The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1β and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.
Fibroblast growth factor (FGF)-2 is one of a family of FGFs that includes 22 structurally related members.1 FGF-2 has been shown to exert a potent angiogenic effect by interacting with tyrosine kinase receptors, FGFR1, FGFR2 and FGFR3, in various cancers including hepatocellular carcinoma (HCC).2–4 Aside from its angiogenic effect, FGF-2 has also been shown to act as a mitogen for HCC cell proliferation via an autocrine mechanism.5 Uematsu et al. reported that the serum FGF-2 of chronic liver disease patients without HCC tended to be higher than that of those with HCC.6 Decrease of serum FGF-2 could be observed prior to the emergence of HCC, and this suggests that FGF-2 may play a critical role in the surveillance of HCC. However, the immunological significance of elevating the FGF-2 levels in chronic liver disease patients remains unclear.
HCC is one of the leading causes of cancer deaths worldwide. Chronic liver disease caused by hepatitis virus infection and nonalcoholic steatohepatitis leads to a predisposition for HCC, with liver cirrhosis (LC), in particular, being considered a premalignant condition.7, 8 The liver contains a large compartment of innate immune cells (NK cells and NKT cells) and acquired immune cells (T cells),9, 10 but the activation process of these immune cells in HCC development remains unclear. A recent study has demonstrated that the innate immune system may play a critical role in tumor surveillance via an NKG2D signal.11 Knowing the details of how to activate the abundant NK cells in the liver could lead to the establishment of attractive new strategies for HCC treatment.
In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. Of importance are the findings that serum FGF-2 levels in patients with CH and LC without HCC were significantly higher than that in those with HCC and that FGF-2 enhanced the NK sensitivity of HCC cells. The present study sheds light on previously unrecognized immunological effects of FGF-2 on HCC cells and thus suggests a role of FGF-2 in HCC development in patients with CH type C.
Material and Methods
Liver tissues and immunohistochemistry
Human HCC tissues (n = 6) and normal liver tissues (n = 2) were obtained at surgical resection. CH tissues (n = 4) and LC tissues (n = 4) were obtained as liver biopsy samples. Informed consent, under an Institutional Review Board-approved protocol, was obtained from all patients before sample acquisition. Liver sections were subjected to immunohistochemical staining using the ABC procedure (Vector Laboratories, Burlingame, CA). The primary antibody (Ab) was antihuman FGF-2 Ab (Abcam, Cambridge, MA). To confirm the specificity of the staining, the primary antibody was incubated with recombinant human FGF-2 protein (R&D Systems, Minneapolis, MN) for 3 hr and then applied onto liver sections in parallel with staining of the primary antibody as the absorption test.
HCC cell lines
HepG2 and PLC/PRF/5, human hepatoma cell lines, were purchased from American Type Culture Collection (Rockville, MD) and were cultured with Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (GIBCO/Life Technologies, Grand Island, NY) in a humidified incubator at 5% CO2 and 37°C.
The sera from CH patients (n = 80), LC patients (n = 84), HCC patients (n = 112, StageI/II n = 51 and StageIII/IV n = 61) and age-matched healthy volunteers (HVs) (n = 24) were subjected to analysis of the FGF-2 level. Clinical backgrounds of patients were summarized in Table 1. Informed consent, under an Institutional Review Board-approved protocol, was obtained from all patients before sample acquisition. The level of FGF-2 and soluble major histocompatibility complex class I-related chain A (MICA) were determined using Quantikine Human FGF basic (R&D Systems) and DuoSet MICA eELISA kit (R&D Systems), respectively.
Table 1. Clinical backgrounds
HCC cells and normal hepatocyts cultures
Both HepG2 and PLC/PRF/5 cells or normal hepatocytes (ScienCell Research Laboratories, Carlsbad, CA) were cultured for 72 hr in the presence or absence of human interleukin-1β (IL-1β) (50 ng/ml, Peprotech, Rocky Hill, NJ), human IL-6 (300 ng/ml, Peprotech), human transforming growth factor-β1 (TGF-β1) (50 ng/ml, R&D Systems) and human tumor necrosis factor-α (TNF-α) (100 ng/ml, Peprotech), and the treated cells were harvested and evaluated for expression of FGF-2. In some experiments, HepG2 and PLC/PRF/5 cells were cultured in the presence or absence of recombinant human FGF-2 protein (250 ng/ml, R&D Systems) with or without antihuman FGFR2 neutralizing Ab (10 μg/ml, R&D Systems) for 48 hr, and the hepatoma cells were harvested and evaluated for the immunological regulation of the NK cells.
