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The cell cycle control system ensures that the genome is properly duplicated and evenly distributed over the daughter cells. This system is highly conserved and regulated by a plethora of genes. Among this regulatory system is the Rb-E2F network, which controls the expression of key cell cycle genes. Thus, it is not surprising that Rb is often lost or mutated in cancer, resulting in hyperactivated E2F transcription and enhanced tumorigenesis. To counteract enhanced E2F transcription, alternative mechanisms that inhibit E2F transcription to efficiently block proliferation of cancer cells are needed. In this thesis, we manipulated E2F-dependent transcription by altering the expression of E2F7 and E2F8 in mouse models. E2F7/8 are atypical cell cycle inhibitors belonging to the E2F family that possess Rb-independent repressive function. We hypothesized that overactivation of atypical E2F repressors efficiently blocks cancer cell proliferation. The studies from this thesis demonstrated that downregulation of E2F transcription by boosting E2F7/8 expression impaired the ability of liver cancer cells to progress through the cell cycle and suppressed tumor growth. In addition, unrestrained E2F dependent transcription due to loss of atypical E2Fs and RB, induced tumorigenesis in a tissue cell-type specific manner via a mechanism that involves maintenance of genomic instability. Lastly, we unravel novel functions of atypical E2Fs in lipid metabolism by controlling liver polyploidization. Taken together, this thesis has contributed to our understanding of the role of atypical E2Fs and Rb in cancer and provides scientific evidence that inhibition of E2F-depedent transcription could be a novel therapeutic option in oncology .

Background and Aims Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking.Approach and Results Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors.Conclusion Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

E2F transcription factors are important regulators of the cell cycle, and unrestrained activation of E2F-dependent transcription is considered to be an important driver of tumor formation and progression. Although highly expressed in normal skin and skin cancer, the role of the atypical E2Fs, E2F7 and E2F8, in keratinocyte homeostasis, regeneration and tumorigenesis is unknown. Surprisingly, keratinocyte-specific deletion of E2F7 and E2F8 in mice did not interfere with skin development and wound healing. However, the rate for successful isolation and establishment of E2f7/8-deficient primary keratinocyte cultures was much higher than for wild-type keratinocytes. Moreover, E2f7/8-deficient primary keratinocytes proliferate more efficiently under stress conditions, such as low/high confluence or DNA damage. Application of in vivo stress using the DMBA/TPA skin carcinogenesis protocol revealed that combined inactivation of E2f7/8 enhanced tumorigenesis and accelerated malignant progression. Loss of atypical E2Fs resulted in increased expression of E2F target genes, including E2f1. Additional loss of E2f1 did not rescue, but worsened skin tumorigenesis. We show that loss of E2F7/8 triggers apoptosis via induction of E2F1 in response to stress, indicating that the tumor-promoting effect of E2F7/8 inactivation can be partially compensated via E2F1-dependent apoptosis. Importantly, E2F7/8 repressed a large set of E2F target genes that are highly expressed in human patients with skin cancer. Together, our studies demonstrate that atypical E2Fs act as tumor suppressors, most likely via transcriptional repression of cell cycle genes in response to stress.

The cell cycle is a series of events that take place in a cell leading to duplication of its DNA and division into two daughter cells. It is an extremely vital process that, is tightly controlled by an evolutionally conserved regulatory network. Among the regulatory pathways that control cell cycle, Rb-E2F signaling plays an important role to manipulate the expression of key cell cycle genes. Therefore, regulation of the Rb-E2F pathway activity is essential to control the cell cycle, and its importance was illustrated by the fact that Rb is among the most frequently mutated genes in cancer cells, leading to increased E2F-mediated transcriptional activity that drives unscheduled proliferation. To date, a total of eight E2F transcription factors have been identified. Notably, E2F7 and E2F8 are “atypical” members of the E2F family for their Rb-independent repressor function and a distinctive mechanism to control gene expression. Atypical E2Fs are known to inhibit a network of genes involved in DNA replication, DNA metabolism, and DNA repair. However, how the activity of atypical E2Fs is controlled in normal cells and cancer cells, was largely unexplored. The studies from this thesis shine a clear light on this matter. We described two proteasomal degradation routes of atypical E2F in G1 and G2, by APC/CCdh1 (anaphase-promoting complex/cyclosome and Cdh1 subunit) and SCFcyclin F (Skp1-Cullin-F-box protein complex and cyclin F), respectively. We proved that these precise and timely regulations of atypical E2Fs are critical for proper cell cycle progression and DNA repair function. In addition, we found that repressor function of atypical E2F could be temporarily attenuated by Chk1 (Checkpoint kinase 1) and 14-3-3 proteins in response to DNA damage. This interplay sustains E2F-mediated transcription to avoid permanent cell death. Importantly, we observed a strong correlation between elevated levels of 14-3-3 proteins and E2F7/8 target gene expression in multiple types of cancers, suggesting that repressor functions of E2F7/8 were compromised to promote uncontrolled cell proliferation. Taken together, this thesis provides novel insight into the regulation of atypical E2Fs, and improved our understanding of cell cycle progression and DNA damage response.

Different types of mature B-cell lymphocytes are overall highly similar. Nevertheless, some B cells proliferate intensively, while others rarely do. Here, we demonstrate that a simple binary classification of gene expression in proliferating vs. resting B cells can identify, with remarkable selectivity, global in vivo regulators of the mammalian cell cycle, many of which are also post-translationally regulated by the APC/C E3 ligase. Consequently, we discover a novel regulatory network between the APC/C and the E2F transcription factors and discuss its potential impact on the G1–S transition of the cell cycle. In addition, by focusing on genes whose expression inversely correlates with proliferation, we demonstrate the inherent ability of our approach to also identify in vivo regulators of cell differentiation, cell survival, and other antiproliferative processes. Relying on data sets of wt, non-transgenic animals, our approach can be applied to other cell lineages and human data sets.

The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/E2f pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential for neuronal regeneration therapies. Recently, two new members of the Rb/E2f pathway have been discovered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse processes such as cell proliferation, DNA-stress response, apoptosis, however, their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical development was largely unaffected by E2f7/E2f8 deficiency, we observed an enlargement of the lateral ventricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at the ependymal cells, which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the absence of atypical E2fs. We found progressive denaturation of the ependymal cell layer, distortion of the ependymal motile cilia and reactive astrocytes within the layer. We identified Gli3, a component of the Sonic hedgehog pathway (Shh), as a target for E2fs, including atypical E2fs. We unravelled a novel mechanism by which atypical E2fs regulate the expression of Gli3, leading to up-regulation of Numb/NumbL, which in consequence destabilizes cadherins organization within the ependymal cell layer. In conclusion, our work suggests that E2f7 and E2f8 transcription factors play an essential role in maintaining the ependymal cell barrier.