E2f8 mediates tumor suppression in postnatal liver development.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.

The DNA damage checkpoint is activated during residual tumour cell survival to methotrexate treatment as an initial step of acquired drug resistance.

In the process of acquired drug resistance, the absence of tumour cell subpopulations already resistant before treatment implies an initial adaptive stage of cell growth following drug exposure that, under the selective pressure of the drug, allows the emergence of stably resistant cell variants. Here, we show that p53-defective HT-29 colon cancer cells overcome methotrexate-induced cell death owing to DNA damage checkpoint-mediated cell survival at the adaptive stage that precedes stable resistance acquisition. HT-29 cell cycle progression was dramatically delayed in the presence of a lethal dose of methotrexate, leading to DNA damage during S-phase transition and to cell death as treated cells progressed to G2 and M phases. As a result, the DNA damage checkpoint was induced as indicated by the presence of activated phosphorylated forms of checkpoint proteins Chk1 and Rad9. As we recently described, in-vitro resistance to methotrexate occurs without cell subpopulations already resistant before treatment, hence resistance is acquired through a multistep process that includes an early stage of transient cell survival. Our present results showed that this acute cell survival stage was due to a minor percentage of cells that could complete the first division cycle after drug exposure. Cell survival was enhanced by drug withdrawal during S-phase transition and suppressed if drug withdrawal was followed by treatment with the checkpoint-inhibitor drug caffeine. These results thus point to checkpoint-mediated transient adaptation as a target to prevent the emergence of acquired resistance to methotrexate.