Overexpression of Romo1 Promotes Production of Reactive Oxygen Species and Invasiveness of Hepatic Tumor Cells

GASTROENTEROLOGY 2012;143:1084 –1094

Overexpression of Romo1 Promotes Production of Reactive Oxygen Species and Invasiveness of Hepatic Tumor Cells JIN SIL CHUNG,* SUNHOO PARK,‡ SEON HO PARK,* EUN–RAN PARK,§ PU–HYEON CHA,§ BU–YEO KIM,§,储 YOUNG MIN CHUNG,* SEON RANG WOO,§ CHUL JU HAN,¶ SANG–BUM KIM,# KYUNG–SUK SUH,** JA–JUNE JANG,‡‡ KYOUNGBUN LEE,‡‡ DONG WOOK CHOI,§§ SORA LEE,* GI YOUNG LEE,* KI BAIK HAHM,储 储 JUNG AR SHIN,*,¶¶ BYUNG SOO KIM,## KYUNG HEE NOH,*** TAE WOO KIM,*** KEE–HO LEE,§ and YOUNG DO YOO* *Laboratory of Molecular Cell Biology, Graduate School of Medicine, Korea University College of Medicine, Seoul; ‡Department of Pathology, §Division of Radiation Cancer Research, ¶Department of Internal Medicine, and #Department of Surgery, Korea Institute of Radiological and Medical Sciences, Seoul; 储Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon; **Department of Surgery and ‡‡Department of Pathology, Seoul National University School of Medicine, Seoul; §§Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; 储 储Lab of Translational Medicine Gachon University Lee Gil Ya Cancer and Diabetes Institute, Incheon; ¶¶Department of Internal Medicine, Yonsei University College of Medicine, Seoul; and ##Department of Internal Medicine and ***Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Seoul, Republic of Korea

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BACKGROUND & AIMS: Chronic oxidative stress from reactive oxygen species (ROS) produced by the mitochondria promotes hepatocarcinogenesis and tumor progression. However, the exact mechanism by which mitochondrial ROS contributes to tumor cell invasion is not known. We investigated the role of ROS modulator 1 (Romo1) in hepatocellular carcinoma (HCC) development and tumor cell invasiveness. METHODS: We performed real-time, semi-quantitative, reverse transcriptase polymerase chain reaction; invasion and luciferase assays; and immunofluorescence and immunohistochemical analyses. The formation of pulmonary metastatic nodules after tumor cell injection was tested in severe combined immunodeficient mice. We analyzed Romo1 expression in HCC cell lines and tissues (n ⫽ 95). RESULTS: Expression of Romo1 was increased in HCC cells, compared with normal human lung fibroblast cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Knockdown of Romo1 in Hep3B and Huh-7 HCC cells reduced their invasive activity in response to stimulation with 12-O-tetradecanoylphorbol13-acetate. Levels of Romo1 were increased compared with normal liver tissues in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced differentiation, and larger tumor size. CONCLUSIONS: Romo1 is a biomarker of HCC progression that might be used in diagnosis. Reagents that inhibit activity of Romo1 and suppress mitochondrial ROS production, rather than eliminate up-regulated intracellular ROS, might be developed as cancer therapies. Keywords: Liver Cancer; Metastasis; Chemotherapy Resistance; Prognostic Factor.

M

atrix metalloproteinases (MMPs) play an important role in tissue remodeling and organ develop1 ment. However, aberrant regulation of MMP activity has been implicated in tumor progression, including tumor

cell invasion, and increased expression of MMP-2 (gelatinase A, type IV collagenase) and MMP-9 (gelatinase B, type IV collagenase) is observed in various malignant tumors.2 MMP function is controlled at various phases, including gene expression, localization, proteolytic activation, and inhibition of proteolytic activity.1 MMP activity is also regulated by reactive oxygen species (ROS) generated by cytokines, growth factors, and tumor promoters.3,4 ROS induce MMP expression through the mitogenactivated protein kinase pathway, and ROS produced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase have a role in tumor invasion.5–7 Chronic oxidative stress from NADPH oxidase is associated with malignant transformation and increased invasive potential of tumor cells.8,9 Mitochondrial ROS production is also associated with MMP gene expression.4,10 The overexpression of Sod2 enhances the steady-state production of H2O2, resulting in increased MMP expression and enhanced metastasis.11,12 Mitochondrial DNA mutations in the gene encoding NADH dehydrogenase subunit 6 cause a deficiency of complex I activity in the mitochondrial electron transport chain, resulting in increased metastatic potential, which is suppressed by treatment with ROS scavengers.13 Thus, ROS produced by NADPH oxidase and/or mitochondria are associated with tumor cell invasion. ROS modulator 1 (Romo1) was first cloned from the tumor tissue of a patient who was initially sensitive to chemotherapy but became resistant after a recurrence. We found that increased Romo1 expression enhanced cellular ROS levels and oxidative DNA damage.14,15 Further invesAbbreviations used in this paper: DPI, diphenyleneiodinium; FITC, fluorescein isothiocyanate; HCC, hepatocellular carcinoma; MMP, matrix metalloproteinase; NAC, N-acetyl-L-cysteine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Romo1, ROS modulator 1; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; TGF-␤, transforming growth factor-␤; TNF-␣, tumor necrosis factor-␣; TPA, 12-O-tetradecanoylphorbol-13-acetate. © 2012 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.06.038

tigation showed that Romo1 functions as a molecular bridge between tumor necrosis factor–␣ (TNF-␣) signaling and the mitochondria for TNF-␣–induced ROS production.16 Romo1, which is induced by c-Myc, is needed for the degradation of c-Myc after the G1 to S transition in the cell cycle.17 Romo1 is responsible for an increase in ROS of cancer cells, and Romo1-derived ROS are needed for proliferation of both cancer and normal cells, such as IMR-90 cells.18,19 Interestingly, Romo1 expression is increased in most cancer cell lines and senescent cells compared with normal cells.14,15,20 ROS affect chronic liver diseases and hepatocarcinogenesis.21,22 Human hepatocellular carcinoma (HCC) is highly invasive and metastatic, and recurrence after surgical resection is frequent.23,24 Vascular invasion is a major determinant in assessing HCC clinical outcomes, including tumor stage.25–27 These findings prompted us to hypothesize that Romo1-derived ROS might affect invasion and prognosis of HCC. In this study, we evaluated the role of Romo1 as a potent invasiveness factor in cancer cells, especially in HCC, and investigated whether Romo1 has an impact on the prognosis of HCC patients.

Methods Patient Specimens HCC and adjacent liver tissue samples were taken from 95 patients who underwent curative surgical resection at Korea Cancer Center Hospital and Seoul National Hospital. All tissues were serologically positive for hepatitis B surface antigen but not for hepatitis C antibody. Liver tissues adjacent to metastatic liver cancers that originated from primary tumors of other organs were included as normal liver tissue controls and had no signs of fibrosis and cirrhosis. Cases with histopathological evidence of either portal vein invasion or microvessel invasion were classified as vascular invasion⫺positive. After surgical resection, a piece of each tissue sample was immediately frozen and stored in liquid nitrogen until RNA extraction. The serologic presence of hepatitis B surface antigen was considered to be positive evidence of hepatitis B serology. Experiments using patient specimens were performed under approval of the Institutional Review Board.

