CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factor stimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

Long Non-Coding RNA Differentiation Antagonizing Nonprotein Coding RNA (DANCR) Promotes Proliferation and Invasion of Pancreatic Cancer by Sponging miR-214-5p to Regulate E2F2 Expression.

BACKGROUND Long non-coding RNA differentiation antagonizing nonprotein coding RNA (lncRNA DANCR) has been reported to act as an oncogene in various human cancers. The role of DANCR in development of pancreatic cancer (PC) is unknown. The aim of our research was to investigate the biological role of DANCR in PC. MATERIAL AND METHODS Expressions of DANCR, miR-214-5p, and E2F2 mRNA in PC tissues and cell lines were examined by qRT-PCR. Western blotting was carried out for detection of E2F2 protein expression in PC cells. Transwell assays were used to examine the metastatic ability of PC cells, while CCK-8 and colony formation assay were applied to evaluate cell proliferation. The effects of DANCR on PC cells were assessed by knockdown in vitro and in vivo. The regulatory mechanism of competitive endogenous RNAs were obtained from bioinformatics prediction and luciferase reporter assay. RESULTS DANCR was markedly upregulated in clinical tissues and cell lines of PC. High DANCR expression exhibited a significant correlation with poor prognosis. DANCR knockdown inhibited growth and metastasis of PC cells. Furthermore, DANCR acted as sponge to regulate miR-214-5p, and miR-214-5p inhibitor reversed the effects of DANCR knockdown on PC cells. Moreover, DANCR positively modulated E2F2 expression through miR-214-5p in PC cells. CONCLUSIONS Collectively, our findings demonstrated that lncRNA DANCR/miR-214-5p/E2F2 axis acts as an oncogene in PC development, which might provide a potential target for PC therapy.

Expression and prognostic value of E2F activators in NSCLC and subtypes: a research based on bioinformatics analysis.

E2F activators (E2F1-3) codify a family of transcription factors (TFs) in higher eukaryotes. E2F activators are involved in the cell cycle regulation and synthesis of DNA in mammalian cells, and their overexpression has been detected in many human cancers. However, their clinical significance has not been deeply researched in non-small-cell lung cancer (NSCLC), and bioinformatics analysis has never been reported to explore their clinical role in NSCLC. In the current study, we investigated the expression and prognostic value of E2F activators in NSCLC patients through the "TCGA datasets" and the "Kaplan-Meier plotter" (KM plotter) database. Hazard ratio (HR), 95 % confidence intervals, and log-rank P were calculated. Compared with normal tissue samples, E2F activators were overexpressed in NSCLC tissues, in lung adenocarcinoma (LUAD) tissues, and in lung squamous cell carcinoma (LUSC) tissues. In NSCLC patients, E2F1 expression was significantly correlated with age, sex, and tumor stage. E2F2 expression was found to be significantly correlated with sex and tumor size. We further demonstrated that E2F1 and E2F2 overexpressions were significantly associated with poor prognosis. In LUAD patients, E2F1 expression was significantly correlated with tumor size and tumor stage. E2F2 expression was significantly correlated with lymph node status and tumor stage. E2F1 and E2F2 overexpression showed a significant association with poor prognosis, while E2F3 overexpression was significantly correlated to better prognosis. In LUSC patients, E2F1 was concluded to be significantly correlated with tumor stage. However, E2F activators were not found to be correlated to prognosis.

MiR-218 Inhibited Growth and Metabolism of Human Glioblastoma Cells by Directly Targeting E2F2.

In recent years, microRNA has become a hotspot in research on diseases, especially in the initiation and progression of different types of cancer. In this study, we found that miR-218 could inhibit growth and metabolism in gliomas by directly targeting E2F2. First, we obtained data from the Chinese Glioma Genome Atlas (CGGA) database to analyze miR-218 expression in different grades of gliomas. The effects of miR-218 on cell cycle progression and cell proliferation in U87 and U251 cell lines were investigated by flow cytometry, specifically CCK8 assay and tablet cloning, respectively. Glucose consumption and lactate production of glioma cell lines were measured by correlative test kits. Furthermore, we used Western blot analysis and luciferase reporter assay to identify the direct and functional target of miR-218. Data from the CGGA database and real-time quantitative reverse transcription-PCR demonstrated that miR-218 was obviously reduced in human glioblastoma tissues, as well as in the cell lines. When miR-218 level was elevated in vitro, cell cycle progression was arrested in the G1 phase, and cell proliferation was dramatically inhibited. Both glucose consumption and lactate production of glioma cells were significantly reduced. Western blot analysis and luciferase reporter assay revealed that E2F2 was a direct target of miR-218 in glioma cells. This investigation demonstrated that elevated E2F2 expression could partly weaken the effect of miR-218 in vitro. This study also showed that miR-218 may be a repressor in glioma by directly targeting E2F2, as well as a potential therapeutic target in gliomas.

Liver × receptor ligands disrupt breast cancer cell proliferation through an E2F-mediated mechanism.

