2 and p38 MAPK pathways

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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGE-mediated ERK1/2 and p38 MAPK pathways Q1

Rui Wua,n, Liang Duanb, Fang Cuia, Ju Caoa, Yu Xianga, Yishu Tanga, Lan Zhoub,nn a

Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Key Laboratory of Diagnostic Medicine Designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China

b

article information

abstract

Article Chronology:

S100A9 belongs to the S100 family of calcium-binding proteins and is over-expressed in many

Received 17 December 2014

human tumors including hepatocellular carcinoma (HCC). Recent study demonstrated that

Received in revised form

S100A9 is significantly elevated and is associated with tumor differentiation and vascular

6 April 2015

invasion in HCC. The functional role of S100A9 is, however, poorly understood. Here, we

Accepted 12 April 2015

demonstrated that S100A9 treatment increased viability, invasiveness and clone formation in three HCC cell lines (HepG2, SMMC-7721 and Huh7). S100A9 also promoted tumor growth in vivo

Keywords:

by a xenograft mouse model. In addition, we observed a co-localization of S100A9 with receptor

S100A9

for advanced glycation end products (RAGE) in human HCC intratumoral tissues and an

Growth

interaction of S100A9 with RAGE in vitro. Treatment with RAGE blocking antibody blocked the

Invasion

enhanced viability, invasion, clone formation and tumor growth in vivo resulted by S100A9,

Hepatocellular carcinoma

suggesting that these effects were mediated via RAGE ligation. In order to investigate the signaling pathways, mitogen-activated protein kinase (MAPK) phosphorylation was characterized. S100A9 caused a significant increase in p-p38 and p-ERK1/2 levels, and inhibition of which blocked enhanced invasion and viability resulted by S100A9, respectively. Furthermore, treatment with RAGE blocking antibodies also abrogated the S100A9-induced p38 and ERK1/2 activation, suggesting that S100A9-induced MAPK activation is mediated via RAGE ligation. Our data demonstrate that S100A9 binds to RAGE and stimulates RAGE-dependent MAPK signaling cascades, promoting cell growth and invasion in HCC. & 2015 Published by Elsevier Inc.

n

Corresponding author. Correspondence to: Medical Laboratory of Chongqing Medical University, No.1 Yi Xue Yuan Road, Yuan Jia Gang, Yu Zhong District, Chongqing, China. E-mail addresses: [email protected] (R. Wu), [email protected] (L. Zhou). nn

http://dx.doi.org/10.1016/j.yexcr.2015.04.008 0014-4827/& 2015 Published by Elsevier Inc.

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Introduction

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MAPK signaling pathway, which could be targeted for HCC prevention and therapy.

Q3 Hepatocellular carcinoma (HCC) is one of the most common

human cancers, being the fifth most prevalent tumor type and the third leading cause of cancer-related deaths worldwide [1]. HCC is thought to result from persistent, nonspecific activation of the immune system within the chronically inflamed liver, causing repeated cycles of tissue damage, repair and regeneration, and eventually carcinogenesis [2,3]. These tumors are highly associated with chronic liver inflammation provoked by several causes such as chronic hepatitis B and C viral infection, chronic alcohol consumption, and aflatoxin B1-contaminated food [4,5]. Additionally, many studies have revealed that aberrant inflammatory molecules and inactivation of inflammatory pathways are major players in liver carcinogenesis [6,7]. Although the pathogenesis underlying HCC has been widely studied, the exact and detailed understanding of factors and mechanisms associated with the processes leading from inflammation to cancer remain obscure. S100A9 is a low molecular protein and a member of the S100 protein family which is characterized by the presence of two Ca2þ-binding sites of the EF-hand type [8]. S100A9 has also been shown to regulate inflammatory processes by serving as leukocyte chemoattractants and by inducing the expression of proinflammatory cytokines [9,10]. Of note, increased S100A9 expression was also detected in various human cancers, and extracellular S100A9 protein exhibits pro-tumor responses in breast cancer, prostate cancer and cutaneous squamous cell carcinoma [11–14]. Recent study demonstrated that S100A9 is not only overexpressed, but also correlates with poor differentiation in HCC [15]. For its biological functions, S100A9 over-expression has been found in the HCC cells of humans and mice and has been shown to protect Hep3B HCC cells from TNF-γ-induced apoptosis [16]. In addition, our recent study also found that S100A9 promotes the proliferation and invasion of HepG2 hepatocellular carcinoma cells [17]. Receptor for advanced glycation end products (RAGE) is a multiligand receptor classified as an immunoglobulin superfamily cell surface molecule [18]. Ligand-RAGE interaction activates multiple signaling pathways that are implicated in both inflammation and cancer, including mitogen-activated protein kinase (MAPK), PI3K/Akt, and nuclear factor-kappa B (NF-κB) pathways [18,19]. Recent studies showed that RAGE is over-expressed in HCC compared to adjacent paraneoplastic liver samples [20], and it mediates HCC progression by affecting caner cell proliferation, migration, invasion, and Angiogenesis [21–23]. RAGE has been reported to serve as a common extracellular S100 receptor because of the common structural features and display sequence homology of S100s [24]. Although several members of S100s such as S100A12, S100B, S100A1, and S100P have been shown to interact with RAGE [24–26], and the direct interaction of S100A9 with RAGE in HCC still remain unclear. In the present study, we investigated the function of exogenous S100A9 on HCC cell lines. We found that exogenous S100A9 activate MAPK signaling pathway, promoting cell survival and invasion as well as tumor growth in an in vivo mouse model system via RAGE ligation. Blocking the interaction of S100A9 and RAGE could inhibit the effects of S100A9 both in vitro and in vivo. Therefore, exogenous S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGE-mediated

