E2F1 Protein Expression Research Articles

CDK4/6 inhibitor ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council CDK4/6 inhibitor Ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression Background Ribociclib, a novel chemotherapeutic agent for metastatic breast cancer, functions as a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor1. Its mechanism of action involves the CDK4/6-Rb-E2F1 pathway. Ribociclib use has very recently been associated with development of heart failure (HF)2, yet the causality of its cardiac effects remains to be explored. Aims This study aims to investigate ribociclib's cardiotoxic effects in dynamic human engineered heart tissues (dyn-EHTs), focusing on the CDK4/6-Rb-E2F1 axis. Methods and Results Dyn-EHTs were generated using human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and treated with repeated doses of ribociclib (7 µM) to mimic clinical drug administration. Post-treatment, dyn-EHTs exhibited a 17.0% (p<0.001) increase in tissue dilatation, an 8.9% (p<0.001) decrease in systolic force generation, and a 22.1% (p<0.001) increase in systolic stress, indicating significant cardiac dysfunction. Additionally, an increase in phosphorylation levels of Rb protein (Ser249 and Thr252) was observed (FC=0.445, p=0.009). Overexpression of E2F1 in iPSCs resulted in a threefold increase in E2F1 protein expression and successfully mitigated the ribociclib-induced tissue dilatation (p=0.007), decreased systolic force generation (p=0.005), and increase in systolic stress (p=0.004) making their performance comparable to healthy controls, as shown by linear regression analysis. Conclusions Our study demonstrates that ribociclib induces significant cardiotoxic effects in dyn-EHTs and iPSC-CMs through the CDK4/6-Rb-E2F1 pathway. The prevention of these effects by E2F1 overexpression highlights the pathway's involvement in ribociclib's cardiotoxic profile. These findings emphasize the need for careful cardiac monitoring in patients treated with CDK4/6 inhibitors and suggest further research into specific mechanisms of cardiotoxicity in cancer therapeutics.Graphical Abstract

E2F1-mediated Up-regulation of NCAPG Promotes Hepatocellular Carcinoma Development by Inhibiting Pyroptosis.

As a subunit of the condensin complex, NCAPG has an important role in maintaining chromosome condensation, but its biological function and regulatory mechanism in hepatocellular carcinoma (HCC) remains undefined. The prognostic ability of NCAPG in HCC patients was examined by univariate and multivariate Cox regression analysis. ROC curves were plotted to compare the predictive ability of NCAPG and AFP. Double luciferase reporter system, and ChIP were used to investigate transcriptional potential of E2F1 to NCAPG. Pyroptosis was observed by scanning electron microscopy. Protein expression of NCAPG, E2F1, and major proteins constituting NLRP3 inflammasome was determined by western blotting and ELISA. An in vivo tumor formation assay was conducted to verify the in vitro results. Up-regulated NCAPG was identified in HCC tissues compared with adjacent tissue and high NCAPG was positively correlated with poor prognosis. Serum NCAPG mRNA level was a prognostic factor in HCC patients and also a diagnostic factor with higher predictive ability compared with AFP [AUROC 0.766 (95% CI: 0.650-0.881) vs. 0.649 (95% CI 0.506-0.793)]. HBx transfection resulted in concomitant up-regulation of E2F1 and NCAPG. E2F1 significantly increased the activity of luciferase reporter fused with NCAPG reporter, and the interaction of E2F1 and NCAPG gene was confirmed by ChIP. Silencing of E2F1 resulted in significant down-regulation of NCAPG. Knockdown of NCAPG promote pyroptosis mediated by NLRP3 inflammasome activation in multiple HCC cell lines and also suppressed tumorigenesis in vitro. We identified a novel role of NCAPG in the regulation of NLRP3 inflammasome-mediated pyroptosis, which was regulated by its upstream transactivator, E2F1. The role of E2F1-NCAPG-NLRP3 regulation of pyroptosis network may be a potential target in HCC treatment.

Synthesis and Antitumor Activity of Panaxadiol Pyrazole and Isooxazole Derivatives.

In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC50 value was as low as 13.44±1.23 μM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3 K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.

SENP5 deteriorates traumatic brain injury via SUMO2-dependent suppression of E2F1 SUMOylation.

