Knockdown Of E2F1 Research Articles

DTL promotes the growth and migration of melanoma cells through the ERK/E2F1/BUB1 axis

Melanoma is the most dangerous form of skin cancer. Hence, a better understanding of molecular mechanisms in melanoma pathogenesis is urgently needed, which provides a new insight into the therapy of melanoma. DTL gene is screened out in melanoma pathogenesis by integrated bioinformatics analysis, and its expression is validated in the tissue and cell samples of melanoma. Forced DTL expression facilitates the proliferation, invasion, migration and EMT of melanoma cells, while DTL knockdown suppresses the biological behavior of melanoma cells. In addition, DTL promotes the malignancy of melanoma in vivo. Mechanistically, BUB1 is the crucial downstream target of DTL. Reduced DTL expression suppresses BUB1 expression, while enhanced DTL expression induces BUB1 upregulation. Rescue experiments showed that growing and migrating of melanoma cells induced by DTL are partially impaired by BUB1 inhibition. In addition, the expression of phosphorylated ERK (p-ERK) and the downstream transcription factor E2F1 are reduced when DTL expression is blocked. Meanwhile, BUB1 levels are decreased when the expression of p-ERK or E2F1 is repressed. Notably, the growth and migration of melanoma cells by inhibition of ERK and knockdown of E2F1 was rescued by overexpressing BUB1. DTL gene may be a prognosis marker and represent a unique potential target for melanoma patients. DTL supports the biologically malignant activity of melanoma cells via the ERK/E2F1/BUB1 axis.

ARHGEF39 targeted by E2F1 fosters hepatocellular carcinoma metastasis by mediating fatty acid metabolism

BackgroundHepatocellular carcinoma (HCC) stands as the prevailing manifestation of primary liver cancer. Previous studies have implicated ARHGEF39 in various cancer progression processes, but its impact on HCC metastasis remains unclear. MethodsBioinformatics analysis and qRT-PCR were employed to test ARHGEF39 expression in HCC tissues and cells, identified enriched pathways associated with ARHGEF39, and investigated its regulatory relationship with E2F1. The impact of ARHGEF39 overexpression or knockdown on cellular phenotypes in HCC was assessed through the implementation of CCK-8 and Transwell assays. Accumulation of neutral lipids was determined by BODIPY 493/503 staining, while levels of triglycerides and phospholipids were measured using specific assay kits. Expression of E-cadherin, Vimentin, MMP-2, MMP-9, and FASN were analyzed by Western blot. The interaction between ARHGEF39 and E2F1 was validated through ChIP and dual-luciferase reporter assays. ResultsOur study demonstrated upregulated expression of both ARHGEF39 and E2F1 in HCC, with ARHGEF39 being associated with fatty acid metabolism (FAM) pathways. Additionally, ARHGEF39 was identified as a downstream target gene of E2F1. Cell-based experiments unmasked that high expression of ARHGEF39 mediated the promotion of HCC cell viability, migration, and invasion via enhanced FAM. Moreover, rescue assays demonstrated that the promotion of HCC cell metastasis by high ARHGEF39 expression was attenuated upon treatment with Orlistat. Conversely, the knockdown of E2F1 suppressed HCC cell metastasis and FAM, while the upregulation of ARHGEF39 counteracted the repressive effects of E2F1 downregulation on the metastatic potential of HCC cells. ConclusionOur findings confirmed the critical role of ARHGEF39 in HCC metastasis and unmasked potential molecular mechanisms through which ARHGEF39 fostered HCC metastasis via FAM, providing a theoretical basis for exploring novel molecular markers and preventive strategies for HCC metastasis.

ZFP36L1 and ZFP36L2 reduce cyclin D1 expression by decreasing expression of E2F1 and long 3'UTR isoform of CCND1 transcripts.