Materials and methods Cell culture and tissues Human hepatocellular carcinoma cell lines HepG2, SMMC-7721, Huh7 and human normal liver cell line L02 were purchased from ATCC (American Type Culture Collection, Manassas, VA). All of them were maintained in the Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS, Hyclone, USA). Cell culture was maintained at 37 1C in a humid atmosphere containing 5% CO2. Hepatocellular carcinoma intratumoral and peritumoral tissues were collected from 10 patients who had undergone HCC resection at the First Affiliated Hospital of Chongqing Medical University. The patients received no chemotherapy, or radiotherapy before surgery, and the written informed consent was received from all participants. This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

Reagents and antibodies The primary antibodies used for this study were as follows: The mouse anti-S100A9 monoclonal antibody (Santa Cruz, Cat#58706, USA), rabbit anti-p38 monoclonal antibody (Cell Signaling Technology, Cat#9212, USA), rabbit anti-phosphor-p38 monoclonal antibody (Cell Signaling Technology, Cat#4511, USA), rabbit antiERK1/2 monoclonal antibody (Cell Signaling Technology, Cat#4695, USA), rabbit anti-phosphor-ERK1/2 monoclonal antibody (Cell Signaling Technology, Cat#3510, USA), rabbit anti-RAGE polyclonal antibody (Santa Cruz Biotechnology, sc-5563, USA), RAGE blocking monoclonal antibody (abcam, ab89911, USA), and mouse anti-βactin monoclonal antibody (Santa Cruz, Cat#47778, USA). Specific inhibitors of p38 (SB203580) and ERK1/2 (PD98059) were obtained from Santa Cruz Biotechnology (California, USA), and were used as per the manufacturer's instructions.

Preparation of the recombinant proteins The pGST-Moluc-S100A9 has been described previously [17]. Briefly, the plasmid was transformed into Escherichia coli (BL21) following the instructions of calcium chloride transformation. Isopropylthio-β-D-galactoside was used to induce the expression of GST-S100A9. After the bacteria were sonicated, the supernatants were collected, spun and incubated with glutathioneSepharose 4B beads, and GST-S100A9 on the beads was eluted by elution buffer with reduced glutathione. Finally, the proteins were filtered via 0.22 μm membrane and stored at  80 1C. The control protein GST was prepared at the same time. Its plasmid is pGSTMoluc.

Cell viability assay MTT (methyl-thiazoldiphenyl tetrazolium) assay was used to assess the cell viability. 2  103 cells were seeded into each well of 96-well culture plates, grown for 12 h. Then added with GST or

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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GST-S100A9 at 20 μg/ml, cells were further cultured in DMEM containing 1% FBS for 4 days, and cell viability was determined by MTT assay as previously described [17]. In the blocking studies, cells were pretreated with RAGE blocking antibody (160 μg/ml) for 1 h, the inhibitor of p38 (SB203580, 10 mM) for 30 min or the inhibitor of ERK1/2 (PD98059, 20 mM) for 30 min before stimulation with GST-S100A9.

Transwell invasion assay The invasion assay was performed as previously described [17]. The chamber of non-type I-collagen-coated 24-well culture insert (MILLIPORE, USA) was used, and the upper side of the insert was coated with ECM gel (SIGMA, USA). Briefly, cells were placed in the upper chamber (1  105 cells) and incubated with GST or GSTS100A9 (20 μg/ml) in serum-free medium, while there was only the medium (600 μl/each insert) with 20% FBS in the lower chamber. After incubation for 24 h, the transmembrane cells were dried, fixed with methanol, stained with 0.1% crystal violet, and counted under microscopy at 150  . The experiment was performed thrice. In the blocking studies, cells were pretreated with RAGE blocking antibody (160 μg/ml) for 1 h, the inhibitor of p38 (SB203580, 10 mM) or ERK1/2 (PD98059, 20 mM) for 30 min before stimulation with GST-S100A9.

