9x104

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Fig. 1. ROS production and redox signaling were compared between more invasive SK-hep1 and HepG2 cells. SK-hep1 and HepG2 cells were collected to measure (A) ROS production using flow cytometry after DCFH-DA staining and (B) superoxide production and (C) Nox activity using chemiluminescence analysis. (D) The lysates of the two cells were collected to analyze TGIF, Nox4, p-SrcY416, and p-AKTS473 expression using RT-PCR or Western blot. *P o 0.05.

3000

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Fig. 2. TGIF-overexpressing HepG2 cells promote HCC tumor growth and metastasis in xenograft tumor model mice. (A) Stable TGIF-overexpressing HepG2 cells (TG) and their vehicle HepG2 cells (control) were used to measure TGIF expression using Western blot. (B) Photographs show nude mice inoculated with TGIF-overexpressing HepG2 cells (T1–T5) (n ¼ 5) and vehicle control cells (C) (n ¼ 5). (C) Photographs show metastatic nodules on the surface of the lungs in nude mice inoculated with TGIFoverexpressing HepG2 cells (T) and vehicle control cells (C). (D) Tumor size was measured in the TG and control groups every 7 days for 4 weeks. The statistical analysis was determined between the two groups using t test at every time point. (E) Total proteins were extracted from the two groups for measuring TGIF and β-actin expression using Western blot. The samples of mouse blood were collected to analyze human β-globin mRNA expression. *P o 0.05, **P o 0.01 compared with control.

Western blot Cell lysates (30 μg) were separated by SDS–PAGE (10% gels) and then transferred onto a polyvinylidene difluoride membrane using a semidry blotting apparatus. The membrane was blocked with

nonfat dried skimmed milk, and antibodies against TGIF, p-SrcY416, p-AKTS473, AKT, Flag, E-cadherin, vimentin, MMP2, MMP9, Nox4, and β-actin were used as the primary antibodies. Rabbit or mouse IgG-specific antibodies conjugated with horseradish peroxidase were used as the secondary antibodies. β-Actin expression was

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used as an internal control. After development with an enhanced chemiluminescence kit (GE Healthcare), the density of the immunoblots was determined using an image analysis system installed with BIO-ID software (Vilber Lourmat, France). Reverse transcription–polymerase chain reaction (RT-PCR) The RT-PCR method was performed according to the manufacturer's instructions with a slight modification. The mixture contained 3 μg of total RNA, 2.5 μM oligo(dT), 1.5 mM MgCl2, 0.01 M dithiothreitol, and 200 units of SuperScript III reverse transcriptase in a total volume of 20 μl. The products were amplified on an Eppendorf MasterCycler under the following conditions. The RT steps consisted of an RT step (55 1C for 50 min) and a denaturation step (95 1C for 5 min). The PCR steps consisted of a denaturation step (95 1C for 30 s), a primer annealing step (58 1C for 30 s), and an elongation step (72 1C for 1 min) for 30 cycles. For the final step, the duration of the elongation step was 7 min. Finally, samples were mixed with loading buffer (50 mM Tris–HCl, 10 mM EDTA, and 0.25% bromophenol blue) and loaded onto a 1% agarose gel containing 0.1 μg/ml ethidium bromide. The agarose gels were run at 135 V for 20 min in a Tris/ acetate/EDTA buffer. The gels were observed and photographed under

