Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, and the mechanisms underlying the development of HCC remain to be elucidated. Forkhead box N3 (FOXN3) is an important member of the FOX family of transcription factors that plays an essential role in several cancers but has not been investigated in HCC. In this study, we demonstrate that FOXN3 is downregulated in human primary HCC tissues compared with their matched adjacent liver tissues. Functional tests of FOXN3 demonstrated that FOXN3 inhibits the proliferation of HCC cells in vitro and in vivo. Additionally, FOXN3 repressed the mRNA and protein expression of E2F5, a reported potential oncogene, by inhibiting the promoter activity of E2F5. Collectively, our findings indicate that FOXN3 functions as a tumor suppressor in HCC by downregulating the expression of E2F5.

The retinoblastoma (Rb) gene family member p130 binds preferentially to the E2F5 transcription factor and forms a repressive E2F5/p130 complex that inhibits cell cycle progression and tumor growth. It is unclear whether E2F5, either alone or in combination with p130, can interfere with the transcriptional activity of other E2F family members, such as E2F1 and E2F3a, in vivo. In this study, we used transgenic mice to test whether overexpression of E2F5 with/without p130 would be sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry.

A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets.

The MYCN oncoprotein induces the proliferation of neuroblastoma cells through regulating gene transcription. The E2F transcription factor 5 (E2F5) plays an oncogenic role in several types of cancer, however, its role in neuroblastoma cells remains poorly characterized. We report here that MYCN positively regulates E2F5 expression in neuroblastoma cells. Analyses of chromatin immunoprecipitation (ChIP) and reporter gene assay demonstrate that MYCN directly binds to a Myc E-Box DNA binding motif within the promoter of E2F5 gene, whereby inducing its transcription. In addition, E2F5 knockdown inhibits the proliferation of MYCN-amplified neuroblastoma cells. Furthermore, E2F5 knockdown inhibits cell cycle progression, which could be attributed to its regulation in the expression of related cyclin-dependent kinases (CDKs), including CDK2 and CDK6. These results suggest that MYCN-regulated E2F5 upregulation is an important mechanism through which MYCN induces the proliferation of neuroblastoma cells. In conclusion, this study identifies E2F5 as a pro-proliferative factor in MYCN-amplified neuroblastoma cells, and also implicates it as a potential target in neuroblastoma treatment.

Liver regeneration is characterized by cell cycle reentrance of hepatocytes. N-Myc, encoded by MYCN, is a member of the Myc family of transcription factors. Elevation of MYCN expression has been noted in the course of liver regeneration whereas the underlying mechanism remains unclear. Here we describe that up-regulation of MYCN expression, as measured by quantitative PCR, Western blotting, and immunohistochemical staining, paralleled liver regeneration in animal and cell models. MYCN expression was up-regulated as a result of transcriptional activation. Ingenuity pathway analysis (IPA) revealed several up-stream transcriptional regulators for MYCN and RNA interference validated E2F5 and TFDP1 as essential for hepatocyte growth factor (HGF)-induced MYCN trans-activation. Further examination showed that deficiency of BRG1, a chromatin remodeling protein, attenuated MYCN induction during liver regeneration. BRG1 interacted with and was recruited by E2F5/TFDP1 to the MYCN promoter. Mechanistically, BRG1 might play a role regulating histone H3 acetylation and H3K4 trimethylation and facilitating/stabilizing the binding of RNA polymerase II surrounding the MYCN promoter. Over-expression of ectopic MYCN in BRG1-null hepatocytes overcame deficiency of proliferation. Importantly, a positive correlation between MYCN expression and BRG1/E2F5/TFDP1 expression was observed in human liver specimens. In conclusion, our data identify a novel epigenetic pathway where an E2F5-TFDP1-BRG1 complex regulates MYCN transcription to promote liver regeneration.

