Knockdown Of E2F1 Research Articles

E2F1 renders prostate cancer cell resistant to ICAM-1 mediated antitumor immunity by NF-κB modulation.

BackgroundE2F1 is the gatekeeper of the cell cycle controlling an analogous balance between proliferation and cell death. E2F1 expression is elevated in advanced prostate cancer. However, it is still unclear that the roles and mechanisms of E2F1 on prostate cancers.MethodsTargeted knockdown by interferon RNA was applied on two prostate cancer and Hela cell lines to examine the inverse correlation expression of E2F1 and ICAM-1. ICAM-1 promoter reporter and ChIP assays were used for analysis of the molecular basis of transcriptional regulation of E2F1 on ICAM-1. Co-IP assays were employed for testing the protein interaction between E2F1 and NF-κB. Tumor xenograft mice model with E2F1 and ICAM-1-knockdown prostate cancer cells were used to investigate the effects of E2F1 and ICAM-1 on antitumor immunity.ResultsE2F1 knockdown by a specific short hairpin RNA increased gene transcription and protein expression of ICAM-1. By using wild type and a series of mutant ICAM-1 promoter luciferase constructs, the NF-κB binding sites were found to be important for E2F1 regulation of ICAM-1 promoter. Targeted knockdown of E2F1 did not affect expression and phosphorylation of NF-κB and IκBα, but facilitated NF-κB binding to the ICAM-1 promoter, subsequently induced ICAM-1 transcription and production in prostate carcinoma cells. Furthermore, knockdown of E2F1 inhibited tumor growth of prostate cancer in vivo through increasing the susceptibility of tumor cells to ICAM-1-mediated anti-tumor immunity including enhancement of monocyte adhesion, leucocytes infiltration, as well as cytotoxicity against tumor cells.ConclusionsE2F1 knockdown inhibited prostate tumor growth in vitro and in vivo through sensitizing tumor cells to ICAM-1 mediated anti-immunity by NF-κB modulation, highlighting the potential of E2F1 as a therapeutic target.

The long non-coding RNA ERIC is regulated by E2F and modulates the cellular response to DNA damage

BackgroundThe human genome encodes thousands of unique long non-coding RNAs (lncRNAs), and these transcripts are emerging as critical regulators of gene expression and cell fate. However, the transcriptional regulation of their expression is not fully understood. The pivotal transcription factor E2F1 which can induce both proliferation and cell death, is a critical downstream target of the tumor suppressor, RB. The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity.ResultsHere, we report that lncRNA XLOC 006942, which we named ERIC, is regulated by E2F1 and, most probably, also E2F3. We show that expression levels of ERIC were elevated upon activation of exogenous E2F1, E2F3 or endogenous E2Fs. Moreover, knockdown of either E2F1 or E2F3 reduced ERIC levels and endogenous E2F1 binds ERIC’s promoter. Expression of ERIC was cell cycle regulated and peaked in G1 in an E2F1-dependent manner. Inhibition of ERIC expression increased E2F1-mediated apoptosis, suggesting that E2F1 and ERIC constitute a negative feedback loop that modulates E2F1 activity. Furthermore, ERIC levels were increased following DNA damage by the chemotherapeutic drug Etoposide, and inhibition of ERIC expression enhanced Etoposide -induced apoptosis.ConclusionsOur data identify ERIC as a novel lncRNA that is transcriptionally regulated by E2Fs, and restricts apoptosis induced by E2F1, as well as by DNA damage.