UV light. The primers used were as follows: tgif, sense primer 50 AGATCTGAATTGTGCCAGTGTTTCTCTTTG-30 and antisense primer 50 CCATGGCGGCGCTTCAGAGTGAG-30 ; nox4, sense primer 50 -CTCAGCGGAATCAATCAGCTGTG-30 and antisense primer 50 -AGAGGAACACGACAATCAGCCTTAG-30 ; akt1, sense primer 50 -ATGAGCGACGTGGCTATTGTGAAG-30 and antisense primer 50 -TCAGGCCGTGCCGCTGGC30 ; mmp9, sense primer 50 -GCTGGCAGAGGAATACCTGTACC-30 and antisense primer 50 -CCGAGTTGGAACCACGACGCCC-30 ; e-cadherin, sense primer 50 -TCCCATCAGCTGCCCAGAAA-30 and antisense primer 50 -TGACTCCTGTGTTCCTGTTA-30 ; and gapdh, sense primer 50 -CCATCACCATCTTCCAGGAG-30 and antisense primer 50 -CCTGCTTCACCACCTTCTTG-30 . The gapdh mRNA expression was used as an internal control. Migration/invasion assay A 6.5-mm Transwell chamber with a pore size of 8 μm (Corning, Corning, NY, USA) was used in the migration and invasion assay. All the treated cells were collected and stained with trypan blue (0.03%) to measure the cell viability. The counted viable cells (1.5  104) were resuspended in serum-free medium and seeded on the upper compartment of the chamber for

300

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Fig. 3. TGIF contributes to the migration and invasion of HCC cells. HepG2 cells were transiently transfected with pcTGIF or control vector (pc3.1). (A and B) Equal numbers of pcTGIF-transfected cells or vector-transfected cells or (C and D) stable TGIF-silencing SK-hep1 transfectants (shTG) or shluc-transfected control cells were plated on the upper chamber of Transwell inserts to detect their (A and C) migration activity or (B and D) invasion activity. (E) HepG2 cells were transiently transfected with pcTGIF expression plasmids or with vector control for 24 h. The cells were fixed and stained with cortactin (red) and Alexa Fluor 488–phalloidin (green) to detect the invadopodia formation. Total RNA was extracted from (F) pcTGIF-transfected HepG2 cells or (H) shTGIF-transfected SK-hep1 cells to measure tgif, mmp2, and mmp9 RNA levels using RTPCR. Total lysates were collected from (G) pcTGIF-transfected HepG2 cells or (I) shTGIF-transfected SK-hep1 cells to determine TGIF, MMP2, and MMP9 protein expression using Western blot. *P o 0.05, ***P o 0.001.

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μg) TGIF (μ

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Fig. 3. (continued)

migration assay or on the upper chamber coated with Matrigel (BD Bioscience, Bedford, MA, USA) for invasion assay. In addition, culture medium supplemented with 10% (v/v) fetal bovine serum (FBS) was added into the bottom chamber to act as a chemoattractant. After incubation at 37 1C for 16 h in the migration assay, or for 24 h in the invasion assay, cells that translocated to the bottom surface of the membrane were fixed with 100% methanol for 10 min, followed by staining with 10% Giemsa for 30 min. Then the stained cells were determined by counting five random fields per chamber and quantifying the results by image analysis with the software ImageJ. The relative migration and invasion ability is the ratio of translocated cells of treated cells to those of vehicle control and is designated as migration (%) and invasion (%), respectively. Means were based on the numbers from the triplicate wells of each reaction.

samples were collected from the sacrificed mice to analyze the human β-globin and mouse gapdh gene expression in each group using RT-PCR. In addition, the proteins were extracted from tumor tissues to detect the TGIF and β-actin expression. Tumor size (V) was calculated weekly, according to the formula V (mm3) ¼ 0.52  (width)2  (length).

Immunofluorescence analysis

Results

HepG2 cells were transfected with various plasmids for 24 h and plated on a glass coverslips. The cells were washed and fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100, and then blocked with 10% FBS in PBS. The cells were then stained with anti-cortactin antibody and/or Alexa Fluor 488–phalloidin (Invitrogen) for detection of invadopodia formation. The images were captured on a Leica TCS SP2 confocal microscope (Leica, Wetzlar, Germany) and analyzed using a 40  oil-immersion objective.

