Formylpeptide receptor 1 mediates the tumorigenicity of human hepatocellular carcinoma cells

abstract G protein-coupled chemoattractant receptors (GPCRs) have been implicated in cancer progression. Formylpeptide receptor 1 (FPR1) was originally identified as a GPCR mediating anti-microbial host defense. However, the role of FPR1 in tumorigenesis remains poorly understood. The current study aims to investigate the potential of FPR1 to regulate human hepatoma growth and invasion. We found the FPR1 gene and protein expression in human intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) specimens and in human hepatoma cell lines. FPR1 activation mediated the migration, calcium mobilization and ERK-dependent IL-8 production by hepatic cancer cells. FPR1 knockdown substantially reduced the tumorigenicity of hepatoma cells in nude mice. Necrotic hepatic tumor cells released factor(s) that activated FPR1 in live tumor cells. Our results indicate a critical role of FPR1 in the progression of malignant human hepatic cancer. FPR1 thus may represent a molecular target for the development of novel anti-hepatoma therapeutics.


Introduction
HCC is the fifth most common cancer with increasing incidence worldwide. 1 It is also one of the most lethal human malignancies due to the difficulty of early detection, rapid progression and chemoresistance. Hepatoma is characterized by vigorous angiogenesis and metastasis which account for high rate of postsurgical recurrence and extremely poor prognosis. 1 HCC generally develops from chronic liver injury, leading to inflammation, matrix remodeling, fibrosis and cirrhosis. 2 Chronic infections by hepatitis viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) are major risk factors for HCC development. 3 FPR1 is a G protein-coupled 7 transmembrane cell surface receptor (GPCR), originally identified in phagocytic leukocytes, that mediates cell chemotaxis and activation in response to the bacterial formylated chemotactic peptides. 4 FPR1 is involved in a broad spectrum of pathophysiologic processes including inflammation, 5 wound healing, 6 glioblastoma progression 7,8 and the host defense against HCV infection. 9 As one of the most vascularized solid tumors, the HCC progression and prognosis correlate with the status of angiogenesis, 10 due largely to elevated production of angiogenic factors, such as interleukin-8 (IL-8, CXCL8) by tumor cells. 11 Previous reports have implicated IL-8 in the growth and angiogenesis of malignant tumors. 12 In cancer models of pancreas, colorectum, melanoma and liver, IL-8 functions as an autocrine growth factor. 11 In human hepatoma, clinical investigation has reported that high level of IL-8 is associated with higher frequency invasion of portal vein venous vessels and bile duct by tumor. 13 IL-8 production was also associated with severe hepatitis and the development of hepatocellular carcinoma. 14 Upregulation of the IL-8 receptor CXC chemokine receptor 2 (CXCR2) was found in HCC and was correlated with intrahepatic metastasis. 15 In vitro experiments showed IL-8 production by HCC cell lines in response to tumor necrosis factor-a (TNF-a). 16 Previous studies reported that N-formylmethionyl-leucyl-phenylalanine (fMLF), a bacterial chemotactic peptide activating FPR1, increased chemotaxis and production of angiogenic factor IL-8 by human gliobstoma. 7,17 FPR1 in glioblastoma cells also interacts with agonists released by necrotic tumor cells, 7 suggesting that tumor cells may utilize FPR1 to recognize agonists produced in the tumor microenviroment for their advantage. Since hepatocarcinogenesis involves a highly orchestrated interplay of injury, chronic inflammation and neovascularization, 2 the multitude of FPR1 suggests that it may also play a role in the development of hepatic cancer. 5-7, 9,17 In the present study, we report that FPR1 was expressed by HCC tissues from patients and the human hepatoma cell lines. Hepatoma cells responded to the FPR1 agonist fMLF by increased motility, proliferation and enhanced IL-8 production. FPR1 small hairpin RNA (shRNA) substantially reduced the tumorigenicity of hepatoma cells in nude mice. Our study thus demonstrates a significant role of FPR1 in the carcinogenesis of human hepatoma.