Traumatic brain injury (TBI) represents a main public health concern during the past decade, attracting considerable interest because of its rising prevalence, wide-ranging risk factors and lifelong familial and societal influence. SUMO2 can conjugate to substrates upon various cellular stresses. Nevertheless, whether and how SUMO2-specific proteases partake in TBI is less understood. The aim of this study is to dissect the effects of SUMO-specific peptidase 5 (SENP5) on accentuating TBI in rats in an effort to unveil its underlying mechanism. SENP5 is overexpressed in hippocampal tissues of TBI rats, and inhibition of SENP5 reduces neurological function scores, decreases brain water content, inhibits apoptosis in hippocampal tissues, and attenuates brain injury caused in rats. Moreover, SENP5 inhibits the SUMOylation level of E2F transcription factor 1 (E2F1) and increases the protein expression of E2F1. Silencing of E2F1 blocks the p53 signaling pathway. Overexpression of E2F1 partially reverses the protective effect of sh-SENP5 on TBI in rats. These findings reveal an essential role of SENP5 and the SUMOylation status of E2F1 in the TBI development.

Therapeutic Effects of Long-Term Administration of Tranilast in an Animal Model for the Treatment of Fibroids.

Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) is an orally administered drug with antiallergic properties and approved in Japan and the Republic of Korea for the treatment of asthma and hypertrophic scars. Previous in vitro studies indicated that tranilast reduced fibroid growth through its inhibitory effects on cell proliferation and induction of apoptosis. The objective of this study was to determine the efficacy of tranilast for treatment of human-derived fibroids in a mouse model. SCID mice (ovariectomized, supplemented with estrogen and progesterone) were implanted with fibroid explants and treated for two months with tranilast (50 m/kg/daily) or the vehicle. After sacrifice, xenografts were excised and analyzed. Tranilast was well tolerated without adverse side effects. There was a 37% reduction in tumor weight along with a significant decrease in staining for Ki67, CCND1, and E2F1; a significant increase in nuclear staining for cleaved caspase 3; and reduced staining for TGF-β3 and Masson's trichrome in the tranilast treated mice. There was a significant inhibition of mRNA and protein expression of fibronectin, COL3A1, CCND1, E2F1, and TGF-β3 in the xenografts from the tranilast-treated mice. These promising therapeutic effects of tranilast warrant additional animal studies and human clinical trials to evaluate its efficacy for treatment of fibroids.

Chronic Alcohol Reduces Bone Mass Through Inhibiting Proliferation and Promoting Aging of Endothelial Cells in Type-H Vessels

Alcohol consumption is regarded as one of the leading risk factors for secondary osteopenia. Angiogenesis and osteogenesis coupled by type-H vessels coordinate the biological process of bone homeostasis to prevent osteopenia. This study aimed to determine whether chronic alcohol inhibits type-H vessel-dependent bone formation. Two-month-old mice were fed with 5% (v/v) alcohol liquid diet (28% of calories) or normal liquid diet every day for 2 months. The tibias were isolated and detected with X-ray and microcomputed tomography. Paraffin-embedded or frozen tibial sections were prepared and used for immunohistochemical or immunofluorescence staining, respectively. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of alcohol, including 0 mM (0%), 8.7 mM (0.5%), 52 mM (3%), or 87 mM (5%) alcohol for 12 h. The conditioned medium of the above HUVEC cells was collected to culture human bone marrow-mesenchymal stem cells (BM-MSCs), which were induced to differentiate into osteoblasts in vitro. The alcoholic diet retarded the bone growth and led to osteoporosis, impaired bone formation of osteoblasts, and decreased CD31hiEMCNhi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells in mice. Alcohol treatment obviously increased the expression of p16, while significantly decreased the expression of Bmi-1, CDK6, Cyclin D, E2F1, and bone morphogenetic protein (BMP)2 compared with vehicle. Alcohol inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. Alcoholic diet impaired CD31hiEMCNhi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells through Bmi-1/p16 signaling, and inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. This study provides a basis for developing a new treatment strategy targeting aging endothelial cells of type-H vessel to prevent alcoholic osteopenia.