E2F1 promotes angiogenesis through the VEGF-C/VEGFR-3 axis in a feedback loop for cooperative induction of PDGF-B

Angiogenesis is essential for primary tumor growth and metastatic dissemination. E2F1, frequently upregulated in advanced cancers, was recently shown to drive malignant progression. In an attempt to decipher the molecular events underlying this behavior, we demonstrate that the tumor cell-associated vascular endothelial growth factor-C/receptor-3 (VEGF-C/VEGFR-3) axis is controlled by E2F1. Activation or forced expression of E2F1 in cancer cells leads to the upregulation of VEGFR-3 and its ligand VEGF-C, whereas E2F1 depletion prevents their expression. E2F1-dependent receptor induction is crucial for tumor cells to enhance formation of capillary tubes and neovascularization in mice. We further provide evidence for a positive feedback loop between E2F1 and VEGFR-3 signaling to stimulate pro-angiogenic platelet-derived growth factor B (PDGF-B). E2F1 or VEGFR-3 knockdown results in reduced PDGF-B levels, while the coexpression synergistically upregulates promoter activity and endogenous protein expression of PDGF-B. Our findings delineate an as yet unrecognized function of E2F1 as enhancer of angiogenesis via regulation of VEGF-C/VEGFR-3 signaling in tumors to cooperatively activate PDGF-B expression. Targeting this pathway might be reasonable to complement standard anti-angiogenic treatment of cancers with deregulated E2F1.

E2F1 positively regulates Trib2 pseudokinase expression and proliferation in acute leukaemia

Deregulation of the transcription factor E2F1 occurs in AML, and it has been shown to induce both cell cycle progression and apoptosis. In normal granulopoiesis, proliferation arrest and differentiation mediated by C/EBPalpha involves repression of E2F1 target genes. Elevated Trib2 expression has been linked with a subset of human AML and dysregulated C/EBPalpha. Using promoter assays, mutational analyses and chromatin immunoprecipitation experiments, we show that E2F1 (and E2F2, E2F3, but not E2F4 or E2F5) are bound directly to the DNA on site-specific regions on the Trib2 promoter in leukaemic cells. Trib2 expression is decreased following siRNA-mediated knockdown of E2F1, and in E2F1 knockout cells as compared to wild type cells. The reintroduction of E2F1 rescued Trib2 expression showing that E2F1 is regulating the expression of endogenous Trib2. Further analyses revealed that this activation of Trib2 by E2F1 is repressed by wild type C/EBPalpha consistent with C/EBPalpha having a negative regulatory role on E2F1, and in normal GMP cells we detect C/EBPalpha bound to the Trib2 promoter. Conversely there was synergistic activation upon coexpression of the oncogenic C/EBPalpha truncated mutant, and Trib2 expression levels were elevated in GMPs from preleukaemic mutant C/EBPalpha mice compared to wild type GMPs. Indeed a positive correlation between Trib2 and E2F1 expression in AML datasets support these findings. Finally, inhibition of the cell cycle pathway in leukaemia cells expressing high endogenous levels of Trib2 protein resulted in G1 arrest with a reduction in E2F1 levels and Trib2 protein levels. Our work indicates that the cell cycle regulator E2F1 plays a key role in the control of Trib2 expression important for the control of cell proliferation and may have important implications for normal and malignant haematopoiesis.

Lysine Acetyltransferase GCN5 Potentiates the Growth of Non-small Cell Lung Cancer via Promotion of E2F1, Cyclin D1, and Cyclin E1 Expression

The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment.

Abstract 5445: Differential roles of E2F1 in melanoma.