Results
The expression of FPR1 on human hepatocellular carcinoma tissues We performed histologic and immune fluorescence staining of FPR1 in tumor tissues from HCC patients. In surgical specimens, hematoxylin-eosin (H&E)-staining revealed poorly (Fig. 1A) and moderately (Fig. 1B) differentiated HCC with a trabecular pattern. In the high-grade intratumor specimens, multiple tumors of intrahepatic metastases and portal vein invasion were observed. The cellular and nuclear pleomorphism, intracellular vacuoles, mitotic patterns, vessel formation and the necrosis in central tumor tissues were also demonstrated (Fig. 1A, upper right panels). The peritumor ( Fig. 1A and B, lower right panels) liver tissue showed a chronic inflammatory infiltration in the fibrous stroma, diagnosed as hepatic cirrhosis. Strong FPR1 signal was detected in grade III HCC specimens (Fig. 1A, left panels), and positive staining was enriched in intratumor area Fig. 1A, upper left panels). In contrast, the lesser aggressive grade II hepatoma specimens showed intermediate staining intensity of FPR1 in intratumoral tissues (Fig. 1B, Figure 1. FPR1 expression in human hepatocellular carcinoma tissues. Sections of 20 samples from grade III (A) and II (B) hepatocellular carcinoma and 10 samples of adjacent normal liver tissues (C) were stained with an antibody against FPR1 (green) and counterstained with DAPI (blue). Representative intratumor (upper panels) and peritumor (lower panels) immunofluorescence staining (left panels) and corresponding H&E staining (right panels) are shown. Bar D 100 mm. (D), quantitative PCR analysis showing the level of FPR1 gene in intratumor or peritumor tissues of grade III and II hepatocellular carcinoma and adjacent normal liver tissues. The data was shown as the mean-fold changes of FPR1 expression levels ( § SEM) after intra-sample normalization to the levels of GAPDH. Ã p <0.05, statistically significant difference vs. adjacent normal liver tissues in values.

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upper left panels) and very low FPR1 expression in peritumoral tissues (Fig. 1B, lower left panels). We then examined whether FPR1 expression is selectively enhanced in hepatocellular carcinoma. Fig. 1C shows that protein was detectable in human normal liver tissues adjacent to HCC. However, the levels were far lower than that in HCC tissues. Very few FPR1-positive cells were found in the adjacent normal liver tissues, demonstrating that FPR1 expression is selective in HCC and in particular in intratumor tissues.
We next measured FPR1 RNA in HCC tissues and found FPR1 mRNA was higher in grade III than in grade II carcinoma specimens. The highest mRNA expression was found in the poorly-differentiated intratumor samples ( Fig. 1D and Fig. S1A and B), consistent with the results obtained with histology analysis. These results demonstrate the expression of FPR1 by human hepatocellular carcinoma.

The expression of FPR1 by human hepatoma cell lines
To further determine the biological function of FPR1 in hepatic cancer cells, we used established human hepatic cancer cell lines HepG2 and Hep3B. Fluorescence-activated cell sorting (FACS) analysis shows that both HepG2 and Hep3B cells expressed FPR1 ( Fig. 2A). HepG2 cells expressed higher levels of FPR1 on cell surface than Hep3B cells ( Fig. 2A). HepG2 and Hep3B cells also expressed FPR1 gene with higher levels in HepG2 cells ( Fig. 2B and Fig. S1C and D).

FPR1 promotes human hepatoma cell chemotaxis and invasion
We next tested the capacity of the FPR1 ligand fMLF to induce directional migration of human hepatoma cell lines. Both HerG2 and Hep3B cells migrated in response to fMLF with a bell-shaped dose-response curve ( Fig. 3A and C and Fig. S2A), typical of the cell response to chemoattractants. 18 We also tested the capacity of Ac2-26, a specific cognate ligand for FPR1, 19 to induce the migration of human hepatoma cell lines. Both HepG2 and Hep3B cells migrated in response to this FPR1 agonist ( Fig. S2B-D). However, addition fo fMLF and Ac2-26 simultaneously to the cells in the upper wells of the chemotaxis chamber abrogated cell migration induced by equal concentrations of fMLF and Ac2-26 in the lower wells (data not shown). Thus, FPR1-induced migration of human hepatoma cell lines was based on chemotaxis rather than chemokinesis. Furthermore, the migration of HepG2 and Hep3B in response to fMLF was completely inhibited by pretreatment of the cells with the Gi protein inhibitor pertussis toxin (PT) and a FPR1-specific antagonist tBoc-MLF, 20 but not cholera toxin (CT) or herbimycin A, a protein tyrosine kinase inhibitor ( Fig. 3B and C and Fig. S2A-E), suggesting the involvement of a G-protein of the Gi-type coupled receptor. 21 To more precisely examine the contribution of FPR1 to liver tumor cell motility and invasiveness, we used a wound-healing model by creating a gap in a confluent HepG2 cell monolayer. HepG2 cells treated with fMLF showed more rapid locomotion than vehicle-treated cells toward the center of the gap on cell monolayer ( Fig. 3D and E). Addition of cyclosporin H (a FPR1 specific antagonist) and PT to the cells blocked the capacity of fMLF to induce human hepatoma cell migration in scratch wound-healing assays, confirming FPR1-mediated cell motility ( Fig. S3A and B). Thus FPR1 activation enables HepG2 cells to exhibit higher motility.
In addition to promoting cell motility, fMLF elicited a potent dose-dependent and PT-sensitive Ca 2C mobilization in HepG2 and Hep3B cells ( Fig. 4A and B and Fig. S4A and B). Sequential stimulation of HepG2 cells with fMLF at high and low concentrations or vice versa resulted in bidirectional desensitization ( Fig. 4C and D). However, pretreatment with FPR1 antagonists cyclosporin H or tBoc-MLF reduced the response of HepG2 and Hep3B cells induced by fMLF ( Fig. S4D-I). These results further support the specificity of FPR1 expressed by HCC cell lines in response to fMLF

