Downregulation Of E2F1 Research Articles

Abstract 1017: Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells

Abstract Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down-regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53-knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs. Citation Format: Paul B. Tchounwou, Sanjay Kumar. Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1017.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells’ susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.

E2F1 promotes proliferation and metastasis of clear cell renal cell carcinoma via activation of SREBP1-dependent fatty acid biosynthesis.

Enhanced synthesis or uptake of lipids contributes to rapid cancer cell proliferation and tumor progression. In recent years, cell cycle regulators have been shown to be involved in the control of lipid synthesis, in addition to their classical function of controlling the cell cycle. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is characterized by lipid-rich cytoplasmic deposition. However, the relationship between altered lipid metabolism and tumor progression in ccRCC is poorly understood. Here, we demonstrated that E2F transcription factor 1 (E2F1), in addition to its key role in regulating the cell cycle, induces extensive lipid accumulation and elevated levels of lipogenic enzymes in ccRCC cells by upregulating sterol regulatory element-binding protein 1 (SREBP1). E2F1 knockdown or SREBP1 suppression attenuated fatty acid (FA) de novo synthesis, cell proliferation and epithelial-mesenchymal transition (EMT) in ccRCC cells. Furthermore, overexpression of E2F1 promoted lipid storage, tumor growth and metastasis in a mouse xenograft model, whereas E2F1 downregulation or SREBP1 inhibition reversed these effects. In ccRCC patients, high levels of E2F1 and SREBP1 were associated with increased lipid accumulation and correlated with poor prognosis. Our results demonstrate that E2F1 can increase proliferation and metastasis through SREBP1-induced aberrant lipid metabolism, which is a novel critical signaling mechanism driving human ccRCC progression.

MicroRNA-939 Promotes the Vitality of Human Breast Cancer Cells via Inhibition of E2F1/P73 Signaling

We assessed miR-939’s role in breast cancer (BC) and its molecular mechanism. PCR was performed to detect miRNA levels. Correlations between miR-939 and patients’ pathological information were analyzed. After transfection of E2F1 plasmid, P73 plasmid, si-E2F1, si-P73, miR-939 mimic or si-miR-939, cell proliferation and apoptosis were measured. The miR-939 target gene was proved by a luciferase assay. Protein and mRNA levels of E2F1 and P73 were detected by immunoblotting and PCR, and corresponding proliferation or apoptosis were assessed. MiR-939 expression was significantly increased in BC and associated with TNM staging, Ki-67 enhancement, and shorter disease-free survival time. In BC clinical samples, E2F1 expression is negatively correlated with miR-939 expressions. Overexpressing miR-939 stimulated growth but suppressed cell apoptosis. Functional analysis indicated E2F1 is the target gene of miR-939, and overexpression of miR-939 significantly downregulated E2F1 and P73. Silencing of E2F1 or P73 significantly promoted MDA-MB-231 cell proliferation and inhibited apoptosis. Overexpression of E2F1 plasmid or P73 plasmid significantly inhibited MDA-MB-231 cell proliferation but induced apoptosis. Transfection of P73 or E2F1 plasmid abolished miR-939’s effects on proliferation and apoptosis. miR-939 promotes breast cancer progression by downregulation of E2F1 to inhibit P73 pathway, thereby promoting proliferation and inhibiting apoptosis.

E2F1 Maintains Gastric Cancer Stemness Properties by Regulating Stemness-Associated Genes.