Semi-Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-Time RT-PCR, and Northern Blotting Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Total RNA concentration and quality were assessed by absorbance at 260/280 nm using a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Reverse transcription was performed with 2 ␮g total RNA treated with iScript reverse transcriptase (Bio-Rad, Hercules, CA) for first-strand complementary DNA synthesis, according to manufacturer’s protocol. Semi-quantitative RT-PCR was performed with Romo1 and 18S ribosomal RNA primers, as described previously.28 Primers and probe sequences for real-time RT-PCR were: Romo1: 5=-CTGTCTCAGGATCGGAATGCG-3= (sense); 5=CATCGGATGCCCATCCCAATG-3= (anti-sense); and 5=-FAMCCATGAATGTGCCAAAGGTGCCGCCA-BHQ-1-3= (probe); 18S ribosomal RNA: 5=-GGAGAGGGAGCCTGAGAAACG-3= (sense);

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5=-TTACAGGGCCTCGAAAGAGTCC-3= (anti-sense); and 5=-FAMTACCACATCCAAGGAAGGCAGCAGGCG-BHQ-1-3= (probe). RTPCR amplification was performed with primers or probes, template complementary DNA, and 2X iQ Supermix (Bio-Rad). Reactions were assayed in triplicate. The relative expression levels of the Romo1 target gene were normalized to the median of the reference gene, 18S ribosomal RNA. Relative Romo1 expression in HCC and adjacent liver tissues was in comparison to normal liver tissues and was analyzed using the comparative threshold cycle (2⫺⌬⌬C(t)) method.29 Northern blotting was performed as described previously.14

Statistical Analysis Patient survival was measured from the time of hepatic resection, with death as the end point. Only death from HCC or liver failure was counted, and living patients were censored to the date of the last follow-up. The threshold ratio for discriminating Romo1 high- and low-risk groups was determined after measuring P values by a log-rank test between 2 groups from all possible combinations based on the Romo1 expression ratio. A 2-fold ratio was identified as the cutoff value that showed the minimum P value in the log-rank test. We evaluated relationships between clinicopathological parameters of HCC and the frequency of Romo1 overexpression using a ␹2 test. For all analyses, a 2-tailed P ⬍ .05 was considered statistically significant. The association of clinicopathological parameters with survival or recurrence time was measured using univariate or multivariate Cox’s proportional hazards modeling. For multivariate analysis, clinical variables were incorporated into the model in a stepwise manner. Overall and disease-free survival rates were estimated by the Kaplan–Meier method with log-rank testing performed using R (version 2.12.0).

Results Romo1 Expression Increases the Invasive Activity of HCC Cells ROS produced in the mitochondria are correlated with tumor invasion.4,10 Because Romo1 expression induces mitochondrial ROS production,14,16 we investigated whether Romo1 expression is associated with HCC cell invasiveness. First, transfection efficiencies of Romo1 small interfering RNA (siRNA) in HCC cells and MCF-7 breast cancer cells were measured by flow cytometry after transfection with Romo1 siRNA-fluorescein isothiocyanate (FITC) transfection for 16 hours. Romo1 siRNA transfection efficiencies in Huh-7, Hep3B, and MCF-7 cells were 96.76%, 97.23%, and 93.08%, respectively. In a previous report, we showed that decreased ROS levels were observed in other cancer cell lines transfected with Romo1 siRNA.19 Therefore, ROS levels were examined in Hep3B cells transfected with Romo1 siRNA-FITC by flow cytometric analysis. Figure 1A shows that Romo1 knockdown down-regulated cellular ROS levels. The left column in Figure 1A represents 2-dimensional dot plots of fluorescence intensities. R3 and R4 contain FITC-positive cells. Cells transfected with Romo1 siRNA-FITC and stained with MitoSOX (a mitochondrial superoxide indicator) were negatively shifted along the y-axis. The same result was obtained with Huh-7 and MCF-7 cells (Figure 1B). Romo1 knockdown by Romo1 siRNA transfection was

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Figure 1. Decreased ROS levels and tumor invasion by Romo1 knockdown. (A) Density plot analysis of decreased ROS levels in Hep3B cells for FITC (FL1) and ROS intensity (FL2). After transfection with Romo1 siRNA-FITC or control siRNA-FITC, cells were stained with MitoSOX, and intracellular ROS levels were measured by flow cytometry. Data are mean and SE of at least 3 independent experiments. ***, ###P ⬍ .001 vs control siRNA by 2-way analysis of variance (ANOVA). (B) Density plot analysis of ROS levels in Huh-7 and MCF-7 cells transfected with Romo1 siRNA-FITC. Data are mean (SE) of at least 3 independent experiments. ***, ###P ⬍ .001 vs control siRNA by 2-way ANOVA. (C) After transfection of Romo1 siRNA for 16 hours, Romo1 knockdown was analyzed by RT-PCR. ␤-actin was used as a loading control. (D) After transfection of Romo1 siRNA for 48 hours, Boyden chamber invasion assay was performed in Huh-7 (1 ⫻ 105) and Hep3B (2 ⫻ 105) cells. Data are mean (SE) of at least 3 independent experiments. ***P ⬍ .001 vs control siRNA by 2-way ANOVA. (E) Conditioned medium was collected and normalized to cell number for gelatin zymography. (F) Huh-7 cells transfected with control siRNA or Romo1 siRNA were intravenously injected into severe combined immunodeficient mice, and the number of lung nodules was counted (n ⫽ 4 for each group). *P ⬍ .05 vs control siRNA by 2-way ANOVA. Ci, control siRNA; Ri, Romo1 siRNA.

confirmed by RT-PCR in Huh-7 and Hep3B cells (Figure 1C). These results demonstrated that Romo1 expression contributed to increased ROS levels in HCC cells. Next, Huh-7 and Hep3B cells were transfected with Romo1 siRNA to knock down Romo1 expression, and a Boyden chamber invasion assay was performed to measure cell invasive activity. As shown in Figure 1D, Romo1 knockdown decreased the invasive activity of the tumor cells. To confirm this result, we performed gelatin zymography assays. Diminished MMP-9 activity was observed in HCC

cells transfected with Romo1 siRNA (Figure 1E). We further confirmed the role of Romo1 in the invasive potential of HCC cells using an in vivo lung metastatic model. Huh-7 cells were transfected with control siRNA or Romo1 siRNA twice for 2 days. Severe combined immunodeficient mice were intravenously injected with the siRNAtransfected cells and examined for the formation of pulmonary metastatic nodules at 8 weeks after tumor cell injection. As shown in Figure 1F, Romo1 knockdown significantly suppressed the formation of visible lung met-

astatic nodules. Taken together, the in vitro invasion assays and the in vivo severe combined immunodeficient mice assays demonstrated that Romo1 expression contributed to the invasive activity of HCC cells.