Activation of FPR1 promotes IL-8 production by human hepatoma HepG2 cells
Since activation of FPR1 induces the production of IL-8, 11,25 we tested whether FPR1 also promotes IL-8 production by hepatoma cells. Untreated HepG2 and Hep3B cells produced low levels of IL-8 protein and treatment of fMLF and Ac2-26 increased IL-8 production ( Fig. 5B and Fig. S5E and F), reaching a maximum at 48 h after stimulation with 100 nM fMLF (Fig. 5B). Addition of the ERK inhibitors PD98059, U0126 and CsH, but not p38 MAPK inhibitor SB203580, inhibited fMLF-and Ac2-26-stimulated IL-8 production by HCC cells (Fig. 5B and Fig. S5E and F). Thus, only the ERK1/2 MAPK pathway appears to be crucial for FPR agonist-induced IL-8 expression by hepatoma cells.
IL-8 contained in the culture medium of fMLF-stimulated HepG2 cells was biologically active. When cultured with conditioned medium from fMLF-treated HepG2 cells and fMLFCSBtreated HepG2 cell, but not fMLFCCsH-nor fMLFCU0126-treated HepG2 cells, HUVECs formed capillary-like structures on a Matrigel surface (Fig. 5C and D and Fig. S5G and H), which was inhibited by the addition of a monoclonal antibody against human IL-8 ( Fig. 5C and D). Thus, IL-8 in the conditioned medium from FPR1-activated HepG2 cells induces endothelial cells to form capillary-like structures, a key event associated with neovascularization.

FPR1 knockdown by shRNA reduces the tumorigenicity of hepatic cancer cells
To evaluate the role of FPR1 in hepatoma tumorigenicity, we used shRNA to delete FPR1 in HepG2 cells. After stable transfection of FPR1 shRNA of HepG2 cells, the expression of FPR1 mRNA (Fig. 6A) and fMLF-induced chemotaxis (Fig. 6B) were abolished. In addition, the ability of fMLF to induce IL-8 production by HepG2 cells (Fig. 6C) was abrogated. Further, the cells failed to respond to the proliferation stimulating activity of fMLF (Fig. 6D).
We then injected HepG2 cells transfected with FPR1 shRNA into the flanks of athymic mice. Tumor nodules formed by HEPG2 cells transfected with FPR1 shRNA appeared later (Fig. 6E) and grew more slowly than those formed by wild-type HepG2 cells or by mock-transfected cells (Fig. 6F and G). By day 52, all mice implanted with wild-type or mock-transfected HepG2 cells were dead. In contrast, 90% of the mice bearing tumors formed by FPR1 shRNA-transfected HepG2 cells survived to the day 66 after implantation (Fig. 6H). To conform the role of FPR1 in HCC progression by in vivo experiments using murine cancer cell lines in immunocompetent mice, we examined the effect of FPR1 tumorigenicity in H22 tumor model of BALB/c mice following the method described by another group. 26 We show that FPR1-shRNA had similar antitumor effects in immunocompetent mice shown in immunocompromised mice with human cell line (Fig. S6A, B and C). These results indicate that depletion of FPR1 markedly reduced the ability of HepG2 cells to form tumors, confirming the contribution of FPR1 to the tumorigenicity of human HCC cells.