Abstract 6298: The novel long noncoding RNA lncNeur promotes neuroblastoma by up-regulating AURKA and Myc expression

Abstract Background: Neuroblastoma is the most common solid tumor in early childhood, and accounts for approximately 15% of childhood cancer death. We have recently analyzed RNA sequencing data from 493 human neuroblastoma tissues, and identified the long noncoding RNA lincNeur as one of the transcripts, high expression of which most considerably correlated with poor prognosis in both MYCN oncogene-amplified and MYCN non-amplified neuroblastoma patients. Aims: To determine the tumorigenic role of lincNeur in neuroblastoma, and to identify the mechanism of action through which lincNeur exerts oncogenic function, also to screen for small molecule compound inhibitors of lincNeur. Methods and results: We performed RNA-binding protein pull-down assay and RNA-immunoprecipitation assay. RALY was identified and validated as one of the binding proteins of lincNeur. We also identified down-stream targets of lincNeur via genome-wide differential gene expression analysis. E2F1 and AURKA were identified as among the transcripts significantly down-regulated after lincNeur knockdown. RT-PCR and western blot analysis showed that lincNeur knockdown reduced E2F1 and AURKA mRNA and protein expression, and reduced N-Myc and c-Myc protein but not mRNA expression. In addition, lincNeur or RALY knockdown reduced, while over-expression of lincNeur or RALY promoted, neuroblastoma cell proliferation. We also demonstrated lincNeur knockdown significantly suppressed RALY protein binding to E2F1 and AURKA mRNAs. Mouse xenograft model experiment showed that lincNeur knockdown suppressed neuroblastoma tumor progression in vivo. Importantly, we have confirmed the binding site between lincNeur and RALY, and are screening small molecule compound libraries for inhibitors of lincNeur and RALY interaction for neuroblastoma therapy. Conclusions: The novel long noncoding RNA lincNeur promotes neuroblastoma by interacting with RALY to up-regulate E2F1 and AURKA mRNA and protein, as well as N-Myc and c-Myc protein expression. Small molecule compound inhibitors of lincNeur and RALY interaction is a promising therapeutic approach. Citation Format: Jing Wu, Pei Yan Liu, Belamy Cheung, Glenn Marshall, Marcel Dinger, Tao Liu. The novel long noncoding RNA lncNeur promotes neuroblastoma by up-regulating AURKA and Myc expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6298.

SY5609, a Potent and Selective CDK7 Inhibitor, Potentiates BTK Inhibitor Activity in Mantle Cell Lymphoma Preclinical Models

Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional (e.g., MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g., loss of RB pathway checkpoint function) mechanisms. SY-5609 is a potent and selective CDK7 inhibitor currently in development in patients with solid tumors (NCT04247126). To assess the potential for SY-5609 development in heme malignancies, we evaluated SY-5609 activity in preclinical models of mantle cell lymphoma (MCL). MCL is an aggressive B cell lymphoma driven by genetic alterations in RB pathway genes (e.g. CCND1, RB1, CDKN2A) and by hyper-activation of B-cell receptor (BCR) signaling leading to activation of NF-kB-dependent transcriptional programs that drive cell proliferation and survival. Our results support evaluation of SY-5609 in MCL patients and highlight the potential for combining SY-5609 with Bruton's Tyrosine Kinase (BTK) inhibitors, inhibitors of BCR signal transduction approved for treatment of MCL.Methods: SY-5609 in vitro antiproliferative activity was assessed in a panel of 7 MCL cell lines by evaluating growth rate inhibition (GR) curve metrics GR 50 (drug concentration at which growth rate is inhibited by 50%) and GR max (maximum depth of response) after 5-days of treatment. SY-5609 pharmacodynamic (PD) responses and combination activity with the BTK inhibitor acalabrutinib were further assessed in MCL cell line Mino-1. PD activity was assessed by measuring transcriptional changes in POLR2A using a NanoString-based PD assay from the SY-5609 solid tumor trial. Combination activity in vitro was assessed by comparing GR curve metrics between acalabrutinib, SY-5609, and acalabrutinib in combination with SY-5609. Expression of CCND1 and E2F1, a key transcriptional regulator of DNA replication commitment and progression, were assayed via western blot. Combination activity in vivo was assessed in mice bearing Mino-1 xenograft tumors by comparing tumor growth rate inhibition (GRI) and body weight change in mice treated with acalabrutinib (15mg/kg, QD) and/or SY-5609 (0.5mg/kg, BID, days 1-7 and 15-21) over 25 days.Results: SY-5609, as a single agent, potently inhibited proliferation of all 7 MCL cell lines tested with a GR 50 geometric-mean of 7 nM (range: 2 to 20 nM) and a GR max geometric-mean of -0.2 (range: -0.6 to 0.2). In MCL cell line Mino-1, SY-5609 induced a dose-dependent increase in POLR2A expression to levels associated with SY-5609 antiproliferative activity in Mino-1 cells in vitro and to levels comparable with those observed in peripheral blood mononuclear cells (PBMCs) collected from SY-5609 solid tumor trial patients. In combination with acalabrutinib, SY-5609 demonstrated synergistic antiproliferative activity in Mino-1 cells in vitro, a dose-dependent decrease in acalabrutinib GR 50 (up to ~2-fold at the highest SY-5609 concentration tested, 5nM), and a dose-dependent increase in GR max (up to ~10-fold). In addition, the combination of SY-5609 and acalabrutinib caused dose-dependent decreases in CCND1 and E2F1 protein expression in vitro, which were not observed with either single agent alone. Finally, in mice bearing Mino-1 xenografts, the combination of SY-5609 and acalabrutinib significantly (p < 0.01) increased tumor GRI relative to each single agent at the doses and regimens tested and was well tolerated (no difference in body weight changes relative to vehicle treated mice).Conclusions: SY-5609 is a potent and selective CDK7 inhibitor that demonstrates antiproliferative activity in MCL cells in vitro, associated with PD changes comparable to those observed in patients enrolled in the SY-5609 solid tumor trial (ENA, 2020). The combination of SY-5609 and acalabrutinib is synergistic in MCL cells in vitro and inhibits expression of key cell cycle regulatory proteins CCND1 and E2F1 at concentrations that are preclinically subtherapeutic for either single agent alone. The combination of SY-5609 and acalabrutinib is also significantly more effective at inhibiting MCL xenograft growth in vivo than either single agent. These results support evaluation of SY-5609, including in combination with BTK inhibitors, in patients with mantle cell lymphoma. DisclosuresJohannessen: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Henry: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sawant: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. D'Ippolito: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Ke: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Lefkowitz: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Eaton: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dworakowski: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Rosario: Syros Pharmaceuticals: Current Employment. Hodgson: Syros Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company.

Long non-coding RNA LINC00630 facilitates hepatocellular carcinoma progression through recruiting transcription factor E2F1 to up-regulate cyclin-dependent kinase 2 expression.

This study is aimed to investigate the role of long non-coding RNA 630 (LINC00630) in hepatocellular carcinoma (HCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine LINC00630 expression in HCC cell lines and tissues. After LINC00630 was overexpressed or depleted in HCC cell lines, cell counting kit-8 (CCK-8) assay, BrdU assay, and flow cytometry were conducted for detecting HCC cell multiplication, apoptosis, and cell cycle progression. The catRAPID database was adopted to predict the binding relationship between LINC00630 and E2F transcription factor 1 (E2F1), and RNA pull-down and RNA immunoprecipitation (RIP) assays were carried out to verify this binding relationship. The binding of E2F1 to the cyclin-dependent kinase 2 (CDK2) promoter region was verified by dual-luciferase reporter gene assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assay. Western blotting was conducted to detect the protein expression of E2F1 and CDK2 in HCC cells. We report that LINC00630 expression was up-regulated in HCC and was significantly correlated with TNM stage and lymph node metastasis. LINC00630 overexpression facilitated HCC cell proliferation and cell cycle progression and inhibited the cell apoptosis, while LINC00630 knockdown had the opposite effects. LINC00630 directly bounds with E2F1. LINC00630 overexpression enhanced the binding of E2F1 to the CDK2 promoter region, thereby promoting CDK2 transcription, whereas knocking down LINC00630 inhibited CDK2 transcription. Collectively, LINC00630 promoted CDK2 transcription by recruiting E2F1 to the promoter region of CDK2, thereby promoting the malignant progression of HCC. Our data suggest that LINC00630 is a promising molecular target for HCC.

MCTS1 promotes the development of lung adenocarcinoma by regulating E2F1 expression.