The CCND1 mRNA possesses at least two distinct lengths of the 3'-untranslated region (3'UTR), with the long isoform containing multiple AU-rich elements (AREs). The tandem zinc finger (TZF) domains of human ZFP36 family members have the capacity to bind to AREs and promote mRNA degradation. Our previous study demonstrated that mutations in the TZF domain of ZFP36L1 or ZFP36L2 increased the CCND1 expression. In this study, we investigated whether ZFP36L1 and ZFP36L2 could downregulate the expression of the long 3'UTR isoform of CCND1 mRNA in human colorectal cancer (CRC) cells. Firstly, the Gene Expression Profiling Interactive Analysis 2 database indicated downregulation of ZFP36 and ZFP36L1, while E2F1 and CCND1 were upregulated in human CRC tissues compared to normal colorectal tissues. Overexpression of ZFP36L1 and/or ZFP36L2 in T-REx-293, DLD-1, and HCT116 cells led to a decrease in the total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Conversely, knockdown of ZFP36L1 and ZFP36L2 in HCT116 cells resulted in an increase in total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Knockdown of E2F1 decreased CCND1 expression, indicating a potential role for E2F1 in regulating CCND1 expression at the transcriptional level. These findings suggest that ZFP36L1 and ZFP36L2 play a negative role in CCND1 expression. The underlying mechanisms might involve the reduction of E2F1 transactivation at the transcriptional level and the promotion of AREs-mediated decay of the long 3'UTR isoform of CCND1 through posttranscriptional processes.

E2F1 Facilitates the Proliferation and Stemness of Gastric Cancer Cells by Activating CDC25B Transcription and Modulating the MAPK Pathway.

Gastric cancer (GC) is a health problem that concerns people around the world. CDC25B is an essential cell cycle regulatory factor that is overexpressed in a variety of tumor cells. CDC25B plays a vital part in the progression and proliferation of malignant tumors. However, it is not yet clear that how CDC25B affects the stemness of GC cells. The study used bioinformatics to detect the expression of E2F1 and CDC25B in GC tissues and their correlation, as well as pathways enriched by CDC25B. We detected the expression of E2F1 and CDC25B in GC cell lines using quantitative reverse transcription polymerase chain reaction and tested the combination relationship between E2F1 and CDC25B using chromatin immunoprecipitation (ChIP) and dual-luciferase assays. We measured cell viability using CCK-8 assay, evaluated sphere-forming efficiency using sphere formation assay, and determined cell proliferation ability using colony formation assay. We also analyzed the expression of stemness markers and MAPK pathway-related proteins using western blot. In GC tissues and cells, CDC25B was upregulated. Silencing CDC25B could affect the MAPK pathway, thereby repressing the proliferation and stemness of GC cells. As predicted by bioinformatics, CDC25B had an upstream transcription factor, E2F1, which also had a high expression level in GC. Dual-luciferase and ChIP assays confirmed the combination relationship between the two. Rescue experiments uncovered that overexpression of CDC25B could reverse the impact induced by E2F1 knockdown on proliferation and stemness of cells. In conclusion, E2F1 could activate CDC25B transcription to regulate the MAPK pathway and enhance the proliferation and stemness of GC cells. We revealed a potential regulatory pathway of stemness of GC cells that was mediated by CDC25B, providing new ideas for improving and innovating GC treatment.

Curcumin suppresses the malignant phenotype of laryngeal squamous cell carcinoma through downregulating E2F1 to inhibit FLNA.

Curcumin is a kind of polyphenol substance extracted from the rhizome of Curcuma longa. Because of its good biological activity and pharmacological effects, it has been used in anti-tumor research. The aim of this study was to investigate the anti-cancer mechanism of curcumin on laryngeal squamous cell carcinoma (LSCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to check the expression level of transcription factor E2F1 (E2F1) and filamin A (FLNA) mRNA. E2F1 and FLNA protein and proliferation-associated protein were detected through western blot. Cell viability was showed by MTT assay, and flow cytometry was used to exhibit cell cycle distribution and cell apoptosis. Tube formation assay was used to detect the angiogenesis ability of cells. Transwell was used as a method to observe cell migration and invasion. The online website JASPAR predicted the binding site of E2F1 and FLNA promoter, and chromatin immunoprecipitation (ChIP) and dual-luciferase report experiment verified the combination. Curcumin treatment made LSCC cells viability reduce, cell cycle retardant, angiogenesis decrease, metastasis inhibition and apoptosis increase. And curcumin treatment could downregulate the expression of E2F1, and E2F1 overexpression would reverse the influence of curcumin treatment in LSCC cells. Moreover, E2F1 could bind to FLAN promoter and promote FLNA expression. The expression level of FLNA was higher in LSCC tissue and cells compared with normal tissue and cells. E2F1 knockdown inhibited malignant phenotype of LSCC cells, which would be reversed by FLNA addition. In addition, FLNA had high level in LSCC tissue and cells. Curcumin regulated FLNA expression via inhibiting E2F1. Finally, in vivo assay showed that curcumin inhibition restrained LSCC tumor formation. Curcumin downregulated FLNA expression through inhibiting E2F1, thereby suppressing the malignant phenotype and angiogenesis of LSCC cells, which was a new regulatory pathway in LSCC.