Purpose To determine the regulatory role of E2F1 in maintaining gastric cancer stemness properties and the clinical significance of E2F1 in gastric cancer. Materials and Methods We conducted a tumor spheroid formation assay to enrich gastric cancer stem-like cells. The protein and mRNA expression levels of genes were measured using Western Blot and qRT-PCR. Lentivirus-mediated overexpression and downregulation of E2F1 were performed to evaluate the effect of E2F1 on the stemness properties of gastric cancer cells. The effect of E2F1 on gastric cancer cell sensitivity of 5-Fu was evaluated using cell viability assay and TdT-mediated dUTP Nick-End Labeling staining. We also analyzed the association between E2F1 expression and clinical characteristics in gastric cancer patients. The KM plotter database was used to analyze the relationship between E2F1 and overall survival in GC patients. Results We found that E2F1 expression was significantly higher in gastric cancer tissues than in the paired adjacent normal tissues (p < 0.05) and was positively correlated with tumor size (p < 0.05), T stage (p < 0.05), and differentiation degree (p < 0.05). KM plotter database demonstrated a close association between higher E2F1 expression level and worse overall survival of gastric cancer patients (p < 0.05). In vitro assay illustrated that E2F1 could regulate the expression of stemness-associated genes, such as BMI1, OCT4, Nanog, and CD44, and maintain the tumor spheroid formation ability of gastric cancer cells. E2F1 enhanced 5-Fu resistance in gastric cancer cells, and the E2F1 expression level was correlated with the prognosis of gastric cancer patients receiving 5-Fu therapy. The expression levels of stemness-associated genes were also significantly higher in gastric cancer tissues than the paired adjacent normal tissues (p < 0.05). A positive correlation was observed between E2F1 and BMI1 (r = 0.422, p < 0.05), CD44 (r = 0.634, p < 0.05), OCT4 (r = 0.456, p < 0.05), and Nanog (r = 0.337, p < 0.05) in gastric cancer tissues. The co-overexpression of E2F1 and stemness-associated genes was associated with worse overall survival. Conclusion E2F1 plays a significant role in gastric cancer progression by maintaining gastric cancer stemness properties through the regulation of stemness-associated genes. The close association between E2F1 and poor prognosis of patients suggests that E2F1 could serve as a prognostic biomarker and a therapeutic target in gastric cancer patients.

Abstract MP17: Upregulating E2F1 Mitigates Senescence-Associated Phenotypes in Cultured Primary Endothelial Cells

Vascular contributions to cognitive impairment and dementia (VCID) includes multiple disorders that are identified by cognitive deficits secondary to cerebrovascular pathology. The risk of VCID is higher in people after the age of 70, and, currently, there is no effective treatment. Vascular endothelial cells (VEC) are critical components of the brain vasculature and neurovascular unit and their health is vital to the capacity of the brain vasculature to respond to stressors. However, aged VEC may enter an irreversible replicative-arrest state (senescence), which has been associated with dementia. E2F transcription factor 1 (E2F1) regulates cell cycle progression and DNA damage repair. Importantly, E2F1 deficiency is associated with cell senescence. We hypothesized that E2F1 downregulation contributes to senescence in the cerebral endothelium during aging. We used cultured primary VEC from young (4-months old, mo) and aged (18-mo) male and female mice for RNA sequencing, plasmid-based gene delivery, high-resolution microscopy, and (4-, 12-, and 18-mo) mice of the bilateral carotid artery stenosis (BCAS) model, which produces chronic cerebral hypoperfusion and recapitulates some of the features seen in patients with VCID. We found that overexpression of E2F1 reduced the levels of senescence-associated phenotypes in cultured VEC from young mice that were exposed to oxygen and glucose deprivation (p&lt;0.001), which induces endothelial senescence. Our RNA seq data showed that the expression of E2f1 was reduced (~40%) in cultured primary VEC from aged mouse brains compared with young cells (p&lt;0.001). E2F1 levels were reduced in the brains of aged mice. Interestingly, we found sex differences in E2F1 levels, with less protein levels (~30%) in males vs females (p&lt;0.05), independently of age. Also, aged BCAS mice (1 month after surgery) had more severe senescence phenotypes, reduced cerebral blood flow, and worse memory deficits compared with control mice (p&lt;0.05). The effect of BCAS was more prominent in aged mice compared with younger (4- and 12-mo) mice. In conclusion , our study identifies E2F1 as a potential regulator of endothelial senescence in mice and highlights the contribution of aging as an important factor in losing endothelial resilience.