For the detection of membrane-bound MICA, cells were incubated with anti-MICA specific Ab (2C10, Santa Cruz Biotechnology, Santa Cruz, CA) and stained with Goat F(ab′)2 fragment anti-Mouse IgG(H+L)-PE (Beckman Coulter, Fullerton, CA) as a secondary reagent and then subjected to flow cytometric analysis. For the detection of human leukocyte antigen (HLA) class I, cells were incubated with PE-conjugated antihuman HLA-A,B,C Ab (w6/32, BD Biosciences, San Jose, CA). Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
The total cellular protein was electrophoretically separated using sodium dodecyl sulfate-12% polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked in Tris-buffered saline-Tween20 containing 5% skim milk for 1 hr and then probed with rabbit polyclonal Ab to FGF-2 (Abcam) at room temperature overnight. Horseradish peroxidase-conjugated anti-rabbit IgG and SuperSignal West Pico System (Pierce, Rockford, IL) were used for the detection of blots.
Total RNA was isolated using RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan) and was reverse transcribed using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Foster City, CA). The mRNA levels were evaluated using ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Ready-to-use assay (Applied Biosystems) was used for the quantification of FGF-2 (ID: Hs00960934_m1), MICA (Hs00792195_m1) and β-actin (Hs:99999903_m1) mRNAs according to the manufacturer's instructions. β-actin mRNA from each sample was quantified as endogenous control of internal RNA.
NK cell analysis
NK cells were isolated from human peripheral blood mononuclear cells by magnetic cell sorting using CD56 MicroBeads (Miltenyi Biotech, Auburn, CA).12 The cytolytic ability of NK cells against FGF-2-treated HepG2 and PLC/PRF/5 cells was assessed by 4-hr 51Cr-release assay with or without antihuman MICA/B Ab (BD Biosciences) as previously described.12 The expressions of NKG2D and NKG2A on NK cells were analyzed by flow cytometry with PE-conjugated antihuman NKG2D Ab (BD Biosciences) and PE-conjugated IgG antihuman NKG2A Ab (R&D Systems).
For human sample data, values were expressed as the median and interquartile range using box plots and the 10th and 90th percentiles as horizontal bars. For comparison of more than two groups, the Kruskal–Wallis rank sum test was used. If the Kruskal–Wallis test was significant, post hoc multiple comparisons were carried out using the Steel-Dwass procedure. Differences between retreatment and post-treatment values were tested by the paired t-test. FGF-2 mRNA values were expressed as the mean and SD, and the statistical significance of differences between the groups was determined by applying Student's t test after each group had been tested with equal variance and Fisher's exact probability test. We defined statistical significance as p < 0.05.
FGF-2 is expressed in the liver and serum of patients with chronic liver diseases
We first examined the FGF-2 expressions in the livers of normal volunteers and the patients with chronic liver diseases. Immunohistochemical analysis revealed that FGF-2 was not expressed in normal liver tissues. In contrast, the expressions of FGF-2 were detected in chronic liver tissues (Fig. 1a). We evaluated the serum FGF-2 levels by specific ELISA. All of the chronic liver disease patients were hepatitis C virus (HCV)-RNA positive. As shown in Figure 1b, the serum FGF-2 levels in CH and LC patients were significantly higher than those of HV, but those in HCC patients were not. Those in CH patients were also significantly higher than those in LC or HCC patients. Those in LC patients tended to be higher than those in HCC patients, although this was not significant. The serum FGF-2 levels in HCC patients were low and significant difference between StageI/II patients and III/IV patients was not observed (data not shown). We compared the serum FGF-2 levels before and after the development of HCC in six chronic liver disease patients. The mean follow-up period was nine years. The serum FGF-2 levels of the patients before the occurrence of HCC were significantly higher than those of the same patients after the occurrence of HCC (Fig. 1c). These results demonstrated that the serum FGF-2 levels were highest in CH patients and significantly decreased as the liver disease progressed.