Tumor Promoter Stimulates ROS Generation and Tumor Invasion Through Romo1 Expression 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ROS production that is mediated by NADPH oxidase or mitochondria. ROS induced by TPA are associated with tumor cell invasion.4,5 We examined whether TPAtriggered ROS generation is mediated by Romo1 using HCC cells after Romo1 depletion. Consistent with a previous report,5 TPA increased ROS generation in HepG2 (Figure 2A) and Chang cells (Supplementary Figure 1A). Interestingly, Romo1 knockdown blocked mitochondrial ROS production in HepG2 cells stimulated by TPA, as determined by MitoSOX (Figure 2B). We also examined TPA-induced cellular ROS generation by staining cells with dihydroethidine (an indicator of intracellular superoxide levels). TPA treatment enhanced superoxide production in HepG2 cells and this increase was blocked by Romo1 depletion (Supplementary Figure 1B). In a previous article, we showed that myxothiazol, an inhibitor of the electron transport at complex III, suppressed Romo1-mediated ROS production.15 Therefore, we examined whether myxothiazol treatment also suppressed mitochondrial ROS production induced by TPA. As shown in Supplementary Figure 1C, TPA-induced ROS generation was suppressed by myxothiazol treatment in HepG2 cells. The same result was obtained in cells treated with N-acetyl-L-cysteine (NAC, an antioxidant) or diphenyleneiodinium (DPI, a flavoprotein inhibitor). Romo1 dependency on TPA-induced ROS generation was also observed in MCF-7 and HeLa cells (Supplementary Figure 1D). Thus, Romo1 mediated TPA-induced ROS generation. Next, we investigated whether TPA-triggered tumor cell invasion was also mediated by Romo1. Romo1 depletion significantly suppressed the invasive activity of HepG2 cells that had been stimulated by TPA (Figure 2C). Treatment with NAC also reduced the TPA-stimulated tumor cell invasion. To further examine Romo1 involvement in TPA-induced tumor cell invasion, we examined the activity of MMP-9, which is also controlled by TPA. As shown in Figure 2D, TPA treatment enhanced the MMP-9 activity in HepG2 cells, but the increased MMP-9 activation was reversed by Romo1 depletion. Romo1-mediated MMP-9 activation was also clearly observed in MCF-7 cells. In contrast to MMP-9, MMP-2 activity was not changed by TPA treatment (Figure 2D). To confirm this result, a MMP-9 promoter assay was performed. Romo1 siRNA and a pGL2-MMP-9 luciferase reporter plasmid were transfected into HepG2, Huh-7, and MCF-7 cells before TPA treatment. As expected from the partial reduction of MMP-9 activity, Romo1 depletion also partially inhibited TPA-induced stimulation of MMP-9 promoter

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activity in HepG2 and Huh-7 cells (Figure 2E). Inhibition of MMP-9 promoter activity by Romo1 siRNA was more severe in MCF-7 cells. To observe the involvement of NADPH oxidase in TPA-induced MMP-9 promoter activity, cells were treated with apocynin, a specific inhibitor of NADPH oxidase, and the MMP-9 promoter assay was performed. As shown in Figure 2E, apocynin partially reduced TPA-induced MMP-9 promoter activity. Another inhibitor of NADPH oxidase, DPI, which also inhibits mitochondrial ROS production, was also used. As shown in Figure 2E, DPI completely blocked TPA-triggered MMP-9 promoter activity. These results demonstrated that TPA induced tumor cell invasion through mitochondria as well as NADPH oxidase. Romo1 knockdown by Romo1 siRNA in HepG2 cells was confirmed by RT-PCR (Supplementary Figure 1E). Growth factors and cytokines increase cellular ROS levels by activating cellular enzymes including NADPH oxidase or other factors.30 –32 Therefore, we investigated Romo1 involvement in the invasive capacity of HCC cells in response to cytokines and growth factors. For response to growth factors, we examined whether transforming growth factor (TGF)–␤ enhanced the invasive activity of HCC cells via Romo1. HepG2 cells were depleted of Romo1 by transfection with Romo1 siRNA, then treated with TGF-␤ for 48 hours. TGF-␤ treatment increased the invasive activity of HCC cells as reported previously.3 However, Romo1 depletion failed to suppress TGF-␤– induced tumor cell invasion (Supplementary Figure 2). We examined whether other factors, such as hepatocyte growth factor, epidermal growth factor, and TGF-␣, also required Romo1 to increase the invasiveness of HCC cells. In contrast to TGF-␤ and TGF-␣, Romo1 knockdown partially suppressed hepatocyte growth factor– and epidermal growth factor–triggered tumor cell invasion (Supplementary Figure 2). The exact mechanism of Romo1 involvement in hepatocyte growth factor– and epidermal growth factor–induced tumor cell invasion remains to be studied. To assess whether TPA treatment induced Romo1 expression, Chang cells were treated with TPA and examined by Western blotting. As shown in Figure 3A, with TPA treatment, Romo1 expression increased after 15 minutes and decreased at 30 minutes. TPA-stimulated Romo1 expression was confirmed by confocal microscopy. As shown in Figure 3B, TPA treatment enhanced Romo1 expression. Red Romo1 signals overlapped with the mitochondria-specific MitoTracker green fluorescent dye (Figure 3B, left panel). ROS production also increased with TPA treatment (Figure 3B, right panel). Next, we conducted a Boyden chamber invasion assay with Chang cells. Romo1 expression enhanced the invasive activity of Chang cells (Figures 3C, D). To examine whether Romo1-mediated ROS production was involved in HCC cell invasion, antioxidants were added to the cells after Romo1 overexpression. As shown in Figures 3C and D, treatment with NAC as well as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

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Figure 2. Blockage of TPA-induced ROS production and invasion by Romo1 knockdown. (A) HepG2 cells were treated with TPA (50 nM) or dimethyl sulfoxide as control for the indicated times. Cells were stained with 2=,7=-dichlorofluorescein diacetate, and ROS generation was analyzed by flow cytometry. *P ⬍ .05; ##P ⬍ .01 vs control by 2-way analysis of variance (ANOVA). (B) Density plot analysis of ROS levels using flow cytometry. After transfection with Romo1 siRNA-FITC, HepG2 cells were treated with TPA, and ROS levels were measured by flow cytometry. Data are mean (SE) of at least 3 independent experiments. **P ⬍ .01; ***, ###, ¥¥¥P ⬍ .001 vs control siRNA by 2-way ANOVA. (C) After HepG2 cells were transfected with Romo1 siRNA, cell invasion was measured by Boyden chamber invasion assay in the presence or absence of either TPA or NAC (5 mM). Data are means of at least 3 independent experiments. ##P ⬍ .01; ***, ###P ⬍ .001 vs control siRNA by 2-way ANOVA. (D) HepG2 and MCF-7 cells were transfected with Romo1 siRNA, and gelatin zymography was performed with or without addition of TPA or TPA coupled with NAC for 24 hours. (E) HepG2, Huh-7, and MCF-7 cells were cotransfected with Romo1 siRNA, and a MMP-9 promoter-containing reporter vector and ␤-galactosidase plasmid, and treated with TPA in the presence of apocynin (100 ␮M) or DPI (10 ␮M) for 24 hours. Cells were lysed, and luciferase activity was measured. Data are means of at least 3 separate experiments. #P ⬍ .05; ##P ⬍ .01; ***, ###P ⬍ .001 vs control siRNA by 2-way ANOVA. Apo, apocynin.

Figure 3. TPA-stimulated Romo1 expression and tumor invasion. (A) Chang cells were treated with TPA, and Romo1 induction was detected by Western blotting using anti-Romo1 antibody. ␤-actin was used as a loading control. (B) Chang cells were stained with anti-Romo1 antibody (red) and MitoTracker (green) after TPA treatment for the indicated times and observed by confocal microscopy (left panel). Images were quantified by MetaMorph software (Universal Imaging). After staining with 2=,7=-dichlorofluorescein diacetate, cells were observed by confocal microscopy and quantified by MetaMorph software (right panel). Data are means of at least 3 separate experiments. ***P ⬍ .001 vs control by 2-way analysis of variance (ANOVA) (C) Chang cells were transfected with Flagtagged Romo1 for 6 hours and were treated with NAC or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid for 6 hours. Cell invasion was measured by Boyden chamber invasion assay. (D) Invading cells were stained and counted in at least 3 fields per filter. Data represent the mean (SE) of at least 3 independent experiments. #P ⬍ .05; **, ##P ⬍ .01; ###P ⬍ .001 vs vector by 2-way ANOVA. (E) Flag-tagged Romo1 expression was examined by Western blotting with anti-Flag antibody. ␤-actin was used as a loading control.