MTORC2 deploys the mRNA binding protein IGF2BP1 to regulate c-MYC expression and promote cell survival

mTORC2 promotes cell survival by phosphorylating AKT and enhancing its activity. Inactivation of mTORC2 reduces viability through down-regulation of E2F1 caused by up-regulation of c-MYC. An additional target of mTORC2 is IGF2BP1, an oncofetal RNA binding protein expressed de novo in a wide array of malignancies. IGF2BP1 enhances c-MYC expression by protecting the coding region instability sequence (CRD) of its mRNA from endonucleolytic cleavage. Here we show that repression of mTORC2 signalling and prevention of Ser181 phosphorylation of IGF2BP1 enhanced translation and destabilization of the endogenous c-myc mRNA as well as the mRNA of reporter transcripts carrying the CRD sequence in frame. The consequent increase in c-MYC protein was accompanied by the emergence of an apoptotic c-MYC overexpressing population. On the other hand, preventing phosphorylation of IGF2BP1 on Tyr396 by Src kinase caused the accumulation of translationally silent transcripts through sequestration by IGF2BP1 into cytoplasmic granules. The apoptotic effect of mTORC2 signalling deprivation was augmented when preceded by inhibition of IGF2BP1 phosphorylation by the Src kinase in concert with further increase of c-MYC levels because of enhanced translation of the previously stored mRNA only in the presence of IGF2BP1. Furthermore, the combined administration of mTORC2 and Src inhibitors exhibited synergism in delaying xenograft growth in female NOD.CB17-Prkdcscid/J mice. The above in vitro and in vivo findings may be applied for the induction of targeted apoptosis of cells expressing de novo the oncofetal protein IGF2BP1, a feature of aggressive malignancies resulting in a more focused anticancer therapeutic approach.

Metformin Alleviated Bone Loss in Ovariectomized Mice Through Inhibition of Autophagy of Osteoclast Precursors Mediated by E2F1

Background: Studies about autophagic regulation during osteoclastogenesis under estrogen deficiency condition are very restricted. Evidence from clinical practice defines a bone beneficial effect of metformin, but the potential mechanisms are still vague. Our study is designed to conduct an investigation whether metformin performs its protective role against OVX-induced bone loss via the autophagy machinery. Methods: Bone resorption markers in the serum from postmenopausal women and ovariectomized mice with or without metformin were measured using an ELISA. Ovariectomized mice were treatment with metformin, and osteoclast precursors undergone administration treatment in vitro and vivo were collected to examine the differentiation potential and autophagy level. The mechanism of involving the study was investigated by infection with lentivirus-mediated BNIP3 or E2F1 overexpression. Findings: We observed a marked inhibition of autophagosomes synthesis and osteoclasts formation and activity. Treatment with RAPA, an autophagy activator, abrogated the metformin-mediated autophagy downregulation and inhibition of osteoclastogenesis. Additionally, overexpression of E2F1 demonstrated that reduction of OVX-upregulated autophagy mediated by metformin was E2F1 dependent. Mechanistically, metformin-mediated downregulation of E2F1 in ovariectomized mice could downregulated the BNIP3 level, which subsequently perturbed the blinding of BECN1 to BCL2. Furthermore, the disconnection between BECN1 and BCL2 was revealed by BNIP3 overexpression. Interpretation: The study demonstrates the mechanism of the protective effect of metformin in OVX-induced bone loss, which could be, at least in part, ascribed to its role in downregulating autophagy during osteoclastogenesis via E2F1 dependent. Furthermore, the study also provides the science support for the research and development of new drugs with bone-targeting property. Funding Information: National Science Foundation of China (No. 82002313，No. 82072444), the National Key Research & Development Program of China (No.2018YFC2001502, 2018YFB1105705); Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration (No.2020kqhm008), the Health Commission of Hubei Province (No. WJ2019Z009), and the Wuhan Union Hospital “Pharmaceutical Technology nursing” special fund (No. 2019xhyn021). Declaration of Interests: All authors declare that they have no competing financial interests. Ethics Approval Statement: This study was carried out in full compliance with per the Declaration of Helsinki, and authorized by Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, with the approval number [2018] S431. All samples were attained with signed informed consent. All animal studies were performed following protocols approved by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology and were carried out as regulated by the Tongji Medical College Animal Care and Use Committee.

Transcription Factor E2F1 Aggravates Neurological Injury in Ischemic Stroke via microRNA-122-Targeted Sprouty2.