Invasive SK-hep1 cells have higher superoxide production, Nox activity, c-SrcY416 and AKTS473 phosphorylation, and TGIF and Nox4 expression than do HepG2 cells

Xenograft tumor model Stable TGIF-overexpressing HepG2 transfectants (TG) or mock (control) cells (2  106) were suspended with 0.2 ml PBS (pH 7.4) and mixed with 20% (v/v) Matrigel for xenograft tumorigenesis assay. The cells were then subcutaneously inoculated into the backs of 8-week-old nude mice (Balb/C). The mice were monitored for overall health, tumor volume, and total body weight. Tumor growth was measured every 7 days for 4 weeks. The blood

Statistical analysis Representative results are presented. All experiments were performed at least three times. The statistical analysis was determined using t test. All values are displayed as means 7 SD for three determinations. P o 0.05 was taken as statistically significant.

TGIF and ROS levels were compared between HepG2 cells (higher differentiated grade and lower invasive ability) and SKhep1 cells (lower differentiated grade and higher invasive ability). ROS levels (Fig. 1A), superoxide production (Fig. 1B), and Nox activity (Fig. 1C) were elevated in SK-hep1 cells about twofold over that of HepG2 cells. SK-hep1 cells also exhibited more phosphorylated c-SrcY416 and AKTS473 and higher levels of TGIF and Nox4 expression than did HepG2 cells (Fig. 1D). TGIF overexpression in HepG2 xenograft tumors markedly accelerates tumor growth and metastasis To investigate the role of TGIF in tumor growth and metastasis in vivo, a xenograft tumor model derived from stable TG cells was used. Mock-transfected cells served as controls (Fig. 2A). As shown

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Fig. 4. TGIF-induced ROS were produced from Nox4 in HCC cells. (A–C) HepG2 cells were transfected with pcTGIF or control vector, respectively. (D–F) Stable transfectants of shluc control cells and shTG cells were used. (A and D) The tgif, nox4, and gapdh mRNA expression was detected using RT-PCR. (B and E) The superoxide production and (C and F) the NADPH oxidase activity were determined by lucigenin chemiluminescence using a high-performance chemiluminescence analyzer. **P o 0.01, ***P o 0.001.

in Figs. 2B and D, the tumor volume of the TG group was about twofold greater than that of the control group at 4 weeks postinoculation (Fig. 2D). In addition, TGIF expression in the harvested tumor tissues of the TG group was higher than that of the control group (Fig. 2E), indicating that TGIF overexpression increases HCC tumor growth. To investigate the effect of TGIF overexpression on tumor metastasis in vivo, we measured the metastatic nodules formed in lungs. No metastatic nodules were found in the lungs of control animals. However, metastatic nodules were observed on the surface of the lung in two (T1 and T5) of five mice (40%) in the TG group (Fig. 2C). Blood samples were collected to extract genomic DNA for detecting the human β-globin gene by PCR. Expression of the human β-globin gene was also detected in the T1 and T5 mice (Fig. 2E), indicating that TGIF increases the metastatic potential of HCC cells, allowing them to extravasate from the circulation to form metastatic nodules in the lungs of xenograft tumor model mice. TGIF overexpression increases migration/invasiveness of HCC cells To further investigate the effect of TGIF overexpression on tumor metastasis, we performed in vitro migration/invasion assays using a Transwell chamber. Overexpression of TGIF in HepG2 cells markedly enhanced cell migration (Fig. 3A) and invasion ability about twofold over those of controls (Fig. 3B). Conversely, knockdown of TGIF by its specific shRNA (pLKO.1-TGIF-shRNA) (shTG) in SK-hep1 cells suppressed cell migration (Fig. 3C), invasion (Fig. 3D), and protein expression by Western blot analysis (data