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Electrophoresis Documentation (Gel Doc 1000, Bio-Rad, USA) and Quantity One Version 4.5.0.

Real time quantitative PCR analysis Cells were incubated with GST-S100A9 (20 μg/ml) or GST for 24 h and then lysed with Trizol (Invitrogen, Carlsbad, CA, USA). Complementary single-stranded DNA was synthesized from total RNA by reverse transcription (TaKaRa, Japan). Primers were also synthesized by Invitrogen. Real time PCR was performed using SYBR Green master mix (TaKaRa, Japan). Quantification of cDNA targets was performed on Roche LightCycler480. GAPDH was used as an internal control. The PCR conditions and primers sequence were as follows: RAGE primers (forward) 50 -CACCTTCTCCTGTAGCTTCA-30 and (reverse) 50 -TGCCACAAGATGACCCCAAT-30 ; EpCAM primers (forward). 50 -CGCAGCTCAGGAAGAATGTG-30 and (reverse) 50 -TGAAGTACACTGGCATTGACG-30 ; CD133 primers (forward) 50 -CAGAGTACAACGCCAAACCA-30 and (reverse) 50 -AAATCACGA TGAGGGTCAGC-30 ; GAPDH primers (forward) 50 -CAGCGACACCCACTCCTC-30 and (reverse) 50 TGAGGTCCACCACCCTGT-30 . Gene expression was determined by normalization against GAPDH expression. In the blocking studies, cells were pretreated with RAGE blocking antibody (160 μg/ml) for 1 h for before stimulation with GST-S100A9.

Enzyme-linked immunosorbent assay (ELISA) Immunohistochemical procedures For immunohistochemistry (IHC) analysis, the sections from the formalin fixed, paraffin-embedded tissues were deparaffinized and rehydrated. Then the sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 0.3% H2O2 in methanol to block endogenous peroxidase. And the sections were incubated with the anti-S100A9 or anti-RAGE antibody (1:200 dilution), followed by incubation with secondary antibody tagged with the peroxidase enzyme and were visualized with 0.05% DAB until the desired brown reaction product was obtained. All slides were observed under a Nikon E400 Light Microscope and representative photographs were taken. For double immunofluorescence staining, the sections were incubated with anti-S100A9 (1:50) together with anti-RAGE (1:50) antibodies, followed by incubation with secondary antibodies Alexa fluor 647-conjugated anti-mouse IgG (1:200) and FITC-conjugated anti-rabbit IgG (1:200). These sections were also stained with 10 μg/ml DAPI. The fluorescent images were then observed and analyzed using a multi-laser confocal microscope.

Western blot analysis Western blot analysis was applied to evaluate levels of S100A9, RAGE, p38, p-p38, ERK1/2, and p-ERK1/2 in cells. Briefly, the cells were collected and lysed on ice in radio immunoprecipitation assay (RIPA) buffer. Samples containing equal amount of proteins were separated in 10% SDS-PAGE and blotted onto the PVDF membranes. Then the membranes were blocked with 5% bovine serum albumin and incubated with anti-RAGE, anti-phosphor-p38, anti-p38, anti-phosphor-ERK1/2, anti-ERK1/2, or anti-β-actin antibody (1:1000 dilution, respectively), followed by incubation with secondary antibodies conjugated with horseradish peroxidase. The proteins of interest were detected using the SuperSignal West Pico Chemiluminescent Substrate kit. The results were recorded by the Bio-Rad

S100A9 concentrations in the cell culture medium of HCC cells (HepG2, SMMC-7721 and Huh7) and normal liver cells (L02) were measured using a human S100A9 ELISA kit (DGE10839, China) according to the manufacturer's instructions. The ELISA was repeated three times.

Co-immunoprecipitation Co-immunoprecipitation assay was used to analyze the interaction of S100A9 with RAGE according to instructions of BeaverBeadsTM Mag sProtein A/G Immunoprecipitation Kit ( BEAVER, USA). Briefly, HepG2, SMMC-7721 and Huh7 cells were incubated with GST-S100A9 or GST and bound proteins were immunoprecipitated with anti-S100A9-coated immunomagnetic beads or an irrelevant control IgG. The immunoprecipitated proteins on the beads were finally added in the SDS-PAGE loading buffer, and were analyzed for RAGE by SDS-PAGE and Western blots analysis.

Soft agar assay Log-phase HCC cells were collected and seeded in triplicate in a soft agar medium at 500 cells/well in 6 well plates for 10–14 days. When the clone was observed, the cells were washed by PBS and fixed by methanol. Then the clone was stained by crystal violet and was counted. The colony-forming rate was obtained as: (colony number/seeded cell number)  100%.