Abstract Melanoma, the malignant tumor of melanocytes, accounts for 4% of all skin cancer incidence but approximately 78% of skin cancer related deaths. No current treatment significantly enhances disease-free survival once distant metastases have established. The 5 year survival rate drops dramatically from 99.9% to 9% when melanoma progresses. To facilitate the development of effective treatment strategies, it is imperative that we obtain a better understanding of the biology of melanoma cells as they progress through the various stages of disease. Previous studies have shown that E2F1, a transcription factor, is over-expressed in melanoma cells. Interaction between E2Fs and tumor suppressor, Rb is essential for regulation of cell cycle progression. E2F1 is known to be involved in many other important biological processes, including DNA damage, DNA repair, differentiation, development, autophagy and apoptosis. Involvement of E2F1 in so many diverse functions underscores the importance of this protein under normal and pathologic conditions including melanoma. Previously we have found that primary melanoma cells have high level of E2F1 message and protein, and that the acetylation of E2F1 (active form) is higher in late stage melanoma cells. This led us to propose that E2F1 may function differentially in various stages of melanoma progression. Therefore we generated stable E2F1 knockdown cells in early stage primary and advanced metastatic melanoma cells. Our results show that E2F1 reduction works more effectively to inhibit cell growth in primary melanoma cells. Further, we also found differential effects on apoptosis and invasion in early and late stage melanoma cells. There is increased spontaneous cell death in primary melanoma cells after E2F1 knockdown, while there is increased apoptosis resistance in response to UV or valinomycin. These results taken together suggest that E2F1 has pleotropic effects in melanoma. It can switch between oncogenic or tumor suppressive functions during melanoma progression. A better understanding of the mechanism involved in this switch may allow combining gene therapies with other therapies for improved therapeutics for malignant melanoma. Supported by R21 CA125719 (RG). Citation Format: Peng Meng, Rita Ghosh. Differential roles of E2F1 in melanoma. [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 5445. doi:10.1158/1538-7445.AM2013-5445

Effect of E2F1 Knockdown on proliferation and invasion of clear cell renal cell carcinoma cell line Caki-2

Objective To study the effect of E2F1 knockdown on proliferation,migration and invasion of clear cell renal cell carcinoma cell line Caki-2.Methods Small interfering RNA (siRNA)-E2F1and negative control sequences were transfected into Caki-2 cells by Lipo2000.The E2F1 expression levels were detected by using real-time quantitative polymerase chain reaction (PCR) and Western blotting respectively after transfection.MTS and Transwell assays were used to detect variations in cell proliferation,migration and invasion.Results Compared to untransfected group (130.100 ± 6.301) × 10-5 and negative control group (134.600 ± 7.163) × 10-5,the mRNA levels in siRNA-E2F1 group (37.770 ± 3.782) × 10-5were significantly reduced (P ＜0.01).The protein levels were consistent with mRNA levels.MTS assay demonstrated the absorbance in siRNA-E2F1 group was significantly decreased at the time points of 24,48,72 and 96 h after transfection (P ＜ 0.05),and Transwell assay revealed that the number of migrating cells in siRNA-E2F1 group (38.330 ±4.041) was significantly reduced at 48 h after transfection as compared with untransfected group (83.000 ± 8.888) and negative control group (87.330 ± 6.429) (P ＜0.01),and the number of invasive cells in siRNA-E2F1 group (78.330 ±7.572) was strongly reduced at 48 h after transfection as compared with untransfected group (143.30 ± 10.066) and negative control group (147.000 ± 14.000) (P ＜0.01).Conclusion Knockdown of E2F1 significantly decreased proliferation,migration and invasion of Caki-2 cells. Key words: Clear cell renal cell carcinoma; E2F1 gene ; Proliferation; Migration; Invasion

Lysyl Oxidase Is Induced by Cell Density-Mediated Cell Cycle Suppression via RB-E2F1-HIF-1α Axis

Remodeling of the matrix surrounding tumor cells plays a crucial role in the development and maintenance of cancer. Lysyl oxidase (LOX), a matrix remodeling factor, is induced by HIF-1α under hypoxic conditions and associated with tumor growth and metastasis. Here, we report that high cell density induces HIF-1α expression under normoxic condition, resulting in the promotion of LOX expression. This phenomenon was observed in the retinoblastoma tumor suppressor (RB)-proficient breast cancer cells but not in RB-deficient cells. In RB-proficient cancer cells, the cell cycle regulator E2F1 was down-regulated and cell cycle progression was inhibited at high density culture condition. Knockdown of E2F1 stabilized HIF-1α and promoted LOX expression, while knockdown of both E2F1 and HIF-1α prevented the up-regulation of LOX. These findings suggest that elevated cell density enhances cell cycle arrest and matrix remodeling via RB-E2F1-HIF-1α axis.

