Fibroblast growth factor 19 expression correlates with tumor progression and poorer prognosis of hepatocellular carcinoma

Abstract

Background: Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice, its involvement in human hepatocellular carcinoma (HCC) has not been well investigated. FGF19, a member of the FGF family, has unique specificity for its receptor FGFR4. This study aimed to clarify the involvement of FGF19 in the development of HCC.

Methods: We investigated human FGF19 and FGFR4 expression in 40 hepatocellular carcinoma specimens using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Moreover, we examined the expression and the distribution of FGF19 and FGFR4 in 5 hepatocellular carcinoma cell lines (HepG2, HuH7, HLE, HLF, and JHH7) using RT-PCR and immunohistochemistry. To test the role of the FGF19/FGFR4 system in tumor progression, we used recombinant FGF19 protein and small interfering RNA (siRNA) of FGF19 and FGFR4 to regulate their concentrations.

Results: We found that FGF19 was significantly overexpressed in HCCs as compared with corresponding noncancerous liver tissue (P < 0.05). Univariate and multivariate analyses revealed that the tumor FGF19 mRNA expression was an independent prognostic factor for overall and disease-free survival. Moreover, we found that the FGF19 recombinant protein could increase the proliferation (P < 0.01, n = 12) and invasion (P < 0.01, n = 6) capabilities of human hepatocellular carcinoma cell lines and inhibited their apoptosis (P < 0.01, n = 12). Inversely, decreasing FGF19 and FGFR4 expression by siRNA significantly inhibited proliferation and increased apoptosis in JHH7 cells (P < 0.01, n = 12). The postoperative serum FGF19 levels in HCC patients was significantly lower than the preoperative levels (P < 0.01, n = 29).

Conclusions: FGF19 is critically involved in the development of HCCs. Targeting FGF19 inhibition is an attractive potential therapeutic strategy for HCC.

Figure 1

Real-time quantitative RT-PCR analysis, immunohistochemical staining of representative specimens from HCC patients of 40 HCC samples. (A): Ratio of average FGF19/GAPDH expression in HCC (T) compared with corresponding noncancerous hepatic tissues (N). The average FGF19/GAPDH level in HCCs. (B): The average FGFR4/GAPDH level in HCCs. (C): Immunohistochemistry using anti-FGF19 monoclonal antibodies; HCC tissue (lower) and noncancerous hepatocytes (upper). (D): Immunohistochemistry using anti-FGFR4 monoclonal antibodies; HCC tissue (lower) and noncancerous tissue (upper). (Original magnifications: ×40 (upper); ×40 (lower)). RT-PCR; reverse transcription polymerase chain reaction; HCC, hepatocellular carcinoma.

Figure 2

Survival rates of 40 patients with HCC. (A) Disease-free survival rates of patients with HCCs. (B) Overall survival rates of patients with HCCs. High expression of FGF19 mRNA was significantly associated with poorer prognosis.

Figure 3

(A) FGF19 mRNA expression in cell extracts from HuH7, HepG2, PLC/PRF/5, HLE, HLF, JHH1, JHH2, JHH5, JHH6, and JHH7 cell lines was examined using quantitative RT-PCR (n = 8). FGF19 was expressed in all cell lines. (B) FGF19 protein levels in the supernatant media from ten cell lines and normal hepatocytes (normal HC) were assayed by ELISA. FGF19 was detected in the supernatant of all of them. (C) Using anti-FGF19 monoclonal antibodies, diffuse positive staining was demonstrated in the cytoplasm of HuH7, HepG2, HLE, HLF, and JHH7 cells. The closed rectangles indicate the same cells stained Immunohistochemically using control antibody. (D) FGFR4 mRNA expression in cell extracts from HuH7, HepG2, PLC/PRF/5, HLE, HLF, JHH1, JHH2, JHH5, JHH6, and JHH7 cell lines was examined using quantitative RT-PCR (n = 8). FGFR4 was expressed in all cell lines. (E) Using anti-FGFR4 monoclonal antibodies, positive staining was demonstrated in the cell membranes of all of the above HCC lines. (Original magnifications (C) × 400), (E) × 400)) The closed rectangles indicate the same cells stained Immunohistochemically using control antibody.

Figure 4

(A) Proliferation changes after addition of various concentration of the FGF19 recombinant protein. (B) The proliferation index (PI) was defined as the OD values of cells treated with recombinant protein divided by those of untreated cells. The PI increased after addition of FGF19 recombinant protein (0.01-10 ng/mL final concentration) to culture media and culturing for 96 h. PI, proliferation index.

Figure 5

(A) Apoptosis assay at 72 h after FGF19 recombinant protein treatment. (left) 5-FU treatment; (right) 5-FU and recombinant protein treatment. The apoptosis index (AI) was defined as the OD values of cells treated with these agents divided by those of untreated cells. (B) (upper) Invasion assay after FGF19 recombinant protein treatment (100× magnification). (lower) Invasion assay 48 h after FGF19 recombinant protein treatment after FGF19 recombinant protein treatment. (C) FGF19 enhanced cell migration ability in JHH7 cell line. The transwell system was used to evaluate migratory ability. Migrated cells were counted and the quantitative results are shown (n = 6). The data presented are from a representative experiment, being quantitatively similar in the replicate experiments.

Figure 6

Suppression of FGF19 expression was confirmed at transcript level by RT-PCR. (A) and at protein level by ELISA analysis (B). Analysis was performed at 48 h and 72 h following siRNA transfection for mRNA and protein level, respectively. FGF19 mRNA expression was suppressed to 30% of control siRNA in FGF19-specific siRNA-treated cells. n = 12, P < 0.05 at top of the Figure 6A, 6B, and 6C (like figure 6E and 6D). FGFR4 gene silencing by siRNA. Suppression of FGFR4 expression was confirmed at transcript level by RT-PCR (C) and at protein level by flow cytometry (D). Analysis was performed at 48 h and 72 h following siRNA transfection for mRNA and protein levels, respectively. FGFR4 mRNA expression was suppressed to 40% of control siRNA in FGFR4-specific siRNA-treated cells. n = 12, P < 0.05 at top of the Figure 6A, 6B, and 6C (like figure 6E and 6D). (E) Proliferation assay in JHH7 cells at 72 h after FGF19/FGFR4 siRNA treatment. (F) Apoptosis assay in JHH7 cells at 96 h after FGF19/FGFR4 siRNA treatment. PI, Proliferation Index. AI, Apoptosis Index.

Figure 7

The changes in serum FGF19 levels, as measured by ELISA, of the 29 patients with HCCs from 1^st day and the 8^th day after curative resection surgery. Normal shows serum FGF19 levels of the 10 health subjects.