Expression and prognostic role of E2F transcription factors in high-grade glioma.

INTRODUCTION: Patients with high-grade glioma (HGG) suffered poor survival due to inherent or acquired therapeutic resistance and refractory recurrence. The outcome of HGG patients has improved little during the past decade. Therefore, molecular signatures are urgently needed for improving diagnosis, survival prediction and identification of therapeutic targets for HGG. E2F transcription factors (E2Fs), a family of transcription factors recognized as master regulators of cell proliferation, have been found to be involved in the pathogenesis of various tumor types. AIMS: To investigate the expression of E2Fs and their prognosis value in high-grade glioma (HGG). RESULTS: Expression of E2Fs was analyzed in 394 HGG samples from TCGA dataset. E2Fs were generally expressed in HGG. Except for E2F3 and E2F5, expression of E2Fs was significantly upregulated and linked with grade progression. E2F1, E2F2, E2F7, and E2F8 were highly correlated with aggressive proliferation oncogenes, as well as potential therapeutic resistance oncogenes. Elevated E2Fs (not E2F3) were associated with adverse tumor features and poorer outcome. E2F7 and E2F8 exhibited superior outcome prediction performance compared with other E2Fs. Additionally, E2F7 and E2F8 independently predicted poorer survival in HGG patients. Gene set enrichment analysis identified a variety of critical oncogenic pathways that were tightly associated with E2F7 or E2F8, including epithelial-mesenchymal transition, NFκB, STAT3, angiogenesis pathways. Furthermore, elevated expression of E2F7 indicated worse therapeutic response of HGG to irradiation and silencing of E2F7 conferred higher cell-killing effect when combined with irradiation treatment. Mechanically, E2F7 directly regulates the transcriptional activity of EZH2 via binding at the corresponding promoter area. CONCLUSIONS: E2Fs (except for E2F3 and E2F5) are highly expressed in HGG and indicate adverse outcome. E2F7 and E2F8 were identified as novel potential prognostic markers in HGG. E2F7 was further validated to be closely associated with radioresistance of HGG and a critical transcriptional regulator of EZH2.

Emerging links between E2F control and mitochondrial function.

The family of E2F transcription factors is the key downstream target of the retinoblastoma tumor suppressor protein (pRB), which is frequently inactivated in human cancer. E2F is best known for its role in cell-cycle regulation and triggering apoptosis. However, E2F binds to thousands of genes and, thus, could directly influence a number of biologic processes. Given the plethora of potential E2F targets, the major challenge in the field is to identify specific processes in which E2F plays a functional role and the contexts in which a particular subset of E2F targets dictates a biologic outcome. Recent studies implicated E2F in regulation of expression of mitochondria-associated genes. The loss of such regulation results in severe mitochondrial defects. The consequences become evident during irradiation-induced apoptosis, where E2F-deficient cells are insensitive to cell death despite induction of canonical apoptotic genes. Thus, this novel function of E2F may have a major impact on cell viability, and it is independent of induction of apoptotic genes. Here, we discuss the implications of these findings in cancer biology.

Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote.

Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain unknown. Here, using the unicellular red alga Cyanidioschyzon merolae, we show that the G1/S regulator RBR-E2F-DP complex links the G1/S transition to circadian rhythms. Time-dependent E2F phosphorylation promotes the G1/S transition during subjective night and this phosphorylation event occurs independently of cell cycle progression, even under continuous dark or when cytosolic translation is inhibited. Constitutive expression of a phospho-mimic of E2F or depletion of RBR unlinks cell cycle progression from circadian rhythms. These transgenic lines are exposed to higher oxidative stress than the wild type. Circadian inhibition of cell cycle progression during the daytime by RBR-E2F-DP pathway likely protects cells from photosynthetic oxidative stress by temporally compartmentalizing photosynthesis and cell cycle progression.

Transcription of the Geminin gene is regulated by a negative-feedback loop.

