High real-world effectiveness of 12-week elbasvir/grazoprevir without resistance testing in the treatment of patients with HCV genotype 1a infection in Norway.

BACKGROUND AND AIMS: The recommended treatment duration of hepatitis C virus (HCV) genotype 1a (GT1a) infection with elbasvir/grazoprevir (EBR/GZR) in the presence of a high baseline viral load and resistance associated substitutions (RAS) is 16 weeks with ribavirin added. The objective of this study was to evaluate the real-world effectiveness of 12 weeks of EBR/GZR without ribavirin and regardless of baseline viral load and RAS testing. METHOD: This retrospective, observational cohort study was performed at five Norwegian hospitals that did not systematically utilize RAS testing. All adult patients with chronic HCV GT1a and compensated liver disease who had received 12 weeks of EBR/GZR without ribavirin and baseline RAS testing, were included. The primary endpoint was sustained virologic response at week 12 (SVR12), or if not available, at week 4 (SVR4). RESULTS: We included 433 patients and attained SVR data on 388. The mean age was 45.7 years (22-73 years). 67.2% were male. HIV co-infection was present in 3.8% (16/424) and cirrhosis in 4% (17/424). The viral load was >800 000 IU/mL in 55.0% (235/427) of patients. Overall SVR was achieved in 97.2% (377/388). SVR was achieved in 98.3% (169/172) of those with viral load ≤800 000 IU/mL and in 96.2% (202/210) of those with viral load >800 000 IU/mL. CONCLUSION: We observed high SVR rates among patients with HCV GT1a infection treated with EBR/GZR for 12 weeks without ribavirin, with no regard to baseline viral load and no RAS testing.

FAK regulates Cdk2 in EGF-stimulated primary cultures of hepatocytes.

In this study, we report a novel role of FAK as a regulator of Cdk2 in anchorage-dependent primary cultured hepatocytes. In response to EGF, we found that S-phase entry was reduced upon FAK inhibition. This correlated with decreased protein expression and nuclear accumulation of the G1/S-phase regulator Cdk2. Further, nuclear accumulation of the Cdk2 partner cyclinE, was reduced, but not its protein level. Also, protein levels of Cdk2 were inversely linked with increased expression of the Cdk2 inhibitor p27, known to be degraded in a Cdk2-dependent manner. Also, cyclinD1 was regulated by FAK, but to a lesser extent than Cdk2. To assess the mechanism in which FAK mediates Cdk2-regulation, FAK mutants were used: FAKY397F, mutated at its integrin-regulated site, and two others mutated at docking sites for Grb2-ERK-activation (FAKY925F) and for p130Cas-Rac1-activation (FAKY861F). All three sites were central for EGF-induced ERK-activity and Cdk2 expression. In addition, FAK was important for HGF-mediated proliferation, suggesting a general mechanism for anchorage-dependent growth. Moreover, growth factor-induced cell spreading, but not survival, required FAK. Hence, integrins and growth factors cooperate in anchorage-dependent signaling events leading to proliferation and motility. In conclusion, our data suggest that FAK acts as a central coordinator of integrin and growth factor-mediated S-phase entry by its ability to regulate Cdk2.

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase.

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

Distinct functions of H-Ras and K-Ras in proliferation and survival of primary hepatocytes due to selective activation of ERK and PI3K.

Ras proteins mediate signals both via extracellular signal-regulated kinase 1 and 2 (ERK), and phosphoinositide 3-kinase (PI3K). These signals are key events in cell protection and compensatory cell growth after exposure to cell damaging and pro-apoptotic stimuli, thus maintaining homeostasis. By transfection techniques, we found that both H-Ras and K-Ras were expressed and appeared functionally active in primary hepatocytes. We compared the ability of H-Ras and K-Ras homologues to preferentially activate one of the two pathways, thereby differentially controlling cell survival and growth. We found that ectopic expression of dominant negative (DN) H-RasN17, but not DN K-RasN17, efficiently inhibited both phosphorylation and translocation of ERK to the nuclear compartment, which are prerequisites for cell cycle progression. Furthermore, ectopic expression of constitutive active (CA) H-RasV12, but not CA K-RasV12, potentiated EGF-induced proliferation. We also found that expression of CA mutants of either H-Ras or K-Ras protected hepatocytes from transforming growth factor-beta1 (TGF-beta1)-induced apoptosis. However, H-Ras-induced survival was mediated by ERK/RSK as well as by PI3K, whereas K-Ras-induced survival was mediated by PI3K only. In conclusion, H-Ras and K-Ras had differential functions in proliferation and survival of primary hepatocytes. H-Ras was the major mediator of ERK-induced proliferation and survival, whereas H-Ras and K-Ras both mediated PI3K-induced survival.

Cytoplasmic retention of peroxide-activated ERK provides survival in primary cultures of rat hepatocytes.

Reactive oxygen species (ROS) are implicated in tissue damage causing primary hepatic dysfunction following ischemia/reperfusion injury and during inflammatory liver diseases. A potential role of extracellular signal-regulated kinase (ERK) as a mediator of survival signals during oxidative stress was investigated in primary cultures of hepatocytes exposed to ROS. Hydrogen peroxide (H(2)O(2)) induced a dose-dependent activation of ERK, which was dependent on MEK activation. The ERK activation pattern was transient compared with the ERK activation seen after stimulation with epidermal growth factor (EGF). Nuclear accumulation of ERK was found after EGF stimulation, but not after H(2)O(2) exposure. A slow import/rapid export mechanism was excluded through the use of leptomycin B, an inhibitor of nuclear export sequence-dependent nuclear export. Reduced survival of hepatocytes during ROS exposure was observed when ERK activation was inhibited. Ribosomal S6 kinase (RSK), a cytoplasmic ERK substrate involved in cell survival, was activated and located in the nucleus of H(2)O(2)-exposed hepatocytes. The activation was abolished when ERK was inhibited with U0126. In conclusion, our results indicate that activity of ERK in the cytoplasm is important for survival during oxidative stress in hepatocytes and that RSK is activated downstream of ERK. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).