Xenograft nude mice model studies The in vivo experiments were performed in accordance with the guidelines established by the Animal Care and Use Committee, University Laboratory Animal Research. Female 5- to 6-week-old nude mice were injected subcutaneously in right flank with 1  107 HepG2 cells/mouse suspended in 100 mL of medium without serum and antibiotics. About a week later, mice bearing tumors around

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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100 mm3 were selected and then randomly divided into 4 groups (5 mice per groups). GST (20 mg/ml), anti-RAGE (160 mg/ml), GSTS100A9 (20 mg/ml) and GST-S100A9 (20 mg/ml)þanti-RAGE (160 mg/ml) diluted in 50 mL of PBS were injected intratumorally. Subcutaneous tumor growth was recorded every 5 days with vernier calipers. Tumor volume was calculated using the formula 1/2a  b2, where a stands for the long diameter and b is the short diameter. Mice were sacrificed 20 days after injected intratumorally with proteins, and tumors were collected, fixed in buffered formaldehyde, embedded in paraffin, and sectioned for further histological and immunohistochemical analysis. Sections were stained with hematoxylin and eosin (H&E) or immunostained with antibodies against RAGE, p-p38 or p-ERK1/2.

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treat cells for our subsequent studies. After treating three HCC cell lines HepG2, SMMC-7721, Huh7 and a normal liver cell line L02 with GST-S100A9 (20 mg/ml) for 4 days, we found that GSTS100A9 had no effect on the viability of normal liver cell line L02 (Fig. 2A) but enhanced the viability in all these three HCC cell lines at day 3 (po0.05) and day 4 (po0.01) (Fig. 2B–D). Cell invasion plays a crucial role in the process of tumor metastasis. Transwell invasion assay was used to detect the change in cellular invasiveness induced by GST-S100A9. After treatment for 24 h, no significant effect on invasion was found in normal liver cell line L02 (Fig. 2E). Contrarily, the number of transmembrane cells of the HepG2, SMMC-7721 and Huh7 increased by 65.2% (po0.05), 95.4% (po0.01), 53.7% (po0.05) (Fig. 2F–H).

Statistical analysis All values in the text and figures are presented as the mean7standard deviation (SD). The differences were analyzed using oneway ANOVA followed by the Student–Newman–Keuls test, and all statistical analyses were performed using GraphPad Prism software (GraphPad Software, CA, USA). Statistical differences are presented at probability levels of po0.05, po0.01 and po0.001.

Results S100A9 stimulates cell survival and invasiveness of HCC cell lines HepG2, SMMC-7721 and Huh7 but not in normal liver cell line L02 Extracellular S100A9 represents danger signal and triggers cellular responses in the dose-dependent manner in tumors. To further evaluate its influence on the viability and invasiveness of HCC cells, we prepared recombinant protein GST-S100A9 to treat cells, and GST was used as the control. Their purities were all over 90% (by Quantity One Software after SDS-PAGE, Fig. 1A and B). GSTS100A9 was identified by specific anti-S100A9 antibody (Fig. 1B). The putative cell viability-increasing activity of S100A9 was investigated by MTT assay. Our previous study showed that GSTS100A9 at 20 μg/ml had a strongest effect on cell viability and invasion in HepG2 cells, so we chose GST-S100A9 at 20 μg/ml to

Elevated expression of RAGE in human HCC tissues and HCC cell lines RAGE ligation can activate multiple signaling pathways. Previous studies revealed that several S100 family members can extracellularly function through RAGE ligation [27]. So we presume that RAGE could be the receptor responsible for S100A9-induced cell effects in HCC. To test the hypothesis, we first examined the expression of S100A9 and RAGE in sections from 10 pairs of HCC samples (peritumoral and intratumoral tissues) using immunohistochemical (IHC) staining. The results showed that both S100A9 and RAGE expression was significantly higher in tumor cell surface of intratumoral tissues than in peritumoral tissues (Fig. 3A). And the enhanced immunoreactivity for S100A9 and RAGE was also found in all examined samples. In addition, we also examined the endogenous expression of S100A9 and RAGE in HCC cell lines (HepG2, SMMC-7721 and Huh7) and normal L02 cells. We observed the elevated expression of S100A9 and RAGE in all HCC cell lines compared with that in L02 cells (Fig. 3B). Further, the high concentration of S100A9 was also detected in the cultural supernatants of HCC cell lines (HepG2, SMMC-7721 and Huh7) compared with that of normal L02 cells (Fig. S1), suggesting that S100A9 could secreted by HCC cells into the culture supernatant. We further studied the effect of S100A9 on the expression of RAGE in HCC cells and normal L02 cells. After treatment for 24 h,

