Novel chemotherapeutic agent FX-9 activates NF-κB signaling and induces G1 phase arrest by activating CDKN1A in a human prostate cancer cell line.

BACKGROUND: The aminoisoquinoline FX-9 shows pro-apoptotic and antimitotic effects against lymphoblastic leukemia cells and prostate adenocarcinoma cells. In contrast, decreased cytotoxic effects against non-neoplastic blood cells, chondrocytes, and fibroblasts were observed. However, the actual FX-9 molecular mode of action is currently not fully understood. METHODS: In this study, microarray gene expression analysis comparing FX-9 exposed and unexposed prostate cancer cells (PC-3 representing castration-resistant prostate cancer), followed by pathway analysis and gene annotation to functional processes were performed. Immunocytochemistry staining was performed with selected targets. RESULTS: Expression analysis revealed 0.83% of 21,448 differential expressed genes (DEGs) after 6-h exposure of FX-9 and 0.68% DEGs after 12-h exposure thereof. Functional annotation showed that FX-9 primarily caused an activation of inflammatory response by non-canonical nuclear factor-kappa B (NF-κB) signaling. The 6-h samples showed activation of the cell cycle inhibitor CDKN1A which might be involved in the secondary response in 12-h samples. This secondary response predominantly consisted of cell cycle-related changes, with further activation of CDKN1A and inhibition of the transcription factor E2F1, including downstream target genes, resulting in G1-phase arrest. Matching our previous observations on cellular level senescence signaling pathways were also found enriched. To verify these results immunocytochemical staining of p21 Waf1/Cip1 (CDKN1A), E2F1 (E2F1), PAI-1 (SERPNE1), and NFkB2/NFkB p 100 (NFKB2) was performed. Increased expression of p21 Waf1/Cip1 and NFkB2/NFkB p 100 after 24-h exposure to FX-9 was shown. E2F1 and PAI-1 showed no increased expression. CONCLUSIONS: FX-9 induced G1-phase arrest of PC-3 cells through activation of the cell cycle inhibitor CDKN1A, which was initiated by an inflammatory response of noncanonical NF-κB signaling.

KDM5D inhibits the transcriptional activation of FKBP4 by suppressing the expression of E2F1 in colorectal cancer in males.

Colorectal cancer (CRC) remains the most frequently diagnosed malignancy and also a major contributor to cancer-related death throughout the world. Here, we first revealed the role of histone lysine-specific demethylase 5D (KDM5D) in CRC in males. KDM5D expression in tumor and adjacent tissues of male CRC patients was investigated using immunohistochemistry and RT-qPCR, and the correlation between its expression and patients' prognosis was analyzed. Downregulation of KDM5D in CRC patients was associated with poor prognoses. Overexpression of KDM5D significantly inhibited the growth and metastasis of CRC in vitro and in vivo. The downstream mechanism of KDM5D in CRC was investigated using bioinformatics analysis, and the regulatory relationship was confirmed by ChIP-qPCR and luciferase reporter assays. KDM5D suppressed E2F1 expression by mediating H3K4me3 demethylation. E2F1, highly expressed in CRC, promoted the expression of FKBP4 at the transcriptional level by binding to the FKBP4 promoter. Finally, rescue experiments revealed that overexpression of FKBP4 significantly reversed the inhibitory effect of KDM5D on CRC growth and metastasis. Collectively, KDM5D exerted an anti-tumor and anti-metastatic in CRC through demethylation in E2F1 and suppression of FKBP4 transcription, which might represent a novel target in CRC treatment in male.

E2F1-mediated repression of WNT5A expression promotes brain metastasis dependent on the ERK1/2 pathway in EGFR-mutant non-small cell lung cancer.

Brain metastasis (BM) is associated with poor prognosis in patients with advanced non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutation reportedly enhances the development of BM. However, the exact mechanism of how EGFR-mutant NSCLC contributes to BM remains unknown. Herein, we found the protein WNT5A, was significantly downregulated in BM tissues and EGFR-mutant samples. In addition, the overexpression of WNT5A inhibited the growth, migration, and invasion of EGFR-mutant cells in vitro and retarded tumor growth and metastasis in vivo compared with the EGFR wide-type cells. We demonstrated a molecular mechanism whereby WNT5A be negatively regulated by transcription factor E2F1, and ERK1/2 inhibitor (U0126) suppressed E2F1's regulation of WNT5A expression in EGFR-mutant cells. Furthermore, WNT5A inhibited ß-catenin activity and the transcriptional levels of its downstream genes in cancer progression. Our research revealed the role of WNT5A in NSCLC BM with EGFR mutation, and proved that E2F1-mediated repression of WNT5A was dependent on the ERK1/2 pathway, supporting the notion that targeting the ERK1/2-E2F1-WNT5A pathway could be an effective strategy for treating BM in EGFR-mutant NSCLC.