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Figure 4. Romo1 expression in HCC cell lines and tumor tissues. (A) Western blotting was performed on HCC cell lines (Huh-7, HepG2, Hep3B, SK-Hep1, and SNU-449), normal lung fibroblast (IMR-90), other cell lines (HeLa, SiHa, MCF-7, and HEK 293), and Chang cells. (B) Northern blotting was performed on rat liver cancer tissues. Fresh specimens of precancerous and cancerous tissues were obtained from a rat model of HCC. Three animals were used for each sample. RKO cells were used as a positive control for Romo1. The Romo1 expression levels of each paired sample are represented as a number (ratio of Romo1/18S ribosomal RNA) calculated using densitometry analysis (ImageJ, National Institutes of Health).

(an antioxidant) blocked Romo1-induced tumor cell invasion. Romo1 expression was examined by Western blotting (Figure 3E). As in other cell lines, TPA stimulated Romo1 expression and ROS production, as seen by confocal microscopy of SNU-449 and Huh-7 cells (Supplementary Figure 3A). NAC treatment also suppressed Romo1-induced tumor invasion in these cells (Supplementary Figure 3B). Romo1 expression was examined by Western blotting (Supplementary Figure 3C). These results demonstrated that TPA treatment increased mitochondrial ROS generation and tumor cell invasion through Romo1 induction.

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Increased Romo1 Expression in HCC Cell Lines and Tissues A previous report demonstrated that Romo1 expression is increased in various cancer cell lines.14 Therefore, we examined whether Romo1 expression was enhanced in HCC cell lines. As shown in Figure 4A, enhanced Romo1 expression was detected in most HCC cell lines compared with normal human lung fibroblast cells (IMR-90). Furthermore, to document the expression of Romo1 in HCC tissues, fresh specimens were obtained from a rat model of HCC that continuously received carcinogen through an implanted osmotic pump. As expected, Romo1 expression was observed in the precancerous and cancer tissues from the HCC rat model (Figure 4B).

Romo1 Is Overexpressed in HCC and Overexpression Is an Adverse Risk Factor of HCC Patient Survival To determine whether Romo1-mediated oxidative stress and invasion have a clinical impact on human HCC, we examined the levels of Romo1 expression in 30 pairmatched HCC and adjacent liver tissue samples positive for hepatitis B virus antigen by semi-quantitative RT-PCR.

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Pair-matched comparison revealed that 18 HCC samples (60%) had higher levels of Romo1 expression than their corresponding adjacent liver tissues (Figure 5A). Romo1 overexpression in HCC was further confirmed by Northern blotting (Figure 5B). To further explore Romo1 overexpression in HCC, we performed real-time RT-PCR using 95 HCC and 39 adjacent liver tissue samples positive for hepatitis B virus antigen, and evaluated the level of Romo1 expression compared with normal liver. Similar to the results of semi-quantitative RT-PCR, real-time RTPCR analysis showed that Romo1 expression of HCC was significantly higher than in adjacent liver tissues (t test: P ⬍ .001) (Figure 5C). Median increases in Romo1 expression were 2.48-fold in HCC and 1.20-fold in adjacent liver tissues (Figure 5C). These findings indicated that Romo1 was involved in the pathogenesis of malignant progression rather than in fibrosis and cirrhosis of the liver. When we examined Romo1 expression in paraffin-embedded specimens by immunohistochemistry, HCC tissues reacted with antibody against Romo1 (Figure 5D). As expected, based on Romo1 localization in mitochondria, all HCC tissues positive for Romo1 showed cytoplasmic staining. Increased invasiveness leads to poor prognosis in HCC patients.25–27 Romo1 overexpression increased the invasion rate of cancer cells and was found in HCC, suggesting that Romo1 overexpression might affect the clinical outcomes in HCC patients after surgery. To explore this possibility, we examined the cutoff point of Romo1 overexpression to evaluate the prognostic value of Romo1 in HCC. After measuring the P values of all possible combinations, based on the expression ratio of Romo1 between patients with and without Romo1 overexpression, a 2-fold ratio was found to be the cutoff value showing the minimum P value (Supplementary Figure 4). Under conditions of a ⬎2.0-fold increase compared with normal liver, Romo1 was considered to be overexpressed. A Kaplan– Meier plot showed significant separation of overall and disease-free survival between patients with (n ⫽ 63) and without Romo1 overexpression (n ⫽ 32) (Figure 5E, logrank test: P ⫽ .0003 and P ⫽ .0013, respectively). As expected from the results that Romo1 overexpression increased invasive properties, the survival of patients with Romo1 overexpression (high-risk group) was poorer than with patients without overexpression (low-risk group) (Figure 5E). Comparing high- and low-risk patients defined by Romo1 expression, median overall survival periods were 100.4 vs 38.2 months, and the disease-free survival periods were 65.9 vs 11.3 months. To reduce the possibility that the prognostic evaluation of Romo1 was biased because of clinical aspects rather than Romo1 per se, we performed the survival analysis after eliminating patients whose liver function or tumor stage was poor. When we excluded 7 patients with Child score B or C who suffered from poor liver function, we observed sharp separations of overall and disease-free survival between patients with and without Romo1 overexpression (Supplementary Figure 5A, log-rank test: P ⫽ .0016 and P ⫽

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.0068, respectively). The exclusion of 4 patients with TNM stage IV did not affect the mode of overall and disease-free survival according to Romo1 overexpression (Supplementary Figure 5B, log-rank test: P ⫽ .0007 and P ⫽ .0024, respectively). These analyses further support that Romo1 had prognostic significance in HCC patients.

Association Between Romo1 Overexpression and Clinicopathological Parameters Next, we analyzed the correlation between Romo1 overexpression and clinicopathological parameters to determine whether Romo1 overexpression and poor prognosis were associated with clinical features. Consistent with Romo1 regulation of cancer cell invasion, Romo1 overexpression correlated closely with

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Figure 5. Romo1 overexpression and poor prognosis in HCC patients. (A) Representative image of Romo1 expression levels in 12 pair-matched HCC (T) and corresponding adjacent nontumor liver tissues (N), from 30 analyzed by semi-quantitative RT-PCR. Relative Romo1 expression levels of HCC compared with adjacent liver tissues adjusted by 18S ribosomal RNA (rRNA) levels in pair-matched tissue samples. (B) Representative image of Romo1 expression in HCC (T) and adjacent nontumor liver tissues (N) analyzed by Northern blotting using a Romo1 probe. Patient sample numbers (1–12) are marked on the top of panels A and B. (C) Mean Romo1 expression in HCC (n ⫽ 95) and adjacent liver tissues (n ⫽ 39) compared with normal liver tissues by real-time RTPCR. 18S rRNA was used as a control (A to C). P value was determined by t test. (D) Immunohistochemical analysis of Romo1 expression in HCCs with and without Romo1 expression. (E) The cumulative overall and disease-free survival rates of HCC patients by Romo1 expression level based on real-time RTPCR, analyzed by a Kaplan– Meier curve. P values were determined by the log-rank test.