BackgroundIt has been documented that microRNAs (miRs) assume a pivotal role in the development of ischemic stroke (IS). However, it remains poorly identified about the role of miR-122 in IS. Herein, this study was intended to explore the mechanism of E2F1-orchestrated miR-122 in IS.Patients and MethodsE2F1, miR-122, and SPRY2 expression in serum from patients with IS and oxygen-glucose deprivation (OGD)-treated N2a cells was detected by RT-qPCR. After gain- and loss-of-function approaches in OGD-induced N2a cells, GAFP staining, flow cytometry, and Western blot analysis were adopted to assess neuronal viability, cell cycle and apoptosis, and expression of apoptosis- and autophagy-related proteins, respectively. Meanwhile, mice with IS were induced, in which E2F1, miR-122, and SPRY2 were overexpressed, followed by evaluation of neurological deficit and cerebral infarction area. The MAPK pathway activity in tissues of mice and cells was determined.ResultsmiR-122 was down-regulated, and E2F1 and SPRY2 were up-regulated in IS patients and OGD-induced N2a cells. E2F1 inhibited miR-122 transcription, while miR-122 targeted SPRY2. Overexpression (OE) of miR-122 or down-regulation of E2F1 or SPRY2 increased viability, but decreased apoptosis, cell cycle arrest, and autophagy in OGD-induced N2a cells. In IS mice, the neurological deficit score and cerebral infarction area were elevated, which was aggravated by up-regulating E2F1 or SPRY2 but attenuated by overexpressing miR-122. E2F1/miR-122/SPRY2 axis mediated the MAPK pathway in vivo and in vitro.ConclusionCollectively, E2F1 reduced miR-122 transcription to up-regulate SPRY2, which inactivated MAPK pathway and promoted neurological deficit in IS.

MicroRNA‑34a expression affects breast cancer invasion invitro and patient survival via downregulation of E2F1 and E2F3 expression.

Breast cancer is the most common cancer type and the leading cause of cancer‑associated mortality in women across the majority of countries. In general, the incidence of breast cancer has been decreasing in developed countries over the previous 20 years, while it has increased in the other areas, such as the Asian‑Pacific region. MicroRNA‑34a (miR‑34a) targets stem cell‑associated transcription factors E2F1/E2F3, and may have clinical relevance in breast cancer. The present study aimed to investigate the association between miR‑34a/E2F1/E2F3 and patient survival in breast cancer, as well as the underlying molecular mechanism of miR‑34a in suppressing factors associated with tumor aggressiveness invitro. Kaplan‑Meier survival curves were constructed and a meta‑analysis was performed to analyze the association of miR‑34a, E2F1 and E2F3 expression and overall survival in breast cancer, and the differential expression levels of E2F1 and E2F3 between breast cancer and normal breast tissues was assessed using publicly accessed datasets. Then 2D and 3D experiments on cell cultures were performed invitro on both T‑47D and MDA‑MB‑231 cells to investigate the cancer biology of miR‑34a and its effect on E2F1 and E2F3 expression using reverse transcription‑quantitative PCR. Then, caspase‑3 (CASP3) activity was measured using a CaspACE™ assay system. E2F1 and E2F3 expression levels were upregulated in breast cancer, compared with normal breast tissues. Both high miR‑34a, and low E2F1 and E2F3 mRNA levels were positively associated with longer survival times in patients with breast cancer. The invitro 2D and 3D cell experiments revealed that overexpression of miR‑34a significantly downregulated the expression of E2F1 and E2F3, and increased CASP3 activity in both T‑47D and MDA‑MB‑231 cells, and that miR‑34a treatment inhibited tumor cell proliferation, migration and invasiveness, as well as 3D spheroid formation. Thus, miR‑34a influences the aggressiveness of breast cancer and patient survival, and is a potential therapeutic tool in the clinical management of breast cancer.

Dual functionalized brain-targeting nanoinhibitors restrain temozolomide-resistant glioma via attenuating EGFR and MET signaling pathways

Activation of receptor tyrosine kinase (RTK) protein is frequently observed in malignant progression of gliomas. In this study, the crosstalk activation of epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) signaling pathways is demonstrated to contribute to temozolomide (TMZ) resistance, resulting in an unfavorable prognosis for patients with glioblastoma. To simultaneously mitigate EGFR and MET activation, a dual functionalized brain-targeting nanoinhibitor, BIP-MPC-NP, is developed by conjugating Inherbin3 and cMBP on the surface of NHS-PEG8-Mal modified MPC-nanoparticles. In the presence of BIP-MPC-NP, DNA damage repair is attenuated and TMZ sensitivity is enhanced via the down-regulation of E2F1 mediated by TTP in TMZ resistant glioma. In vivo magnetic resonance imaging (MRI) shows a significant repression in tumor growth and a prolonged survival of mice after injection of the BIP-MPC-NP and TMZ. These results demonstrate the promise of this nanoinhibitor as a feasible strategy overcoming TMZ resistance in glioma.