CAMK2N1 suppresses hepatoma growth through inhibiting E2F1-mediated cell-cycle signaling.

Human kinome/phosphatome screen identified CAMK2N1 genes suppressing the development of human hepatocellular carcinoma (HCC). CAMK2N1 downregulation was found in 47% HCCs and associated with poor prognosis. The downregulation was mainly attributed to its genome deletion (28.4%) and DNA hypermethylation of its promoter (12.5%). Silencing and ectopic expression of CAMK2N1 respectively enhanced and suppressed cell proliferation, colony formation, and xenograft tumor growth in nude mice. Comparative proteomics revealed that CAMK2N1 silencing transcriptionally deregulated the genes regulated by E2F1 (89 out of the 114 E2F-signaling targets, P = 8.8E-240). The promoter assays revealed that CAMK2N1 suppressed E2F1-mediated transcriptional activities. CAMK2N1 silencing induced cyclins D/E expression, whereas its ectopic expression induced P27/KIP1 expression and suppressed the cell cycle. CAMK2N1 was translocated from the nuclei to the cytoplasm when cell proliferation reached the stationary phase, where its functions as an endogenous inhibitor of CAMK2. In conclusion, CAMK2NA is a novel 1p36 tumor suppressor gene that inhibits E2F1 transcriptional activities and induces P27/KIP1 expression. CAMK2N1-CAMK2 signaling forms a mechanism that restricts the cell cycle progression. Its deregulation could lead to tumorigenesis and might serve as promising therapeutic targets.

DNMT3A inhibits E2F1-induced arterial marker expression and impairs angiogenesis in human umbilical artery endothelial cells.

Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.

p53 inhibits the osteogenic differentiation but does not induce senescence in human dental follicle cells.

Replicative senescence causes a reduced osteogenic differentiation potential of senescent dental follicle cells (DFCs). The transcription factor p53 is often involved in the induction of cellular senescence, but little is known about its role in DFCs. This study examined for the first time the role of p53 compared to its pro-proliferative antagonist E2F-1 in terms of osteogenic differentiation potential and induction of senescence. Protein expression of E2F-1 decreased during cell aging, while p53 was expressed constitutively. Gene silencing of E2F1 (E2F-1) inhibited the proliferation rate of DFCs and increased the induction of cellular senescence. The induction of cellular senescence is regulated independently of the gene expression of TP53 (p53), since its gene expression depends on the expression of E2F1. Moreover, gene silencing of TP53 induced E2F1 gene expression and increased cell proliferation, but did not affect the rate of induction of cellular senescence. TP53 knockdown further induced the alkaline phosphatase and mineralization in DFCs. However, the simultaneous silencing of TP53 and E2F1 did not inhibit the inductive effect of TP53 knockdown on osteogenic differentiation, indicating that this effect is independent of E2F-1. In summary, our results suggest that p53 inhibits osteogenic differentiation and cell proliferation in senescent DFCs, but is not significantly involved in senescence induction.

A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtype-specific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy.

A significant role of transcription factors E2F in inflammation and tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor from head and neck with characteristics in remarkable geographic and racial distribution worldwide, which has the important features of vigorous proliferation and inflammatory cells infiltration. By analyzing the expression profile data of NPC, we found that the E2F-related gene sets were highly enriched in NPC tissues. E2F transcription factor family is an important cycle regulator, which can promote the malignant proliferation and tumorigenesis. Here, we showed that E2Fs accelerated malignant phenotypes of NPC cells. RNA sequencing revealed that E2Fs can significantly up-regulate the inflammatory pathways in NPC cells. E2F1, as a transcription factor, can active the transcription activity of IL-6 promoter, and modulate macrophage function through a microenvironment manner. Thus, this study characterized a significant role of E2Fs in inflammation and tumorigenesis of NPC, which provided a promising anti-tumor target in NPC, since E2Fs are highly expressed and activated in NPC.

Differential network analysis depicts regulatory mechanisms for hepatocellular carcinoma from diverse backgrounds.

