Organ size control is dominant over deregulation of the core cell cycle machinery to restrict hepatocyte proliferation in vivo

Introduction: The retinoblastoma (RB) tumor suppressor pathway is inactivated via genetic and epigenetic mechanisms in the large majority of human cancers, including hepatocellular carcinoma (HCC). Direct inhibition of RB pathway components by HBV and HCV viral proteins may also play an important role in HCC development following viral hepatitis. In mammals, RB family members (RB itself, p130, and p107) are the downstream mediators of the RB pathway and critical regulators of the G1/S transition of the cell cycle. Inactivation of the RB pathway by deletion of the three Rb family genes in the liver of adult mice leads to development of HCCs that arise from liver stem/progenitor cell populations. Mature hepatocytes do enter the cell cycle after acute deletion of the RB family, but surprisingly rapidly arrest, which correlates with activation of the Hippo signaling pathway. Uncovering the mechanisms that prevent unrestricted cell proliferation in RB family mutant hepatocytes in vivo are important to understand tumor initiation and may identify novel targets in HCC.

Methods: Rblox/lox;p130lox/lox;p107-/-;Alb-CreERT2 (triple knockout, TKO) and control mice were treated with tamoxifen to delete the RB family in adult mice. Liver-specific deletion of the RB family was also achieved by in vivo transfection of hepatocytes with CreER recombinase using a sleeping-beauty transposon. Liver tissue was harvested 5–150 days after tamoxifen treatment. Cell cycle regulators were quantified by RT-PCR, 3'RNAseq, immunoblot, and immunostaining.

Results: After acute deletion of the RB family, mature hepatocytes enter the cell cycle, replicate their DNA, and undergo mitosis. However, these divisions mostly lead to limited endoreplication. Furthermore, this phenotype is transient and no DNA replication is observed at later time points. Studies in quadruple knockout mice demonstrate that this cell cycle exit in TKO hepatocytes is independent of the p53/p21 axis. Unbiased gene expression profile experiments showed that the cell cycle arrest in TKO hepatocytes correlates with activation of the Hippo pathway, a signaling pathway known to regulate organ size. Importantly, reducing liver size by partial hepatectomy in TKO mice results in inhibition of Hippo signaling and cell cycle re-entry in TKO hepatocytes.

Conclusions: Deletion of the RB family mimics the deregulation of the RB pathway observed in HCC and leads to transient cell cycle entry in hepatocytes followed by cell cycle arrest. Our data show for the first time that mechanisms exist in differentiated mammalian cells to induce cell cycle arrest independent of the RB family. These experiments identify a novel level of regulation of the cell cycle in vivo and indicate that inactivation of the Hippo pathway is required for complete cell cycle deregulation and possibly tumor initiation in hepatocytes.