Fig. 1 – Identification of GST-S100A9 protein. (A) Recombinant protein GST-S100A9 was about 39 kDa, and GST was about 26 kDa; their purities were 490% (by Quantity One Software). (B) GST-hS100A9 was recognized by anti-S100A9 antibody by Western blot analysis. Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 2 – The influence of S100A9 on the viability and invasiveness of HCC cells and liver normal cells. (A–D) L02 (A), HepG2 (B), SMMC-7721 (C) and Huh7 (D) cells were seeded at a density of 2  103 cells per well in a 96 well plate and treated with GST or GSTS100A9 at 20 μg/ml. Cell viability was measured 3 days later by MTT assay. Results are expressed as the mean absorbances7SD of three independent experiments. (E–H) L02 (E), HepG2 (F), SMMC-7721 (G) and Huh7 (H) cells (1  105 per well) were seeded in the upper chamber of a 24-well plate as described in the Materials and methods Section. The number of transmembrane cells were determined after 24 h. Each experiment was done in triplicate. The representative images of transmembrane cells are shown in the left panel, the mean numbers of transmembrane cells7SD per microscopic field of three independent experiments are quantified in the right panels. Magnification, 150  . npo0.05, nnpo0.01, all vs. GST control. the mRNA level of RAGE was significantly up-regulated in S100A9-treated HepG2 (po0.01), SMMC-7721 (po0.001) and Huh7 cells (po0.001) than the control GST treated cells, while no obvious up-regulation was found in S100A9-treated normal L02 cells (Fig. 3C).

RAGE and MAPK are involved in S100A9-induced increase in cell viability and invasion To explore whether RAGE was involved in cell viability and invasion driven by S100A9, the HCC cells (HepG2, SMMC-7721 and Huh7) were stimulated by S100A9 with or without anti-RAGE blocking antibody. And the viability and invasiveness were

RAGE is the receptor for S100A9 on HCC cells

measured. The cell viability-promoting activity of S100A9 was significantly

To investigate whether S100A9 can interact with RAGE, we performed confocal microscopy to observe the possible colocalization of S100A9 with RAGE in human HCC tissues. We observed a co-localization of S100A9 with RAGE in the membranes of tumor cells in human HCC tissues (Fig. 3D). To further investigate whether RAGE provided binding sites for S100A9 on HCC cells, we performed co-immunoprecipitation assay to detect the possible interaction. Lysates from HepG2 cells incubated with GST-S100A9 were immunoprecipitated with anti-S100A9 antibody and the immunoprecipitated proteins were separated by electrophoresis and immunoblotted with anti-RAGE. We detect RAGE in lysates of cells incubated with GST-S100A9 (Fig. 3E). RAGE was also present in immunoprecipitates from cells incubated with GST or not, suggesting that RAGE on the cell surface could exist as a complex with endogenous S100A9. Similar results were also found in SMMC-7721 and Huh7 cells (Fig. 3F and G). These results suggest that RAGE could be the receptor for S100A9 on HCC cells.

suppressed

by

anti-RAGE

antibody

(po0.05)

(Fig. 4A–C). Similarly, the cell invasion-promoting activity of S100A9 was also significantly suppressed by anti-RAGE antibody (po0.05) (Fig. 4D–F). These results indicate that RAGE is responsible for S100A9-induced cell survival and invasion. Our previous studies demonstrated that S100A9 could promote growth and invasion of HepG2 cells by activating ERK1/2 and p38 MAP kinases respectively [17]. Ligand-RAGE interaction activates multiple signaling pathways including MAPK pathway [18,19]. We therefore reasoned that RAGE could be the receptor responsible for enhanced activity of p38 and ERK1/2 MAPKs driven by S100A9 in HepG2 cells. We detected and analyzed phosphorylation of p38 and ERK1/2 in cell lysates of HepG2 cells treated with GST-S100A9 (20 mg/ml) with or without anti-RAGE blocking antibody by Western blot. Our results showed that S100A9-stimulated p38 and ERK1/2 phosphorylation was significantly suppressed by blockade of RAGE (Fig. 5A). Furthermore, our results also showed that S100A9 enhanced p-p38 and p-ERK1/2 expression in SMMC7721 and Huh7 cells, and which were also significantly