Aim: To elucidate the integrative combinational gene regulatory network landscape of hepatocellular carcinoma (HCC) molecular carcinogenesis from diverse background. Materials & methods: Modified gene regulatory network analysis was used to prioritize differentially regulated genes and links. Integrative comparisons using bioinformatics methods were applied to identify potential critical molecules and pathways in HCC with different backgrounds. Results: E2F1 with its surrounding regulatory links were identified to play different key roles in the HCC risk factor dysregulation mechanisms. Hsa-mir-19a was identified as showed different effects in the three HCC differential regulation networks, and showed vital regulatory role in HBV-related HCC. Conclusion: We describe in detail the regulatory networks involved in HCC with different backgrounds. E2F1 may serve as a universal target for HCC treatment.

An investigation of targeted inhibition of transcription factor activity with pyrrole imidazole polyamide (PA) in chronic myeloid leukemia (CML) blast crisis cells.

Tyrosine kinase inhibitor (TKI) therapy is the standard treatment for chronic phase (CP)-chronic myeloid leukemia (CML), yet patients in blast crisis (BC) phase of CML are unlikely to respond to TKI therapy. The transcription factor E2F1 is a down-stream target of the tyrosine kinase BCR-ABL1 and is up-regulated in TKI-resistant leukemia stem cells (LSC). Pyrrole imidazole polyamides (PA) are minor groove binders which can be programmed to target DNA sequences in a gene-selective manner. This manuscript describes such an approach with a PA designed to down-regulate E2F1 controlled gene expression by targeting a DNA sequence within 100 base pairs (bp) upstream of the E2F1 consensus sequence. Human BC-CML KCL22 cells were assessed after treatment with PA, TKI or their combination. Our PA inhibited BC-CML cell expansion based on cell density analysis compared to an untreated control after a 48-hour time-course of PA treatment. However, no evidence of cell cycle arrest was observed among BC-CML cells treated with PA, with respect to their no drug control counterparts. Thus, this work demonstrates that PAs are effective in inhibiting E2F1 TF activity which results in a temporal reduction in BC-CML cell number. We envisage that PAs could be used in the future to map genes under E2F1 control in CML LSCs.

Long non-coding RNA-HAGLR suppressed tumor growth of lung adenocarcinoma through epigenetically silencing E2F1.

Emerging evidence indicates that long noncoding RNAs (LncRNAs) are new players in gene regulation but their mechanisms of action are mainly undocumented. In this study, we investigated LncRNA alterations that contribute to lung cancer by analyzing published microarray data in Gene Expression Obminus (GEO) and The Cancer Genome Atlas RNA (TCGA) sequencing data. Here, we reported that HAGLR (also called HOXD-AS1) was frequently down-regulated in lung adenocarcinoma (LUAD) tissues, and decreased HAGLR expression was clinically associated with shorter survival of LUAD patients. Preclinical studies using multiple LUAD cells and in vivo mouse model indicated that HAGLR could attenuate LUAD cell growth in vitro and in vivo. Mechanistically, HAGLR could physically interact with DNMT1, and recruit DNMT1 on E2F1 promoter to increase local DNA methylation. Overall, our study demonstrated that HAGLR promoted LUAD progression by recruiting DNMT1 to modulate the promoter methylation and expression of E2F1, which expanded potential therapeutic strategies for LUAD treatment.

HBX-6, Standardized Cornus officinalis and Psoralea corylifolia L. Extracts, Suppresses Benign Prostate Hyperplasia by Attenuating E2F1 Activation.

BACKGROUND: The aim of this study was to simplify and identify the contents of the herbal formula, HBX-5. This study was carried out to evaluate the therapeutic effects of HBX-6 in a mouse model of benign prostatic hyperplasia (BPH). Based on in vitro, we selected a candidate, reconstituted an experimental agent and investigated the effects on testosterone-induced BPH rats. Cell viability was determined by MTT assay in RWPE-1 and WPMY-1 cells. The expression of androgen receptor (AR) was measured in dihydrotestosterone-stimulated RWPE-1 and WPMY-1 cells. BPH was induced in mice by a subcutaneous injection of testosterone propionate for four weeks. Animals were divided into six groups: Group 1, control mice; Group 2, mice with BPH; Group 3, mice with BPH treated with finasteride; Group 4, mice with BPH treated with 200 mg/kg HBX-5; Group 5, mice with BPH treated with 100 mg/kg HBX-6; and Group 6, mice with BPH treated with 200 mg/kg HBX-6. Changes in prostate weight were measured after treatments, and the thickness of the epithelium was evaluated. The expression levels of proteins associated with prostatic cell proliferation and cell cycle-related proteins were determined. Based on previous reports and in vitro results, we selected Cornus officinalis and Psoralea corylifolia among HBX-5 components and reconstituted the experimental agent, and named it HBX-6. The result represented a new herbal formula, HBX-6 that suppressed the pathological alterations in BPH and showed a marked reduction in proliferation-related protein expression compared to mice with BPH. Our results indicate that HBX-6 has a better therapeutic effect in the BPH murine model than those of HBX-5 and finasteride, suggesting the role of HBX-6 as a new BPH remedial agent.