Circular RNA (circRNA) is a key regulator of tumor progression. However, the role of circFOXM1 in glioblastoma (GBM) progression is unclear. The aim of this study was to investigate the role of circFOXM1 in GBM progression. The expression levels of circFOXM1, miR-577, and E2F transcription factor 5 (E2F5) were examined by real-time quantitative polymerase chain reaction. Cell counting kit 8 assay, EdU staining, and transwell assay were used to detect cell proliferation, migration, and invasion. The levels of glutamine, glutamate, and α-ketoglutarate were determined to evaluate the glutaminolysis ability of cells. Protein expression was tested by Western blot analysis. Dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay were employed to verify the interaction between miR-577 and circFOXM1 or E2F5. Mice xenograft model for GBM was constructed to perform in vivo experiments. Our results showed that circFOXM1 was highly expressed in GBM tumor tissues and cells. Silencing of cir FOXM1 inhibited GBM cell proliferation, migration, invasion, glutaminolysis, as well as tumor growth. MiR-577 could be sponged by circFOXM1, and its inhibitor could reverse the suppressive effect of circFOXM1 downregulation on GBM progression. E2F5 was a target of miR-577, and the effect of its knockdown on GBM progression was consistent with that of circFOXM1 silencing. CircFOXM1 positively regulated E2F5 expression, while miR-577 negatively regulated E2F5 expression. In conclusion, our data confirmed that circFOXM1 could serve as a sponge of miR-577 to enhance the progression of GBM by targeting E2F5, which revealed that circFOXM1 might be a biomarker for GBM treatment.

Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. MicroRNA (miRNA)-544 is an important cancer-associated RNA that is downregulated in multiple types of cancer. However, the role of miR-544 in ESCC progression remains unknown. In the present study, miR-544 expression level was determined via RT-qPCR in 30 pairs of ESCC and adjacent normal tissues and in a panel of ESCC cell lines. Cell proliferation and cell apoptosis were assessed by MTT and flow cytometry assays. Luciferase reporter assay and western blot analysis were conducted to verify E2F transcription factor 5 (E2F5), an oncogene in ESCC, as a novel target gene of miR-544. The results illustrated that miR-544 is frequently downregulated in ESCC tissues and cell lines. Overexpression of miR-544 in ESCC cells resulted in decreased cell proliferation and increased cell apoptosis. Thus, E2F5 was identified as a target of miR-544, and its expression was negatively correlated with miR-544 expression in clinical ESCC tissues. More importantly, overexpression of miR-544 led to increased sensitivity of ESCC cells to cisplatin, an anticancer drug. Overall, these findings indicate that miR-544 serves as a tumor suppressor by targeting E2F5; thus, miR-544 may be a therapeutic target for the treatment of ESCC.

E2Fs, as a family of pivotal transcription factors, have been implicated in multiple biological functions in human cancer; however, the expression and prognostic significance of E2Fs in breast cancer remains unknown. In the present study, the mRNA expression patterns of E2Fs in breast cancer were investigated with Oncomine and The Cancer Genome Atlas data. Prognostic values of E2Fs for patients with breast cancer were determined using the Kaplan-Meier plotter database. The results strongly indicated that E2F1, E2F2, E2F3, E2F5, E2F7 and E2F8 were overexpressed in patients with breast cancer, whereas E2F4 and E2F6 exhibited no expression difference between patients with cancer and healthy controls. In survival analyses, elevated E2F1, E2F3, E2F5, E2F7 and E2F8 expression levels were significantly associated with lower overall survival, relapse-free survival (RFS), distant metastasis-free survival (DMFS) or post-progression survival for patients with breast cancer. Furthermore, high expression of E2F4 indicated improved RFS but reduced DMFS. Subgroup analyses based on four clinicopathological factors further revealed that E2Fs were associated with the prognosis of patients with breast cancer in an estrogen receptor-, progesterone receptor-, human epidermal growth factor 2- and lymph node status-specific manner. These data indicated that E2Fs may serve as promising biomarkers and therapeutic targets for breast cancer.

Multiciliated cells play critical roles in the airway, reproductive organs, and brain. Generation of multiple cilia requires both activation of a specialized transcriptional program and subsequent massive amplification of centrioles within the cytoplasm. The E2F4 transcription factor is required for both roles and consequently for multiciliogenesis. Here we establish that E2F4 associates with two distinct components of the centriole replication machinery, Deup1 and SAS6, targeting nonhomologous domains in these proteins. We map Deup1 and SAS6 binding to E2F4's N-terminus and show that this domain is sufficient to mediate E2F4's cytoplasmic role in multiciliogenesis. This sequence is highly conserved across the E2F family, but the ability to bind Deup1 and SAS6 is specific to E2F4 and E2F5, consistent with their shared roles in multiciliogenesis. By generating E2F4/E2F1 chimeras, we identify a six-residue motif that is critical for Deup1 and SAS6 binding. We propose that the ability of E2F4 and E2F5 to recruit Deup1 and/or SAS6, and enable centriole replication, contributes to their cytoplasmic roles in multiciliogenesis.

