E2F Family Of Transcription Factors Research Articles

Abstract 5785: Shedding light on PARP1-E2F1 interaction by Cryo-EM

Abstract Cell division is orchestrated by a complex network of interactions between proteins involved in numerous signaling pathways that control cell proliferation. One of these main players is the E2F family of transcription factors. The role of these key players is to regulate the expression of several genes important in cell proliferation, particularly those involved in progression through G1 and into the S-phase of the cell cycle. In fact, cell cycle deregulation is a hallmark of cancer, and multiple studies have highlighted the critical role of E2F hyperactivation in tumor progression, angiogenesis, and metastasis. However, targeting a master regulator of cell growth will also have a pernicious effect on non-cancerous cells, thus limiting its applications. In order to overwhelm these restrictions, we propose a therapeutic approach in which E2F transcriptional activity is specifically reduced in cells with hyperactivation of the pathway, such as cancer cells.We and others have shown that poly (ADP-ribose) polymerase 1 (PARP1) is a transcriptional co-activator of E2F1 and, owing to this, loss of PARP results in cell cycle re-regulation and reduction of tumor growth. Interestingly, this effect of PARP1 does not depend on its enzymatic activity, thus opening a completely new strategic approach. Furthermore, considering that most oncogenic processes are associated with cell cycle deregulation, disruption of PARP1-E2F1 interaction could provide a new therapeutic target with a wide spectrum of indications in cancer. Currently, we lack comprehensive structural details regarding the PARP-1/E2F1 protein-protein interaction. To address this, we used cryo-EM to investigate both the standalone structure of PARP1 (full length) and its interaction with E2F1. This approach yielded high-resolution structural insights into the PARP1-E2F1 complex, enabling precise identification of the protein-protein interaction interface (PPI). Through this exploration of structural and molecular determinants governing this interaction, we aim to develop innovative strategies for designing drugs targeted at disrupting this complex. This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF. Citation Format: Pablo Iglesias, Ester Casajus-Pelegay, Maria Moreno-Morcillo, Lara González-Rendo, Laura Porres-Ventin, Victor M. Arce, Rafael Fernandez-Leiro, Jose A. Costoya. Shedding light on PARP1-E2F1 interaction by Cryo-EM [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 5785.

Abstract 1648: RB represses cohesin-dependent loop formation and activates E2F-independent transcription

Abstract The retinoblastoma protein (RB) suppresses the activity of the E2F transcription factor family, controlling cellular proliferation. Recent studies, however, indicate that RB's role in chromatin organization, which is not yet fully understood, might be distinct from this E2F-dependent regulation. Employing chromosome conformation capture at a single nucleosome resolution, we identified RB as a cell cycle-regulated repressor of cohesin-dependent loop formation at topologically associating domain (TAD) boundaries. RB depletion increased the number and size of cohesin-dependent loops and strengthened topologically associating domains (TADs). This phenomenon was specific to the G1 phase and was not observed in the S phase, indicating that this novel RB function is regulated by the cell cycle. Mechanistically, RB showed extensive colocalization with cohesin in the human genome, and it impacted cohesin’s distribution on the chromatin. Active RB reduced cohesin from RB-bound TAD boundaries and decreased cohesin activity therein, as assessed by the increased K105/106 acetylation of the cohesin subunit SMC3. This led to reduced insulation in chromosome conformation capture assays. Importantly, by weakening the insulation activity of the adjacent insulators, RB non-canonically enhanced the expression of non-E2F target genes salient for cell adhesion and extracellular matrix organization. When RB was lost, cells showed a more rapid cellular detachment rate and an elevated migration rate, indicating that this novel RB function controls specific transcriptional programs rather than arbitrary genes to regulate the process of cell adhesion and migration. Overall, we conclude that RB has a central role in the interplay between cell cycle and chromatin organization, by repressing the cohesin-dependent loop formation at TAD boundaries. This RB function safeguards E2F-independent transcriptional programs driven by active enhancers and helps maintain cellular adhesion to the extracellular matrix. Citation Format: Hanjun Lee, Ioanna-Maria Gkotinakou, Badri Krishnan, Nicholas J. Dyson, Michael S. Lawrence, Ioannis Sanidas. RB represses cohesin-dependent loop formation and activates E2F-independent transcription [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 1648.

The role of E2F2 in cancer progression and its value as a therapeutic target.

The E2F family of transcription factors plays a crucial role in the regulation of cell cycle progression and cell proliferation. Accumulative evidence indicates that aberrant expression or activation of E2F2 is a common phenomenon in malignances. E2F2 has emerged as a key player in the development and progression of various types of tumors. A wealth of research has substantiated that E2F2 could contribute to the enhancement of tumor cell proliferation, angiogenesis, and invasiveness. Moreover, E2F2 exerts its influence on a myriad of cellular processes by engaging with a spectrum of auxiliary factors and downstream targets, including apoptosis and DNA repair. The dysregulation of E2F2 in the context of carcinogenesis may be attributable to a multitude of mechanisms, which encompass modifications in upstream regulatory elements or epigenetic alterations. This review explores the function of E2F2 in cancer progression and both established and emerging therapeutic strategies aiming at targeting this oncogenic pathway, while also providing a strong basis for further research on the biological function and clinical applications of E2F2.