RBM38 Is a Direct Transcriptional Target of E2F1 that Limits E2F1-Induced Proliferation

The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation in higher eukaryotes and is a critical downstream target of the tumor suppressor pRB. The pRB/E2F pathway is defective in most human tumors, resulting in deregulated E2F activity that induces uncontrolled cell proliferation, a hallmark of tumor cells. The RNA-binding protein RBM38, also named RNPC1, induces cell-cycle arrest in G(1), at least in part, via binding to and stabilizing the mRNA of the cyclin-dependent kinase inhibitor p21. RBM38 levels are altered in human cancer. Generally, RBM38 is overexpressed in various tumors; however, RBM38 mRNA levels are reduced in some breast tumors due to increased methylation of its promoter region. We show here that expression of RBM38 is regulated by E2F1. Specifically, RBM38 mRNA and protein levels are elevated upon activation of either exogenous E2F1 or endogenous E2Fs. Moreover, endogenous E2F1 binds the human RBM38 promoter and E2F1 knockdown reduces RBM38 levels. Our data raise the possibility that E2F1 together with E2F1-regulated RBM38 constitute a negative feedback loop that modulates E2F1 activity. In support of this, inhibition of RBM38 expression increases E2F1-mediated cell-cycle progression. Moreover, in human ovarian cancer, high correlation between expression of E2F1 and RBM38 is associated with increased survival. Overall, our data identify RBM38 as novel transcriptional target of E2F1 that restricts E2F1-induced proliferation. Furthermore, this negative feedback loop seems to restrict tumor aggressiveness, thereby promoting survival of patients with cancer.

Transcription factor E2F1 suppresses dendritic cell maturation. (167.3)

Abstract Recent studies report that transcription factor E2F1 regulates a number of genes in macrophage in response to immune stimulus, and involves in regulating T cell proliferation and differentiation. These studies indicate that E2F1 has impacts on the immune regulation additionally to its roles of regulating cell cycle and apoptosis. Our work shows that during the lipopolysaccharides (LPS) induced dendritic cell (DC) maturation, the level of E2F1 expression was transiently down regulated. The further study showed that knockdown of E2F1 induced both phenotypic and functional maturation in DCs. Conversely, DCs maintained in immature state when E2F1 was ectopically overexpressed. Moreover, E2F1 induced activation of multiple signaling pathways including PI3K, Erk1/2, NF-kappaB, which are generally thought to be involved in DC maturation. Overall, our study demonstrated that E2F1 served as a negative feedback factor to prevent overreaction in DC maturation.

Abstract 4208: Post-translational modification of E2F1 in malignant melanoma

Abstract Malignant melanoma has the highest fatality among all skin cancers. No current treatment significantly enhances survival once metastasis is established. Previous studies have shown that E2F1, a transcription factor, is over-expressed in melanoma cells. However, the mechanism through which E2F1 is elevated remains unknown. E2F1 is known to be involved in many important biological processes, including cell cycle progression, DNA damage, DNA repair, differentiation, development, autophagy and apoptosis. This underscores the importance of E2F1 in carcinogenesis. Normally in late S phase, E2F1 is recognized by the E3 ligase p45 SKP2 and undergoes ubiquitination and finally degradation in 26S proteasome. Our overall hypothesis is that E2F1 escapes proteasomal degradation in melanoma cells due to changes in post-translational modification. In this study we have used melanoma cells of increasing disease progression. Our results show that although the primary melanoma cells have high level of E2F1 message and protein, it is unstable. On the other hand the more metastatic melanoma cells have low levels of E2F1 which is however very stable. Consistent with this result, post-translational modification of E2F1 including the phosphorylation at Serine 337 and the acetylation at Lysine 117 increase with melanoma progression, which suggests that these post-translational modification sites might play an important role in stabilization of E2F1 and keep it functionally active in the more metastatic cell lines. E2F1 knockdown data also shows that E2F1 KD can inhibit cell proliferation in early stage melanoma cells but not in the more metastatic cells, which also suggest that inhibition of post-translational modifications and stability of E2F1 may be a more effective way to inhibit melanoma progression to more advanced stages. Abrogation of acetylation is expected to cause E2F1 protein instability. On the other hand, if acetylation is potentiated, the stabilized E2F1 will continue to promote progression through the cell cycle. We are currently testing whether the ectopic expression of the continuously acetylated E2F1 protein can rescue proliferation inhibition caused by E2F1 down-regulation, and drive metastasis and a mutant E2F1 with abrogated acetylation can block rescue of the proliferation inhibition in the E2F1 down-regulated melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4208. doi:1538-7445.AM2012-4208

