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. 2021 Mar 5;11(1):5303.
doi: 10.1038/s41598-021-84117-9.

The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Hiroaki Kanzaki  1 , Tetsuhiro Chiba  2 , Junjie Ao  1 , Keisuke Koroki  1 , Kengo Kanayama  1 , Susumu Maruta  1 , Takahiro Maeda  1 , Yuko Kusakabe  1 , Kazufumi Kobayashi  1 , Naoya Kanogawa  1 , Soichiro Kiyono  1 , Masato Nakamura  1 , Takayuki Kondo  1 , Tomoko Saito  1 , Ryo Nakagawa  1 , Sadahisa Ogasawara  1 , Eiichiro Suzuki  1 , Yoshihiko Ooka  1 , Ryosuke Muroyama  3 , Shingo Nakamoto  1 , Shin Yasui  1 , Akinobu Tawada  1 , Makoto Arai  1 , Tatsuo Kanda  4 , Hitoshi Maruyama  5 , Naoya Mimura  6 , Jun Kato  1 , Yoh Zen  7 , Masayuki Ohtsuka  8 , Atsushi Iwama  9 , Naoya Kato  1
Affiliations

The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Hiroaki Kanzaki et al. Sci Rep. .

Abstract

FGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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Conflict of interest statement

Tatsuo Kanda received research support from Eisai. Naoya Kato received grant support, advisory fee, and honoraria from Bayer, Bristol-Myers Squibb, Eisai, Merck & Co., Inc. and Takeda Pharmaceutical Company Limited. The other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Basal expression of FGF19/ FGFR4 and FGFR4-knockdown in HCC cells. (A) FGF19 mRNA expression in HCC cells. (B) FGF19 secretion in HCC cells. (C) FGFR4 protein levels in HCC cells. (D) Summary of the FGF19/FGFR4 expression pattern in HCC cells. (E) Cells transduced with the indicated lentiviruses were subjected to Western blot analyses using anti-FGFR4 antibody and anti-tubulin antibody (loading control). (F) Cell growth inhibition in FGFR4-knockdown HCC cells 60 and 120 h after seeding. These data were from three independent experiments.
Figure 2
Figure 2
In vitro assays of FGF19-stimulated HCC cells. (A) Proliferation of HCC cells transduced with the indicated lentiviruses in culture 96 h after seeding. These data were from three independent experiments. *Significant (p < 0.05). (B) Cells treated with FGF19 and/or a FGFR4 inhibitor were subjected to Western blot analyses using anti-FGFR4, anti-pFRS2α, anti-pERK and anti-tubulin (loading control) antibodies.
Figure 3
Figure 3
FGFR4/ERK signaling inhibition in HCC cells treated with MKIs and a FGFR4 selective inhibitor. (A, B) Cells treated with MKIs (A), and co-treated with MKIs and BLU-9931, a FGFR4 selective inhibitor (B), were subjected to Western blot analyses using anti-FGFR4, anti-pFRS2α, anti-pERK and anti-tubulin (loading control) antibodies. (C) Synergistic inhibitory effects of MKIs and FGFR4 selective inhibitor on HCC cell proliferation 120 h after seeding. These data were from three independent experiments. The combination index (CI) is shown below each graph.
Figure 4
Figure 4
Xenograft transplantation and immunohistochemical analyses of NOD/SCID mice. (A) A total of 2 × 106 Huh7 cells were transplanted into the subcutaneous areas of NOD/SCID mice. Representative images of recipient mice treated with FGFR4 inhibitor (30 mg/Kg) and/or sorafenib (10 mg/Kg) 4 weeks after transplantation (left panel). The tumor volume was monitored weekly after cell transplantation (right panel). (B) Hematoxylin and eosin (H&E) staining and immunohistochemical analyses of subcutaneous tumors. Scale bar = 100 μm.
Figure 5
Figure 5
FGF19/FGFR4 expression in HCC surgical samples. (A) Representative FGF19/FGFR4 immunohistochemical staining of primary HCC tissues (score 0 or 4). Scale bar = 100 μm. (B) Correlation between FGF19 and FGFR4 expression. (C) Clinicopathological features of FGF19+FGFR4+ and FGF19−FGFR4− HCC. (D) Cumulative RFS rate based on the FGF19/FGFR4 expression pattern.
Figure 6
Figure 6
Prognosis of HCC patients treated using MKIs based on serum FGF19 levels before treatment. (A, B) Progression-free survival (PFS) (A) and overall survival (OS) (B) of patients treated using sorafenib based on serum FGF19 levels. (C, D) PFS (C) and OS (D) of patients treated using lenvatinib based on serum FGF19 levels.
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