&lt;p&gt;Identification of the Different Roles and Potential Mechanisms of T Isoforms in the Tumor Recurrence and Cell Cycle of Chordomas&lt;/p&gt;

PurposeThe roles of T (brachyury) isoforms in chordomas remain unclear. This study aimed to investigate the different roles and mechanisms of them in chordomas.Patients and methodsThe expression of T isoforms mRNAs in 57 chordomas was assessed, and a prognosis analysis was conducted. Cell apoptosis, proliferation and cell cycle assays were performed after specific T isoform mRNA knockdown. Whole-transcriptome sequencing, Gene Set Enrichment Analysis, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and competing endogenous RNA (ceRNA) analysis were conducted.ResultsAs revealed in this study, the T-long isoform was a significant risk factor (hazard ratio [HR], 1.09; P=0.018) and the T-short isoform was a protective factor (HR, 0.24; P=0.012) associated with tumor recurrence. After T-long isoform knockdown, the cell cycle was arrested at G0/G1 phase and cell proliferation was significantly inhibited. A bioinformatic analysis revealed that the upregulation of H19, P21 and GADD45B; downregulation of SKP2 and CDK2; and accompanying changes in the P53 signaling pathway consistently contributed to G0/G1 arrest. After T-short isoform knockdown, the cell cycle was arrested at G2/M phase and cell apoptosis tended to increase slightly (P=0.067). The upregulation of YWHAZ and downregulation of E2F1 and its target genes might contribute to cell cycle arrest in G2/M phase and apoptosis. In addition, the ceRNA network, consisting of long noncoding RNAs, mRNAs and microRNAs, was established.ConclusionThe T-long isoform was a risk factor and the T-short isoform was a protective factor for chordoma recurrence. In addition, the cell cycle was the main target of T isoforms knockdown, and the changes in the downstream transcriptome may contribute to the different effects of specific T isoform knockdown on the changes in the cell cycle distributions and apoptosis and proliferation of chordoma cells.

Oxidative Stress Down-Regulates MiR-20b-5p, MiR-106a-5p and E2F1 Expression to Suppress the G1/S Transition of the Cell Cycle in Multipotent Stromal Cells.

Oxidative stress has been linked to senescence and tumorigenesis via modulation of the cell cycle. Using a hydrogen peroxide (H2O2)-induced oxidative stress-induced premature senescence (OSIPS) model previously reported by our group, this study aimed to investigate the effects of oxidative stress on microRNA (miRNA) expression in relation to the G1-to-S-phase (G1/S) transition of the cell cycle and cell proliferation. On global miRNA analysis of the OSIPS cells, twelve significantly up- or down-regulated miRNAs were identified, the target genes of which are frequently associated with cancers. Four down-regulated miR-17 family miRNAs are predicted to target key pro- and anti-proliferative proteins of the p21/cyclin D-dependent kinase (CDK)/E2F1 pathway to modulate G1/S transition. Two miR-17 miRNAs, miR-20-5p and miR-106-5p, were confirmed to be rapidly and stably down-regulated under oxidative stress. While H2O2 treatment hampered G1/S transition and suppressed DNA synthesis, miR-20b-5p/miR-106a-5p over-expression rescued cells from growth arrest in promoting G1/S transition and DNA synthesis. Direct miR-20b-5p/miR-106a-5p regulation of p21, CCND1 and E2F1 was demonstrated by an inverse expression relationship in miRNA mimic-transfected cells. However, under oxidative stress, E2F1 expression was down-regulated, consistent with hampered G1/S transition and suppressed DNA synthesis and cell proliferation. To explain the observed E2F1 down-regulation under oxidative stress, a scheme is proposed which includes miR-20b-5p/miR-106a-5p-dependent regulation, miRNA-E2F1 autoregulatory feedback and E2F1 response to repair oxidative stress-induced DNA damages. The oxidative stress-modulated expression of miR-17 miRNAs and E2F1 may be used to develop strategies to retard or reverse MSC senescence in culture, or senescence in general.