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer that results in the majority of cancer-associated mortality. Multiple copies in T-cell lymphoma-1 (MCTS1) is an oncogene that is expressed at high levels in several types of cancer tissues. However, its exact role and pathomechanism in the development of LUAD remains unknown. Reverse transcription-quantitative PCR analysis was performed to detect MCTS1 expression. Immunohistochemistry analysis was performed to detect MCTS1 expression in LUAD tissues and normal tissues. The MTT, colony formation, EdU, flow cytometry, wound healing and Transwell assays were performed to assess the proliferation, apoptosis, migration and invasion of LUAD cells. Western blot analysis was performed to detect protein expression levels. The present study aimed to investigate the effects of MCTS1 on the progression of LUAD and the potential mechanisms underlying its effects. The results demonstrated that MCTS1 expression was upregulated in LUAD tissues and cells, which was associated with an unfavorable outcome in patients with LUAD. MCTS1 knockdown inhibited LUAD progression by suppressing cell viability and motility, and promoting apoptosis. In addition, E2F1 protein expression was attenuated following MCTS1 knockdown. The silencing MCTS1-induced inhibitory effect on LUAD malignancy was reversed following overexpression of E2F1 by modulating the c-Myc signaling pathway. Taken together, the results of the present study suggest that MCTS1 facilitates cell proliferation and migration, and suppresses apoptosis of LUAD cells by regulating E2F1 expression and the c-Myc signaling pathway.

Long non-coding RNA ZEB1-AS1 promotes proliferation and metastasis of hepatocellular carcinoma cells by targeting miR-299-3p/E2F1 axis.

There is emerging evidence that dysregulation of long non-coding RNAs (lncRNAs) is associated with hepatocellular carcinoma (HCC). Zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) functions as an oncogenic regulator in various malignancies. Nonetheless, the potential role of ZEB1-AS1 in HCC remains poorly elucidated. Herein, qRT-PCR was employed for examining ZEB1-AS1, miR-299-3p and E2F transcription factor 1 (E2F1) mRNA expressions in HCC cells and tissues. MTT assay was performed to evaluate cell proliferation. Transwell assay was utilized for evaluating cancer cell migration and invasion. Western blot was employed for measuring E2F1 protein expression. What's more, dual-luciferase reporter assay was utilized for verifying the targeting relationships between ZEB1-AS1 and miR-299-3p, as well as E2F1 and miR-299-3p. It was demonstrated that, in HCC tissues and cells, ZEB1-AS1 expression was markedly increased, and meanwhile, its high expression level is related to the unfavourable clinicopathologic indicators. ZEB1-AS1 overexpression facilitated HCC cell proliferation, migration and invasion, while its knockdown led to the opposite effects. In terms of mechanism, we discovered that ZEB1-AS1 could decoy miR-299-3p and upregulate E2F1 expression. This work reveals the functions and mechanism of ZEB1-AS1 in HCC tumourigenesis and progression, which provides novel biomarkers and therapeutic targets for HCC.

Phytochemical profiling, antioxidant and antiproliferation potential of Euphorbia milii var.: Experimental analysis and in-silico validation.

This study was aimed to investigate the anticancer potential of Euphorbia milii (E. milii) using an exquisite combination of phytopharmacological and advanced computational techniques. The chloroform fraction (Em-C) of E. milii methanol extract showed the highest antioxidant activity (IC50: 6.41 ± 0.99 µg/ml) among all studied fractions. Likewise, Em-C also showed significant cytotoxicity (IC50: 11.2 ± 0.8 µg/ml) when compared with that of standard compound 5-fluorouracil (5-FU) (IC50: 4.22 ± 0.6 µg/ml) against hepatocarcinoma cell line (HepG2). However, in a human cervical cancer cell line (HeLa), Em-C demonstrated a non-significant difference in cytotoxicity (22.1 ± 0.8 µg/ml) when compared with that of 5-FU (IC50: 6.87 ± 0.5 µg/ml). Furthermore, Western blot and qRT-PCR analysis revealed that the suppression of HepG2 cells was the consequence of a tremendous decrease in CDK2 and E2F1 protein expression. The GC–MS analysis of Em-C revealed the unique presence of cyclobarbital (CBT) and benzodioxole derivative (BAN) as major constituents. Furthermore, molecular docking of compounds BAN, CBT, and MBT into the binding site of different molecular targets i.e. cyclin dependent kinase 2 (CDK2), thymidylate synthase (TS), caspase 3, BCL2 and topoisomerase II was carried out. Compounds BAN and CBT have demonstrated remarkable binding affinity towards CDK2 and thymidylate synthase, respectively. Molecular dynamic simulation studies have further confirmed the finding of docking analysis, suggesting that CDK2 and TS can act as an attractive molecular target for BAN and CBT, respectively. It can be concluded that these E. milii phytoconstituents (BAN and CBT) may likely be responsible for anti-invasive activity against HepG2 cells.