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 3 – RAGE expression in human HCC tissue and HCC cell lines, and its interaction with S100A9. (A) Representative peritumoral and intratumoral immunohistochemical staining for S100A9 and RAGE are shown. Red scale bars¼ 150 lm. (B) The endogenous expression of S100A9 and RAGE in HepG2, SMMC-7721, Huh7 and L02 cells was detected by Western blot. (C) The levels of mRNA for RAGE were determined using real-time PCR. nnpo0.01, nnnpo0.001, GST-S100A9 vs. GST control. (D) Confocal analysis of S100A9 (red) and RAGE (green) in HCC tissue. S100A9 and RAGE were detected using specific antibodies. The fluorescence patterns of the two proteins are overlapping. White scale bars ¼150 lm. (E–G) Receptor on HCC cells for S100A9 was identified by coimmunoprecipitation. HepG2 (E), SMMC-7721 (F) and Huh7 cells (G) were incubated with GST or GST-S100A9, and S100A9 were immunoprecipitated with anti-S100A9 or an irrelevant control rabbit IgG. Whole-cell lysates and immunoprecipitated proteins were separated on SDS-PAGE gels, transferred and immunoblotted with anti-RAGE.

suppressed by blockade of RAGE (Fig. 5B and C). Therefore, RAGE may be a receptor for S100A9 signaling in HCC cells. These three HCC cell lines (HepG2, SMMC-7721 and Huh7) were treated with GST-S100A9 (20 mg/ml) in the presence and absence of specific inhibitors of p38 (SB203580) or ERK1/2 (PD98059) for 72 h. We found that the enhanced viability by S100A9 was blocked by PD98059 (po0.05) (Fig. 5D–F) and that S100A9-induced cell invasion was blocked by SB203580 (po0.05) (Fig. 5G–I). These data indicated that S100A9 promotes cell viability and invasion through ERK1/2 and p38 MAP kinases.

Effects of RAGE on tumorigenesis induced by S100A9 in HCC cells in vitro and in vivo Anchorage-independent growth is an important characteristic of in vitro tumor growth. Therefore, we confirmed whether RAGE is involved in tumor cell growth driven by S100A9 using colony formation assay. The HCC cells (HepG2, SMMC-7721 and Huh7) in soft agar were stimulated by S100A9 with or without anti-RAGE blocking antibody. We found that the HCC cell growth-promoting

activity of S100A9 was significantly suppressed by anti-RAGE antibody (po0.05) (Fig. 6A and B), suggesting that RAGE is responsible for S100A9-induced cell growth in vitro. Further, S100A9 treatment dramatically increased the mRNA levels of epithelial cell adhesion molecule (EpCAM) and CD133 (HCC stemness markers) in HCC cell line SMMC-7721, and which were also partially suppressed by blockade of RAGE (po0.01, Fig. S2), suggesting that interaction of S100A9 and RAGE plays a crucial role in maintaining self-renewal or cancer stem-like properties in HCC. To confirm this effect in vivo, we employed a xenograft nude mice model, divided into 4 groups (GST, anti-RAGE, GST-S100A9 and GST-S100A9þanti-RAGE). Tumor growth was monitored for 20 days after injected intratumorally with proteins. Compared with GST group, GST-S100A9 substantially promoted tumor growth, and which was inhibited by anti-RAGE antibody. The histological examination (H&E staining) in tumor sections from different groups showed that the tumor cells in the four groups have the obvious heterogeneity, large nucleus, high nucleus/ cytoplasma ratio, irregular nuclear shape and variable nuclear

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 4 – The effect of RAGE on S100A9-induced viability and invasion of HCC cells. (A–C) HepG2 (A), SMMC-7721 (B) and Huh7 (C) cells were seeded at a density of 2  103 cells per well in a 96 well plate and pretreated or not with anti-RAGE antibody for 1 h, then cells were treated with GST or GST-S100A9 at 20 μg/ml for 3 days. Cell viability was quantified as in Fig. 1. (D–F) HepG2 (D), SMMC7721 (E) and Huh7 (F) cells (1  105 per well) were seeded in the upper chamber and pretreated or not with anti-RAGE antibody for 1 h, then cells were treated with GST or GST-S100A9 at 20 μg/ml. Cell invasion was quantified as in Fig. 1. npo0.05, nnpo0.01.

size (Fig. 6D). Of note, we found that the immunoreactivity for RAGE in group of GST-S100A9 was much more intense than that in the control GST group (Fig. 6D), suggesting that S100A9 may up-regulate RAGE expression. In addition, we observed the enhanced immunoreactivity for p-p38 and p-ERK1/2 in GSTS100A9 group, and which was significantly suppressed in group of GST-S100A9þanti-RAGE (Fig. 6D).