PTEN Inhibition Protects Against Experimental Intracerebral Hemorrhage-Induced Brain Injury Through PTEN/E2F1/β-Catenin Pathway.

Intracerebral hemorrhage (ICH) is a subtype of stroke with highest mortality and morbidity. We have previously demonstrated that dipotassium bisperoxo (picolinato) oxovanadate (V), (bpV[pic]) inhibits phosphatase and tensin homolog (PTEN) and activates extracellular signal-regulated kinase (ERK)1/2. In this study, we examined the effect of bpV[pic] in the rat ICH model in vivo and the hemin-induced injury model in rat cortical cultures. The rat model of ICH was created by injecting autologous blood into the striatum, and bpV[pic] was intraperitoneally injected. The effects of bpV[pic] were evaluated by neurological tests, Fluoro-Jade C (FJC) staining, and Nissl staining. We demonstrate that bpV[pic] attenuates ICH-induced brain injury in vivo and hemin-induced neuron injury in vitro. The expression of E2F1 was increased, but β-catenin expression was decreased after ICH, and the altered expressions of E2F1 and β-catenin after ICH were blocked by bpV[pic] treatment. Our results further show that bpV[pic] increases β-catenin expression through downregulating E2F1 in cortical neurons and prevents hemin-induced neuronal damage through E2F1 downregulation and subsequent upregulation of β-catenin. By testing the effect of PTEN-siRNA, PTEN cDNA, or combined use of ERK1/2 inhibitor and bpV[pic] in cultured cortical neurons after hemin-induced injury, we provide evidence suggesting that PTEN inhibition by bpV[pic] confers neuroprotection through E2F1 and β-catenin pathway, but the neuroprotective role of ERK1/2 activation by bpV[pic] cannot be excluded.

Akt Inhibition Differently Controls PRC2 Components and Synergizes with Dual EZH2/1 Inhibitor in the Treatment of Multiple Myeloma

PI3K/Akt pathway is constitutively activated in multiple myeloma (MM). A plethora of studies extensively investigated Akt inhibitors, alone or in combination; however, the outcomes in hematological malignancies were largely unsatisfactory, emphasizing the need for critical preclinical evaluations. Polycomb repressive complex 2 (PRC2) components, EZH2 and its related homolog EZH1, induce H3K27me3 to silence the transcription of target genes. Recent studies ensured that EZH2 inhibition alone is not sufficient to completely disrupt the oncogenic functions of PRC2. With the importance of PRC2 as a therapeutic target in MM, we aimed to investigate the mechanisms by which Akt inhibition may impact PRC2 function, and test whether targeting both EZH2 and EZH1 together with Akt inhibition is a promising treatment strategy for MM. We herein evaluated the cytotoxic effect of TAS-117, a potent and selective non-competitive Akt inhibitor, against different MM cell lines and found that responsive cell lines tended to have significant levels of activated Akt, coupled with low/deleted PTEN. Then, we examined signaling-epigenetic crosstalk on EZH2 level. TAS-117 significantly down-regulated EZH2 mRNA and protein in dose- and time-dependent manners, while H3K27me3 levels were rather maintained or elevated, suggesting compensation by EZH1. As EZH2 is a direct target for E2F1, we focused on Rb-E2F pathway as a regulatory mechanism for EZH2. TAS-117 induced marked down-regulation of E2F1 and E2F2. Moreover, TAS-117 induced the up-regulation of CDKN1B, in addition to the inactivation of cyclins and cyclin dependent kinases, hence, hypo-phosphorylated Rb, thereby stabilizing Rb-E2F1 complex and diminishing free E2F1 available for binding to its target genes, including EZH2 promoter. This prompted us to examine the impact of TAS-117 combination with either dual EZH2/1 inhibitor, UNC1999, or selective EZH2 inhibitor, GSK126. In agreement, UNC1999, but not GSK126, synergistically enhanced TAS-117-induced cytotoxicity, confirmed by combination index calculation, and provoked MM cell apoptosis. As we observed an increase in H3K27me3 levels after TAS-117 treatment, we hypothesized that EZH1 function was augmented. Consistently, we found that EZH1 was markedly up-regulated after TAS-117 treatment in dose- and time-dependent manners. Importantly, EZH1 knockdown significantly enhanced the sensitivity of myeloma cells to TAS-117-induced cytotoxicity. To clarify the molecular mechanisms underlying EZH1 up-regulation, we performed RNA-seq followed by KEGG pathway analysis for up-regulated genes in TAS-117-treated group. We focused on FOXO pathway enrichment as it is a crucial target in MM treatment using Akt inhibitors. We then focused on FOXO3 as it was the main FOXO family gene expressed in MM cells according to our RNA-seq data. We examined the nuclear localization of FOXO3 following TAS-117 treatment. We found that TAS-117 significantly enhanced the nuclear accumulation of FOXO3, as depicted by both the immunostaining images and the digital calculations of the nuclear subset of FOXO3. Murine Ezh1 promoter was shown to be bound by Foxo transcription factors (TFs) in neuronal progenitors, T-regulatory cells, CD8+ cells, and pre-B cells. More than 80% of FOXO3-binding sites share the common binding motif, GTAAACAA, which was found both in human EZH1 (+48 from the TSS) and mouse Ezh1 (+77 from the TSS) promoter regions. So, we hypothesized that FOXO3 may be a regulatory partner for human EZH1 gene in myeloma cells in response to TAS-117 treatment. To this end, we performed ChIP-qPCR analysis for TAS-117-treated and -untreated cells. TAS-117 promoted the binding of FOXO3 to EZH1 promoter, in addition to one of the canonical FOXO3 targets, BIM promoter. To further confirm our results, we expressed shRNA against FOXO3 (shFOXO3) in MM cells which, interestingly, induced the down-regulation of EZH1 mRNA. In conclusion, the present results defined novel signaling-epigenetic crosstalk between PI3K/Akt pathway and PRC2 components, EZH2 and EZH1, and demonstrated that Akt inhibition can differently modulates EZH2 and EZH1 levels via Akt downstream effectors, E2F1 and FOXO3, respectively. Therefore, targeting both EZH2 and EZH1 in addition to Akt inhibition may be a promising rationale to eradicate MM, leading to significant advances in treatment. Disclosures No relevant conflicts of interest to declare.