FGF-2 levels were associated with the incidence of HCC in chronic liver disease patients
The earlier results suggested that increased FGF-2 levels might prevent HCC tumor development. We investigated the correlation of the serum FGF-2 level and HCC incidence. The 84 LC patients were divided into two groups according to serum FGF-2 levels, high (serum FGF-2 concentration > 1.8 pg/ml; 40 patients) and low (≦1.8 pg/ml; 44 patients), because the median of FGF-2 levels in these patients was 1.8 pg/ml. We followed these LC patients for three years and compared the rates of HCC-free survival. As shown in Figure 1d, the HCC free ratio of the high FGF-2 patients was significantly higher than that of the low FGF-2 patients. These results suggested that FGF-2 production from chronically diseased liver tissues might be associated with the occurrence of HCC.
Inflammatory cytokines increased FGF-2 expression in HCC cells and normal hepatocytes
Previous reports demonstrated that FGF-2 expressions were detected in both tumor cells and normal hepatocytes in addition to sinusoidal endothelial cells in HCC tissues.5 Some inflammatory cytokines, such as IL-1β, IL-6, TGF-β and TNF-α, are known to increase in CH patients.13–15 To examine the effect of such inflammatory cytokines on FGF-2 expression in liver cells, we cultured HepG2 and PLC/PRF/5 HCC cells for 72 hr in the presence or absence of these cytokines. As shown in Figure 2a, IL-1β and IL-6 increased FGF-2 protein levels in both HepG2 and PLC/PRF/5 cells. FGF-2 mRNA levels in HepG2 and PLC/PRF/5 cells treated with IL-1β and IL-6 were significantly higher than those in nontreated control HCC cells (Fig. 2b). We also examined FGF-2 levels in the supernatants of the HCC cells cocultured with inflammatory cytokines. FGF-2 levels of IL-1β- or IL-6-treated HepG2 cells or PLC/PRF/5 cells tended to increase compared with those of nontreated HCC cells (data not shown). FGF-2 mRNA levels in normal hepatocytes treated with IL-1β, but not IL-6, were also significantly higher than those in nontreated control cells (Fig. 2c). These results suggested that both IL-1β and IL-6 were capable of inducing FGF-2 expression in HCC cells and normal hepatocytes. We also examined whether TGF-β1 and TNF-α could induce FGF-2 expressions on HCC cells. We found that FGF-2 expression levels in treated HCC cells did not change in Western blotting or real-time RT-PCR analysis (data not shown).
FGF-2 induced the expression of membrane-bound MICA and suppressed the expression of HLA class I on HCC cells, but FGF-2 did not change the expressions of NKG2D and NKG2A on NK cell
The above findings suggested that decreasing FGF-2 might affect the HCC development in the patients with chronic liver disease. To investigate whether or not FGF-2 protein directly activates NK cells, we examined whether FGF-2 affected the expression of NKG2D (activating receptor) or NKG2A (inhibitory receptor) on NK cells. We cultured CD56+ NK cells obtained from HVs with FGF-2 for 24 hr and then subjected them to flow cytometric analysis. The expressions of both NKG2D and NKG2A on NK cells did not change by adding FGF-2 protein (Fig. 3a), suggesting that FGF-2 did not have a direct effect on NK cells. We next examined the immunological modification of human HCC cells by adding human FGF-2 protein. We evaluated the expressions of membrane-bound MICA (NK activating molecule) and HLA class I (NK inhibitory molecule) in HepG2 and PLC/PRF/5 cells by flow cytometry. The expressions of MICA on FGF-2-treated cells were higher than those on nontreated cells in both HepG2 and PLC/PRF/5 cells (Fig. 3b). In contrast, those of HLA class I on FGF-2-treated cells were lower than those on nontreated cells in both types of HCC cells (Fig. 3b). FGF-2-treatment could modify the expressions of MICA and HLA class I on HCC cells in a dose-dependent manner (data not shown). The mRNA level of MICA in FGF-2-treated HepG2 cells was also significantly higher than that in nontreated HepG2 cells. The mRNA level of MICA in FGF-2-treated PLC/PRF/5 tended to be higher than that in nontreated cells, although the difference was not statistically significant (Fig. 3b). We examined the expressions of MICA and HLA class I on FGF-2-treated normal hepatocytes. The expressions of both molecules did not change in FGF-2-treated normal hepatocytes (Fig. 3c). We also evaluated FGF-2-dependent MICA regulation on a gastric cancer cell line (KATOIII), colon cancer cell lines (HCT116, HT29) and a cervical cancer cell line (Hela). The MICA expression was induced in FGF-2-treated HCT116 cells and weakly in FGF-2-treated Hela cells, but not in the other two cell lines (data not shown). These results suggested that FGF-2 could modify the MICA expressions in several types of cancers.
