Hepatitis B virus X protein (HBx) contributes to Hepatitis B virus (HBV)-related liver cancer. However, its impact on hepatocyte proliferation and genomic stability remains elusive. We studied the role of HBx expression on the progression of cell cycle and liver polyploidization during proliferation and liver carcinogenesis. Full-length HBx transgenic mice (FL-HBx) were developed to investigate liver ploidy as well as hepatocyte proliferation, along normal liver maturation and during cancer initiation (chemical carcinogen treatment). Investigation of postnatal liver development in FL-HBx showed an aberrant G1/S and G2/M transitions, triggered (1) a delay of the formation of hepatocytes binucleation, (2) the early synthesis of polyploidy nuclei (≥4n) and (3) DNA damage appearance. Moreover, HBV infection during hepatocytes proliferation in a humanized liver mouse model led, to modifications in polyploidy of hepatocytes. In initiation of hepatocellular carcinoma, FL-HBx protein decreased ChK1 phosphorylation, Mre11 and Rad51 expression, upregulated IL-6 expression and impaired apoptosis. This was related to DNA damage accumulation in FL-HBx mice. At day 75 after initiation of hepatocellular carcinoma, FL-HBx mice revealed significant cell cycle changes related to the increased amount of 4n nuclei and of markers of cancer progenitor cells. Finally, PLK1 upregulation and p38/ERK activation in FL-HBx mice were implicated in aberrant polyploidization favoring DNA damage propagation and hepatocyte transformation. In conclusion, our data indicate that FL-HBx protein increases DNA damage through the hijack of hepatocyte polyploidization. That leads to enhancement of hepatocellular carcinoma initiation in an inflammatory context.
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We thank S. Berissi and N. Gadessaud (Inserm US24/CNRS UMS3633) for tissue processing and slide staining, S. Morosan and O. Bregerie (UMS28, phénotyage du petit animal, UPMC-Paris VI) for animal care.
This work was supported by grants from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS) (AO 2014-1 16032), Ligue Contre le Cancer (2016, RS16/75-7 and 2017 RS17/75-23), Institut National de la Santé et de la Recherche Médicale (Inserm) and Université Pierre et Marie Curie (UPMC). J.A. was supported by an ANRS doctoral grant.
DK, JA, and CD contributed to conception of the study and design. JA, MB, CC, and JM performed experiments. All authors contributed to the interpretation of results. DK, JA, and CD drafted the manuscript.