A TRAIL-TL1A Paracrine Network Involving Adipocytes, Macrophages, and Lymphocytes Induces Adipose Tissue Dysfunction Downstream of E2F1 in Human Obesity.

Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)–MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high. While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity.

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.

Biomarker Discovery for TNBC and CRC Tumor Sensitivity to Novel CDK8 Inhibitors

BackgroundPrevious work done by the Russu lab developed a novel piperazinylpyrimidine compound, Q12, which acts as a competitive inhibitor for the ATP binding pocket in cyclin‐dependent kinase 8 (CDK8). Q12 was tested for its ability to selectively inhibit the growth of various tumor cell lines within the NCI‐60 cell line panel. Those results revealed that HCT‐116, a colorectal cancer cell line, and MDA‐MB‐468, a triple negative breast cancer (TNBC) cell line, showed the highest sensitivity to Q12 with GI50 values of 0.27M and 0.06M respectively. Q12 was studied in the context of these two cell lines through in vitro analysis. We hypothesize that Q12 can be used as a molecular probe for biomarker discovery in tumor types that may benefit from clinical CDK8 inhibition.Materials and methodsWe performed in vitro analysis of the cancer cell lines previously mentioned: MDA‐MB‐468 and HCT‐116. A MUSE Cell Analyzer was used to perform cell viability and apoptosis assays. Western blot analysis was performed to test Q12’s effect on various proteins including: β‐catenin, STATs, and E2F1. Genetic knockdown experiments of E2F1 were performed using siRNA transfection in the MDA‐MB‐468 cell line and results were obtained through western blot analysis and the MUSE Cell Analyzer. Conditioned media and co‐treatment of Q12 and cryptotanshinone (CPT) was performed to test the regulation of STAT3 phosphorylation.ResultsOur results showed that Q12 reduced cellular viability and p‐STAT1(Ser727) expression as well as induced apoptosis across both cell lines. For HCT‐116, we found that Q12 reduced ‐catenin, and p‐STAT3(Tyr 705) levels. However, the MDA‐MB‐468 cells showed no observable decrease in ‐catenin levels and a significant increase in p‐STAT3(Tyr 705) levels. The conditioned media experiments showed upregulation of p‐STAT3(Tyr 705) protein in the MDA‐MB‐468 cells. Co‐treatment with Q12 and CPT showed retention of cell viability even after Q12 treatment in MDA‐MB‐468 cells. Treatment of MDA‐MB‐468 cells with Q12 also revealed upregulation of total E2F1 protein expression. siRNA knockdown of E2F1 revealed a retention of cell viability in MDA‐MB‐468 cells after treatment with Q12.ConclusionOur results showed that, while both HCT‐116 and MDA‐MB‐468 are both sensitive to CDK8 inhibition, the mechanism through which Q12 induces apoptosis may stem from different biochemical pathways. HCT‐116 harbors a ‐catenin stabilizing mutation which contributes to its cellular proliferation. The results of our in vitro studies on the colon cancer cell lines are consistent with the literature indicating wnt/catenin signaling may be the primary biochemical pathway involved in HCT‐116 cell proliferation and likely associated with its sensitivity to Q12. MDA‐MB‐468 cells, on the other hand, harbor a deletion of the RB1 tumor suppressor gene which, under normal conditions, inhibits the E2F1 transcription factor. Our results indicated that interactions between E2F1, STAT3 and the RB biochemical pathway may be more significantly associated with Q12 sensitivity in the MDA‐MB‐468 cell line. There is also the possibility of autocrine/paracrine apoptosis induction from secreted cytokines which can be tested through an IL Family ELISA assay in both HCT‐116 and MDA‐MB‐468 cells.

