Novel findings in an old model: Overexpression of c-myc in hepatocytes promotes the activation of hepatic stellate cells and facilitates the onset of liver fibrosis

Background and Aims: Overexpression of the proto-oncogene c-myc is frequently found in human and murine hepatocellular carcinoma. In mice, transgenic overexpression of c-myc in hepatocytes alb-myctg is sufficient to initiate liver cancer. However, the impact of c-myc for initiation of pathological precursor stages such as liver fibrosis is less defined. Liver fibrogenesis involves cell proliferation of hepatic cell populations such as hepatocytes and hepatic stellate cells (HSC). In the present study we aimed to determine the potential role of c-myc in this process.

Methods: Expression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by quantitative real-time PCR (qPCR). Liver fibrosis in wildtype (WT) and alb-myctg mice was induced by periodic CCl4 treatment. Primary HSC were isolated from WT and alb-myctg mice and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy.

Results: In patients with advanced (F3) liver fibrosis, hepatic c-myc was tenfold upregulated compared to healthy controls. Similarly, c-myc was also induced in murine liver in CCl4 -mediated fibrogenesis. Immunohistochemistry revealed an accumulation of c-myc-expressing cells in areas of fiber formation. To dissect the potential pro-fibrotic functions of c-myc in HSC and hepatocytes, we next analyzed the expression profile of c-myc in primary WT HSC in vitro demonstrating that c-myc induction was transient and restricted to day 1 after plating. However, onset of HSC proliferation and transdifferentiation into myofibroblasts was observed at later time points suggesting that c-myc is not essential for this process. We thus analyzed profibrotic functions of c-myc in hepatocytes using alb-myctg mice. These animals displayed an age dependent increase of liver collagen deposition in comparison to WT controls but lacked typical fiber formation. Interestingly, alb-myctg mice revealed an accelerated onset of liver fibrosis already after two weeks of chronic CCl4 treatment, which was associated with increased expression of pro-fibrotic proteins (α-SMA, collagen I) along with induction of cell-cycle activators (PCNA, Ki-67). Importantly, primary HSC derived from alb-myctg mice showed only transient c-myc expression identical to WT cells but revealed stronger proliferation and differentiated much faster into myofibroblasts.

Conclusion: Overexpression of c-myc is a novel hallmark of liver fibrosis in man and mice. From our data we conclude that prolonged induction of c-myc especially in hepatocytes has the potential to prime resident HSC for activation, proliferation and myofibroblast differentiation.