The signal via FGF-2/FGF-receptor2 is essential for the induction of MICA and HLA class I expressions on HCC cells
We examined the FGF receptors (FGFR1, FGFR2, and FGFR3) on both types of HCC cells by flow cytometry. The expressions of FGFR2 were high for both cell types. While FGF-2 has cross-reactivity with FGFR1 and FGFR3, the expressions of FGFR1 and FGFR3 were very low on both types of HCC cells (Fig. 4a). To examine whether the interaction between FGF-2 and FGFR2 could induce the expressions of MICA and HLA class I on both types of HCC cells, we evaluated the expressions of both molecules on FGF-2-treated HCC cells with anti-FGFR2 neutralizing Ab. The anti-FGFR2 Ab blocks the ability of FGF-2 to modulate MICA and HLA class I on both HepG2 and PLC/PRF/5 cells (Fig. 4b).
FGF-2 enhanced susceptibility to NK cells of HCC cells and the correlation of serum FGF-2 and soluble MICA levels in patients with chronic liver disease
The earlier results suggested that FGF-2 might enhance the susceptibility to NK cells of HCC cells. We next examined whether FGF-2 could modify the NK sensitivity of human HCC cells. The cytolytic activities of NK cells against FGF-2-treated HepG2 and FGF-2-treated PLC/PRF/5 cells were higher than those against nontreated HCC cells (Fig. 5a). The cytolytic activity against FGF-2-treated HCC cells decreased to the control levels on addition of anti-MICA/B blocking antibody (Fig. 5a) but not on addition of isotype IgG antibody (Fig. 5b). These results demonstrated that adding FGF-2 enhanced the NK sensitivity of HCC cells via increased expression of membrane-bound MICA. We next examined the correlation of serum FGF-2 and soluble MICA in patients with chronic liver disease. Serum FGF-2 levels in patients with chronic liver disease correlated with soluble MICA levels (Fig. 5c). These results suggested that high FGF-2 levels in patients with chronic liver disease may prevent the shedding of MICA in liver tissues.
The FGF-2 levels in chronic liver disease, a premalignant condition, have not been well studied. Uematsu et al. reported that the serum FGF-2 levels of patients with LC or HCC were significantly higher than those of HVs, and serum FGF-2 levels of HCC patients tended to be lower than those of LC patients without HCC.6 In contrast, Jinno et al. reported that the circulating FGF-2 levels in HCC patients were significantly higher than those in CH and LC patients.16 In the present study, we analyzed the serum FGF-2 levels on a larger scale for patients with chronic liver disease. Consistent with Uematsu's report, the serum FGF-2 levels significantly decreased along the progression of chronic liver disease and those in HCC patients were significantly lower than those in CH or LC patients. These results suggested that decreasing FGF-2 levels might be associated with the occurrence of HCC during the progression of chronic liver disease. FGF-2 has been shown to act as a potent angiogenic factor in a number of cell lines and solid tumors.1, 2 As for HCC development, FGF-2 has been reported to augment vascular endothelial growth factor (VEGF)-mediated angiogenesis in HCC development.17 However, at present, in contrast to the clear roles of VEGF in the angiogenesis of HCC, the roles of FGF-2 in the HCC development are still controversial and should be elucidated.
Immunohistochemical analysis revealed that hepatocytes in patients with chronic liver diseases seemed to produce FGF-2, but those in healthy donors did not. This suggested that inflammatory responses in liver tissues might have roles in the production of FGF-2. Some inflammatory cytokines, such as IL-1β and IL-6, increased in CH or LC patients.13–15 Aside from liver cells, IL-6 could induce FGF-2 expressions in basal cell carcinoma cell line18 or Kaposi's sarcoma cell and human umbilical vein endothelial cells.19 On the basis of these reports, we examined the effect of such inflammatory cytokines on FGF-2 expression in HCC cells and normal hepatocytes. The FGF-2 expression could be, at least in part, induced by IL-1β and IL-6. Both IL-1β and IL-6 are produced mainly by local immune cells, including activated Kupffer cells.20 Although the detail mechanism of the induction of FGF-2 expression in HCC cells and normal hepatocytes is little known, the production of these cytokines might contribute to preventing HCC development via promoting FGF-2 expression in the liver.
Guerra et al. reported that NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy,11 suggesting that NK-dependent immune-surveillance might play a critical role in tumor development. However, the mechanism of tumor surveillance of NK cells remains unclear in HCC development. We previously demonstrated that membrane-bound MICA on HCC cells plays essential roles in the NK sensitivity of HCC cells.21 We therefore evaluated the MICA (activating molecule of NK cells) and HLA class I (inhibitory molecule of NK cells) on HCC cells treated with FGF-2. This treatment resulted in increasing MICA expression and decreasing HLA class I on HCC cells. Consistent with these results, the cytolytic activity of NK cells against FGF-2-treated HCC cells was higher than that against nontreated HCC cells. These results suggested that FGF-2 enhanced the NK sensitivity of HCC cells by upregulating MICA expression and downregulating HLA class I on the cellular surface. Interestingly, adding FGF-2 did not change the expressions of MICA and HLA class I on normal hepatocytes. These demonstrated that FGF-2 could enhance the NK sensitivity of HCC cells but not that of normal hepatocytes. We also evaluated the expressions of MICA and HLA class I on other growth factors (such as VEGF or PDGF)-treated HCC cells. The expressions of MICA and HLA class I on VEGF- or PDGF-treated HCC cells were similar to those on nontreated HCC cells (Tsunematsu H, unpublished data). In this study, we demonstrated that FGF-2 production from liver tissues decreased along the progression of chronic liver disease. FGF-2 production from liver tissues might prevent the occurrence of HCC by eliminating HCC cell by enhancing NK sensitivity. If the innate immunity of the liver can be efficiently activated, preventing the occurrence of HCC could be expected. We previously demonstrated that anti-HCC chemotherapy and molecular targeted therapy using sorafenib resulted in enhancing NK sensitivity of HCC cells via upregulation of membrane-bound MICA on HCC cells.12, 22 These results suggested the possibility of new routes for chemoprevention of HCC, which could improve the prognosis of chronic liver disease patients. Also, on the basis of our results, FGF-2 supplementation therapy may be a rational approach for eliminating HCC cells in the chronic liver disease.
The concentration of FGF-2 in our in vitro study was high compared with the serum FGF-2 concentration level. Previous reports demonstrated that FGF-2 produced in the liver tissues acts in an autocrine or paracrine fashion.2, 5 We demonstrated that serum FGF-2 levels in chronic liver disease were significantly higher than those in HVs and that serum FGF-2 levels decrease with the progression of liver disease. These results suggested that FGF-2 production from liver tissues might also decrease with the progression of liver disease. Although the local FGF-2 concentration in the liver tissues still remains unknown and may differ from the serum FGF-2 concentration, our results have at least demonstrated that FGF-2 could enhance NK sensitivity of HCC cells via modification of the activating and inhibitory molecules on HCC cells.
The expression of NKG2D has been reported in all NK cells. However, this has also been reported in most NKT cells, subsets of γδ T cells and all human CD8+ T cells and a subset of CD4+ T cells.23 In addition to NK cells, the MICA-NKG2D pathway plays roles in the costimulation or recognition of each cell. Our results demonstrated that FGF-2 might increase the membrane-bound MICA on HCC cells. It might be possible that the increased expression of MICA may also activate other lymphocytes expressing NKG2D and that these cells may also contribute to the elimination of HCC cells.
The earlier results suggested that FGF-2 levels might contribute to the eradication of HCC cells in liver tissues, which would prevent the incidence of HCC in chronic liver disease. Our patients' data demonstrated that HCC occurrence of the patients with high levels of FGF-2 was significantly lower than that with low levels of FGF-2, which is consistent with the results of NK sensitivity of FGF-2-treated HCC cells. Moreover, the FGF-2 levels in patients before HCC occurrence were significantly higher than those in the same patients after HCC occurrence. The decreasing levels of serum FGF-2 may be a prediction factor for the occurrence of HCC in chronic liver disease.
Despite recent progress in understanding HCC development, unknown mechanisms remain. We have shown here that FGF-2 levels in chronic liver disease were significantly higher than those in HVs, and serum FGF-2 levels decreases along the progression of liver disease. Importantly, FGF-2 enhances NK sensitivity of HCC cells via modification of the activating and inhibitory molecules on HCC cells. These findings suggested that FGF-2 might play roles in eliminating occurring HCC cells by innate immunity.