E2F-mediated POLA2 upregulation is correlated with macrophage infiltration and poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the major histological type of primary liver cancer with a relatively poor prognosis, because most HCC cases are usually diagnosed at an advanced stage. Thus, it is essential to explore novel candidate biomarkers for early diagnosis and prognosis predication of HCC. HCC transcription sequencing data were downloaded and analyzed. POLA2 expression pattern was characterized in tumor development process. POLA2 expression was evaluated by western blotting. Kaplan-Meier plot was utilized to evaluate POLA2's ability for prognosis predication. Correlation analysis and enrichment analysis were performed to explore the functional role of POLA2 in HCC development. Knockdown of E2F1 or E2F4 was used to evaluate their ability in regulating POLA2 expression. CYBERSORT (https://cibersortx.stanford.edu/) was used to estimate the infiltration levels of immune cells. POLA2 was overexpressed in HCC. Moreover, western blotting results showed that POLA2 was upregulated by transcription factors E2F1 rather than E2F4 in HCC. POLA2 facilitated cell cycle progression, DNA replication and DNA repair. High POLA2 expression was correlated with poor overall survival in HCC patients. POLA2 induced macrophage infiltration in the tumor microenvironment by upregulating the expression CSF1 and VEGFA expression. POLA2 is a novel diagnostic and prognostic biomarker of HCC with potential clinical value.

Abstract 6814: E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma

Abstract Melanoma is a cancer that is metastatic, drug-refractory and fatal if managed late or inadequately. The transcription factor E2F1 is a key player in melanoma pathology, promoting metastatic traits like EMT, neoangiogenesis, chemoresistance and evasion of immune surveillance. Particularly, high levels of E2F1 trigger tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a prometastatic gene regulatory network (GRN). We aimed to investigate the extent to which E2F1-expressing melanoma cells influence the immune response. Methods: We combined transcriptomic approaches with bioinformatics and structural modeling to search for target genes involved in E2F1-activated immunomodulatory and prometastatic GRNs in aggressive melanoma. Immunoprecipitation and ChIP were performed to verify both PPI and protein-DNA interactions. Functional analyses, including qRT-PCR, immunoblotting and luciferase assays, were performed to verify expression and regulation of downstream targets. Additionally, we established a melanoma-immune cell coculture system with patient-derived CD4+ and CD8+ T cells. Cytokine arrays and ELISA were used for measurement of cytokine secretion to analyze the E2F1-induced tumor-immuno-crosstalk. Results: We identified an E2F1-dependent GRN with IL-6 as a central factor mediating EMT and immune regulation. Mechanistically, E2F1-induced activation and secretion of IL-6 leads to an autocrine inflammatory forward-feedback loop, which in turn further promotes invasiveness and EMT marker expression in melanoma cells. Interestingly, IL6 activated STAT3 associates with E2F1 in a structure-dependent manner, forming a complex that enhances IL-6 expression by binding to a composite E2F1:STAT3-responsive promoter element. Moreover, we observed that the E2F1-IL6 axis dispenses immunomodulatory effects on surrounding CD4+ and CD8+ T cells, further stimulating IL-6 release, but also leading to secretion of IL-10 into the TME. Although IL-10 has an immunosuppressive function, it also showed anti-metastatic properties in melanoma cells. Thus, in contrast to IL-6, IL-10 decreased cell motility and reduced the expression of Vim, Slug, and Snai1, while simultaneous knockdown of E2F1 increased these effects. The findings are supported by clinical data sets showing that melanoma patients who have high levels of E2F1 and IL-6 and low IL-10 exhibit poor survival. Conclusion: Our results unveil a novel gene regulatory program by which E2F1-associated IL-6 release induces the formation of an E2F1:STAT3 complex, resulting in a forward-feedback loop that promotes cancer invasion and regulates T cells response through intercellular cytokine signaling in the tumor microenvironment. Citation Format: Prabir Dhar, Krishna P. Singh, Shailendra K. Gupta, Dhanya Peringot, Alf Spitschak, Brigitte M. Pützer. E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune cell crosstalk that regulates metastatic properties in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6814.