MicroRNA-26a inhibits multiple myeloma cell growth by suppressing cyclin-dependent kinase 6 expression.

MicroRNA-26a (miR-26a) has been reported to be involved in the tumorigenesis of several tumors, but its biological function and molecular mechanism in multiple myeloma (MM) are still unknown. In this study, we found that overexpression of miR-26a obviously inhibited MM cell growth, and delayed tumor growth in xenografts. Further studies showed that overexpression of miR-26a induced cell cycle arrest at G0/G1 phase in MM cells. MiR-26a mimic down-regulated the expression levels of CDK6 and E2F1, but up-regulated p53 and p21 expression. In contrast, overexpression of CDK6 decreased the effect of miR-26a mimic on MM cell survival. Moreover, miR-26a targeted CDK6 mRNA and thus suppressed CDK6 protein expression. Overexpression of miR-26a also enhanced the cytotoxic action of doxorubicin against MM. These results demonstrated that miR-26a was involved in the development of MM through regulating CDK6 signaling pathway, and indicated that miR-26a could be as a novel target for anti-tumor therapy in clinic as a single strategy or in combination with other anti-tumor drugs in MM.

Improved synthesis, resolution, absolute configuration determination and biological evaluation of HLM006474 enantiomers.

An improved green synthesis of the E2F inhibitor HLM0066474 is described, using solvent-free and microwave irradiation conditions. The two enantiomers are separated using semi-preparative separation on Chiralpak ID and their absolute configuration is determined by vibrational circular dichroism (VCD) analysis. Biological evaluation of both enantiomers on E2F1 transcriptional activity reveals that the (+)-R, but not the (-)-S enantiomer is biologically active in repressing E2F1 transcriptional activity.

Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress.

We have shown that hydrogen peroxide (H2O2) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H2O2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H2O2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H2O2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H2O2 on DHFR promoter activity. H2O2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H2O2. E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)-infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H2O2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.

The RASSF6 Tumor Suppressor Protein Regulates Apoptosis and Cell Cycle Progression via Retinoblastoma Protein.

RASSF6 is a member of the tumor suppressor Ras association domain family (RASSF) proteins. RASSF6 is frequently suppressed in human cancers, and its low expression level is associated with poor prognosis. RASSF6 regulates cell cycle arrest and apoptosis and plays a tumor suppressor role. Mechanistically, RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. However, RASSF6 also induces cell cycle arrest and apoptosis in a p53-negative background, which implies that the tumor suppressor function of RASSF6 does not depend solely on p53. In this study, we revealed that RASSF6 mediates cell cycle arrest and apoptosis via pRb. RASSF6 enhances the interaction between pRb and protein phosphatase. RASSF6 also enhances P16INK4A and P14ARF expression by suppressing BMI1. In this way, RASSF6 increases unphosphorylated pRb and augments the interaction between pRb and E2F1. Moreover, RASSF6 induces TP73 target genes via pRb and E2F1 in a p53-negative background. Finally, we confirmed that RASSF6 depletion induces polyploid cells in p53-negative HCT116 cells. In conclusion, RASSF6 behaves as a tumor suppressor in cancers with loss of function of p53, and pRb is implicated in this function of RASSF6.

Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma.

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

E2F1 inhibition mediates cell death of metastatic melanoma.

Melanoma is one of the most lethal cancers when it reaches a metastatic stage. Despite advancements in targeted therapies (BRAF inhibitors) or immunotherapies (anti-CTLA-4 or anti-PD1), most patients with melanoma will need additional treatment. Thus, there is an urgent need to develop new therapeutical approaches to bypass resistance and achieve more prolonged responses. In this context, we were interested in E2F1, a transcription factor that plays a major role in the control of cell cycle under physiological and pathological conditions. Here we confirmed that E2F1 is highly expressed in melanoma cells. Inhibition of E2F1 activity further increased melanoma cell death and senescence, both in vitro and in vivo. Moreover, blocking E2F1 also induced death of melanoma cells resistant to BRAF inhibitors. In conclusion, our studies suggest that targeting the E2F1 signaling pathway may be therapeutically relevant for melanoma.