Effect Of E2F1 Research Articles

Study of the Role of E2F1 and TMEM132A in Prostate Cancer Development.

Identify transcription factors and target genes associated with prostate cancer, offering new therapy approaches. Gene Set Enrichment Analysis (GSEA) investigates early 2 factor (E2F) transcription factor family roles in prostate cancer using the TCGA database. Survival analysis examined E2F factors and patient survival connections. Dataset analysis identified E2F1-involved key genes. Quantitative Real-time PCR (qPCR), which combines ultrasound-guided methods to collect clinical samples from prostate cancer patients, was utilized to determine the expression levels of E2F1 and its target genes in patient samples and cancer cells. The effect of E2F1 and its target gene expression alterations on prostate cell proliferation was examined utilizing the cell counting kit-8 (CCK8) technique. Double fluorescence enzyme experiment verified E2F1-target gene connections. E2F family genes induce prostate cancer and show correlated co-expression. E2F1, E2F2, E2F3, E2F5, and E2F7 were considerably over-expressed in prostate cancer tissues. While E2F4 and E2F6 were notably underexpressed, there was no statistically important change in the E2F8 expression between prostate cancer and surrounding tissues. High expression of E2F genes is associated with lower patient survival. The transmemrane protein 132 (TMEM132A) was identified as a key gene for E2F1 action and is associated with poor prognosis in patients. The essential gene for E2F1 function, TMEM132A, was discovered. According to the qPCR results, E2F1 and TMEM132A are considerably expressed in cancer cells and patient samples. Interfering with its expression significantly inhibited the proliferation ability of cancer cells. The double luciferase experiment showed that E2F1 regulates the expression level in phase by binding directly to the TMEM132A promoter. The E2F transcription factor family induces prostate cancer and correlates with poor prognosis. E2F1 directly regulates TMEM132A by binding its promoter and controlling the degree of protein expression, thereby affecting cancer cell growth.

Blocking the E2F transcription factor 1/high-mobility group box 2 pathway enhances the intervention effects of α-santalol on the malignant behaviors of liver cancer cells

In view of the tumor-inhibiting effect of α-santalol in various cancers and the role of E2F transcription factor 1 (E2F1) as an important target for anticancer research, this study investigates the relation between α-santalol and E2F1, as well as the effect of α-santalol on liver cancer progression and the corresponding mechanism. Concretely, liver cancer cells were treated with different concentrations of α-santalol. The IC50 value of α-santalol was determined using Probit regression analysis. Then, transcription factors that are targeted by α-santalol and differentially expressed in liver cancer were screened out. The clinicopathological impact of E2F1 and its targets were evaluated and predicted. The expressions of E2F1 and high-mobility group box 2 (HMGB2) and their correlation in the liver cancer tissues were analyzed by bioinformatics. The effects of E2F1 and HMGB2 on the biological characteristics of liver cancer cells were examined through loss/gain-of-function and molecular assays. With the extension of treatment time, the inhibitory effects of 10 μmol/L and 20 μmol/L α-santalol on cancer cell survival rate were enhanced (P < 0.001). E2F1 and HMGB2 were highly expressed and positively correlated in liver cancer tissues (P < 0.05). High E2F1 expression was correlated with large tumors and high TNM stages (P < 0.05). E2F1 knockdown promoted the effects of α-santalol on dose-dependently inhibiting viability, colony formation, invasion and migration (P < 0.05). Moreover, E2F1 knockdown reduced the IC50 value and HMGB2 level, while HMGB2 overexpression produced opposite effects. HMGB2 overexpression and E2F1 knockdown mutually counteracted their effects on the IC50 value and on the viability and apoptosis of α-santalol-treated liver cancer cells (P < 0.01). Collectively, blocking the E2F1/HMGB2 pathway enhances the intervention effects of α-santalol on the proliferation, migration and invasion of liver cancer cells.

ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype

ObjectiveObesity variably disrupts human health, but molecular-based patients' health-risk stratification is limited. Adipose tissue (AT) stresses may link obesity with metabolic dysfunction, but how they signal in humans remains poorly-characterized. We hypothesized that a transcriptional AT stress-signaling cascade involving E2F1 and ASK1 (MAP3K5) molecularly defines high-risk obese subtype. MethodsASK1 expression in human AT biopsies was determined by real-time PCR analysis, and chromatin immunoprecipitation (ChIP) adopted to AT explants was used to evaluate the binding of E2F1 to the ASK1 promoter. Dual luciferase assay was used to measure ASK1 promoter activity in HEK293 cells. Effects of E2F1 knockout/knockdown in adipocytes was assessed utilizing mouse-embryonal-fibroblasts (MEF)-derived adipocyte-like cells from WT and E2F1−/− mice and by siRNA, respectively. ASK1 depletion in adipocytes was studied in MEF-derived adipocyte-like cells from WT and adipose tissue-specific ASK1 knockout mice (ASK1-ATKO). ResultsHuman visceral-AT ASK1 mRNA (N = 436) was associated with parameters of obesity-related cardio-metabolic morbidity. Adjustment for E2F1 expression attenuated the association of ASK1 with fasting glucose, insulin resistance, circulating IL-6, and lipids (triglycerides, HDL-cholesterol), even after adjusting for BMI. Chromatin-immunoprecipitation in human-AT explants revealed BMI-associated increased occupancy of the ASK1 promoter by E2F1 (r2 = 0.847, p < 0.01). In adipocytes, siRNA-mediated E2F1-knockdown, and MEF-derived adipocytes of E2F1-knockout mice, demonstrated decreased ASK1 expression and signaling to JNK. Mutation/truncation of an E2F1 binding site in hASK1 promoter decreased E2F1-induced ASK1 promoter activity, whereas E2F1-mediated sensitization of ASK1 promoter to further activation by TNFα was inhibited by JNK-inhibitor. Finally, MEF-derived adipocytes from adipocyte-specific ASK1-knockout mice exhibited lower leptin and higher adiponectin expression and secretion, and resistance to the effects of TNFα. ConclusionsAT E2F1 –ASK1 molecularly defines a metabolically-detrimental obese sub-phenotype. Functionally, it may negatively affect AT endocrine function, linking AT stress to whole-body metabolic dysfunction.

Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation

Tumour suppressor miR-34c deficiency resulted from hyper-methylation in its promoter is believed to be one of the main causes of colorectal cancer (CRC). Till date, miR-34c has been validated as a direct target of p53; but previous evidence suggested other transcription factor(s) must be involved in miR-34c transcription. In the present study, we in the first place identified a core promoter region (-1118 to -883bp) of pre-miR-34c which was embedded within a hyper-methylated CpG island. Secondly, E2F1 promoted miR-34c transcription by physical interaction with the miR-34c promoter at site -897 to -889bp. The transcriptional activating effect of E2F1 on miR-34c was in a p53 independent manner but profoundly promoted in the presence of p53 with exposure to 5-aza-2'-deoxycytidine (DAC). Thirdly, stem cell factor (SCF), a miR-34c target, was specifically reduced upon an introduction of E2F1 which lead to suppression of CRC cell proliferation. The E2F1-suppressed cell proliferation was partially abrogated by additional miR-34c inhibitor, indicating that the anti-proliferation effect of E2F1 was probably through activating miR-34c-SCF axis. Finally, SCF/KIT signalling increased E2F1 production by reducing its proteosomal degradation dependent on PI3K/Akt-GSK3β pathway. In conclusion, our results suggested the existence of E2F1-miR-34c-SCF negative feedback loop which was interrupted by the hyper-methylation of miR-34c promoter in CRC cells and increased cell proliferation.

Abstract 4389: E2F7 regulates sensitivity to the cytotoxic actions of anthracyclins in squamous cell carcinoma.

Abstract Squamous cell carcinoma (SCC) of the skin and head and neck region is a common and potentially fatal malignancy. Current chemotherapy treatments are generally not curative. Therefore, there is considerable need to develop new and targeted therapies to treat SCC. There is abundant data to show that E2Fs play an important role in many KC functions such as differentiation, growth and apoptosis. However, the identification of two new inhibitory forms of E2F, E2F7 and E2F8 has caused a rethink of our previous models. In this study, we examined the effects of E2F1, E2F7 and E2F8 on cytotoxic responses to chemotherapeutic agents. For in vitro experiments, we generated murine KCs from E2F7 fl/fl or E2F8 fl/fl mice that were made deficient in E2F7 and E2F8 following infection with Cre expressing adenoviral constructs. In addition, we had KCs generated from E2F1 KO mice. Our results showed that E2F8 deficient KCs displayed unaltered responses to 3 structurally different classes of chemotherapeutics represented by doxorubicin (anthracycline), cisplatin (platinum) or etoposide (topoisomerase II inhibitor). Similarly, E2F1 and E2F7-deficient KCs retained apoptotic responses to etoposide and cisplatin. In contrast, E2F7-deficient murine KCs were selectively sensitised to the cytotoxic actions of doxorubicin whilst E2F1-deficiency protected against doxorubicin. We showed that the sensitivity to doxorubicin was E2F7 specific since the reintroduction of E2F7 in E2F7 deficient KCs had a clear protective effect against doxorubicin. These results indicate that E2F7 regulate cytotoxic responses of SCC to doxorubicin, and doxorubicin toxicity is mediated, in part, by an E2F1-dependent mechanism. We also looked at the possible mechanism to drive E2F7-mediated doxorubicin induced cell death. Our results showed activation of caspase-3 in E2F7 deficient KCs mediates the doxorubicin-induced reduction in cell viability. Since E2F7 modulates sensitivity to doxorubicin, we looked at different SCC cells in their response to cytotoxic action of doxorubicin. The results suggested that SCC cells vary in their sensitivity to doxorubicin and this correlates with E2F7/E2F1 protein expression ratio in these SCC cells. The most sensitive SCC cell lines, KJD, had a very low E2F7/E2F1 ratio whilst the most insensitive SCC cell line, SCC25, had a very high E2F7/E2F1 ratio. These data showed that E2F7/E2F1 ratio is disrupted in SCC and this contributes to the insensitivity to cytotoxic actions of doxorubicin. Combined, these data indicate the existence of a novel, non-redundant and isoform-specific activities of E2F7 in KCs that have implications for our understanding of chemotherapeutic sensitivity in SCC. Our findings would suggest that if we sensitise SCC to the effects of anthracyclines, this would have a significant clinical value and add to therapeutic options available to treat SCC patients where there is a critical need. Citation Format: Mehlika Hazar Rethinam, Nicholas Saunders, Fiona Simpson. E2F7 regulates sensitivity to the cytotoxic actions of anthracyclins in squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4389. doi:10.1158/1538-7445.AM2013-4389