MiR-331-3p Inhibition of the Hepatocellular Carcinoma (HCC) Bel-7402 Cell Line by Down-Regulation of E2F1

MicroRNAs play an important role in cell proliferation and migration in hepatocellular carcinoma (HCC). In this study, the expression of MiR-331-3p in the liver cancer tissues of 116 patients, and in one normal (HL-7702) and five HCC cell lines (HepG2, SMMC-7721, Bel-7402, QGY-7701 and QGY-7703) was determined by qRT-PCR. Results indicated that MiR-331-3p was significantly repressed in HCC patients and in HCC cell lines, especially in the Bel-7402 cells when compared to normal liver tissue and the normal hepatocyte line, HL-7702. Therefore, artificial MiR-331-3p mimic computationally targeting E2F1 and anti-sense MiR-331-3p inhibitor were designed and used to transfect the Bel-7402 lines. Stable expression and significant inhibition of MiR-331-3p, with clear changes in cell morphology, proliferation and motility, were detected in Bel-7402 cells transfected with MiR-331-3p mimic or inhibitor, respectively. As E2F1 was computationally recognized as the target of MiR-331-3p, the expression of E2F1 in transfected Bel-7402 cells was analyzed by qRT-PCR and Western blotting. Results showed a strong inhibition of E2F1 by MiR-331-3p and further promotion of E2F1 by MiR-331-3p inhibitor in transfected Bel-7402 cells. Flow cytometry analysis showed that the cell ratio was restored to S to G0/1 cell number. Inhibition of E2F1 by MiR-331-3p mimic promoted cell proliferation and migration in Bel-7402. These results demonstrated that MiR-331-3p down-regulated E2F1 to promote cell proliferation and migration in HCC.

A Derivate of Benzimidazole-Isoquinolinone Induces SKP2 Transcriptional Inhibition to Exert Anti-Tumor Activity in Glioblastoma Cells.