higher frequency of vascular invasion (Table 1, ␹2 test: P ⫽ .014). Poorer differentiation (P ⫽ .019) and larger tumor size (P ⫽ .004) were also associated with Romo1 overexpression. In our cohort, no significant correlation was found between Romo1 expression and other variables, including sex, age, tumor number, encapsulation, TNM stage, Child score, fibrosis, cirrhosis, and preoperative serum levels of ␣-fetoprotein, aspartate aminotransferase, alanine aminotransferase, total bilirubin, or prothrombin (Table 1). To further evaluate the impact of Romo1 overexpression and clinical variables on patient prognosis, we performed univariate and multivariate survival analyses of Romo1 overexpression and clinicopathological parame-

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Table 1. Romo1 Expression With Clinicopathologic Parameters in 95 HCC Specimens

Table 2. Effects of Romo1 Expression and Clinicopathological Parameters on Overall and Disease-Free Survival of HCC Patients in Multivariate Cox-Proportional Hazard Regression Analysis

Romo1 expression Variables

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Sex Male Female Age, y Younger than 52 52 or older AFP, ng/mL ⬍20 ⱖ20 AST, U/L ⬍40 ⱖ40 ALT, U/L ⬍40 ⱖ40 Prothrombin, % ⬍90 ⱖ90 Child–Pugh clssification A B&C Tumor number Solitary Multiple Satellite nodule No Yes Tumor size, cm ⬍5 ⱖ5 Tumor grade I⫺II III⫺IV Tumor stage I⫺II III⫺IV Necrosis No Yes Macroscopic vascular invasion No Yes Microscopic vascular invasion No Yes Margin, cm ⬍2 ⱖ2 Encapsulation No Yes Fibrosis No Yes Cirrhosis No Yes

n

Low

High

79 16

26 6

53 10

39 56

12 20

27 36

44 50

19 13

25 37

46 49

20 12

26 37

44 51

18 14

26 37

42 52

16 15

26 37

P valuea

Factor

.949

Overall survival AST Tumor grade Macroscopic vascular invasion Romo1 Disease-free survival Tumor grade Macroscopic vascular invasion Romo1

.779

.524

Hazard ratio (95% CI)

P valuea

1.824 (1.015⫺3.276) 2.769 (1.557⫺4.925) 3.805 (1.750⫺8.270) 2.537 (1.283⫺5.016)

.044 ⬍.001 ⬍.001 .007

2.467 (1.443⫺4.214) 3.648 (1.617⫺8.231) 2.114 (1.192⫺3.749)

⬍.001 .002 .010

.082 AST, aspartate aminotransferase; CI, confidence interval. multivariate analysis based on the Cox proportional hazard model was performed using forward stepwise selection in univariate analysis (inclusion P ⬍ .10).

aA

.244

.467

.871 88 7

30 2

58 5

84 11

27 5

57 6

83 12

30 2

53 10

44 51

22 10

22 41

63 31

27 5

36 26

67 28

25 7

42 21

44 51

19 13

25 38

86 9

31 1

55 8

22 73

10 22

12 51

65 30

22 10

43 20

20 74

10 21

10 53

5 89

0 31

5 58

53 41

18 13

35 28

.590

.314

.004

.019

.155

.543

.256

ters. Univariate analysis revealed that macroscopic and microscopic vascular invasions and tumor grade were significantly associated with both overall and disease-free survival, whereas tumor size, aspartate aminotransferase, ␣-fetoprotein, and Child score were associated with overall survival only (Supplementary Table 1). With these significant clinicopathological parameters, Romo1 overexpression in univariate analysis was a significant risk factor for patient prognosis (hazard ratio ⫽ 3.22 for overall survival and 2.40 for disease-free survival). When these significant factors (P ⬍ .10) were entered into stepwisemultivariate Cox regression analysis, Romo1 overexpression (hazard ratio ⫽ 2.53 for overall survival and 2.11 for disease-free survival), together with poorer differentiation (hazard ratio ⫽ 2.76 overall and 2.46 disease-free) and macroscopic vascular invasion (hazard ratio ⫽ 3.80 overall and 3.64 disease-free), independently contributed to the prognosis for both overall and disease-free survival (Table 2). Romo1 was a significant independent predictor of HCC patient prognosis, and played a role in controlling the invasiveness of HCC.

.014

Discussion .644

.120

.261

.992

AFP, ␣-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase. aThe significance of Romo1 expression in clinicopathological parameters was calculated by ␹2 test.

High levels of ROS contribute to tumor cell invasion through MMP production.4 Although many reports demonstrated that ROS derived from NADPH oxidase contribute to tumor invasion, ROS produced by mitochondria also appear to contribute to tumor invasion.4 This is consistent with our results. Romo1 knockdown caused down-regulation of ROS levels, resulting in the reduced invasive activity of several HCC cell lines. This result suggested that increased Romo1 expression was responsible for the enhanced invasive activity of HCC cells. Romo1 also mediated TPA-induced tumor cell invasion. However, Romo1 depletion did not completely block TPA-induced tumor cell invasion in HepG2 cells. TPA is reported to induce MMP-9 activity through NADPH oxidase– derived ROS.33 Consistent with this report, we ob-

served that treatment with apocynin inhibited the TPAinduced MMP-9 promoter activity (Figure 2E). However, this inhibitor failed to completely block TPA-induced MMP-9 promoter activity, demonstrating that TPA did not induce tumor cell invasion through a single pathway in HCC cells. Therefore, another inhibitor of NADPH oxidase, DPI, which also inhibits mitochondrial ROS production, was used to inhibit cellular ROS production from NADPH oxidase as well as mitochondria. DPI treatment of HCC cells completely blocked TPA-triggered MMP-9 promoter activity (Figure 2E). These results demonstrated that TPA increased cellular ROS levels through NADPH oxidase as well as mitochondria, with Romo1 being important for the mitochondrial ROS production required for TPA-induced tumor cell invasion. The exact mechanism by which TPA triggered mitochondrial ROS generation remains to be studied. One possibility is that TPA enhanced Romo1 expression to increase ROS levels. We showed that TPA treatment increased Romo1 expression and concurrent ROS generation in HCC cells (Figure 3). Our results suggest that endogenous Romo1 expression is up-regulated in response to tumor promoters, such as TPA during carcinogenesis, and increased Romo1 protein confers invasive potential to tumor cells. Several reports have demonstrated Romo1 function. The down-regulation of Romo1 expression by Romo1 siRNA transfection decreases ROS levels and causes cell growth inhibition through inhibition of Erk activation and p27Kip1 expression in various normal and cancer cell lines.18,19 Romo1 seems to be indispensible for the redox homeostasis for cell survival. Romo1 is also suggested to be a molecular bridge between TNF-␣ signaling and the mitochondria, for ROS production that triggers TNF-␣–mediated apoptosis.16,31 A recent report demonstrated that Romo1 is important in c-Myc turnover.17 The c-Myc protein is up-regulated in response to growth-stimulating signals that trigger the cell-cycle progression. Subsequently, c-Myc stimulates Romo1 expression. ROS derived from Romo1 expression then triggers Skp2-mediated c-Myc degradation in a negative feedback mechanism. This study identified an important role for Romo1 in tumor cell invasion, which contributes to cancer progression. In fact, invasion is a high risk factor for HCC patient survival.25,34 –36 We found that Romo1 was overexpressed in tumor tissues of HCC patients; Romo1 overexpression was detected in 66.3% of HCC samples (Figure 4C). The correlation between Romo1 overexpression and clinicopathological parameters also suggested that Romo1 overexpression was associated with tumor invasion in HCC patients, and contributed to poor prognosis. Univariate and multivariate analyses revealed that Romo1 overexpression was an independent risk factor in predicting both overall and disease-free survival. ROS affect the progression of both chronic liver diseases and HCC.21,22 The fact that Romo1 overexpression was rare in adjacent liver tissues, however, indicated that Romo1-derived ROS induced oxidative stress in HCC but not in adjacent liver tissues. Therefore, our data indicated that Romo1-in-