Geminin performs a central function in regulating cellular proliferation and differentiation in development and also in stem cells. Of interest, down-regulation of Geminin induces gene transcription regulated by E2F, indicating that Geminin is involved in regulation of E2F-mediated transcriptional activity. Because transcription of the Geminin gene is reportedly regulated via an E2F-responsive region (E2F-R) located in the first intron, we first used a reporter vector to examine the effect of Geminin on E2F-mediated transcriptional regulation. We found that Geminin transfection suppressed E2F1- and E2F2-mediated transcriptional activation and also mildly suppressed such activity in synergy with E2F5, 6, and 7, suggesting that Geminin constitutes a negative-feedback loop for the Geminin promoter. Of interest, Geminin also suppressed nuclease accessibility, acetylation of histone H3, and trimethylation of histone H3 at lysine 4, which were induced by E2F1 overexpression, and enhanced tri-methylation of histone H3 at lysine 27 and monoubiquitination of histone H2A at lysine 119 in E2F-R. However, Geminin5EQ, which does not interact with Brahma or Brg1, did not suppress accessibility to nuclease digestion or transcription but had an overall dominant-negative effect. These findings suggest that E2F-mediated activation of Geminin transcription is negatively regulated by Geminin through the inhibition of chromatin remodeling.

Integrative analysis reveals an outcome-associated and targetable pattern of p53 and cell cycle deregulation in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease with a high proliferation rate. By integrating copy number data with transcriptional profiles and performing pathway analysis in primary DLBCLs, we identified a comprehensive set of copy number alterations (CNAs) that decreased p53 activity and perturbed cell cycle regulation. Primary tumors either had multiple complementary alterations of p53 and cell cycle components or largely lacked these lesions. DLBCLs with p53 and cell cycle pathway CNAs had decreased abundance of p53 target transcripts and increased expression of E2F target genes and the Ki67 proliferation marker. CNAs of the CDKN2A-TP53-RB-E2F axis provide a structural basis for increased proliferation in DLBCL, predict outcome with current therapy, and suggest targeted treatment approaches.

Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. METHODS: Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR) and protein (Western blot) levels. The phosphorylation status of cyclin-dependent kinases (CDKs) and retinoblastoma (Rb) protein was also examined using Western blot analysis. RESULTS: Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1), pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. CONCLUSION: Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

Estradiol-regulated microRNAs control estradiol response in breast cancer cells.

Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2-inducible proteins c-Myc and E2Fs are required for optimal ERalpha activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERalpha-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERalpha-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.

Growth inhibition of human MDA-mB-231 breast cancer cells by delta-tocotrienol is associated with loss of cyclin D1/CDK4 expression and accompanying changes in the state of phosphorylation of the retinoblastoma tumor suppressor gene product.

Tocotrienols, a subgroup within the vitamin E family of compounds, have shown antiproliferative and anticancer properties, however, the molecular basis of these effects remains to be elucidated. In this study, the effect of 3-tocotrienol on cell cycle arrest was assessed by studying the retinoblastoma protein (Rb) levels and phosphorylation status, levels of E2F (a transcription factor critically involved in the G1/S-phase transition of the mammalian cell cycle; originally identified as a DNA-binding protein essential for early region 1A-dependent activation of the adenovirus promoter designated E2), and other cell cycle controlling proteins in estrogen receptor-negative MDA-MB-231 breast cancer cells. The cell growth assay demonstrated that exposure of the MDA-MB-231 cells to 6-tocotrienol (1-20 microM) resulted in a dose- and time-dependent inhibition of cell growth as compared with vehicle treated cells and the magnitude of growth inhibition was higher at 10 and 20 microM treatment for 48 and 72 h. The phosphorylation status of Rb plays a central role in the control of the cell cycle at the G0/G1-phase. delta-Tocotrienol treatment reduced the total Rb and its phosphorylation at the Ser780, Ser795, Ser 807/811 and Thr826 positions in a dose- and time-dependent fashion. The site-specific inhibition of the phosphorylation of Rb by delta-tocotrienol was tightly associated with a marked reduction in the expression of cyclin D1 and its regulatory partner cyclin-dependant kinase 4 (CDK4), which is responsible for the phosphorylation of Rb at Ser780, Ser795, Ser 807/811 and Thr826. In addition, delta-tocotrienol also reduced the expression of E2F that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. Interestingly, delta-tocotrienol also caused a marked reduction in the expression of G2/M regulatory proteins including cyclin B1 and CDK1. To the best of our knowledge, this study was the first to reveal that the target of cell proliferative inhibitory action of delta-tocotrienol in a model estrogen receptor-negative human breast cancer cell line MDA-MB-231 is mediated by the loss of cyclin D1 and associated suppression of site-specific Rb phosphorylation, suggesting its future development and use as an anticancer agent.