We have previously shown that compound-7g inhibits colorectal cancer cell proliferation and survival by inducing cell cycle arrest and PI3K/AKT/mTOR pathway blockage. However, whether it has the ability to exert antitumor activity in other cancer cells and what is the exact molecular mechanism for its antiproliferation effect remained to be determined. In the present study, compound-7g exhibited strong activity in suppressing proliferation and growth of glioblastoma cells. The inhibitor selectively downregulated F-box protein SKP2 expression and upregulated cell cycle inhibitor p27, and then resulted in G1 cell cycle arrest. Mechanism analysis revealed that compound-7g also provokes the down-regulation of E2F-1, which acts as a transcriptional factor of SKP2. Further results indicated that compound-7g induced an increase of LC3B-II and p62, which causes a suppression of fusion between autophagosome and lysosome. Moreover, compound-7g mediated autophagic flux blockage promoted accumulation of ubiquitinated proteins and then led to endoplasmic reticulum stress. Our study thus demonstrated that pharmacological inactivation of E2F-1-SKP2-p27 axis is a promising target for restricting cancer progression.

Glioma cell proliferation is inhibited by miR-342-3p, miR-377 / E2F1 signaling pathway.

Recent years, micoRNAs (miRNAs) have been reported to be critical regulators to influence tumor genesis or further progression by directly targeting downstream tumor related genes in glioma. However, there're still many underlying mechanisms related to miRNAs signaling pathway remain to be uncovered in glioma. In the present study, we found that miR-342-3p and miR-377 inhibited the glioma cell line proliferation and arrested the cell cycle at G1 phase. Inhibition of the function of miR-342-3p and miR-377 promoted the cell proliferation. miR-342-3p and miR-377 target the E2F1 3'UTR to repress its expression on both mRNA and protein level. Downregulation of E2F1 inhibited the cell proliferation and arrested the cell cycle. Overexpression of E2F1 blocked the proliferation repression caused by miR-342-3p or miR-377 in glioma cells. This study showed the function of miR-342-3p, miR-377/E2F1 axis in regulating glioma cells proliferation and provided the potential therapeutic target.

Tumor suppressive function of E2F-1 on PTEN-induced serrated colorectal carcinogenesis.

Many human cancers present Phosphatase and tensin homolog (PTEN) deficiency and between 20 and 30% of colorectal tumors show PTEN loss. The transcription factor, E2 promoter binding factor 1 (E2F-1), exhibits tumor promoter or suppressive functions depending on cellular type and tissue context, but its role in the progression and development of colorectal carcinogenesis was largely unknown. Here, using a tamoxifen-inducible PTEN knockout mouse model, we have demonstrated that loss of PTEN leads to the development of colorectal tumorigenesis through the serrated pathway. Next, we studied PTEN loss-driven colorectal lesions in the context of E2F-1 deficiency in vivo. Our results revealed that monoallelic and biallelic absence of E2F-1 led to an increased incidence and progression of serrated tumorigenesis induced by PTEN loss. Finally, we investigated the mechanisms by which double PTEN/E2F-1 deficiency leads to enhanced tumorigenesis. We found that colorectal tumors from PTEN/E2F-1 double knockout mice and the human colorectal carcinoma cell line HT29 with shRNA-mediated downregulation of PTEN and E2F-1 exhibit hyperactivation of the RAS-MAPK pathway, accumulation of DNA damage and resistance to apoptosis. To date, this is the first preclinical study evaluating the effect of genetic deletion of E2F-1 in colorectal malignancies driven by PTEN deficiency. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Valproic acid suppresses Warburg effect and tumor progression in neuroblastoma

Altered glucose metabolism is a hallmark for cancer, which is characterized by a unique metabolic phenotype known as Warburg effect or aerobic glycolysis. Emerging studies show that valproic acid (VPA), an established histone deacetylase inhibitor, possesses tumor suppressive properties. However, the effects of VPA on the regulation of Warburg effect in neuroblastoma (NB), the most common extracranial malignancy in childhood, still remain elusive. In this study, we show that VPA inhibits the aerobic glycolysis in NB cells by decreasing glucose uptake and reducing lactate and ATP production. Mechanistically, VPA suppresses aerobic glycolysis via reducing the levels of E2F transcription factor 1 (E2F1), resulting in repressed expression of glycolytic genes glucose-6-phosphate isomerase (GPI) and phosphoglycerate pinase 1 (PGK1). Rescue experiments show that VPA inhibits the aerobic glycolysis and NB progression through down-regulation of E2F1. These results demonstrate that VPA suppresses the Warburg effect and tumor progression, indicating a novel therapeutic strategy for NB.