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway promotes the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest NIK expression observed in the most invasive cells. GBM cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote GBM cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting GBM pathogenesis.

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b~25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b~25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b~25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b~25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b~25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway increases the invasiveness of glioma cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK ( MAP3K14 ) is upregulated at the transcriptional level in invading cell populations, with the highest expression observed in the most invasive cells. Glioma cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote glioma cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting glioma pathogenesis.

The p16INK4a-RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a-RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a-RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a-RB pathway.

The E2F family of transcription factors consists of nine members with both distinct and overlapping functions. These factors are situated downstream of growth factor signaling cascades, where they play a central role in cell growth and proliferation through their ability to regulate genes involved in cell cycle progression. For this reason, it is likely that the members of the E2F family play a critical role during oncogenesis. Consistent with this idea is the observation that some tumors exhibit deregulated expression of E2F proteins. In order to systematically compare the oncogenic capacity of these family members, we stably over-expressed E2F1 through 6 in non-transformed 3T3 fibroblasts and assessed the ability of these transgenic cell lines to grow under conditions of low serum, as well as to form colonies in soft agar. Our results show that these six E2F family members can be divided into three groups that exhibit differential oncogenic capacity. The first group consists of E2F2 and E2F3a, both of which have strong oncogenic capacity. The second group consists of E2F1 and E2F6, which were neutral in our assays when compared to control cells transduced with vector alone. The third group consists of E2F4 and E2F5, which generally act to repress E2F-responsive genes, and in our assays demonstrated a strong capacity to inhibit transformation. Our results imply that the pattern of expression of these six E2F family members in a cell could exert a strong influence over its susceptibility to oncogenic transformation.

APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.

Background: The mechanisms underlying low intensity ultrasound (LIUS) mediated suppression of inflammation and tumorigenesis remain poorly determined. Methods: We used microarray datasets from NCBI GEO Dataset databases and conducted a comprehensive data mining analyses, where we studied the gene expression of 299 cell death regulators that regulate 13 different cell death types (cell death regulatome) in cells treated with LIUS. Results: We made the following findings: (1) LIUS exerts a profound effect on the expression of cell death regulatome in cancer cells and non-cancer cells. Of note, LIUS has the tendency to downregulate the gene expression of cell death regulators in non-cancer cells. Most of the cell death regulator genes downregulated by LIUS in non-cancer cells are responsible for mediating inflammatory signaling pathways; (2) LIUS activates different cell death transcription factors in cancer and non-cancer cells. Transcription factors TP-53 and SRF- were induced by LIUS exposure in cancer cells and non-cancer cells, respectively; (3) As two well-accepted mechanisms of LIUS, mild hyperthermia and oscillatory shear stress induce changes in the expression of cell death regulators, therefore, may be responsible for inducing LIUS mediated changes in gene expression patterns of cell death regulators in cells; (4) LIUS exposure may change the redox status of the cells. LIUS may induce more of antioxidant effects in non-cancer cells compared to cancer cells; and (5) The genes modulated by LIUS in cancer cells have distinct chromatin long range interaction (CLRI) patterns to that of non-cancer cells. Conclusions: Our analysis suggests novel molecular mechanisms that may be utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Smoking is responsible for 90% of lung cancer cases. There is currently no clinically available gene test for early detection of lung cancer in smokers, or an effective patient selection strategy for adjuvant chemotherapy in lung cancer treatment. In this study, concurrent coexpression with multiple signaling pathways was modeled among a set of genes associated with smoking and lung cancer survival. This approach identified and validated a 7-gene signature for lung cancer diagnosis and prognosis in smokers using patient transcriptional profiles (n=847). The smoking-associated gene coexpression networks in lung adenocarcinoma tumors (n=442) were highly significant in terms of biological relevance (network precision = 0.91, FDR<0.01) when evaluated with numerous databases containing multi-level molecular associations. The gene coexpression network in smoking lung adenocarcinoma patients was confirmed in qRT-PCR assays of the identified biomarkers and involved signaling pathway genes in human lung adenocarcinoma cells (H23) treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Furthermore, the western blotting results of p53, phospho‑p53, Rb and EGFR in NNK-treated H23 and transformed normal human lung epithelial cells (BEAS-2B) support their functional involvement in smoking-induced lung cancer carcinogenesis and progression.