The E2F-regulated gene Chk1 is highly expressed in triple-negative estrogen receptor /progesterone receptor /HER-2 breast carcinomas.

We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D(3), a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER-/PR-/HER-2- phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present.

E2F-6 suppresses growth-associated apoptosis of human hematopoietic progenitor cells by counteracting proapoptotic activity of E2F-1.

E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation. Disclosure of potential conflicts of interest is found at the end of this article.

Germ line gain of function with SOS1 mutation in hereditary gingival fibromatosis.

Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G(1) to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.

Transcriptome profiling of the C. elegans Rb ortholog reveals diverse developmental roles.

LIN-35 is the single C. elegans ortholog of the mammalian pocket protein family members, pRb, p107, and p130. To gain insight into the roles of pocket proteins during development, a microarray analysis was performed with lin-35 mutants. Stage-specific regulation patterns were revealed, indicating that LIN-35 plays diverse roles at distinct developmental stages. LIN-35 was found to repress the expression of many genes involved in cell proliferation in larvae, an activity that is carried out in conjunction with E2F. In addition, LIN-35 was found to regulate neuronal genes during embryogenesis and targets of the intestinal-specific GATA transcription factor, ELT-2, at multiple developmental stages. Additional findings suggest that LIN-35 functions in cell cycle regulation in embryos in a manner that is independent of E2F. A comparison of LIN-35-regulated genes with known fly and mammalian pocket protein targets revealed a high degree of overlap, indicating strong conservation of pocket protein functions in diverse phyla. Based on microarray results and our refinement of the C. elegans E2F consensus sequence, we were able to generate a comprehensive list of putative E2F-regulated genes in C. elegans. These results implicate a large number of genes previously unconnected to cell cycle control as having potential roles in this process.

cDNA analysis of gene expression associated with DNA-dependent protein kinase activity.

DNA-dependent protein kinase (DNA-PK) is thought to play a pivotal role in DNA double-strand break repair. We recently demonstrated the association of DNA-PK activity in peripheral blood lymphocytes (PBL) with the incidence of chromosomal aberrations and the risk of cancer. In this study, we applied cDNA array technology to find the expression of genes which are associated with DNA-PK activity in PBLs with various levels of DNA-PK activity. Most genes correlated with DNA-PK activity involved cell cycle regulation. Moreover, the transcription factor E2F1, which plays an important role in cell cycle progression, exhibited strong correlation with the DNA-PK activity and Rbp130, which is considered a negative regulator of E2F, showed inverse correlation with DNA-PK activity. In silico promoter analyses showed the presence of at least one E2F binding site in the promoter regions of Ku70, Ku86, DNA-PKcs and genes associated with DNA-PK activity. In order to examine the relationship among the E2F1 expression, the expression of genes related with DNA-PK activity, and DNA-PK activity, we activated PMLs by PHA to progress the cell cycle. After PHA activation of PML, the expression of E2F1 and DNA-PK activity increased. The expression of most genes in PHA-stimulated PBLs had a similar relationship with DNA-PK activity to that without PHA stimulation. These results indicate that the E2F transcription factor may regulate the concerted expression of genes related with DNA-PK activity.

Clinical relevance of E2F family members in ovarian cancer--an evaluation in a training set of 77 patients.