not shown). The formation of F-actin and cortactin-positive invadopodia increased in TGIF-overexpressing HepG2 cells (Fig. 3E). Furthermore, the mRNA and protein expression of MMP2 and MMP9 was elevated in TGIF-overexpressing HepG2 cells (Figs. 3F and G) and lower in TGIF-silenced SK-hep1 cells (Figs. 3H and I). Collectively, these data indicate TGIF contributes to the migration and invasion of HCC cells in vitro. TGIF overexpression increases Nox4 expression and superoxide production in HCC cells We further evaluated whether superoxide production and Nox activation are involved in the TGIF-mediated migration/invasion ability of HCC cells using chemiluminescence analysis. Overexpression of TGIF in HepG2 cells induced nox4 mRNA expression (Fig. 4A), superoxide production (Fig. 4B), and Nox activity in a dose-dependent manner (Fig. 4C). Conversely, in TGIF-silenced SK-hep1 cells, nox4 mRNA expression (Fig. 4D), superoxide production (Fig. 4E), and Nox activity (Fig. 4F) were markedly downregulated. These results indicate that TGIF overexpression increases Nox4 activity to produce superoxides in HCC cells. Nox4-generated superoxide mediates TGIF-induced cellular migration/invasion ability in HCC cells We further addressed the role of Nox4-generated superoxides in the TGIF-induced cellular migration/invasion ability. TGIFinduced superoxide production was inhibited by Nox4 shRNA

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(Fig. 5A). In addition, TGIF increased mmp9 promoter activity in a dose-dependent manner (Fig. 5B). TGIF-induced mmp9 promoter activation was inhibited by treatment with the superoxide scavenger tiron (Fig. 5C) and by knockdown of Nox4 expression using shNox4 (Fig. 5D). Furthermore, knockdown of Nox4 (Figs. 5E and 5F) and treatment with tiron markedly suppressed the TGIFinduced migration and invasion ability of HCC cells (Fig. 5G).

enhanced the migration ability of HepG2 cells; conversely, knockdown of TGIF greatly inhibited c-Src-induced cellular migration (Fig. 7D). These results suggest c-Src activates the PI3K/AKT pathway to increase TGIF, Nox4, and MMP2/9 expression, thereby promoting cellular migration of HCC cells.

Discussion AKT regulates TGIF expression to induce Nox4-generated superoxide production and cellular migration In addition, overexpression of wild-type human AKT1 (HAAKT1) in HepG2 cells increased TGIF, Nox4, and MMP2/9 expression (Fig. 6A) and also increased superoxide production in a dosedependent manner (Fig. 6B). In addition, AKT1-induced superoxide production and cellular migration were inhibited by shNox4 (Figs. 6C and D). These results indicate that AKT1-induced superoxide production and cellular migration are modulated by Nox4. Furthermore, knockdown of TGIF using shTG also suppressed AKT1-induced MMP2 and MMP9 expression (Fig. 6E). c-Src activates AKT signaling to modulate TGIF-induced Nox4 activation and cellular migration

Fold chaange of ssuperoxide production n p

3.0

Fold d change off LUC activ vity

In addition, we transfected wild-type c-Src (FlagSrc) into HepG2 cells and found increased phosphorylation of c-SrcY416 and AKTS473 and increased expression of TGIF, Nox4, MMP2, and MMP9 (Fig. 7A). Pretreatment with the PI3K-specific pharmacological inhibitor wortmannin blocked c-Src-induced phosphorylation of AKTS473 and expression of TGIF and MMP2/9 protein (Fig. 7B). Conversely, overexpression of dominant negative c-Src kinase (dnSrc) in SK-hep1 cells inhibited phosphorylation of AKTS473 and TGIF expression (Fig. 7C). Overexpression of FlagSrc