Inhibitory role of E2F-1 in the regulation of tumor suppressor p53 during DNA damage response

Appropriate regulation of DNA damage response is pivotal for maintaining genome stability. p53 as well as E2F-1 plays a critical role during DNA damage response, however, the physiological significance of their interaction has been elusive. In the present study, we found that E2F-1 has an inhibitory effect on p53 during adriamycin (ADR)-mediated DNA damage response. Upon ADR exposure, p53 and E2F-1 were markedly induced at protein and mRNA levels in p53-procifient U2OS and HCT116 cells, and formed a stable complex as examined by co-immunoprecipitation experiments. Of note, chromatin immunoprecipitation (ChIP) experiments revealed that ADR-mediated induction coincides with the efficient recruitment of p53 and E2F-1 onto the promoters of p53-target genes, such as p21WAF1 and BAX. Subsequent RT-PCR and luciferase reporter assays demonstrated that E2F-1 strongly attenuates p53-dependent transactivation of p53-target genes. Importantly, siRNA-mediated knockdown of E2F-1 stimulated apoptosis in response to ADR, which was associated with an accelerated response of p21WAF1 and BAX. Collectively, our present findings suggest that E2F-1 participates in p53-mediated DNA damage response and might have a checkpoint function to limit overactive p53.

E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas.

Insulin-like growth factor 1 activates methionine adenosyltransferase 2A transcription by multiple pathways in human colon cancer cells

We have previously reported that the expression of MAT2A (methionine adenosyltransferase 2A) is increased in human colon cancer and in colon cancer cells treated with IGF-1 (insulin-like growth factor-1), which was required for its mitogenic effect. The aim of the present study was to elucidate the molecular mechanisms of IGF-1-mediated MAT2A induction. Nuclear run-on analysis confirmed that the increase in MAT2A expression lies at the transcriptional level. DNase I footprinting of the MAT2A promoter region revealed a similar protein-binding pattern in colon cancer and IGF-1-treated RKO cells. IGF-1 induced MAT2A promoter activity and increased nuclear protein binding to USF (upstream stimulatory factor)/c-Myb, YY1 (Yin and Yang 1), E2F, AP-1 (activator protein 1) and NF-κB (nuclear factor κB) consensus elements. IGF-1 increased the expression of c-Jun, FosB, MafG, p65, c-Myb, E2F-1 and YY1 at the pre-translational level. Knockdown of p65, MafG, c-Myb or E2F-1 lowered basal MAT2A expression and blunted the inductive effect of IGF-1 on MAT2A, whereas knockdown of YY1 increased basal MAT2A expression and had no effect on IGF-1-mediated MAT2A induction. Consistently, mutation of AP-1, NF-κB, E2F and USF/c-Myb elements individually blunted the IGF-1-mediated increase in MAT2A promoter activity, and combined mutations completely prevented the increase. In conclusion, IGF-1 activates MAT2A transcription by both known and novel pathways. YY1 represses MAT2A expression.

Resveratrol induces apoptosis in breast cancer cells by E2F1-mediated up-regulation of ASPP1

