Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell Cycle Exit after DNA Damage

Abstract

E2F transcription factors control the expression of a large network of cell cycle genes and are essential for S-phase entry. Cancers often show upregulation of E2F target gene expression, which can be partially explained by an increased percentage of replicating cells. However, we now demonstrate in human cancer biopsies that individual cycling neoplastic cells display abnormally high levels of E2F-dependent transcription using single cell RNA sequencing. Since cancer patients are treated with DNA damaging drugs, we determined whether elevated E2F activity has impact on the response to DNA damage. We specifically increased the expression of E2F target genes during S and G2-phase by deleting the atypical E2F transcriptional repressors (E2F7/8) in untransformed cells. Live cell imaging revealed that cells with elevated E2F activity in S-G2 failed to arrest and underwent unscheduled mitosis after neocarzinostatin-induced DNA damage. In contrast, cells with physiological E2F-activity completed S-phase and subsequently exit the cell cycle in response to DNA damage. This exit into a G1-like arrest is dependent on transcriptional repression of the E2F-target Emi1 resulting in activation of APC/C^Cdh1. Interestingly, ~30% of these arrested 4N-G1 cells could eventually inactivate APC/C^Cdh1 to execute a second round of DNA replication and mitosis, resulting in the formation of tetraploid cells. In summary, we show that a subpopulation of cancer cells display extreme high E2F activity. This unrestricted E2F activity can prevent cell cycle exit after DNA damage and likely increases the chance to acquire more genetic changes and therefore potentially promote malignant progression and reduce sensitivity to anti-cancer drugs.

Keywords: Cell Cycle, Anaphase-Promoting Complex/Cyclosome, E2F transcription, Live cell imaging, Single Cell Sequencing, DNA damage, cancer, P53, Emi1, Tetraploidy