The results of this study indicate three novel findings. First, TGIF contributes to the tumor growth and progression of HCC cells. Second, Nox4-generated superoxide production regulated by TGIF is involved in the malignant progression of HCC cells. Third, the c-Src/AKT pathway regulates TGIF expression to enhance superoxide production and malignant progression. In our previous study, immunohistochemical analysis of upper tract urothelial carcinoma specimens from 168 patients showed that TGIF expression correlates with worse prognosis and progression of upper tract urothelial carcinoma [21]. In the present study, we demonstrated that overexpression of TGIF promotes the growth of HepG2 xenograft tumors and metastasis to the lungs (Fig. 2). Our in vitro studies also show that TGIF overexpression in HepG2 cells increased MMP expression, invadopodia formation, and cellular migration/invasion (Fig. 3). These results indicate that TGIF plays an important role in the malignant progression of HCC cells. According to a survey of the Oncomine database (www.oncomine.org), TGIF expression in HCC specimens is elevated approximately 1.75-fold over that of noncancerous tissues [26]. Accordingly, human HCC patients' specimens will be collected to perform immunohistochemical staining to further prove the clinical roles of TGIF in HCC. As shown in Fig. 1, invasive SK-hep1 cells exhibit greater superoxide and Nox activity than do HepG2 cells. In addition, we

*

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m m p9 - l u c

Fold change of LUC activ F vity

Fold change of LUC actiivity F

2.5

*** **

2.5 2.0

** 1.5 1.0 0.5

*** **

2.5 2.0 1.5 1.0 0.5 0.0

0.0

TGIF (μg) 0

mmp9-lu c 3.0

0.1

0.3

0.5

TGIF (μg) 0 shNOX4 (μg) 0

0.5 0

0.5 0.3

Fig. 5. Nox4-produced superoxide is involved in TGIF-induced cellular migration, invasion, and mmp9 promoter activation. (A) HepG2 cells were transiently transfected with pcTGIF plasmids, pLKO.1-Nox4-shRNA, or vector control for 24 h and then collected to determine the superoxide production. The promoter mmp9-luc was used to cotransfect HepG2 cells (B) with various amounts of pcTGIF for 24 h or (C) with 0.5 μg of pcTGIF for 24 h followed by treatment with tiron for 3 h or (D) with 0.5 μg of pcTGIF and various amounts of pLKO.1-Nox4-shRNA for 24 h. Cell lysates were harvested to measure the luciferase activity. In addition, HepG2 cells were transiently (E, F) cotransfected with pcTGIF and pLKO.1-Nox4-shRNA for 24 h or (G) transfected with pcTGIF for 24 h followed by treatment with tiron for 3 h, and viable cells were counted for (E) migration and (F, G) invasion assay. *P o 0.05, **P o 0.01, ***P o 0.001.

Z.-M. Liu et al. / Free Radical Biology and Medicine 84 (2015) 54–64

Control

TGIF+shNox4

TGIF

Control

shNox4

TGIF+shNox4

250

** Invasion (% %)

Migration (%) (

shNox4

*

200

150 100

150 100

50

50 0

0

TGIF (μg) 0 shNox4 (μg) 0

TGIF

250

200

61

1 0

1 1

0 1

TGIF (μg) 0 shNox4 (μg) 0

Control

TGIF

TGIF+Tiron

Tiron

1 0

1 1

0 1

200

Invasion n (%)

*** 150

100

50

0

pcTGIF (μg) 0 Tiron (μM) 0

1 0

1 80

0 80

Fig. 5. (continued)

also proved that Nox4 contributes to the TGIF-induced migration/ invasion of HCC cells (Figs. 4 and 5). Nox4 distributes ubiquitously in tissue but is highly expressed in kidney [10]. Unlike other members of the Nox family, Nox4 can produce superoxide constitutively. The activity of Nox4 is thus thought to be regulated, at least in part, at the transcriptional level [9]. Several transcription factors have been found to regulate Nox4 through its promoter, including Nrf2 [27], E2F1 [28], HIF-1α, and NF-κB [29]. Our search of the TRANSFAC database revealed that the TGIF consensus core sequence (50 -TGTCA-30 ) is present in the Nox4 promoter, suggesting that TGIF might regulate Nox4 expression via direct binding to the consensus sequence or indirect cross talk to other transcriptional factors. Elucidation of the actual mechanism requires further exploration. Elevated oxidative status and ROS production contribute to tumor progression and metastasis [6]. Numerous studies have demonstrated that superoxide production via Nox4 correlates with