Discussion S100A9 have gained interest because of its differential expression in tumor tissues and its involvement in cancer progression and metastasis [11,12,15,16,28]. Comparing S100A9 expression in 70 HCC with non-carcinomatous hepatocytes indicated exclusive immunoreactivity in tumor cells [15]. These findings hint towards a neo-expression in differentiated malignant hepatocytes, which is correlated with tumor differentiation [29]. S100A9 is one of NFκB target genes in HCC cells during inflammation-associated liver carcinogenesis [16]. While S100A9 is seldom found in normal liver tissues, their expression increases with malignant transformation and facilitates tumor progression. The underlying molecular mechanism is still pending. Here we provide data supporting a tumor-promoting role for extracellular S100A9 in HCC development and that the RAGE-dependent MAPKs signaling is involved in these functions of S100A9. Previous studies demonstrate that S100A9 promotes tumorigenesis and cancer metastasis by creating a proinflammatory microenvironment. S100A9 binds to RAGE on myeloid derived suppressor cells (MDSC), which mainly include immature granulocytes, macrophages and dendritic cells, and promotes MDSC

migration to the tumor site and represses host-mediated antitumor immune response against cancer cells, thereby facilitating carcinogenesis and tumor progression [30,31]. Also, a number of growth factors produced by tumor cells including TNF-α, TGF-β and VEGF-A can stimulate S100A9 expression in pre-metastatic lung, thus attracting MDSC to the pre-metastatic location contributing to the establishment of a “pre-metastatic niche”, and thereby promoting metastasis formation [30,32]. Of note, recent studies showed that extracellular S100A9 protein also functions as danger associated molecular pattern ligand for cell surface receptors on tumor cells, activating signaling cascades and triggering tumor cell responses [33,34]. With regard to S100A9 secretion status in HCC, we detected its level in culture medium of HCC cell lines, and found that S100A9 was able to be secreted into the culture medium of HCC cells. For the functions of extracellular S100A9 in HCC, we used recombinant S100A9 protein (20 mg/ml) to treat three HCC cell lines (HepG2, SMMC-7721 and Huh7) and found an increase of cell viability and invasion in these cell lines. These findings are in agreement with previous data, showing that extracellular S100A9 protein promoted cell viability and invasion in breast cancer and gastric cancer cells [35,36]. So S100A9 in tumor microenvironment exerts their biological roles not only by regulating MDSC but also targeting on tumor cells directly, facilitating tumor growth and metastasis. Interestingly, no obvious change of viability and invasion was found in S100A9-treated normal liver cell line L02. Upon further analyses of the mechanisms that may mediate the promotive effect of S100A9 on viability and invasion in HCC cell lines but not in normal liver cell line L02, we focused on the possible receptor RAGE, which has been reported to function as a signal-transducing receptor for S100A9 in breast cancer, prostate cancer, and colorectal

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 5 – Activated MAPK signaling resulted by S100A9 was RAGE-dependent. (A–C) HepG2 (A), SMMC-7721 (B) and Huh7 (C) cells were pretreated or not with anti-RAGE antibody for 1 h prior to incubation with GST-S100A9 for 30 min. The protein levels of phosphorylated p38 and phosphorylated ERK1/2 were analyzed by Western blot. Total p38, ERK1/2, and β-actin were included as loading controls. (D–F) HepG2 (D), SMMC-7721 (E) and Huh7 (F) cells were pretreated or not with PD98059 (20 lM) for 30 min followed by further incubation for 3 days in the presence or absence of GST-S100A9. Cell viability was quantified as in Fig. 1. (G–I) HepG2 (G), SMMC-7721 (H) and Huh7 (I) cells were pretreated or not with SB203580 (10 lM) for 30 min followed by further incubation for 24 h in the presence or absence of GST-S100A9. Cell invasion was quantified as in Fig. 1. npo0.05, nnpo0.01.

cancer [12,13,28]. We observed an elevated expression of RAGE in HCC intratumoral tissues and cell lines compared to the peritumoral tissues and a normal liver cell line, which is in line with the previous report, demonstrating that RAGE is over-expressed in HCC compared to adjacent para-neoplastic liver samples [20]. By using confocal microscopy and co-immunoprecipitation analysis, we observed the co-localization and interaction of S100A9 with RAGE. Furthermore, S100A9-mediated survival, invasion and tumor growth in vivo were prominently inhibited by anti-RAGE blocking antibody, which provided further evidence that RAGE is the receptor for S100A9. Therefore, we speculate that RAGE may be responsible for the effect of S100A9 on viability and invasion in HCC cell lines but not in normal liver cell line L02. MAPK signaling has been reported to be abnormally activated in liver carcinogenesis [37–39]. It has been shown that RAGE triggered by extracellular S100 proteins transmit three canonical MAPKs (ERK1/2, p38 MAPK and JNK) [40,41]. Our previous study also demonstrated that activated ERK1/2 and p38 MAPK are involved in S100A9-mediated survival and invasion of HepG2