Resveratrol is a natural polyphenolic compound with cancer chemopreventive activity. However, our understanding of the molecular mechanism responsible for resveratrol-induced apoptosis is still very limited. Here, we used MCF-7 and MDA-MB231 breast cancer cells as a model to demonstrate that resveratrol induced the expression of ASPP1, a new member of the ASPP (apoptosis stimulation protein of p53) family, which plays an important role in the regulation of apoptosis. Moreover, resveratrol enhanced apoptosis of MCF-7/ASPP1 cells, accompanied by higher expression of bax and p21. In contrast, siRNA-mediated knockdown of ASPP1 inhibited apoptosis in MB231 cells. Furthermore, we found that higher levels of ASPP1 were associated with adenovirus-mediated overexpression of E2F1 while siRNA-mediated E2F1 knockdown led to down-regulation of ASPP1. In conclusion, our results demonstrate that overexpression of ASPP1 rendered MCF-7 and MDA-MB231 breast cancer cells more sensitive to resveratrol-mediated apoptosis via the E2F pathway, thus suggesting that ASPP1 may represent a novel therapeutic target for resveratrol in human breast cancer.

Dampened activity of E2F1–DP and Myb–MuvB transcription factors inDrosophilaendocycling cells

The endocycle is a variant cell cycle comprised of alternating gap (G) and DNA synthesis (S) phases (endoreplication) without mitosis (M), which results in DNA polyploidy and large cell size. Endocycles occur widely in nature, but much remains to be learned about the regulation of this modified cell cycle. Here, we compared gene expression profiles of mitotic cycling larval brain and disc cells with the endocycling cells of fat body and salivary gland of the Drosophila larva. The results indicated that many genes that are positively regulated by the heterodimeric E2F1-DP or Myb-MuvB complex transcription factors are expressed at lower levels in endocycling cells. Many of these target genes have functions in M phase, suggesting that dampened E2F1 and Myb activity promote endocycles. Many other E2F1 target genes that are required for DNA replication were also repressed in endocycling cells, an unexpected result given that these cells must duplicate up to thousands of genome copies during each S phase. For some EF2-regulated genes, the lower level of mRNA in endocycling cells resulted in lower protein concentration, whereas for other genes it did not, suggesting a contribution of post-transcriptional regulation. Both knockdown and overexpression of E2F1-DP and Myb-MuvB impaired endocycles, indicating that transcriptional activation and repression must be balanced. Our data suggest that dampened transcriptional activation by E2F1-DP and Myb-MuvB is important to repress mitosis and coordinate the endocycle transcriptional and protein stability oscillators.

E2F-1 Directly Regulates Thrombospondin 1 Expression

Thrombospondin 1 (TSP1) has been shown to play a critical role in inhibiting angiogenesis, resulting in inhibition of tumor growth and metastases. To figure out TSP1's regulators will lead to reveal its biological function mechanistically. In this study, we show that E2F-1 could activate the transcription of TSP1 by both promoter assays and Northern blot. Analysis of various TSP1 promoter mutant constructs showed that a sequence located −144/−137 up-stream of the transcriptional initiation site, related to the consensus E2F-responsive sequence, is necessary for the activation. In consistence with up-regulation of TSP-1 activity by over-expression of E2F-1, the knockdown of endogenous E2F-1 inhibited TSP-1 promoter activity significantly, implying that E2F-1 mediated regulation of TSP-1 is relevant in vivo. In addition, E2F-1 could also directly bind to the TSP1 promoter region covering −144/−137 region as revealed by ChIP assays. Furthermore, the E2F-1-induced activation of TSP1 gene transcription is suppressed by pRB1 in a dose-dependent manner. Taken together, the results demonstrate that TSP1 is a novel target for E2F1, which might imply that E2F-1 can affect angiogenesis by modulating TSP1 expression.

Mitogenic Sonic hedgehog signaling drives E2F1-dependent lipogenesis in progenitor cells and medulloblastoma