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duced ROS generation and the subsequent enhancement of invasiveness of HCC affected the clinical outcomes of HCC patients. Because unwanted ROS stress is harmful to normal cells, inducing a variety of pathological disorders, many trials have attempted to prevent or cure diseases related to chronic ROS stresses.37 A variety of antioxidants have been used to prevent or cure tumor invasion and inflammatory disorders. However, these clinical trials were not successful.37 Antioxidants have been used in trials to scavenge ROS produced intracellularly. Another approach for prevention of tumor cell invasion would be controlling mitochondrial ROS production rather than removing the cellular ROS already produced by mitochondria. This approach could provide a new strategy for development of drugs for diseases caused by chronic oxidative stress, such as tumor invasion and inflammation. For this purpose, Romo1 could be a promising target for development of anticancer drugs. Romo1 localizes in the outer mitochondrial membrane and induces mitochondrial ROS generation through the mitochondrial electron transport chain.16 Because Romo1 contains a highly conserved tetrad of the GxxxG motif and this motif is important for membrane channel formation,38 Romo1 might regulate mitochondrial ROS release into the cytosol by forming a channel. However, no direct evidence supports that Romo1 regulates mitochondrial ROS release into the cytosol. A more extensive effort to determine the exact function of Romo1 remains to be studied. In this work, we suggest that Romo1-derived ROS might be important in pathological disorders induced by chronic oxidative stress. If Romo1 inhibitors can be developed, they might prevent, at least in part, the progression of tumor cells. Moreover, Romo1 inhibitors could be used as anticancer agents in combination with existing chemotherapeutic drugs to provide better outcomes of chemotherapy.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2012.06.038. References 1. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010;141:52– 67. 2. Johnsen M, Lund LR, Romer J, et al. Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol 1998;10:667– 671. 3. Leivonen SK, Kahari VM. Transforming growth factor-beta signaling in cancer invasion and metastasis. Int J Cancer 2007;121: 2119 –2124. 4. Wu WS. The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev 2006;25:695–705. 5. Frost JA, Geppert TD, Cobb MH, et al. A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum. Proc Natl Acad Sci U S A 1994;91:3844 –3848.

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6. Han Z, Boyle DL, Chang L, et al. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:73– 81. 7. Westermarck J, Kahari VM. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 1999;13:781–792. 8. Kumar B, Koul S, Khandrika L, et al. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res 2008;68:1777–1785. 9. Mori K, Shibanuma M, Nose K. Invasive potential induced under long-term oxidative stress in mammary epithelial cells. Cancer Res 2004;64:7464 –7472. 10. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. Free Radic Biol Med 2004;37:768 –784. 11. Nelson KK, Ranganathan AC, Mansouri J, et al. Elevated sod2 activity augments matrix metalloproteinase expression: evidence for the involvement of endogenous hydrogen peroxide in regulating metastasis. Clin Cancer Res 2003;9:424 – 432. 12. Wenk J, Brenneisen P, Wlaschek M, et al. Stable overexpression of manganese superoxide dismutase in mitochondria identifies hydrogen peroxide as a major oxidant in the AP-1-mediated induction of matrix-degrading metalloprotease-1. J Biol Chem 1999; 274:25869 –25876. 13. Ishikawa K, Takenaga K, Akimoto M, et al. ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 2008;320:661– 664. 14. Chung YM, Kim JS, Yoo YD. A novel protein, Romo1, induces ROS production in the mitochondria. Biochem Biophys Res Commun 2006;347:649 – 655. 15. Chung YM, Lee SB, Kim HJ, et al. Replicative senescence induced by Romo1-derived reactive oxygen species. J Biol Chem 2008; 283:33763–33771. 16. Kim JJ, Lee SB, Park JK, et al. TNF-alpha-induced ROS production triggering apoptosis is directly linked to Romo1 and Bcl-X(L). Cell Death Differ 2010;17:1420 –1434. 17. Lee SB, Kim JJ, Chung JS, et al. Romo1 is a negative-feedback regulator of Myc. J Cell Sci 2011;124:1911–1924. 18. Chung JS, Lee SB, Park SH, et al. Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27(Kip1) expression. Free Radic Res 2009;43:729 –737. 19. Na AR, Chung YM, Lee SB, et al. A critical role for Romo1-derived ROS in cell proliferation. Biochem Biophys Res Commun 2008; 369:672– 678. 20. Hwang IT, Chung YM, Kim JJ, et al. Drug resistance to 5-FU linked to reactive oxygen species modulator 1. Biochem Biophys Res Commun 2007;359:304 –310. 21. Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: present concepts. J Gastroenterol Hepatol 2011; 26(Suppl 1):173–179. 22. Tien Kuo M, Savaraj N. Roles of reactive oxygen species in hepatocarcinogenesis and drug resistance gene expression in liver cancers. Mol Carcinog 2006;45:701–709. 23. Qin LX, Tang ZY. The prognostic molecular markers in hepatocellular carcinoma. World J Gastroenterol 2002;8:385–392. 24. Vauthey JN, Lauwers GY, Esnaola NF, et al. Simplified staging for hepatocellular carcinoma. J Clin Oncol 2002;20:1527–1536. 25. Fujita N, Aishima S, Iguchi T, et al. Histologic classification of microscopic portal venous invasion to predict prognosis in hepatocellular carcinoma. Hum Pathol 2011;42:1531–1538. 26. Pawlik TM, Delman KA, Vauthey JN, et al. Tumor size predicts vascular invasion and histologic grade: implications for selection

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of surgical treatment for hepatocellular carcinoma. Liver Transpl 2005;11:1086 –1092. Sakata J, Shirai Y, Wakai T, et al. Preoperative predictors of vascular invasion in hepatocellular carcinoma. Eur J Surg Oncol 2008;34:900 –905. Lee SB, Kim JJ, Kim TW, et al. Serum deprivation-induced reactive oxygen species production is mediated by Romo1. Apoptosis 2010;15:204 –218. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402– 408. Novo E, Parola M. Redox mechanisms in hepatic chronic wound healing and fibrogenesis. Fibrogenesis Tissue Repair 2008;1:5. Bae YS, Oh H, Rhee SG, et al. Regulation of reactive oxygen species generation in cell signaling. Mol Cells 2011;32:491–509. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000;279:L1005–L1028. Steinbrenner H, Ramos MC, Stuhlmann D, et al. Tumor promoter TPA stimulates MMP-9 secretion from human keratinocytes by activation of superoxide-producing NADPH oxidase. Free Radic Res 2005;39:245–253. Arii S, Tanaka J, Yamazoe Y, et al. Predictive factors for intrahepatic recurrence of hepatocellular carcinoma after partial hepatectomy. Cancer 1992;69:913–919. Nagasue N, Ono T, Yamanoi A, et al. Prognostic factors and survival after hepatic resection for hepatocellular carcinoma without cirrhosis. Br J Surg 2001;88:515–522. Shirabe K, Kajiyama K, Harimoto N, et al. Prognosis of hepatocellular carcinoma accompanied by microscopic portal vein invasion. World J Gastroenterol 2009;15:2632–2637. Ladas EJ, Jacobson JS, Kennedy DD, et al. Antioxidants and cancer therapy: a systematic review. J Clin Oncol 2004;22:517–528. Russ WP, Engelman DM. The GxxxG motif: a framework for transmembrane helix-helix association. J Mol Biol 2000;296:911–919.

