Cell fate analysis of zone 3 hepatocytes in liver injury and tumorigenesis

Shigeyuki Kurosaki; Hayato Nakagawa; Yuki Hayata; Satoshi Kawamura; Yuki Matsushita; Tomoharu Yamada; Koji Uchino; Yoku Hayakawa; Nobumi Suzuki; Masahiro Hata; Mayo Tsuboi; Hiroto Kinoshita; Yasuo Tanaka; Takuma Nakatsuka; Yoshihiro Hirata; Keisuke Tateishi; Kazuhiko Koike

doi:10.1016/j.jhepr.2021.100315

Cell fate analysis of zone 3 hepatocytes in liver injury and tumorigenesis

JHEP Rep. 2021 May 27;3(4):100315. doi: 10.1016/j.jhepr.2021.100315. eCollection 2021 Aug.

Authors

Shigeyuki Kurosaki¹, Hayato Nakagawa¹, Yuki Hayata¹, Satoshi Kawamura¹, Yuki Matsushita¹, Tomoharu Yamada¹, Koji Uchino¹, Yoku Hayakawa¹, Nobumi Suzuki¹, Masahiro Hata¹, Mayo Tsuboi¹, Hiroto Kinoshita², Yasuo Tanaka¹, Takuma Nakatsuka¹, Yoshihiro Hirata³, Keisuke Tateishi¹, Kazuhiko Koike¹

Affiliations

¹ Department of Gastroenterology, The University of Tokyo, Tokyo, Japan.
² Division of Gastroenterology, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan.
³ Division of Advanced Genome Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Abstract

Background & aims: Liver lobules are typically subdivided into 3 metabolic zones: zones 1, 2, and 3. However, the contribution of zonal differences in hepatocytes to liver regeneration, as well as to carcinogenic susceptibility, remains unclear.

Methods: We developed a new method for sustained genetic labelling of zone 3 hepatocytes and performed fate tracing to monitor these cells in multiple mouse liver tumour models.

Results: We first examined changes in the zonal distribution of the Wnt target gene Axin2 over time using Axin2-Cre ^ERT2 ;Rosa26-Lox-Stop-Lox-tdTomato mice (Axin2;tdTomato). We found that following tamoxifen administration at 3 weeks of age, approximately one-third of total hepatocytes that correspond to zone 3 were labelled in Axin2;tdTomato mice; the tdTomato⁺ cell distribution closely matched that of the zone 3 marker CYP2E1. Cell fate analysis revealed that zone 3 hepatocytes maintained their own lineage but rarely proliferated beyond their liver zonation during homoeostasis; this indicated that our protocol enabled persistent genetic labelling of zone 3 hepatocytes. Using this system, we found that zone 3 hepatocytes generally had high neoplastic potential, which was promoted by constitutive activation of Wnt/β-catenin signalling in the pericentral area. However, the frequency of zone 3 hepatocyte-derived tumours varied depending on the regeneration pattern of the liver parenchyma in response to liver injury. Notably, Axin2-expressing hepatocytes undergoing chronic liver injury significantly contributed to liver regeneration and possessed high neoplastic potential. Additionally, we revealed that the metabolic phenotypes of liver tumours were acquired during tumorigenesis, irrespective of their spatial origin.

Conclusions: Hepatocytes receiving Wnt/β-catenin signalling from their microenvironment have high neoplastic potential, and Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma.

Lay summary: Lineage tracing revealed that zone 3 hepatocytes residing in the pericentral niche have high neoplastic potential. Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling broadly exist across all hepatic zones and significantly contribute to liver tumorigenesis as well as liver regeneration. Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma.

Keywords: CDAHFD, choline-deficient l-amino acid-defined, high-fat diet; CPS1, carbamoyl phosphate synthetase 1; CYP2E1, cytochrome P450 subfamily 2E1; DEN, diethylnitrosamine; GS, glutamine synthetase; HAL, histidine ammonia lyase; HCC, hepatocellular carcinoma; HFD, high-fat diet; Hepatocellular carcinoma; IF, immunofluorescence; ISH, in situ hybridisation; Liver regeneration; MAFLD, metabolic dysfunction-associated fatty liver disease; MUP, major urinary protein; Metabolic dysfunction-associated fatty liver disease; Metabolic zonation; ND, normal diet; PIK3CATg, hepatocyte-specific transgenic mice harbouring mutant PIK3CA variant; PP, periportal; PV, perivenous; RFP, red fluorescent protein; TAM, tamoxifen; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling; WT, wild-type; Wnt/β-catenin signal.