E2F1 Regulates Cellular Growth by mTORC1 Signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

Identification of E2F1 as a positive transcriptional regulator for δ-catenin

δ-Catenin is upregulated in human carcinomas. However, little is known about the potential transcriptional factors that regulate δ-catenin expression in cancer. Using a human δ-catenin reporter system, we have screened several nuclear signaling modulators to test whether they can affect δ-catenin transcription. Among β-catenin/LEF-1, Notch1, and E2F1, E2F1 dramatically increased δ-catenin–luciferase activities while β-catenin/LEF-1 induced only a marginal increase. Rb suppressed the upregulation of δ-catenin–luciferase activities induced by E2F1 but did not interact with δ-catenin. RT-PCR and Western blot analyses in 4 different prostate cancer cell lines revealed that regulation of δ-catenin expression is controlled mainly at the transcriptional level. Interestingly, the effects of E2F1 on δ-catenin expression were observed only in human cancer cells expressing abundant endogenous δ-catenin. These studies identify E2F1 as a positive transcriptional regulator for δ-catenin, but further suggest the presence of strong negative regulator(s) for δ-catenin in prostate cancer cells with minimal endogenous δ-catenin expression.

Adenovirus-mediated E2F-1 gene transfer in nonsmall-cell lung cancer induces cell growth arrest and apoptosis.

Since overexpression of E2F-1 has been shown to induce apoptosis, the ability of adenovirus-mediated transfer of E2F-1 to inhibit tumour growth in nonsmall-cell lung cancer cell lines was investigated. Three cell lines with various genomic status were infected with AdE2F. Cell proliferation and viability were determined by trypan blue exclusion. Apoptosis induction was assessed by flow cytometry and poly-adenosine diphosphate-ribose-polymerase cleavage assay. In vivo, the effect of E2F-1 on tumour growth was determined in severe combined immunodeficiency (SCID) mice. The current experiments showed that overexpression of E2F-1 suppressed tumour cell growth. The population of apoptotic cells was dramatically increased 96 h after infection with AdE2F. Inhibition of cell growth and induction of apoptosis was not dependent on genomic status. Moreover, treatment of implanted tumours in SCID mice with AdE2F inhibited tumour growth. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of nonsmall-cell lung cancer.

E2F4 and E2F1 Have Similar Proliferative Properties but Different Apoptotic and Oncogenic Properties In Vivo

Loss of retinoblastoma (Rb) tumor suppressor function, as occurs in many cancers, leads to uncontrolled proliferation, an increased propensity to undergo apoptosis, and tumorigenesis. Rb negatively regulates multiple E2F transcription factors, but the role of the different E2F family members in manifesting the cellular response to Rb inactivation is unclear. To study the effect of deregulated E2F4 activity on cell growth control and tumorigenesis, transgenic mouse lines expressing the E2F4 gene under the control of a keratin 5 (K5) promoter were developed, and their phenotypes were compared to those of previously generated K5 E2F1 transgenic mice. In contrast to what has been observed in vitro, ectopically expressed E2F4 was found to localize to the nucleus and induce proliferation to an extent similar to that induced by E2F1 in transgenic tissue. Unlike E2F1, E2F4 does not induce apoptosis, and this correlates with the differential abilities of these two E2F species to stimulatep19ARF expression in vivo. To examine the role of E2F4 in tumor development, the mouse skin two-stage carcinogenesis model was utilized. Unlike E2F1 transgenic mice, E2F4 transgenic mice developed skin tumors with a decreased latency and increased incidence compared to those characteristics in wild-type controls. These findings demonstrate that while the effects of E2F1 and E2F4 on cell proliferation in vivo are similar, their apoptotic and oncogenic properties are quite different.

The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction.

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).

81 - Effect of E2F-1 and E2F-1-mutants on the cellular survival and radiosensitivity

81 - Effect of E2F-1 and E2F-1-mutants on the cellular survival and radiosensitivity