Selective inhibitor of Wnt/β-catenin/CBP signaling ameliorates hepatitis C virus-induced liver fibrosis in mouse model

Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/β-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a β-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of β-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.

tetrachloride (CCl 4 ) treatment 7 . Additionally, hepatic macrophages are involved in the regression of hepatic fibrosis 8 ; cells of this type also have been reported to produce MMPs 9 .
Wnt signaling affects developmental processes during embryogenesis and has an important role in tissue homeostasis in adults. Following Wnt activation, β-catenin translocates to the nucleus, where β-catenin binds to the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) to induce the transcription of Wnt target genes 10,11 . Nuclear β-catenin/TCF then assembles a transcriptionally active complex by recruiting the transcriptional coactivators cyclic AMP response element binding protein (CREB) binding protein (CBP) or the closely related protein p300, as well as other components of the basal transcriptional machinery, to stimulate the transcription of target genes. The canonical Wnt signaling pathway has been implicated in the pathogenesis of a variety of tissue fibroses, including liver fibrosis [12][13][14] , and it has been shown that CBP/β-catenin antagonists are efficacious in a variety of injury models, including pulmonary and renal fibrosis 15,16 . PRI-724 is a second-generation CBP/β-catenin-specific antagonist, a selective small-molecule inhibitor of β-catenin/CBP interaction, developed by PRISM Pharma Co., Ltd. (Kanagawa, Japan) 17 . PRI-724 treatment reduces liver fibrosis induced by CCl 4 or bile duct ligation in mice 18 . In the present study, we examined whether PRI-724 has therapeutic potential for use in the treatment of liver fibrosis using an HCV transgenic mouse model. The results suggest that PRI-724 may be a candidate for a new anti-fibrotic drug.

Results
Intraperitoneal PRI-724 injection ameliorates hepatitis C virus-induced liver fibrosis. To evaluate the anti-fibrotic activity of PRI-724 on liver fibrosis induced by HCV, HCV transgenic mice were injected intraperitoneally with PRI-724 once daily for 6 weeks at a dose of 5 or 20 mg/kg body weight or 1 week ON/OFF at a dose of 5 mg/kg body weight for 6 weeks (total of three cycles, each consisting of 1 week of once-daily dosing followed by 1 week without dosing) (Fig. S1A). In HCV transgenic mice, S100A4 expression, which is controlled by CBP/β-catenin, was increased; this induction was attenuated by the administration of PRI-724 (Fig. S2A). In vehicle-treated mice, abnormalities of liver plate arrangement and hepatocellular morphology with collagen deposition in the liver were observed, as shown by hematoxylin and eosin (H&E) and Masson's trichrome staining ( Fig. 1A) without ALT elevation (Fig. S3A). On the other hand, in PRI-724-treated mice, these abnormalities were attenuated and the area of collagen fibrils was reduced without reduction of HCV core protein expression (Fig. S3B). In particular, PRI-724 reduced collagen fibrils even in the case of 1 week ON/OFF treatment, suggesting that short-term discontinuation is tolerated for anti-fibrotic treatment by PRI-724. Sirius Red staining revealed that the increased area of collagen fibrils in the liver induced by HCV (3.4%; Sirius Red positive area/ total area) was significantly attenuated (to 1.9 to 2.0%) by PRI-724 treatment ( Fig. 1B and C). Similarly, the increase of hepatic hydroxyproline by HCV induction was attenuated following treatment with PRI-724 (Fig. 1D). These results clearly indicated that CBP/β-catenin signaling is activated in HCV-expressing liver, and that inhibition of CBP/β-catenin by PRI-724 is effective in counteracting HCV-induced liver fibrosis.

PRI-724 inhibits HSC activation and promotes ECM degradation.
To investigate whether PRI-724 alters the activation status of HSCs in the liver, expression of αSMA was examined. αSMA expression was increased in HCV transgenic mice compared to control mice, and the induction of αSMA expression was attenuated by PRI-724 treatment (Fig. S2B). Similarly, immunohistochemistry revealed that the number of αSMA-expressing cells increased in HCV transgenic mice compared to control mice; this induction was attenuated by PRI-724 treatment ( Fig. 2A). Moreover, the increase of collagen type 3 α1-encoding mRNA and type I collagen (Col-1) expression levels in the HCV transgenic mice also was attenuated by PRI-724 (Fig. 2B,D and E). These results suggested that HSC activation and collagen production in the transgenic mice is inhibited by PRI-724. In addition to the inhibitory effect of PRI-724 on HSC activation, expression of MMP-8-encoding mRNA was found to be enhanced 7.4-fold by PRI-724 treatment (Fig. 2C). Conversely, the HCV-related induction of an mRNA encoding TIMP-1 (an endogenous inhibitor of MMP-8) was attenuated by PRI-724 (Fig. 2C). Measurement of MMP-8 activities in the liver revealed that the total MMP-8 level (pro-MMP-8 plus active MMP-8) was increased by PRI-724 treatment and that the HCV-related reduction of endogenous active MMP-8 was attenuated by PRI-724 (Fig. 2F), suggesting that PRI-724 also is involved in the degradation of ECM by MMP-8. In contrast to the case with the Mmp8 transcript, mRNA expression levels of Mmp2, Mmp9, and Mmp13 were not affected by PRI-724 (Fig. 2C). However, total MMP-9 level was increased by PRI-724 treatment (Fig. S4), suggesting that MMP-9 may also contribute to remission of fibrosis by PRI-724. To identify the MMP-8-producing cells, immunostaining was performed. The numbers of F4/80-, Ly-6C-, and Gr-1-positive cells in HCV transgenic mice were elevated following treatment with PRI-724 (Fig. 3A). Moreover, MMP-8-positive cells also stained with both F4/80 and Gr-1 (Fig. 3B), suggesting that macrophages/monocytes and neutrophils are mobilized by PRI-724 and that these cells are producing MMP-8. Increased chemokine levels were not observed in PRI-724-treated animals ( Table S1). The mechanism of macrophage and neutrophil recruitment in the livers of the PRI-724-treated mice will require further investigation.

Intermittent administration of PRI-724 is sufficient for anti-fibrotic activity on hepatitis C virus-induced liver fibrosis.
In clinical use, daily intraperitoneal injection is not feasible. Thus, the anti-fibrotic activity of intermittently administered PRI-724 was evaluated. HCV transgenic mice were intraperitoneally administered with PRI-724 (15 mg/kg body weight) at a frequency of once or twice per week (Fig. S1A). At six weeks after the initiation of treatment, the area of collagen fibrils in the liver was decreased in the PRI-724-treated group, as indicated by Masson's trichrome staining (Fig. 4A). Sirius Red staining confirmed that both twice-weekly and once-weekly PRI-724 treatment provided a statistically significant attenuation in the HCV-induced increase in the area of collagen fibrils ( Fig. 4B and C).
Scientific RepoRts | 7: 325 | DOI:10.1038/s41598-017-00282-w Subcutaneous PRI-724 injection ameliorates hepatitis C virus-induced liver fibrosis. As more suitable option for clinical use, we assessed the anti-fibrotic activity of PRI-724 using continuous subcutaneous infusion (Fig. S1B). As in the intraperitoneal injection model, subcutaneous administration of PRI-724 (1 mg/ kg/day) effectively reduced the area of collagen fibrils in the liver, as assessed by silver staining and Masson's trichrome staining (Fig. 5A). Administration of PRI-724 at 0.3 mg/kg/day also reduced the area of collagen fibrils  in the liver, whereas the administration at 0.1 mg/kg/day did not show anti-fibrotic effect (Fig. S5), suggesting that PRI-724's efficacy is dose dependent. As seen with intraperitoneal injection, subcutaneous dosing with PRI-724 yielded increases in the level of Mmp8 mRNA expression and in the numbers of F4/80-, Ly-6C-, and Gr-1-positive cells (Fig. 5B,C and D). FACS analyses of intrahepatic leukocytes revealed that the numbers of both M1 (F4/80 + CD11b + CD11c + CD206 − cells) and M2 macrophages (F4/80 + CD11b + CD11c − CD206 + cells) were increased (Fig. S6). Further analysis will be needed to determine the identity and mechanism of the macrophages that contribute to the remission of fibrosis.

Discussion
Cirrhosis is an increasing cause of morbidity and mortality in developed countries, being the 14 th -most-common cause of death worldwide 1 . Organ fibrosis and cirrhosis are common end-stage diseases of the liver, resulting in life-threatening complications such as gastrointestinal bleeding and hepatocellular carcinoma 19 . The only available curative treatment for end-stage liver disease is transplantation of the liver, emphasizing the urgent need to identify new therapeutic strategies for the treatment of liver cirrhosis. Here, we demonstrated that liver   fibrosis in HCV transgenic mice was significantly attenuated by treatment with PRI-724, a specific inhibitor of Wnt/β-catenin/CBP-driven transcription. These findings are consistent with a recent report that blockade of Wnt/β-catenin/CBP signaling by ICG-001 prevents bleomycin-induced pulmonary fibrosis in mice 16 . There is accumulating evidence of a role for Wnt/β-catenin signaling in the regulation of HSCs, a class of cells that have been implicated as key players in liver fibrosis 14,20 . However, to our knowledge, the present study represents the first evidence that an inhibitor of the β-catenin/CBP interaction is capable of ameliorating liver fibrosis in the HCV transgenic mouse model.
The HCV transgenic mice used in the present study express multiple HCV proteins via the Cre/loxP switching system, resulting in the induction of persistent liver injury. As in humans, these mice subsequently develop steatosis, liver fibrosis, and hepatocellular carcinoma 21,22 , suggesting that these mice serve as a good model of human HCV infectious liver diseases. Although there have been reports of experimental fibrosis models established by repeated administration of CCl 4 23 or by ligation of the bile duct 24 , these experimental models appear to be unsuitable for examining the effects of drugs on liver fibrosis, since these models do not involve the immunological responses, such as lymphocyte infiltration and interferon-γ synthesis, seen in human cases. In this context, we note that PRI-724 has relevant effects on the liver fibrosis observed in HCV transgenic mice, including the following: (1) PRI-724 regresses liver fibrosis in this model even after 18 months of continuous liver damage. (2) PRI-724 has anti-fibrotic effects even in older mice, which generally exhibit defects in tissue repair 25 . (3) PRI-724 does not induce liver damage when administered in healthy animals, and does not further damage HCV transgenic animals. Collectively, the above findings suggested that PRI-724 may be a promising therapy for the treatment of human liver fibrosis.
In the injured liver, HSC activation and fibrogenesis are mediated by complex cross-talk between damaged parenchymal and non-parenchymal cells. In our model, induction of αSMA expression by HCV was attenuated by PRI-724 treatment. Wnt signaling is stimulated in activated HSCs compared to that in quiescent cells, and the inhibition of Wnt signaling by the transduction of the adenoviral Wnt co-receptor antagonist Dickkopf-1 restores HSC quiescence and increases apoptosis in cultured HSCs 19 . Thus, HSCs might be the target cells of PRI-724 activity. To determine how PRI-724 exerts an anti-fibrotic function, we assessed the effect of this compound on expression of Mmp8 mRNA, which encodes a matrix metalloproteinase that degrades ECM proteins in the liver. We observed significantly increased accumulation of the Mmp8 transcript following PRI-724 treatment. Furthermore, immunohistochemical analysis revealed that macrophages and neutrophils in the liver produced MMP-8, suggesting that PRI-724 treatment induces the migration of these inflammatory cells into the liver. We postulate that the organ-localized activity of these cells facilitates regression of liver fibrosis. Recently, Ramachandran et al. reported that the CD11b hi F4/80 int Ly-6C lo subset of macrophages was most abundant in livers during maximal fibrosis resolution, and that this population represented the principal MMP-expressing subset 9 . Consistent with that report, our results also suggested that F4/80 macrophages and neutrophils are responsible for the resolution of liver fibrosis in HCV transgenic mice. However, the mechanism of inflammatory cell migration/recruitment to the liver in response to PRI-724 remains unclear.
Previously we reported the anti-fibrosis effects of PRI-724 in liver fibrosis induced by CCl 4 or bile duct ligation in mice 18 . In this study, the anti-fibrosis effects of PRI-724 was confirmed in HCV-induced liver fibrosis, suggesting that PRI-724 is also effective in mouse model mimicking HCV infected patients. Moreover, our studies provide a proof of principle that the selective blockade of β-catenin/CBP signaling has potential as a novel therapeutic strategy for the treatment of liver fibrosis induced by various causes. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. Inhibition of HSC activation and induction of fibrolytic cells expressing certain matrix metalloproteinases might contribute to the anti-fibrotic effects of PRI-724. Thus, PRI-724 can be a candidate of anti-fibrotic drug.

Methods
Ethics Statement. This study was carried out in strict accordance with both the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science and the recommendations in the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. All protocols were approved by the ethics committee of Tokyo Metropolitan Institute of Medical Science. HCV transgenic mice. HCV GT1b transgenic mice (MxCre +/− /CN2-29 +/− ) were prepared as previously described 20 . HCV transgenic mice were injected intraperitoneally with 300 μg/mouse of polyinosinic acid-polycytidylic acid (poly(I)·poly(C)) (GE Healthcare, Buckinghamshire, UK) three times at 48-hour intervals to induce the expression of HCV proteins. Injection of poly(I)·poly(C) induces interferon production and the expression of CN2-29 gene products (HCV core, E1, E2, p7, and NS2) in hepatocytes, non-mesenchymal cells (mainly in Kupffer cells and lymphocytes), and spleen, but not in most other tissues; induction is mediated via the Cre/loxP switching system and mimics chronic HCV infection. The HCV core protein was expressed consistently in hepatocytes for at least 600 days, but not expressed consistently in Kupffer cells, lymphocytes and spleen as previously described 20 . HCV transgenic mice were used in the present study after long-term (18-to 20-month) HCV expression had led to the development of liver fibrosis. Age-matched controls (MxCre −/− /CN2-29 +/− ) were used and are designated below as non-transgenic control or control animals. PRI-724 treatment. PRI-724 (PRISM Pharma Co., Ltd.) was diluted in phosphate-buffered saline (PBS) and injected intraperitoneally once daily at a concentration of 5 or 20 mg/kg body weight for 6 weeks. For the 1 week ON/OFF experiment, the reagent (5 mg/kg/day) was administered intraperitoneally for 6 weeks during which animals were treated for a total of three cycles, each consisting of 1 week of once-daily dosing followed by 1 week without dosing. In a separate experiment, PRI-724 was administered intraperitoneally at 15 mg/kg body weight at a frequency of once or twice per week for 6 weeks. Alternatively, PRI-724 was dosed by subcutaneous infusion