tumorigenesis. Increased superoxide production by Nox4 induces angiogenesis and tumor progression via hypoxia-inducible factor 1 and vascular endothelial growth factor expression in ovarian cancer cells [30], promotes survival of pancreatic cancer cells via AKT signaling [31], and modulates G2–M cell cycle progression in melanoma [32]. In addition, Nox4 expression is higher in HCC tissue specimens than in adjacent nontumorous tissues [33]. The authors suggest that Nox4 is not an independent predictor of HCC prognosis after hepatectomy. However, in HCC patients, high levels of Nox4 correlate significantly with the occurrence of satellite nodules [33], an invasive and poor prognostic marker of HCC [34]. Collectively, these results suggest that Nox4-mediated superoxide production contributes to the development of HCC. Our previous studies demonstrated the EGFR/PI3K/AKT-induced binding of human antigen R to the TGIF mRNA 30 -untranslated region stabilizes TGIF mRNA in response to arsenic trioxide in HepG2 cells [36]. In the present study, AKT-induced TGIF expression increases the

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Z.-M. Liu et al. / Free Radical Biology and Medicine 84 (2015) 54–64

HA-AKT1 (μg) 0

1

2

akt1

10

10 1.0

11.5 5

Fold ch hange of uperoxidee productioon su

RT-PCR 11.88

tgif

RT-PCR 1.0

1.6

2.0

nox4

RT-PCR 1.0

1.3

1.4

mmp9

RT-PCR 1.0

3.3

4.1 RT-PCR

gapdh 1.0

1.0 WB

AKT

WB 1.0

4.0

1.0

1.5

2.5 WB

1.0

1.4

1.6

MMP2

WB 1.0

1.6

1.7

MMP9

WB 1.0

1.6

1.9

β-Actin 0.9

2

1.5 1.0 0.5 0.0

0.9

Control

1

2.0

HA-AKT1 (μg) 0 shNox4 (μg) 0

WB 1.0

2

2.5

WB

NOX4

4

HA-AKT1 (μg) 0

5.1

TGIF

6

0

HA

Fold change c of superoxid de producttion

1.0

8

HA-AKT1

1 0

1 1

HA-AKT1 (μg) 0 shTG (μg) 0

0 1

1 0

1 1

0 1

2.6

2.7

0.7

HA HA-AKT1+shNox4 HA AKT1+shNox4

p-AKT p AKTS473

shNox4

1.0

AKT

Miggration (% %)

1.0

3.4

3.6

1.1

1.0

1.6

1.1

0.7

1.0

1.8

1.1

0.7

1.0

1.7

1.0

0.8

1.0

1.1

1.1

1.0

TGIF

400

* MMP2

300

MMP9 200

β-Actin Actin 100

0

HA-AKT1 (μg) 0 0 shNox4 (μg)

1 0

1 1

0 1

Fig. 6. Overexpression of AKT1 increases superoxide production and cellular migration ability. HepG2 cells were transiently transfected with various amounts of HA-AKT1 for 24 h to (A) measure TGIF, Nox4, and MMP expression at their mRNA and protein levels using RT-PCR and Western blot, respectively, or (B) detect superoxide production using a chemiluminescence analyzer. The HA-AKT1 plasmids (1 μg) were used to cotransfect HepG2 cells (C and D) with pLKO.1-Nox4-shRNA (1 μg) for 24 h and the cells were then collected (C) to determine the superoxide production or (D) for cellular migration assay or (E) with pLKO.1-TGIF-shRNA (1 μg) for 24 h and the cell lysates were harvested to determine HA, p-AKTS473, AKT1, TGIF, MMP2/9, and β-actin expression. *P o 0.05.