MiR-449a/b negatively regulates E2F1 to suppress proliferation of gastric cancer cells

To investigate the possible role of miR-449a/b in the occurrence of gastric cancer. The expression of miR-449a/b and E2F1 mRNA in gastric cancer cells BGC-823 and gastric mucosal cells GES-1 were detected with qRT-PCR. miR-449a/b mimics or a negative control was transiently transfected into BGC-823 cells, and the changes in cell proliferation, apoptosis, and migration ability were assessed using CCK-8 assay, flow cytometry, and scratch wound healing assay, respectively. Western blotting was used to observe the effects of miR-449a/b upregulation on its target gene expression. The effects of transfection with an E2F1-over-expressing plasmid or an empty plasmid were analyzed on the expression level of miR-449a/b in BGC-823 cells using qRT-PCR and digital PCR. The gastric cancer cell line BGC-823 showed significantly a lowered expression of miR-449a/b compared with the normal gastric mucosal cell line GES-1 (P < 0.01). Overexpression of miR-449a/b obviously inhibited the proliferation and migration and promoted apoptosis of BGC-823 cells (P < 0.05). Overexpression E2F1 in the cells resulted in significantly up-regulated expression of miR-449a/b (P < 0.001). Upregulation of miR-449a/b caused significant down-regulation of its direct target genes CDK4 and CDK6 and also of the expression of E2F1 protein via the CDKs-pRb-E2F1 signaling pathway. The low expression of miR-449a/b and the high expression of E2F1 are both involved in the occurrence and progression of gastric cancer, and miR-449a/b negatively regulates E2F1 to inhibit the proliferation of gastric cancer cells.

A Novel Expression Profile of Cell Cycle and DNA Repair Proteins in Nonfunctioning Pituitary Adenomas.

The molecular mechanisms underlying the formation of nonfunctioning pituitary adenomas (NFAs) are largely unknown. In this study, we aimed to understand the relationship between NFAs and functional pituitary adenomas and the possible role of proteins involved in cell cycle, senescence, and DNA damage control mechanisms in the etiology of NFA. We analyzed pATM-S1981, pRb-S608, Rb, pE2F1-S364, p16, E2F1, p73, cyclin D1, and CHEK2 protein expression (in a group of 20 patients with acromegaly, 18 patients with Cushing's disease (CD), and 29 NFA patients) by immunohistochemistry and their relevant mRNA expression by qRT-PCR (in a group of 7 patients with acromegaly, 7 patients with CD, and 7 NFA patients). The clinical and histopathological results on the patients were statistically evaluated. pE2F1-S364 protein expression in the CD group was significantly lower than that in the NFA and acromegaly groups (p = 0.025, p = 0.034, respectively). However, the expression of the p16 protein was lower than in the NFA group than in the CD and acromegaly groups (p = 0.030, p = 0.033, respectively), and E2F1 protein expression was significantly higher in the NFA group than in the CD group (p = 0.025). p73 protein expression in patients with acromegaly was significantly higher (p = 0.031) than that in the CD group. CHEK2 mRNA expression in the CD group was significantly higher than that in the acromegaly group (p = 0.012). The selective and tumor-specific associations between E2F1, pE2F1-S364, CHEK2, and p73 mRNA and protein levels indicate their involvement in pituitary adenoma formation in NFA, CD, and acromegaly patients.

Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors.

The response to acute and long-term arginine starvation results in a conditional adaptive metabolic reprogramming that can be harnessed for therapeutic opportunities in ASS1-negative tumors. Here, we investigate the underlying biology of priming ASS1- tumors with arginine deiminase (ADI-PEG20) before treatment with gemcitabine (GEM) and docetaxel (DTX) in sarcoma, pancreatic cancer, and melanoma cell lines. ASS1- tumor cell lines were treated to create LTAT (long-term ADI treated) cell lines (ASS1+) and used for drug combination studies. Protein expression of ASS1, dCK, RRM2, E2F1, c-MYC, and hENT1 was measured. c-MYC activity was determined, live-cell immunofluorescent studies for hENT1, uptake assays of FITC-cytosine probe, and rescue studies with a c-MYC inhibitor were all determined in the presence or absence of the ADI-PEG20:GEM:DTX. In examining modulations within the pyrimidine pathway, we identified that the addition of DTX to cells treated with ADI-PEG20 resulted in translocation of stabilized c-Myc to the nucleus. This resulted in an increase of hENT1 cell-surface expression and rendered the cells susceptible to GEM. In vivo studies demonstrate that the combination of ADI-PEG20:GEM:DTX was optimal for tumor growth inhibition, providing the preclinical mechanism and justification for the ongoing clinical trial of ADI-PEG20, GEM, and DTX in sarcoma. The priming of tumors with ADI-PEG20 and DTX results in the stabilization of c-MYC potentiating the effect of GEM treatment via an increase in hENT1 expression. This finding is applicable to ASS1-deficient cancers that are currently treated with GEM.