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

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

Mechanism of E2F1 in the proliferation, migration, and invasion of endometrial carcinoma cells via the regulation of BMI1 transcription.

Endometrial carcinoma (EC) is the most prevalent gynecological cancer. Transcription factor (TF) regulates a large number of downstream target genes and is a key determinant of all physiological activities, including cell proliferation, differentiation, apoptosis, and cell cycle. The transcription factor E2F1 shows prominent roles in EC. BMI1 is a member of Polycomb suppressor Complex 1 (PRC1) and has been shown to be associated with EC invasiveness. It is currently unclear whether E2F1 can participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription. We investigated whether E2F1 could participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription, in order to further clarify the pathogenesis and etiology of EC, and provide reference for identifying potential therapeutic targets and developing effective prevention and treatment strategies for this disease. Human endometrial epithelial cells (hEECs) and human EC cell lines were selected. E2F1 expression was assessed by Western blot. E2F1 was silenced in AN3CA or overexpressed in HEC-1 by transfections, or E2F1 was silenced and BMI1 was overexpressed in AN3CA by cotransfection. Cell proliferation, migration, and invasion were detected by MTT, wound healing, and Transwell assays. The binding sites between E2F1 and BMI1 promoters were predicted through JASPAR website, and the targeted binding was verified by dual-luciferase report and ChIP assays. E2F1 was up-regulated in human EC cell lines, with its expression highest in AN3CA, and lowest in HEC-1. AN3CA invasion, migration, and proliferation were repressed by E2F1 knockdown, while those of HEC-1 cells were promoted by E2F1 overexpression. E2F1 overexpression increased the activity of wild type BMI1 reporter vector promoter, while this promotion was weakened after mutation of the predicted binding site in the BMI1 promoter. In the precipitated E2F1, BMI1 promoter site level was higher than that of IgG immunoprecipitant. BMI1 silencing suppressed AN3CA cell growth. BMI1 overexpression partially abrogated E2F1 silencing-inhibited EC cell growth. E2F1 promoted EC cell proliferation, invasion, and migration by promoting the transcription of BMI1.

Ubiquitin specific peptidase 47 contributes to liver regeneration

AimsHepatocytes resume proliferation following liver injuries to compensate for the loss of liver mass. Robust liver regeneration is an intrinsic and pivotal process that facilitates restoration of liver anatomy and function. In the present study we investigated the role of ubiquitin-specific peptidase 47 (USP47) in liver regeneration. Methods and materialsProliferation of hepatocytes was evaluated by Ki67 staining in vivo and EdU incorporation in vitro. DNA-protein interaction was evaluated by chromatin immunoprecipitation (ChIP). ResultsUSP47 expression was up-regulated in hepatocytes isolated from mice subjected to partial hepatectomy (PHx) or exposed to HGF treatment. Ingenuity pathway analysis revealed E2F1 as a primary regulator of USP47 transcription. Reporter assay and ChIP assay confirmed that E2F1 directly bound to the USP47 promoter and activated USP47 transcription. Consistently, E2F1 knockdown abrogated USP47 induction by HGF. Compared to the wild type littermates, USP47 knockout mice displayed compromised liver regeneration following PHx. In addition, USP47 inhibition by a small-molecule compound impaired liver regeneration in mice. On the contrary, USP47 over-expression enhanced proliferation of hepatocytes in vitro and promoted liver regeneration in mice. Importantly, a positive correlation between USP47 expression and hepatocyte proliferation was identified in patients with acute liver failure (ALF). SignificanceOur data suggest that USP47, transcriptionally activated by E2F1, plays an essential role in liver regeneration.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