Received December 21, 2011. Accepted June 22, 2012. Reprint requests Address requests for reprints to: Young Do Yoo, PhD, Korea University College of Medicine, 126-1, 5ka, Anam-dong, Sungbuk-ku, Seoul 136-705, Korea. e-mail: [email protected]; fax: (822) 9205762, or Kee-Ho Lee, PhD, Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, 139706, Korea. e-mail: [email protected]; fax: (822) 970-2417. Acknowledgments Jin Sil Chung and Sunhoo Park contributed equally to this work. Conflicts of interest The authors disclose no conflicts. Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0025760), by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (1020180), and a grant from the National Nuclear R&D Program of the Korean Ministry of Science and Technology.

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Supplementary Materials and Methods Reagents NAC, 2=,7=-dichlorofluorescein diacetate, dihydroethidine, DPI, myxothiazol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and TPA were from SigmaAldrich (St Louis, MO). MitoSOX and MitoTracker were obtained from Molecular Probes (Eugene, OR). Lipofectamine was from Gibco-Invitrogen (Grand Island, NY). Luciferase and galactosidase assay systems were from Promega (Madison, WI). Human recombinant TGF-␤1, epidermal growth factor, and hepatocyte growth factor were from Prospec (Rehovot, Israel). Human recombinant TGF-␣ was from Peprotech (London, UK) and apocynin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Invasion Assay The invasion assay was performed using polycarbonate nucleopore membranes (Corning, Corning NY). Matrigel (100 ␮g/cm2) was loaded on the upper side of the Transwell (6.5 mm in diameter, 8.0 ␮m pore size). Cells (1 ⫻ 105) transfected with Romo1 siRNA (100 nM) for 48 hours were suspended in serum-free media with 0.1% filtered bovine serum albumin. Cells were plated in the Matrigel-coated upper chamber of the Transwell in the presence of TPA. Normal cell culture media was added to the lower chamber of the Transwell, and cells were incubated for 24 hours at 37°C. Invading cells were fixed and stained with deep quick solution hemacolor (Merck, Darmstadt, Germany). The number of invading cells was determined by counting cells that migrated from the upper chamber into the lower chamber of the Transwell using light microscopy. Invading cells were counted in at least 3 fields per filter.

Gelatin Zymography Cells were transfected with Romo1 siRNA for 24 hours and incubated with serum-free media for 24 hours with or without TPA. Conditioned media were collected, mixed with nonreducing sample buffer, and loaded into 10% polyacrylamide gel electrophoresis with gelatin (15 mg/mL). The gel was denatured in 2.5% Triton X-100 solution for 1 hour and developed in 50 mM Tris (pH 7.5), 20 mM NaCl, 10 mM CaCl2, 1 ␮M ZnCl2, 0.1% NaN3 for 24 hours at 37°C.

Lung Nodule Formation Huh-7 cells were transfected daily for 2 days with control siRNA or Romo1 siRNA using Lipofectamine 2000. Severe combined immunodeficient mice were injected intravenously with 1 ⫻106 cells. Mice were sacrificed 8 weeks later, and lungs were isolated and fixed with 3.7% paraformaldehyde for 24 hours. Metastatic nodules was counted.

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Immunohistochemistry Immunohistochemical staining for mouse antihuman Romo1 was performed on formalin-fixed, paraffin-embedded human HCC specimens by Leica Bondmax automated immunostainer (Leica Microsystems, Bannockburn, IL) with 4-␮m sections from blocks deparaffinized in a dry oven at 60°C for 1 hour, dewaxed at 72°C, and rehydrated in graded alcohol washes. Heat pretreatment for Romo1 antigen retrieval was accomplished in a steamer with citrate buffer (pH 6.0; Dako, Glostrup, Denmark) at 100°C for 20 minutes. Endogenous peroxidase activity of specimens was blocked with 0.3% H2O2 in methanol for 5 minutes. Sections were incubated with primary Romo1 antibody at 1:100 dilution for 15 minutes at room temperature. Antibody binding for Romo1 was detected with a Bond polymer refine detection kit (Leica Microsystems) for 8 minutes and a diaminobenzidine tetrahydrochloride kit (Biogenex, San Ramon, CA) for 10 minutes. The developed sections were counterstained with hematoxylin for 10 seconds.

siRNA Transfection Romo1 siRNA sequences were described previously.1,2 Control siRNA sequence and Romo1 siRNA were from Bioneer (Taejon, Republic of Korea). Cells (4 ⫻ 105) were seeded into 60-mm plates and transfected with Romo1 siRNA using Lipofectamine (Gibco-Invitrogen).

Measurement of ROS Production HCC cells were transfected with Romo1 siRNAFITC (100 nM) or control siRNA-FITC (100 nM) for 16 hours and cells were stained with MitoSOX (5 ␮M) or 2=,7=-dichlorofluorescein diacetate (50 ␮M) or dihydroethidine (20 ␮M) for 30 minutes. Intracellular ROS levels were measured by FACScan flow cytometer (Becton Dickson, San Jose, CA) with gating for 10,000 cells/sample or by a fluorescence microscope (Olympus LX71 microscope, Olympus, Tokyo, Japan). ROS levels measured by a fluorescence microscope were analyzed using MetaMorph software (Universal Imaging, Westchester, PA) for quantification as described earlier.2,3 Intracellular ROS levels were also measured by a FACScan flow cytometer as described previously.3

Luciferase Reporter Gene Assay Cells were cotransfected with 1 ␮g MMP-9 (pGL2MMP-9WT) promoter-luciferase reporter constructs4 and 1 ␮g pCMV-␤-galactosidase plasmid for 5 hours using Lipofectamine reagent (Gibco-Invitrogen). After transfection, cells were cultured in normal cell culture media with TPA for 24 hours. Luciferase and ␤-galactosidase activities were assayed according to the manufacturer’s instructions (Promega). Luciferase activity was normalized to ␤-galactosidase activity.