The production of FPR1 agonist activity by necrotic hepatoma cells
Necrotic cell death has been reported to promote hepatocarcinogenesis, 27,28 and mitochondria of ruptured cells contain chemotactic formylpeptides that activate FPR1 in myeloid cells. 29 We thus investigated whether necrotic hepatoma cells and tissues might produce agonist(s) recognized by FPR1 on live hepatic cancer cells. HepG2 cells and HepG2 tumors formed in athymic mice released potent chemotactic activity for live HepG2 cells (Fig. 7A and B) and ETFR cells overexpressing FPR1 (data not shown). The FPR1 agonist activity released by necrotic HepG2 cells and tumor tissues was blocked by an anti-FPR1 antibody or by the FPR1-specific antagonist tBoc-MLF (Fig. 7B, and data not shown). Necrotic hepatoma cell supernatant also induced a robust intracellular Ca 2C mobilization in live HepG2 cells (Fig. 7C) which attenuated HepG2 cell to response to subsequently administered fMLF (Fig. 7D). These results suggest that the agonist(s) contained in the supernatants of necrotic HepG2 cells shares FPR1 with fMLF on HepG2. 30 We additionally observed that necrotic hepatoma supernatants inhibited the expression of FPR1 on the surface of ETFR cells with an efficacy comparable to that of 10 3 nM fMLF (Fig. 7E). Thus, necrotic hepatic cancer cells produce FPR1 agonist(s) that interacts with FPR1 on live tumor cells.

Discussion
In this article, we showed that the classic leukocyte chemoattractant receptor FPR1 is expressed by human HCC tissues and hepatoma cell lines. To our knowledge, this is the first demonstration that FPR1 may contribute to the progression of HCC by mediating tumor cell chemotaxis, proliferation, and production of IL-8 in response to endogenous agonist(s). A growing body of evidence has suggested important roles of chemoattractant GPCRs in cancer initiation and progression by influencing aberrant cell growth and survival. 31 GPCRs are also implicated in the invasion and metastasis of cancer cells and contribute to the establishment and maintenance of a permissive tumor microenvironment. 31 The FPR1 involvement in cancer malignancy has been shown by its roles in glioblastoma tumorigenicity. 7 FPR1 expressed by highly malignant human glioma cells promoted the motility, growth, and the production of angiogenic factors. 7,32 The glioblastoma-promoting activity of FPR1 is mediated in part by transactivation of the epidermal growth factor receptor (EGFR), 8 associated with increased methylation of p53 gene. 33 FPR1 in glioblastoma cells is stimulated by Annexin 1 released by necrotic tumor cells. 19 Our present study extended the functional scope of FPR1 to its potential role in promoting the growth of malignant human hepatoma.
As a primary malignancy that emerges on a background of chronic liver diseases, 34 HCC is the fifth most common cancer worldwide with an extremely poor prognosis. 35 Chronic inflammation and improper wound healing following hepatic injury are crucial causative factors for cancer in the liver. 2 The link between an inflammatory state and cancer can be viewed from an extrinsic perspective for which infection and subsequent chronic inflammation drive oncogenesis. 36 In that case, cancers associated with inflammation are generally aggressive. 11 Functional FPR1 has reportedly been detected in hepatocytes, and it promoted the production of acute phase proteins in response to fMLF. 37 Thus, FPR1 expressed on hepatocytes might participate in the inflammatory courses in liver. It has been shown that the expression levels of chemokines in the liver are positively correlated with the severity of hepatic inflammation in chronic HCV infection. 38,39 The elevated expression of chemokines in a severely inflamed liver could efficiently attract leukocytes to the liver. 39,40 Our previous studies also revealed that HCV peptide (C5A) is able to activate FPR1 on phagocytic leukocytes. 9 Given that HCC is an example of inflammation-related cancer, and the chronic infections with HBV and HCV are major risk factors for HCC development, 41 our findings unveiling the hepatoma-promoting properties of FPR1 may represent a novel mechanism by which the proinflammatory response is linked to tumor initiation.
A hallmark in the progression of malignant tumors is increased angiogenesis. The hyper-vasculature in chronic liver diseases facilitates the progression from small dysplastic nodules, through neoplastic lesions to large hepatocellular carcinoma. 34 It has been reported that FPR2 (FPRL1) in human corneal epithelial cells was activated by an anti-microbial peptide LL-37 to stimulated IL-8 production by the cells resulting in enhanced vascularization. 42 As a proinflammatory chemokine, IL-8 mediates the recruitment of leukocytes, which in turn produce angiogenic factors recruiting vascular endothelial cells and promoting their proliferation. 11 IL-8 has been shown to increase the motility of a variety of tumor cells from glioblastoma, melanoma, pancreatic cancer to colon cancer. 11,17 IL-8 production was correlated with the severity of hepatitis and the development of hepatocellular carcinoma. IL-8 was elevated in patients with advanced HCC with distant metastasis. 14,43 In addition, upregulated IL-8 in the drainage vein of colorectal cancer is linked to the occurrence of hepatic metastasis. 44 Our present study showing the induction of IL-8 by FPR1 activation in human hepatoma cells supports the adverse effect of this chemokine on hepatoma growth and angiogenesis.
The expression of IL-8 in normal cells is low, but it was highly inducible by a variety of pro-inflammatory stimulants. 45 In primary human hepatocytes, IL-8 is induced by adipokine, 46 whereas HCC produced IL-8 after stimulation by TNF-a. 16 In rat cornea, IL-8 stimulates angiogenesis by promoting the chemotaxis and growth of vascular endothelial cells. 47 IL-8 also inhibits the apoptosis of endothelial cells, upregulates endothelial cell production of matrix metalloproteinase-2 and -9, mimics the function of vascular endothelial growth factor (VEGF), and trans-activates VEGF-R2. 11 In vitro mitochondrial proteins from necrotic HepG2 cells stimulate FPR1 in monocyte to release IL-8. 25 Thus, the current study provides evidence for IL-8 as a molecule down-stream of FPR1 signaling pathway to amplify the effect of FPR1 on hepatic tumor growth.
Malignant tumors exploit their microenvironment to favor their survival, growth, invasion, and metastasis. 48 For instance, tumor cells produce aberrant levels of regulatory molecules to increase their proliferation. Tumor cells also produce high levels of IL-8 to enable cancer cells to survive and to recruit endothelial cells for vascularization. 11 In addition, malignant tumor cells express receptors that interact with agonists that are present in the vicinity of the tumor or produced by distant organs to increase tumor cell motility to favor tumor cell invasion and metastasis. In the present study, FPR1 was expressed at a higher level by advanced hepatocellular carcinoma. A positive correlation between FPR1 expression and the grade of HCC suggests FPR1 as a biomarker for more aggressive tumor cells. Identification of FPR1 agonist(s) in the supernatants of necrotic tumor cells provides evidence that this receptor may interact with endogenous agonists produced in tumor lesions, presumably in the necrotic area frequently associated with highly malignant hepatoma or in surrounding tissues that are compressed by growing tumor in a limited anatomical compartment. Previous studies have demonstrated FPR1-dependent necrotaxis as the molecular mechanisms underlying the neutrophil recruitment to sites of sterile focal hepatic necrosis. 49 It is thus plausible that FPR1 in live tumor cells may serve as a sensor for the agonists produced in a "paracrine" manner in the tumor microenvironment to promote cell migration, survival, proliferation and the production of IL-8.
Further study is required to more precisely define the relationship between the FPR1 expression and the progression of human primary HCC and to identify the mechanistic basis for the control of FPR1 expression in highly malignant human hepatic cancer cells. In addition, the pathogenesis of human hepatoma is likely to be complex, and FPR1 may not be the sole factor that regulates the progression of malignant liver tumor. Additional experiments are needed to address the possible cross-talk between FPR1 and other growth receptors such as EGFR 8 in exacerbating the malignant phenotype of cancer cells. Also, the relationship between FPR1 expression and the survival of hepatoma patients after treatment remains to be established. Nevertheless, the present study implicates the role of FPR1 in the rapid progression of highly malignant human hepatocellular carcinoma, thus raises the possibility that FPR1 may be a candidate molecular target for developing novel therapeutics.