INTRODUCTION: Liver × receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors and have established functions as regulators of cholesterol, glucose, and fatty acid metabolism and inflammatory responses. Published reports of anti-proliferative effects of synthetic LXR ligands on breast, prostate, ovarian, lung, skin, and colorectal cancer cells suggest that LXRs are potential targets in cancer prevention and treatment. METHODS: To further determine the effects of LXR ligands and identify their potential mechanisms of action in breast cancer cells, we carried out microarray analysis of gene expression in four breast cancer cell lines following treatments with the synthetic LXR ligand GW3965. Differentially expressed genes were further subjected to gene ontology and pathway analyses, and their expression profiles and associations with disease parameters and outcomes were examined in clinical samples. Response of E2F target genes were validated by real-time PCR, and the posited role of E2F2 in breast cancer cell proliferation was tested by RNA interference experiments. RESULTS: We observed cell line-specific transcriptional responses as well as a set of common responsive genes. In the common responsive gene set, upregulated genes tend to function in the known metabolic effects of LXR ligands and LXRs whereas the downregulated genes mostly include those which function in cell cycle regulation, DNA replication, and other cell proliferation-related processes. Transcription factor binding site analysis of the downregulated genes revealed an enrichment of E2F binding site sequence motifs. Correspondingly, E2F2 transcript levels are downregulated following LXR ligand treatment. Knockdown of E2F2 expression, similar to LXR ligand treatment, resulted in a significant disruption of estrogen receptor positive breast cancer cell proliferation. Ligand treatment also decreased E2F2 binding to cis-regulatory regions of target genes. Hierarchical clustering of breast cancer patients based on the expression profiles of the commonly downregulated LXR ligand-responsive genes showed a strong association of these genes with patient survival. CONCLUSIONS: Taken together, these results indicate that LXR ligands target gene networks, including those regulated by E2F family members, are critical for tumor biology and disease progression and merit further consideration as potential agents in the prevention and treatment of breast cancers.

Expression of HOXC9 and E2F2 are up-regulated in CD133(+) cells isolated from human astrocytomas and associate with transformation of human astrocytes.

Comparative analysis of cancer stem cells with their neoplastic and non-neoplastic counterparts should help better understand the underlying molecular events leading to transformation and tumor dissemination. Here, we report a molecular signature comprised by genes with exclusive aberrant expression in CD133(+) cells, a reported subpopulation of tumorigenic stem-like cells, isolated from human glioblastomas. Microarrays covering 55,000 transcripts were used to compare gene expression profiles in purified subpopulations of CD133(+) and CD133(-) GBM cells. Sixteen genes, many of which not previously associated with astrocytomas, were found aberrantly expressed in CD133(+) cells, but not in CD133(-), when compared with corresponding non-neoplastic controls. Up-regulation of two of such genes, E2F2 and HOXC9, was detected in a set of 54 astrocytomas of different grades and significantly associated with malignancy. Due to their distinctive expression in CD133(+) cells, the use of E2F2 and HOXC9 as therapeutic targets for tumor eradication is suggested.

Human papillomavirus 16 E6 modulates the expression of miR-496 in oropharyngeal cancer.

Human papillomavirus (HPV), notably type 16, is a risk factor for up to 75% of oropharyngeal squamous cell carcinomas (SCC). It has been demonstrated that small non-coding RNAs known as microRNAs play a vital role in the cellular transformation process. In this study, we used an LNA array to further investigate the impact of HPV16 on the expression of microRNAs in oropharyngeal (tonsillar) cancer. A number of miRNAs were found to be deregulated, with miR-496 showing a four-fold decrease. Over-expression of the high risk E6 oncoprotein down-regulated miR-496, impacting upon the post-transcriptional control of the transcription factor E2F2. These HPV specific miRNAs were integrated with the HPV16 interactome to identify possible mechanistic pathways. These analyses provide insights into novel molecular interactions between HPV16 and miRNAs in oropharyngeal cancers.

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.

E2F2 induction in related to cell proliferation and poor prognosis in non-small cell lung carcinoma.

E2F transcription factors regulate a wide range of biological processes, including cell cycle, apoptosis and DNA damage response. In the present study, we examined whether E2F2 is related to the poor prognosis of NSCLC and its role in progress of NSCLC. Firstly, we analyzed 86 NSCLC samples by immunohistochemistry and found that E2F2 expression was markedly increased in 62.8% (54/86) of all samples compared with the normal tissues. Further study showed that E2F2 expression was closely associated with clinical stage (P = 0.039) and tumor size (P = 0.045). Furthermore, Kaplan-Meier analysis indicated that high Bad expression was significantly correlated to overall survival (P = 0.045) but not disease-free survival (P = 0.288). In addition, our results showed that knockdown E2F2 expression could reduce cell viability and colony formation in NSCLC cells. The results in our study for the first time revealed that E2F2 act as an activator in tumor progress of NSCLC and could become a promising marker for the prognosis of patients with NSCLC.

4-hexylresorcinol stimulates the differentiation of SCC-9 cells through the suppression of E2F2, E2F3 and Sp3 expression and the promotion of Sp1 expression.

The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway.