FAK is required for c-Met/β-catenin-driven hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide and most patients with HCC have limited treatment options. Focal adhesion kinase (FAK) is overexpressed in many HCC specimens, offering a potential target for HCC treatment. However, the role of FAK in hepatocarcinogenesis remains elusive. Establishing whether FAK expression plays a role in HCC development is necessary to determine whether it is a viable therapeutic target. In this study, we generated mice with hepatocyte-specific deletion of Fak and investigated the role of Fak in an oncogenic (c-MET/β-catenin, MET/CAT)-driven HCC model. We found that deletion of Fak in hepatocytes did not affect morphology, proliferation, or apoptosis. However, Fak deficiency significantly repressed MET/CAT-induced tumor development and prolonged survival of animals with MET/CAT-induced HCC. In mouse livers and HCC cell lines, Fak was activated by MET, which induced the activation of Akt/Erk and up-regulated cyclin D1 and tumor cell proliferation. CAT enhanced MET-stimulated FAK activation and synergistically induced the activation of the AKT/ERK-cyclin D1 signaling pathway in a FAK kinase-dependent manner. In addition, FAK was required for CAT-induced cyclin D1 expression in a kinase-independent fashion.

Conclusion: Fak is required for c-Met/β-catenin-driven hepatocarcinogenesis. Inhibition of FAK provides a potential strategy to treat HCC.

Figure 1. Deletion of Fak in hepatocytes does not affect morphology, histology, proliferation or apoptosis in mouse liver

(A) Genotyping of Alb-Cre, Alb-Cre; FAK^flox/+, and Alb-Cre; FAK^flox/flox mice. (B) Protein expression of Fak and Gapdh in whole livers and isolated hepatocytes of Alb-Cre (Hep^WT) and Alb-Cre; Fak^flox/flox (Hep^ΔFak) mice was determined by Western blotting. (C) Photographs of livers from Hep^WT and Hep^ΔFak mice at 7 weeks of age. (D) Representative pictures of H&E-stained sections for (C). (E) Left, hepatic proliferation in the livers of 7 week old Hep^WT and Hep^ΔFak mice was examined by immunohistochemistry for Ki67 protein expression. Right, quantification of Ki67 staining (n=3). (F) Left, hepatic apoptosis in the livers of 7 week old Hep^WT and Hep^ΔFak mice was examined by TUNEL staining. Right, quantification of TUNEL staining for (E) (n=3).

Figure 2. Deletion of Fak suppresses tumor development and prolongs survival in the MET/CAT-induced HCC mouse model

(A) Representative pictures of co-staining of c-MET and β-catenin protein expression from the livers of Hep^WT mice 3 days after hydrodynamic injection of MET/CAT (magnification, ×200). (B) Photographs of livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT. (C) Liver weight/body weight ratio was analyzed in the mice from (B) (n=6). (D) Histological analysis of the livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT by H&E staining. (E) HCC in the livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT was examined by immunohistochemistry for AFP. (F) Survival curves of Hep^WT and Hep^ΔFak mice after of MET/CAT (n=4).

Figure 3. Fak deficiency decreases hepatocyte proliferation but does not affect apoptosis in MET/CAT-induced liver tumors

(A) Apoptosis in the livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT was examined by TUNEL staining. (B) Quantification of TUNEL staining for (A) (n=3). (C) Hepatocyte proliferation in the livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT was examined by immunohistochemistry for Ki67 and immunofluorescence for BrdU. (D) Quantification of Ki67 and BrdU staining for (C) (n=3).

Figure 4. Fak activation is increased in mouse livers after delivery of MET/CAT or MET alone

(A) Protein expression of p-Fak (Y397), p-Fak (Y576/577) and Gapdh in the livers of Hep^WT mice 7 weeks after hydrodynamic injection of MET/CAT or pT3 control. (B) Protein expression of p-Fak (Y397), c-Met, β-catenin and Gapdh in the livers of Hep^WT mice with or without introduction of MET/CAT assayed 3 days after hydrodynamic injection. (C) Protein expression of p-Fak (Y397), c-Met and Gapdh in the livers of Hep^WT mice 3 days after hydrodynamic injection with MET or pT3 control. (D) Protein expression of p-Fak (Y397), β-catenin and Gapdh in the livers of Hep^WT mice 3 days after hydrodynamic injection with CAT or pT3 control.

Figure 5. FAK inhibition abrogates HGF/c-MET-mediated AKT and ERK activation in HCC cells

(A) Protein expression of Fak, p-Akt, Akt, p-Erk, Erk and Gapdh in the livers of Hep^WT and Hep^ΔFak mice 7 weeks after injection of MET/CAT. (B) Protein expression of FAK, p-FAK, p-AKT, AKT, p-ERK, ERK and GAPDH in Huh7 cells infected with scrambled shRNA, FAK shRNA#1 or FAK shRNA#2 lentiviral particles and then treated with 20ng/ml HGF for 15 or 60 minutes. (C) Protein expression of FAK, p-AKT, AKT, p-ERK, ERK and GAPDH in HEPG2 cells treated as in (B), except with FAK shRNA#3 instead of FAK shRNA#2.

Figure 6. The combination of CAT and MET has a synergistic effect on FAK activation in mouse liver and HCC cells

(A) Protein expression of p-FAK, FAK, p-AKT, AKT, p-ERK, ERK and GAPDH in Huh7 and HEP3B cells transfected with CAT or control plasmids. (B) Protein expression of FAK, p-FAK, β-catenin, p-β-catenin (Y654) and GAPDH in Huh7 cells transfected with CAT or control plasmids, and after 24 hours treated with 20ng/ml HGF for 60 minutes. (C) Protein expression of p-Fak, p-β-catenin (Y654), and Gapdh in the livers of Hep^WT and Hep^ΔFak mice 3 days after injection of pT3, MET, CAT or MET/CAT.

Figure 7. FAK mediates Cyclin D1 regulation by MET or CAT in HCC cells

(A) Cyclin D1 mRNA expression level in the livers of Hep^WT and Hep^ΔFAK mice 7 weeks after injection of MET/CAT. (B) Protein expression of FAK, Cyclin D1 and GAPDH in Huh7 and HEPG2 cells infected with scrambled shRNA or FAK shRNA#1 lentiviral particles, then treated with 20ng/ml HGF for 15 or 60 minutes. (C) Protein expression of p-AKT, AKT, p-ERK, ERK, Cyclin D1 and GAPDH in Huh7 cells treated with 10 µM LY294002 or 500 nM GSK1120212 for 24 hours. (D) Protein expression of FAK, Cyclin D1 and GAPDH in Huh7 cells infected with scrambled shRNA, FAK shRNA#1 or FAK shRNA#2 lentiviral particles, then transfected with CAT or control plasmids. (E) A schematic model: hydrodynamic injection of MET into hepatocytes results in Fak activation. CAT does not activate FAK directly but the combination of CAT and MET has a synergistic effect on Fak activation. Fak activation regulates MET- or CAT-mediated Cyclin D1 induction, acting through either Akt- and Erk-dependent or -independent pathways, thereby promoting hepatocarcinogenesis.