Deregulation of the Rb/E2F tumor suppressor complex and aberrantion of Sonic hedgehog (Shh) signaling are documented across the spectrum of human malignancies. Exaggerated de novo lipid synthesis is also found in certain highly proliferative, aggressive tumors. Here, we show that in Shh-driven medulloblastomas, Rb is inactivated and E2F1 is up-regulated, promoting lipogenesis. Extensive lipid accumulation and elevated levels of the lipogenic enzyme FASN mark those tumors. In primary cerebellar granule neuron precursors (CGNPs), proposed Shh-associated medulloblastoma cells-of-origin, Shh signaling triggers E2F1 and FASN expression while suppressing fatty acid oxidation (FAO), in a Smoothened-dependent manner. In the developing cerebellum, E2F1 and FASN co-localize in proliferating CGNPs. In vivo and in vitro, E2F1 is required for FASN expression and CGNP proliferation, and E2F1 knockdown impairs Shh-mediated FAO inhibition. Pharmacologic blockade of Rb inactivation and/or lipogenesis inhibits CGNP proliferation, drives medulloblastoma cell death, and extends survival of medulloblastoma-bearing animals in vivo. These findings identify a novel mechanism through which Shh signaling links cell cycle progression to lipid synthesis, through E2F1-dependent regulation of lipogenic enzymes. These findings pertinent to the etiology of tumor metabolism also underscore the key role of the Shh→E2F1→FASN axis in regulating de novo lipid synthesis in cancers, and as such its value as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Mdm2 inhibition induces apoptosis in p53 deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and Siva-1

Camptothecin (CPT) and Nutlin-3 caused apoptosis by increasing p53 protein and its activation in intestinal epithelial cells (IEC-6). We studied the effectiveness of these inducers on apoptosis in human colon cancer cells (Caco2) lacking p53 expression. CPT failed to activate caspase-3 and cause apoptosis in these cells. The absence of p53 expression, higher basal Bcl-xL and lower Bax proteins prevented CPT-induced apoptosis. However, the Mdm2 antagonist Nutlin-3 induced apoptosis in a dose dependent manner by activating caspases-9 and -3. Nutlin-3 prevented the activation of AKT via PTEN-mediated inhibition of the PI3K pathway. Nutlin-3 increased the phosphorylation of retinoblastoma protein causing E2F1 release leading to induction of Siva-1. Nutlin-3-mediated degradation of Mdm2 caused the accumulation of p73, which induced the expression of p53 up-regulated modulator of apoptosis (PUMA). E2F1 and p73 knockdown decreased the expression of Siva and PUMA, respectively and abolished Nutlin-3-induced caspase-3 activation. Cycloheximide (CHX) inhibited Nutlin-3-induced Siva, Noxa, and PUMA expression and inhibited apoptosis in IEC-6 and Caco2 cells. These results indicate that translation of mRNAs induced by Nutlin-3 is critical for apoptosis. In summary, apoptosis in Caco2 cells lacking functional p53 occurred following the disruption of Mdm2 binding with p73 and Rb leading to the expression of pro-apoptotic proteins, PUMA, Noxa, and Siva-1.

The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

The development of small-molecule activators of p53 is currently focused on malignancies containing a wild-type p53 genotype, which is present in most leukemias. JNJ-26854165 is one such p53-activating agent, but its mechanism of action remains to be elucidated. Here, we report the effects of JNJ-26854165 in acute leukemias. JNJ-26854165 treatment induced p53-mediated apoptosis in acute leukemia cells with wild-type p53, in which p53 rapidly drives transcription-independent apoptosis followed by activation of a transcription-dependent pathway. JNJ-26854165 accelerated the proteasome-mediated degradation of p21 and antagonized the transcriptional induction of p21 by p53. Interestingly, JNJ-26854165 induced S-phase delay and upregulated E2F1 expression in p53 mutant cells, resulting in apoptosis preferentially of S-phase cells. E2F1 knockdown blocked apoptosis induced by JNJ-26854165 in p53 mutant cells. Apoptotic activity of JNJ-26854165 against primary acute leukemia cells was maintained in leukemia/stroma cocultures, unlike doxorubicin, which has reduced cytrotoxicity in coculture systems. JNJ-26854165 synergizes with 1-β-arabinofuranosylcytosine or doxorubicin to induce p53-mediated apoptosis. Our data suggest that JNJ-26854165 may provide a novel therapeutic approach for the treatment of acute leukemias. The presence of p53-independent apoptotic activity in addition to p53-mediated apoptosis induction, if operational in vivo, may prevent the selection of p53 mutant subclones during therapy.