migration/invasion activity of HCC (Figs. 6 and 7). Activation of the PI3K/AKT/mTOR pathway and increased MMP9 expression are associated with tumor stage and metastasis of HCC as shown by immunohistochemical analysis of tissue microarrays [35]. In addition, activation of that PI3K/AKT pathway and HIF-1α contributes to the

hypoxia-induced epithelial–mesenchymal transition and chemoresistance of HCC [37]. c-Src, a well-known tyrosine kinase, markedly contributes to the development of HCC [38–41]. It can modulate proline-rich tyrosine kinase 2, a nonreceptor tyrosine kinase belonging to the

Z.-M. Liu et al. / Free Radical Biology and Medicine 84 (2015) 54–64

FlagSrc (μ μg) 0

1

FlagSrc (μg) 0 Wort. (nM) 0

2

Flag

2 0

63

2 0 100 100

p-SrcY416 p-SrcY416 1.0

4.8

7.2

Src 1.0

5.3

8.5

1.5

1.0

0.8

1.5

1.7

MMP9

3.3

β-Actin

Nox4 1.0

2.5

0.8

1.0

1.7

0.9

0.8

10 1.0

0.9 09

0.9 09

0.9 09

1.0

1.6

0.6

0.6

1.0

1.5

0.8

0.6

1.0

1.7

0.8

0.5

MMP2

TGIF 1.0

6.5

TGIF

1.9

AKT 1.0

6.0

AKT

p-AKT p AKTS473 1.0

1.0

p-AKTS473 p

1.0

MMP2 1.0

1.9

2.1

1.0

2.0

2.2

1.0

1.0

1.0

0.9

0.9

0.9

MMP9

β-Actin

dnSrc (μg)

0

1

2

1.0

0.6

0.3

1.0

0.5

0.2

1.0

1.0

0.9

1.0

0.8

0.7

1.0

0.9

0.9

p-SrcY416

FlagSrc (μg) 0 μg 0 shTG (μg)

1 0

1 1

p-AKT p AKTS473 AKT TGIF

β-Actin

p-SrcY416 1.0

3.3

3.0

1.0

2.5

0.8

1.0

1.0

1.0

TGIF

β-Actin

Fig. 7. The c-Src/AKT signaling modulates TGIF-induced cellular migration. (A) HepG2 cells were transfected with various amounts of wild-type c-Src expression plasmids (FlagSrc) for 24 h or (B) with 2 μg of FlagSrc followed by treatment with the PI3K-specific pharmacological inhibitor wortmannin for 2 h. The cell lysates were harvested to detect p-SrcY416, p-AKTS473, TGIF, Nox4, and MMP2/9 expression using Western blot. (C) HepG2 cells were transfected with various amounts of dominant negative c-Src plasmids (dnSrc) for 24 h, and the cell lysates were collected to determine p-SrcY416, p-AKTS473, AKT, and TGIF expression using Western blot. (D) The FlagSrc plasmids were used to cotransfect HepG2 cells with pLKO.1-TGIF-shRNA for 24 h, and the viable cells were counted to measure the migration activity.

FAK family, thereby promoting the proliferation and invasion of HCC cells [40]. c-Src phosphorylates EGFR at Y845, leading to enhanced migration and invasion of cancer cells [42]. Phosphorylated EGFR-Y845 has been identified in most HCC specimens [43], which might be regulated by the interaction of c-Src with transmembrane 4 L six family member 5 to enhance migration and invasion of HCC cells [44]. Therefore, we propose that TGIFregulated superoxide production via Nox4 results from c-Src/ EGFR/AKT signaling, which thereby mediates tumorigenesis in HCC. Accordingly, we believe that TGIF not only plays a role in the resistance to arsenic trioxide-induced apoptosis [23] but also

contributes to the regulation of superoxide production from Nox4 to induce malignant progression of HCC. In patients with advanced HCC, TGIF might be a promising therapeutic target and a critical biomarker for poor prognosis.

Acknowledgment This work was supported by grants from the National Science Council (Taipei, Taiwan; NSC102-2321-B-006-019-MY2; NSC1022320-B-006-017).

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