E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice.

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.

CircPRKCI-miR-545/589-E2F7 axis dysregulation mediates hydrogen peroxide-induced neuronal cell injury.

Excessive oxidative stress induces significant injury and cytotoxicity to neuronal cells. The current study tested expression and the potential function of the circular RNA PRKCI (circPRKCI) in oxidative stress-injured neuronal cells. In cultured SH-SY5Y neuronal cells, hydrogen peroxide (H2O2) downregulated circPRKCI expression, causing accumulation of miR-545 and miR-589, but reduction of their target, the transcription factor E2F7. Importantly, ectopic overexpression of circPRKCI in SH-SY5Y cells significantly attenuated H2O2-induced cytotoxicity. Conversely, siRNA-mediated knockdown of circPRKCI induced SH-SY5Y cell death and apoptosis. Further studies demonstrated that H2O2-induced cytotoxicity in SH-SY5Y cells was inhibited by miR-545/589 inhibitors, but mimicked by miR-545/589 mimics. Importantly, CRISPR/Cas9-mediated knockout (KO) of E2F7 induced potent SH-SY5Y cell death and apoptosis. Furthermore, transfection of circPRKCI siRNA or miR-545/589 mimics were ineffective in E2F7 KO cells. In the primary human neurons, H2O2 stimulation similarly induced circPRKCI downregulation, miR-545/589 accumulation and E2F7 reduction. Moreover, H2O2-induced death and apoptosis in the primary neurons were significantly inhibited by circPRKCI overexpression or miR-545/589 inhibitors. Taken together, our results show that dysregulation of circPRKCI-miR-545/589-E2F7 axis mediated H2O2-induced neuronal cell injury. Targeting this novel cascade could be a fine strategy to protect neurons from oxidative stress.

Polyploid Hepatocytes Facilitate Adaptation and Regeneration to Chronic Liver Injury.

The liver contains diploid and polyploid hepatocytes (tetraploid, octaploid, etc.), with polyploids comprising ≥90% of the hepatocyte population in adult mice. Polyploid hepatocytes form multipolar spindles in mitosis, which lead to chromosome gains/losses and random aneuploidy. The effect of aneuploidy on liver function is unclear, and the degree of liver aneuploidy is debated, with reports showing aneuploidy affects 5% to 60% of hepatocytes. To study relationships among liver polyploidy, aneuploidy, and adaptation, mice lacking E2f7 and E2f8 in the liver (LKO), which have a polyploidization defect, were used. Polyploids were reduced fourfold in LKO livers, and LKO hepatocytes remained predominantly diploid after extensive proliferation. Moreover, nearly all LKO hepatocytes were euploid compared with control hepatocytes, suggesting polyploid hepatocytes are required for production of aneuploid progeny. To determine whether reduced polyploidy impairs adaptation, LKO mice were bred onto a tyrosinemia background, a disease model whereby the liver can develop disease-resistant, regenerative nodules. Although tyrosinemic LKO mice were more susceptible to morbidities and death associated with tyrosinemia-induced liver failure, they developed regenerating nodules similar to control mice. Analyses revealed that nodules in the tyrosinemic livers were generated by aneuploidy and inactivating mutations. In summary, we identified new roles for polyploid hepatocytes and demonstrated that they are required for the formation of aneuploid progeny and can facilitate adaptation to chronic liver disease.

Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells.

BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.

Synergistic functions of E2F7 and E2F8 are critical to suppress stress-induced skin cancer.

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.

Atypical E2Fs: new players in the E2F transcription factor family.

As major regulators of the cell cycle, apoptosis and differentiation, E2F transcription factors have been studied extensively in a broad range of organisms. The recent identification of atypical E2F family members further expands our structural, functional and molecular view of the cellular E2F activity. Unlike other family members, atypical E2Fs have a duplicated DNA-binding domain and control gene expression without heterodimerization with dimerization partner proteins. Recently, knockout strategies in plants and mammals have pinpointed that atypical E2Fs have a crucial role in plant cell size control, endocycle regulation, proliferation and apoptotic response upon DNA stress. Their position at the crossroads of proliferation and DNA stress response marks these novel E2F proteins as interesting study objects in the field of tumor biology.

E2F7 and E2F8 keep the E2F family in balance.

An article by Li and colleagues (in this issue of Developmental Cell) shows that the atypical E2Fs, E2F7 and E2F8, are critical for mouse development. One of the important functions of these family members stems from a negative feedback loop in which E2F7 and E2F8 limit the expression of E2F1 and prevent E2F1-dependent apoptosis.

Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development.

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.