PRMT5 up-regulation improves myocardial hypertrophy by mediating E2F-1/NF-κB/NLRP3 pathway

Various protein arginine methyltransferases (PRMTs) have been demonstrated to be aberrantly expressed in cardiovascular disease. This study aimed to investigate the role of PRMT5 in myocardial hypertrophy. Levels of fibrosis markers, NLRP3-ASC-Caspase1, inflammatory factors, myocardial hypertrophy markers and oxidative stress markers were determined in cardiomyocytes. Overexpression or knockdown models of PRMT5 and E2F-1 were constructed, and pharmacological intervention with NF-κB was determine the function of the PRMT5/E2F-1/NF-κB pathway in myocardial hypertrophy. Results shows that PRMT5 was down-regulated in the TAC rat model as well as in an in-vitro model of Ang II-induced myocardial hypertrophy. Overexpression of PRMT5 dramatically reduced Ang II-induced myocardial hypertrophy, fibrosis, inflammatory response, and oxidative stress, whereas knockdown of PRMT5 had the opposite effect. PRMT5 overexpression restrained E2F-1 expression and impaired NF-κB phosphorylation and NLRP3-ASC-Caspase1 inflammasome activation. Mechanistically, PRMT5 knockdown contributed to E2F-1 expression, but E2F-1 knockdown or NF-κB inhibition reversed PRMT5 knockdown-mediated myocardial hypertrophy. PRMT5 attenuated NLRP3 inflammasome activation and ameliorates angiotensin II-induced myocardial hypertrophy by regulating the E2F-1/NF-κB pathway.

MCM6 promotes intrahepatic cholangiocarcinoma progression by upregulating E2F1 and enhancing epithelial-mesenchymal transition.

Minichromosome maintenance complex component 6 (MCM6), a member of the MCM family, plays a pivotal role in DNA replication initiation and genome duplication of proliferating cells. MCM6 is upregulated in multiple malignancies and is considered a novel diagnostic biomarker. However, the functional contributions and prognostic value of MCM6 in intrahepatic cholangiocarcinoma (ICC) remain unexplored. In this study, we investigated the molecular function of MCM6 in ICC. Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO, GSE107943) indicated an upregulation of MCM6 in tumor tissues. Immunohistochemical analysis performed on 115 cases of ICC samples confirmed the upregulation of MCM6 and further suggested that a high level of MCM6 expression predicted shorter overall and disease-free survival in ICC patients. Functional studies suggested that MCM6 knockdown significantly suppressed cell viability, blocked cell cycle progression and inhibited metastasis, while the enhancement of MCM6 expression promoted the proliferation and migration of ICC cells both in vitro and in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) suggested that the epithelial-mesenchymal transition (EMT) and E2F1-correlated genes were enriched in ICC tissues with high MCM6 expression. Further verification indicated that MCM6 promoted the EMT of ICC cells via upregulating E2F1. In addition, E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression. In summary, MCM6 was found to be a novel prognostic and predictive marker for ICC. MCM6 promoted ICC progression via activation of E2F1-mediated EMT.

Transcription Factor E2F1 Regulates the Expression of ADRB2.

Adrenergic beta-2-receptor (ADRB2) is highly expressed in various tissue cells, affecting the susceptibility, development, and drug efficacy of diseases such as bronchial asthma and malignant tumor. However, the transcriptional regulatory mechanism of the human ADRB2 gene remains unclear. This study aimed to clarify whether E2F transcription factor 1 (E2F1) was involved in the transcriptional regulation of the human ADRB2 gene. First, the 5' flanking region of the human ADRB2 gene was cloned, and its activity was detected using A549 and BEAS-2B cells. Second, it was found that the overexpression of E2F1 could increase promoter activity by a dual-luciferase reporter gene assay. In contrast, treatment of knockdown of E2F1 significantly resulted in a decrease in its promoter activity. Moreover, mutation of the binding site of E2F1 greatly reduced the potential of human ADRB2 promoter transcriptional activity to be regulated by E2F1 overexpression and knockdown. Additionally, by real-time quantitative PCR and Western blot analysis, we demonstrated that overexpression of E2F1 elevated the ADRB2 mRNA expression and protein levels while si-E2F1 reduced its expression. Finally, the consequence of the chromatin immunoprecipitation assay showed that E2F1 was able to bind to the promoter region of ADRB2 in vivo. These results confirmed that E2F1 upregulated the expression of the human ADRB2 gene.