Patients and specimens
Tumor samples were obtained from patients with pathologically confirmed HCC at the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. All samples were coded anonymously in accordance with local ethical guidelines (as stipulated by the Declaration of Helsinki), and written informed consent was obtained. The protocol was approved by the Review Board of the First Affiliated Hospital, Sun Yat-sen University. Detailed information is described in Supplementary Materials and Methods.

Cells
Detailed information about cell lines including human hepatic cancer cells HepG2 and Hep3B, rat basophilic leukemia cells stably transfected with epitope-tagged FPR (ETFR), human umbilical cord vein endothelial cells (HUVECs), human peripheral blood mononuclear cells (PBMC) and monocytes is described in Supplementary Materials and Methods.

Chemotaxis and Ca 2C flux
Assays for tumor cell chemotaxis and Ca 2C mobilization were performed according to the described procedures. 18,50 Details can be obtained from Supplementary Materials and Methods.

Tumorigenesis
To generate xenografts with hepatoma cells transfected with FPR1 shRNA, 1 £ 10 6 cells were injected into the flank of each 4-week-old female athymic Ncr-nu/nu mouse. Animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals. Detailed information about FPR1 knockdown in HepG2 cells and tumor implantation is described in Supplementary Materials and Methods.

Additional Materials and Methods
A full description of the methods including histology and immunofluorescence staining, RT-PCR, flow cytometry analysis, wound-healing assays, Western blotting, ELISA, formation of capillary-like structures, MTT assay, generation of tumor cell supernatant and tissue extracts, and statistical analyses can be found in Supplementary Materials and Methods.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.