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Cell Culture and Rat HCC Tissues Huh-7, Hep3B, Sk-Hep1, SNU-449, Chang, SiHa, HeLa, HEK 293, and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium, and HepG2 cells were cultured in Eagle’s minimal essential media. All media contained 10% heat-inactivated fetal bovine serum (Gibco-Invitrogen), sodium bicarbonate (2 mg/mL; Sigma-Aldrich), penicillin (100 U/mL), and streptomycin (100 ␮g/mL; Gibco-Invitrogen). Tissues were collected from rats (18-month-old BALB/c). Fresh specimens of precancerous and cancerous tissues were obtained from a rat model of HCC that received continuous release of carcinogen through an implanted osmotic pump. The HCC model was generated by continuous infusion of diethylnitrosamine in 15 male F344 rats. All animals developed hepatic masses, which histopathology diagnosed as hepatic adenomas to hepatocellular carcinomas. Normal liver tissue was from a control rat that received

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water from an osmotic pump. Total cellular RNA was isolated from normal liver, hepatic adenoma (as precancer), and HCC tissues. References 1. Hwang IT, Chung YM, Kim JJ, et al. Drug resistance to 5-FU linked to reactive oxygen species modulator 1. Biochem Biophys Res Commun 2007;359:304 –310. 2. Lee SB, Kim JJ, Kim TW, et al. Serum deprivation-induced reactive oxygen species production is mediated by Romo1. Apoptosis 2010;15:204 –218. 3. Kim JJ, Lee SB, Park JK, et al. TNF-alpha-induced ROS production triggering apoptosis is directly linked to Romo1 and Bcl-X(L). Cell Death Differ 2010;17:1420 –1434. 4. Moon SK, Cha BY, Kim CH. ERK1/2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: involvement of the ras dependent pathway. J Cell Physiol 2004;198:417– 427.

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Supplementary Figure 1. (A) Chang cells were treated with TPA (50 nM) or dimethyl sulfoxide (control) for the indicated times. Cells were stained with 2=,7=-dichlorofluorescein diacetate for 30 minutes, and ROS generation was analyzed by flow cytometry. Data are the mean of at least 3 independent experiments. ***, ###P ⬍ .001 vs control by 2-way ANOVA. (B) After transfection of Romo1 siRNA-FITC, HepG2 cells were treated with TPA (50 nM) for 15 minutes. Cells were stained with dihydroethidine for 30 minutes, and ROS generation was analyzed by flow cytometry. (C) HepG2 cells were treated with TPA (50 nM) in the presence of NAC (20 mM), myxothiazol (1 ␮M), or DPI (10 ␮M) for 15 minutes. Cells were stained with MitoSOX for 30 minutes, and ROS production was analyzed by flow cytometry. Myxo, myxothiazol. (D) After transfection of Romo1 siRNA for 16 hours, MCF-7 and HeLa cells were treated with TPA (50 nM) for 15 minutes. Cells were stained with MitoSOX for 30 minutes, and ROS levels were measured by flow cytometry. (E) Romo1 knockdown was analyzed by RT-PCR in HepG2 cells. ␤-actin was used as a loading control.

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Supplementary Figure 2. (A) After HepG2 cells were transfected with Romo1 siRNA, cell invasion was measured by Boyden chamber invasion assay in the presence of TGF-␤1 (10 ng/mL), epidermal growth factor (100 ng/mL), hepatocyte growth factor (40 ng/mL), or TGF-␣ (20 ng/mL) for 48 hours. (B) Invading cells were stained and counted in at least 3 fields per filter. Data are means (SE) of at least 3 independent experiments. *P ⬍ .05; ##, ¥¥, §§P ⬍ .01; ***P ⬍ .001 vs control siRNA by 2-way ANOVA.

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Supplementary Figure 3. (A) After SNU-449 and Huh-7 cells were treated with TPA for indicated times, cells were stained with anti-Romo1 antibody (red) and 2=,7=-dichlorofluorescein diacetate (green), and observed by confocal microscopy. ROS production was quantified by MetaMorph software. Data are means of at least 3 independent experiments. *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001 vs control by 2-way ANOVA. (B) After SNU-449 and Huh-7 cells were transfected with Flag-tagged Romo1 for 6 hours, cells were treated with NAC (20 mM) for 6 hours. Cell invasion was measured by Boyden chamber invasion assay. Invading cells were stained and counted in at least 3 fields per filter. Data are means (SE) of at least 3 independent experiments. #P ⬍ .05; **P ⬍ .01; ***, ###P ⬍ .001 vs vector by 2-way ANOVA. (C) Romo1 expression was examined by Western blotting with anti-Flag antibody. ␤-actin was used as a loading control.

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Supplementary Figure 4. Determination of cutoff value for Romo1 overexpression in logrank survival analysis. All possible combinations of Romo1 expression ratios that could discriminate HCC patients into low- and high-risk groups were examined by log-rank test, and the cutoff value was determined from the range of minimum P values.

Supplementary Figure 5. Overall and disease-free survival rates of HCC patients according to Romo1 expression and clinicopathological parameters. (A) Seven HCC patients whose Child score was B or C and (B) 4 HCC patients whose tumor stage was IV were excluded in Kaplan–Meier analysis of the cumulative overall and disease-free survival rates of HCC patients according to Romo1 expression level. Log-rank P values were used to determine significance.

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Supplementary Table 1. Effects of Romo1 Expression and Clinicopathological Parameters on Overall Survival and DiseaseFree Survival of HCC Patients in Univariate Cox-Proportional Hazard Regression Analysis Overall survival

Disease-free survival

Factora

HR (95% CI)

P valueb

HR (95% CI)

P valueb

Sex Age AFP AST ALT Hemoglobin WBC Platelet Prothrombin Child–Pugh classification Tumor number Satellite nodule Tumor size Tumor grade Necrosis Macroscopic vascular invasion Microscopic vascular invasion Margin Encapsulation Fibrosis Cirrhosis Romo1

2.044 (0.895⫺4.670) 0.833 (0.485⫺1.432) 1.709 (0.982⫺2.972) 1.805 (1.030⫺3.163) 0.733 (0.425⫺1.263) 0.878 (0.446⫺1.730) 1.046 (0.575⫺1.904) 1.334 (0.727⫺2.448) 1.025 (0.592⫺1.777) 2.483 (0.981⫺6.286) 0.962 (0.410⫺2.260) 0.954 (0.406⫺2.238) 2.141 (1.228⫺3.732) 3.551 (2.052⫺6.143) 1.436 (0.348⫺5.929) 5.213 (2.450⫺11.090) 1.771 (0.991⫺3.163) 0.854 (0.488⫺1.494) 1.069 (0.534⫺2.140) 0.733 (0.228⫺2.357) 1.405 (0.821⫺2.405) 3.223 (1.667⫺6.230)

.085 .508 .055 .036 .261 .708 .883 .350 .929 .047 .929 .913 .006 ⬍.001 .615 ⬍.001 .050 .579 .850 .600 .213 ⬍.001

1.582 (0.774⫺3.231) 0.984 (0.598⫺1.619) 1.490 (0.908⫺2.445) 1.315 (0.804⫺2.151) 0.788 (0.482⫺1.287) 1.35 (0.715⫺2.548) 0.753 (0.443⫺1.279) 1.043 (0.613⫺1.775) 1.326 (0.798⫺2.205) 1.427 (0.517⫺3.933) 1.329 (0.656⫺2.696) 0.943 (0.429⫺2.070) 1.417 (0.867⫺2.314) 2.898 (1.733⫺4.845) 1.710 (0.535⫺5.464) 5.098 (2.311⫺11.246) 1.739 (1.022⫺2.962) 0.929 (0.561⫺1.537) 0.935 (0.516⫺1.692) 1.007 (0.315⫺3.217) 1.194 (0.729⫺1.958) 2.409 (6.230⫺4.178)

.205 .949 .112 .275 .340 .353 .293 .876 .274 .490 .428 .883 .162 ⬍.001 .360 ⬍.001 .039 .773 .824 .991 .481 ⬍.001

AFP, ␣-fetoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; HR, hazard ratio; WBC, white blood cell. aCriteria of clinicopathological parameters were the same as in Table 1. bUnivariate Cox regression analyses were performed for clinicopathological parameters and Romo1 expression.