cells [17]. Still, the precise signaling pathway of S100A9 in HCC cells has not been clearly elucidated yet. Our present data showed that S100A9 induced phosphorylation of p38 and ERK1/2 in all the three HCC cell lines, which was prominently inhibited by antiRAGE blocking antibody, suggesting that S100A9-enhanced phosphorylation of p38 and ERK1/2 MAPKs in HCC is mediated by RAGE. Further, our results also showed that S100A9-induced cell growth and invasion was reversed by PD98059 (an ERK1/2 inhibitor) and SB203580 (a p38 inhibitor). ALL these effects were also observed in our in vivo experiment. Therefore, the mentioned above suggests that activation of RAGE-dependent MAPKs signaling by S100A9 is involved in HCC development. Interestingly, the gene and protein levels of RAGE in HCC cells and in vivo tumor tissues were obviously elevated compared to the control GST, suggesting that S100A9 may induce HCC cells to increase RAGE expression ensuing a cycle of sustained receptor activation, facilitating S100A9 interaction with HCC cells. Previous study reported that RAGE-mediated cellular stimulation promotes increased expression of the receptor itself, being characterized by

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 6 – Effects of RAGE on tumorigenesis induced by S100A9 in HCC cells in vitro and in vivo. (A and B) Detection of the clonogenic formation in soft agar. The representative images of the colony-forming unit are presented (A), and colony-forming rates for each group are quantified (B). npo0.05, nnpo0.01. (C) Images of representative mice bearing tumors derived from mice injected with HepG2 cells with different treatment. Tumor growth curves of all groups are shown below. Subcutaneous tumor growth was recorded every 5 days with vernier calipers. (D) Immunohistochemical staining analysis for RAGE, phosphorylated p38 and phosphorylated ERK1/2 in xenograft tumor sections. Scale bar¼ 150 lm.

ligand-receptor interaction followed by increased expression of the receptor [42]. And NF-κB transcriptional activation was reported to be involved in the regulation of RAGE expression [43]. Binding of S100s to RAGE activates a range of signaling pathways including MAPK pathway [36,44–46] and NF-κB pathway [36,46]. And p38 and p44/p42 MAPKs activation are also required for RAGE-mediated NF-κB transcriptional activation [47]. Therefore we speculate that the increased expression of RAGE may be mediated by activating its downstream pathways such as MAPK/NF-κB through binding to S100A9, which is required to elucidate in our further studies. Increased MAPK activity has been found in several types of cancers, which affects cell survival, apoptosis and invasion on numerous downstream targets [48]. In HCC, knockdown of ERK1/2 abolishes liver tumor cell proliferation in vitro as well as the growth of xenografted tumors [49]. In addition, it has been reported that activated p38 signal transduction pathway is involved cell invasion by upregulation of the expression of matrix metalloproteinases (MMPs) [50–53]. Therefore, downstream targets that are affected by S100A9-mediated RAGE/MAPK signaling activation need to be investigated in the future.

Cancer stem cells (CSCs) have been shown to have capacities of promoting tumor growth, tumor regeneration, metastatic progression [54–56]. In the upstream of RAGE signaling, there exist some members of S100s closely correlated with CSCs. Overexpression of S100A4 in head and neck squamous cell carcinomas cells enhanced their stemness and tumorigenic properties [57]. S100A6 expression is over-expressed in mouse glioma CSCs [58]. S100A14 is also identified as a potential novel marker of breast cancer cells with tumor-initiating features [59]. Our result showed that S100A9 enhanced their colony forming ability and stemness property in HCC cells via the interaction with RAGE. In all, these findings revealed a crucial role for S100s/RAGE signaling pathway in maintaining the stemness properties and tumorigenicity of CSCs. Hence, S100s/RAGE-mediated downstream signalings in cancer stem cells need to be investigated in the future. In summary, our results indicate that S100A9 promotes cell growth and invasion in HCC by interaction with RAGE and activation of RAGE-dependent ERK1/2 and p38 MAPK signaling cascades (Fig. 7). Collectively, this data provide important information regarding the role of extracellular S100A9 in HCC progression and should be paid much attention considering that

Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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Fig. 7 – RAGE/MAPK pathway was involved in S100A9-regulated growth and invasion in human HCC cells. Extracellular S100A9 binds to RAGE and stimulates RAGE-dependent ERK1/2 and p38 MAPK signaling cascades, promoting cell growth and invasion in HCC.

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S100A9 has been increasingly recognized as a new molecular target for developing cancer therapeutics. [16]

Acknowledgments Q4 The present study was supported by grants from the National

Clinical Key Subject to the Department of Laboratory Medicine of the First Affiliated Hospital of Chongqing Medical University (No. 20100305) and National Natural Science Foundation of China (No. 30772548).

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Appendix A.

Supporting information

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Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2015.04.008. [20]

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Please cite this article as: R. Wu, et al., S100A9 promotes human hepatocellular carcinoma cell growth and invasion through RAGEmediated ERK1/2 and p38 MAPK pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.008

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