Oncogenic FLT3 internal tandem duplication activates E2F1 to regulate purine metabolism in acute myeloid leukaemia

Oncogene FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for 30 % of acute myeloid leukaemia (AML) cases and induces transformation. Previously, we found that E2F transcription factor 1 (E2F1) was involved in AML cell differentiation. Here, we reported that E2F1 expression was aberrantly upregulated in AML patients, especially in AML patients carrying FLT3-ITD. E2F1 knockdown inhibited cell proliferation and increased cell sensitivity to chemotherapy in cultured FLT3-ITD-positive AML cells. E2F1-depleted FLT3-ITD+ AML cells lost their malignancy as shown by the reduced leukaemia burden and prolonged survival in NOD-PrkdcscidIl2rgem1/Smoc mice receiving xenografts. Additionally, FLT3-ITD-driven transformation of human CD34+ hematopoietic stem and progenitor cells was counteracted by E2F1 knockdown. Mechanistically, FLT3-ITD enhanced the expression and nuclear accumulation of E2F1 in AML cells. Further study using chromatin immunoprecipitation-sequencing and metabolomics analyses revealed that ectopic FLT3-ITD promoted the recruitment of E2F1 on genes encoding key enzymatic regulators of purine metabolism and thus supported AML cell proliferation. Together, this study demonstrates that E2F1-activated purine metabolism is a critical downstream process of FLT3-ITD in AML and a potential target for FLT3-ITD+ AML patients.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

LncRNA PTTG3P promotes tumorigenesis and metastasis of NSCLC by binding with ILF3 to maintain mRNA stability and form a positive feedback loop with E2F1.

Non-small cell lung cancer (NSCLC) is a highly lethal disease worldwide. We found the pseudogene-derived lncRNA PTTG3P is upregulated in NSCLC and associated with larger tumor size, advanced staging, and poor prognosis. This study investigated the oncogenic roles and mechanisms of PTTG3P in NSCLC. We demonstrate that PTTG3P promoted NSCLC cell proliferation, migration, tumorigenesis, and metastasis while inhibiting apoptosis in vitro and in vivo. Mechanistically, PTTG3P formed an RNA-protein complex with ILF3 to maintain MAP2K6 and E2F1 mRNA stability, two oncogenic factors involved in NSCLC progression. RNA-seq revealed MAP2K6 and E2F1 were downregulated upon PTTG3P knockdown. RIP and RNA stability assays showed PTTG3P/ILF3 interaction stabilized MAP2K6 and E2F1 transcripts. Interestingly, E2F1 transcriptionally upregulated PTTG3P by binding its promoter, forming a positive feedback loop. Knockdown of E2F1 or PTTG3P attenuated their mutual regulatory effects on cell growth and migration. Thus, a PTTG3P/ILF3/E2F1 axis enhances oncogene expression to promote NSCLC pathogenesis. Our study reveals PTTG3P exerts oncogenic functions in NSCLC via mRNA stabilization and a feedback loop, highlighting its potential as a prognostic biomarker and therapeutic target.

Abstract 13304: Sox17 Deficiency Induces Pulmonary Hypertension Through E2F1/BRD4 Signaling