PURPOSE: The major obstacle in treating ovarian cancer is the rapid development of platinum resistance during therapy. Deregulation of members of the E2F family of transcription factors is crucially involved in carcinogenesis and probably in mechanisms underlying platinum resistance. We therefore investigated the relevance of the whole set of E2F family members in predicting clinical outcome and their significance in predicting platinum resistance. EXPERIMENTAL DESIGN: Real-time PCR of all E2F family members was done from 77 ovarian carcinomas, defined as our training set, and 8 healthy control samples. The correlation with clinicopathologic characteristics, platinum resistance, and survival was investigated. Furthermore, the cross-talk of E2F family members was assessed for its value in predicting survival and platinum resistance. RESULTS: The proliferation-promoting E2F1 and E2F2 were associated with grade 3 tumors and residual disease >2 cm in diameter after initial surgery. Survival analyses showed low expression of E2F1 or E2F2 to be significantly associated with favorable disease-free and overall survival (E2F1, P = 0.039 and 0.047, respectively; E2F2, P = 0.009 and 0.006, respectively). In contrast, high expression of inhibiting E2F4 or E2F7 predicted favorable disease-free and overall survival (E2F4, P = 0.047 and 0.042, respectively; E2F7, P = 0.048 and 0.042, respectively). A high E2F2 to E2F4 ratio was the most valuable prognostic variable for disease-free survival in multivariate analysis (hazard ratio, 6.494; P = 0.002). Tumors considered platinum resistant were associated with lower E2F4 and E2F7 expression (P = 0.012 and 0.009, respectively) compared with platinum-sensitive tumors. Again, ratios of E2F1 or E2F2 to E2F7 were the most favorable variables in predicting platinum resistance. CONCLUSIONS: We here show that deregulation of both proliferation-promoting and proliferation-inhibiting E2F transcription factors and their cross-talk is crucially involved in the tumor biology of ovarian cancer and influences clinical outcome. Furthermore, down-regulation of E2F7 may contribute to mechanisms underlying platinum resistance, and calculation of ratios of proliferation-promoting E2F1 to E2F7 could serve as a putative predictor of platinum resistance.

Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases.

We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.

Overexpression of SERTAD3, a putative oncogene located within the 19q13 amplicon, induces E2F activity and promotes tumor growth.

The amplified region of chromosome 19q13.1-13.2 has been associated with several cancers. The well-characterized oncogene AKT2 is located in this amplicon. Two members of the same gene family (SERTAD1 and SERTAD3) are also located within this region. We report herein the genomic structure and potential functions of SERTAD3. SERTAD3 has two transcript variants with short mRNA half-lives, and one of the variants is tightly regulated throughout G1 and S phases of the cell cycle. Overexpression of SERTAD3 induces cell transformation in vitro and tumor formation in mice, whereas inhibition of SERTAD3 by small interfering RNA (siRNA) results in a reduction in cell growth rate. Furthermore, luciferase assays based on E2F-1 binding indicate that SERTAD3 increases the activity of E2F, which is reduced by inhibition of SERTAD3 by siRNA. Together, our data support that SERTAD3 contributes to oncogenesis, at least in part, via an E2F-dependent mechanism.

Expression of the E2F family of transcription factors and its clinical relevance in ovarian cancer.

The E2F family of transcription factors plays a pivotal role in the regulation of cellular proliferation. On the basis of sequence homology and function, eight distinct members of E2F transcription factors (E2F-1 to E2F-8) have been distinguished to date. The regulation of E2F transcription factors is closely associated with the function of the retinoblastoma family of tumor suppressors (RB pathway). In the last decade various alterations of distinct components of the RB-E2F pathway were found to be associated with tumor progression. However, no data on the role of E2F family members are available in tumor biology of ovarian cancer. Here we describe an expression study of E2F transcription factors in various human ovarian cancer cell lines; its clinical relevance was examined in a training set of 77 ovarian cancer patients. Expression levels of E2F-1, E2F-2, and E2F-8 were elevated in all the ovarian cancer cell lines studied when compared with human peritoneal mesothelial cells (HPMCs). Interestingly, EGF treatment showed a time-dependent upregulation of the activating transcription factor E2F-3 and a simultaneous increase of DP-1, the heterodimeric partner of E2F-3. High expression of E2F-1, E2F-2, and E2F-8 was found to be associated with histopathologic grade 3 tumors and residual tumor over 2 cm in diameter after primary debulking surgery in ovarian cancer patients. Taken together, these data suggest that the proliferation-promoting E2F transcription factors E2F-1 and especially E2F-2 play a pivotal role in tumor biology of ovarian cancer and may be candidates for specific therapeutic targets.