Effect of miRNA-19a antisense oligonucleotide and Ara-C on the proliferation and apoptosis of HL60 cells.

This study aimed to investigate the effects of microRNA19a (miR-19a) antisense oligonucleotide (ASODN) on the proliferation and apoptosis of acute myeloid leukemia cells (HL60). In experiment 1, HL60 cells were divided into the blank control group, the blank transfection group, the scrambled oligonucleotide (SODN) group and the ASODN group. MiR-19a ASODN and SODN were independently transferred into HL60 cells. The miR-19a expression was detected by real-time quantitative RT-PCR (qRT-PCR) after 48-h and 72-h transfection; CCK8 assay was used to detect the proliferation inhibition rate at 48 and 72 h; Hoechst 33258 staining was performed to examine apoptotic cells at 48 h; the apoptosis rate was detected by flow cytometry after AnnexinV/PI staining at 48 and 72 h; the protein expression of E2F1 and Bim was detected by Western blotting at 48 h. In experiment 2, cells were divided into the Ara-C group, the SODN + Ara-C group and the ASODN + Ara-C group. The cell proliferation inhibition rate at 48 and 72 h and apoptosis rate at 72 h were assessed as mentioned above. MiR-19a expression in the miR-19a ASODN group was lower than in the SODN group and the blank control group (P<0.05). MiR-19a ASODN significantly inhibited the growth of HL60 cells (P<0.05) and increased their apoptosis, and the apoptosis rate peaked at 48 h. The protein expression of E2F1 and Bim in the ASODN group was higher than in the blank control group, blank transfection group and SODN group. In addition, Ara-C further inhibited the growth and induced the apoptosis of miR-19a ASODN-transfected cells (P<0.05) in a time dependent manner. The growth inhibition rate and apoptosis rate in the ASODN + Ara-C group were higher than the sum of those in both Ara-C group and ASODN group. miRNA-19a ASODN can inhibit the proliferation and induce apoptosis of HL60 cells and may exert synergistic effects with Ara-C on HL60 cells.

The silencing of long non-coding RNA ANRIL suppresses invasion, and promotes apoptosis of retinoblastoma cells through the ATM-E2F1 signaling pathway.

As one of the most common primary intraocular carcinomas, retinoblastoma generally stems from the inactivation of the retinoblastoma RB1 gene in retinal cells. Antisense non-coding RNA in the INK4 locus (ANRIL), a long non-coding RNA (lncRNA), has been reported to affect tumorigenesis and progression of various cancers, including gastric cancer and non-small cell lung cancer. However, limited investigations emphasized the role of ANRIL in human retinoblastoma. Hence, the current study was intended to investigate the effects of ANRIL on the proliferation, apoptosis, and invasion of retinoblastoma HXO-RB44 and Y79 cells. The lentivirus-based packaging system was designed to aid the up-regulation of ANRIL and ATM expressions or employed for the down-regulation of ANRIL in human retinoblastoma cells. Afterward, ANRIL expression, mRNA and protein expression of ATM and E2F1, and protein expression of INK4b, INK4a, alternate reading frame (ARF), p53 and retinoblastoma protein (pRB) were determined in order to elucidate the regulation effect associated with ANRIL on the ATM-E2F1 signaling pathway. In addition, cell viability, apoptosis, and invasion were detected accordingly. The results indicated that the down-regulation of ANRIL or up-regulation of ATM led to an increase in the expressions of ATM, E2F1, INK4b, INK4a, ARF, p53, and pRB. The silencing of ANRIL or up-regulation of ATM exerted an inhibitory effect on the proliferation and invasion while improving the apoptosis of HXO-RB44 and Y79 cells. In conclusion, the key observations of our study demonstrated that ANRIL depletion could act to suppress retinoblastoma progression by activating the ATM-E2F1 signaling pathway. These results provide a potentially promising basis for the targetted intervention treatment of human retinoblastoma.