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: Genetic SOX17 mutations are associated with PAH. We hypothesized that endothelial SOX17 mutations or loss of function contributes to endothelial dysfunction and the pathogenesis of PAH. Methods: Endothelial-specific deletion of Sox17 ( Sox17iCKO ) mice were generated. Sox17iCKO and Sox17f/f (WT) mice after tamoxifen injection were incubated without or with hypoxia (10% O 2 ) for 3 weeks to induce PH. EC proliferation, apoptosis, cell junction assay, Western blotting, QRT-PCR, RNA-sequencing analysis, and luciferase assays were performed to understand the molecular mechanisms of SOX17 deficiency and mutation in ECs. siRNA and inhibitors were used to determine the involvement of E2F1/BRD4 signaling in SOX17 deficiency ECs. Results: Sox17iCKO mice developed spontaneous mild PH and exaggerated hypoxia-induced PH evidenced by the increase of RVSP and RV hypertrophy compared to WT mice. SOX17 knockdown in human PVECs induced cell proliferation, anti-apoptosis, and impaired cell integrity. SOX17 knockdown reduced BMPR2 signaling. SOX17 knockdown and mutations at the HMG domain lost transcriptional activities and its ability to repress -catenin. RNA-seq analysis demonstrated enrichment of cell cycle pathway after the loss of SOX17 in PVECs. iRegulon analysis revealed the involvement of transcriptional factor E2F1. siRNA-mediated E2F1 knockdown normalized SOX17 deficiency-induced PVECs dysfunction. Knockdown SOX17 significantly upregulated E2F1 promoter activity, which was dependent on the binding site in the E2F1 promoter by luciferase and mutagenesis assay. E2F1 or BRD4 inhibitors attenuated SOX17 deficiency-induced PH in mice. Conclusions: Our study demonstrated that endothelial loss of SOX17 induces PH in mice. Pharmacological inhibition of E2F1/BRD4 signaling attenuated PH development in Sox17iCKO mice.

E2F1/CKS2/PTEN signaling axis regulates malignant phenotypes in pediatric retinoblastoma

Retinoblastoma (RB) is the most common pediatric intraocular malignancy and is a serious vision- and life-threatening disease. The biallelic mutation of the retinoblastoma gene RB1 is the initial event in the malignant transformation of RB, but the exact molecular mechanism is still unclear. E2F transcription factors can be activated by RB1 loss of function and lead to uncontrolled cell division. Among E2F family numbers, E2F1 has higher expression abundance than E2F2 and E2F3 in RB clinical samples. By integrating E2F1 ChIP-seq data, RNA-seq profiling from RB samples and RNA-seq profiling upon E2F1 knockdown, together with pathway analysis, literature searching and experimental validation, we identified Cyclin-dependent kinases regulatory subunit 2 (CKS2) as a novel regulator in regulating tumor-associated phenotypes in RB. CKS2 exhibited aberrantly higher expression in RB. Depletion of CKS2 in Y79 retinoblastoma cell line led to reduced cell proliferation, delayed DNA replication and decreased clonogenic growth. Downregulation of CKS2 also slowed tumor xenograft growth in nude mice. Importantly, reversed expression of CKS2 rescued cancer-associated phenotypes. Mechanistically, transcription factor E2F1 enhanced CKS2 expression through binding to its promoter and CKS2 regulated the cancer-associated PI3K–AKT pathway. This study discovered E2F1/CKS2/PTEN signaling axis regulates malignant phenotypes in pediatric retinoblastoma, and CKS2 may serve as a potential therapeutic target for this disease.

Resveratrol alleviates Ang II-induced vascular smooth muscle cell senescence by upregulating E2F1/SOD2 axis.

Vascular smooth muscle cells (VSMCs) senescence is a crucial factor relevant to accelerate cardiovascular diseases. Resveratrol (RES) has been reported that could obstruct vascular senescence. However, the detailed molecular mechanisms of RES in VSMCs senescence are still indistinct and deserve further investigations. In this study, VSMCs were treated with 100nM angiotensin II (Ang II) for 3days and then followed with a range of different concentrations of RES (0.5, 5, 15, 25, 35, 50μM), and 25μM of RES was chose for following experiments. We found that the E2F1 and SOD2 expressions were reduced in Ang II-induced VSMCs. RES treatment impeded Ang II-induced oxidative stress and mitochondrial dysfunction through elevating E2F1 and SOD2 expression, thereby alleviating VSMCs senescence. Additionally, E2F1 knockdown reversed the protective effects of RES on VSMCs senescence caused by Ang II administration. Ch-IP assay and dual luciferase reporter gene assay validated that E2F1 could bind to the promoter region of SOD2. Furthermore, E2F1 or SOD2 overexpression blocked Ang II-induced on VSMCs senescence. In conclusion, RES mitigated Ang II-induced VSMCs senescence by suppressing oxidative stress and mitochondrial dysfunction through activating E2F1/SOD2 axis. Our study disclosed that RES might be a potential drug and the axis of its regulatory mechanism might be therapeutic targets for postponing vascular senescence.