Predictive role of E2F6 in cancer prognosis and responses of immunotherapy

BackgroundE2F6 is a member of the E2F transcription factor family. Numerous studies have demonstrated that E2F6 is critical to cancer development and progression, but its role in cancer immunotherapy remains unclear. MethodsGenotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases were used to obtain RNA-seq data for cancer and normal tissues, and we utilized the cBioPortal to analyze E2F6 genomic alterations in pan-cancer. The protein localization of E2F6 was obtained using the Human Protein Atlas (HPA), and the upregulation of E2F6 expression in clinical glioblastoma multiforme (GBM) tissues was detected by Western blot analysis. The ComPPI website was used to analyze the protein interaction information of E2F6. To evaluate the role of E2F6 in pan-cancer prognosis, we used univariate Cox regression and Kaplan-Meier methods, and gene set enrichment analysis (GSEA) was utilized to identify markers associated with E2F6 expression in tumors. TIMER 2.0 was used to study E2F6-related immune cell infiltration in tumor tissues, and the correlation of E2F6 with immunotherapy biomarkers was investigated using Spearman correlation analysis. The role of E2F6 in the cell cycle was analyzed by flow cytometry, and the Cell Counting Kit-8 (CCK-8) and colony formation assays were utilized to determine the proliferative ability of cells. ResultsIn most tumor types, E2F6 was highly expressed and was a good predictor of prognosis. E2F6 was significantly related to markers of immune activation, tumor immune cell infiltration, and immune regulators. Furthermore, E2F6 knockdown significantly attenuated the proliferative ability of glioma cells. Finally, E2F6 effectively predicted anti-programmed cell death 1 (PD1) treatment response. ConclusionE2F6 is an effective biomarker that predicts the prognosis of cancer patients treated with anti-immune checkpoint therapy.

Exosomes Derived from E2F1-/- Adipose-Derived Stem Cells Promote Skin Wound Healing via miR-130b-5p/TGFBR3 Axis.

Skin wound is a widespread health problem and brings extraordinary burdens to patients. Exosomes derived from adipose-derived stem cells (ADSC-Exos) are considered promising strategies for repairing skin wounds. E2F1 is a member of the E2F family of transcription factors involved in cell growth and apoptosis. E2F1 deficiency in mice enhances wound healing by improving collagen deposition and angiogenesis. Additionally, E2F1 can regulate the transcription and paracrine activity of multiple miRNAs, which will inevitably reshape the paracrine expression profile of ADSC-Exos. This study aimed to investigate the impact of transcription factor E2F1 deficiency on the functions of ADSC-Exos in promoting wound healing. First, we obtained ADSCs from subcutaneous adipose tissues of WT and E2F1-/- C57BL/6 mice and separated their exosomes, denoted as ADSCWT-Exos and ADSCE2F1-/--Exos. The wound healing effects of ADSCWT-Exos and ADSCE2F1-/--Exos in full-thickness skin wound models were investigated by wound images, H&E staining, and immunohistochemical staining. For the in vitro study, the abilities of ADSCWT-Exos and ADSCE2F1-/--Exos to promote cell activities, collagen formation, and angiogenesis were evaluated. The potential mechanism by which ADSCE2F1-/--Exos promote wound healing was determined by miRNA sequencing, ChIP‒qPCR, and dual-luciferase assays. ADSCE2F1-/--Exos accelerated wound healing by promoting collagen formation and angiogenesis. As a result, compared with the lower wound healing rate of 30.5% within 7 days in the control group and 42.3% in the ADSCWT-Exo group, ADSCE2F1-/--Exos significantly increased the wound healing rate to 72.5%. In vitro, ADSCE2F1-/--Exos activated the function of fibroblasts and vascular endothelial cells. The loss of E2F1 promoted miR-130b-5p expression in ADSCE2F1-/--Exos through transcriptional regulation. MiRNA high-throughput sequencing identified 12 differently expressed miRNAs between ADSCE2F1-/- and ADSCWT. ADSCE2F1-/--Exos enhanced fibroblast activities via the miR-130b-5p/TGFBR3 axis and TGF-β activation. Our results indicated that ADSCE2F1-/--Exos effectively promoted wound healing by regulating the miR-130b-5p/TGFBR3 axis, thus providing a novel strategy of gene-engineered stem cell exosomes for accelerating wound healing.

Expression and Regulatory Roles of Small Nucleolar RNA Host Gene 4 in Gastric Cancer.

The role of SNHG4 in the initiation and development of gastric cancer. Gastric cancer is one of the leading causes of cancer death worldwide. Studies have shown that lncRNAs have a regulatory function in human diseases, particularly cancers. Small nuclear RNA host gene 4 (SNHG4) has been known as an oncogenic long noncoding RNA (lncRNA) in various cancers, and its dysregulation can lead to tumorigenesis and cancer progression. The present study aims to determine the molecular mechanism of SNHG4 and the effects of its expression on the development of GC. Based on the bioinformatics investigations, we studied gene expression analysis, Kaplan-Meier survival, Gene ontology (GO), KEGG pathway enrichment, microRNA targets, transcription factor targets, and proteins interacting with SNHG4. During the experimental phase, SNHG4 expression was examined by quantitative real-time PCR (qRTPCR) in 40 paired gastric adenocarcinoma tissues and normal neighboring tissues. Also, we investigated the correlation between SNHG4 expression and patients' clinicopathological characteristics. Alteration of SNHG4 expression in gastric cancer and its correlation whit with clinical features of patients with stomach cancer; also, the accomplishment of bioinformatic analysis to find the potential pathways which could be impressed by changes in SNHG4 RNA expression. Increased SNHG4 expression was detected in GC tissues, which is significantly associated with the TNM stage, grade group, tumor size, and metastatic status. Evaluation survival analysis demonstrated that overexpression of SNHG4 in GC tissues is remarkably related to poor overall survival (OS). SNHG4 is closely related to miR-490 and E2F family transcription factors. GO analysis suggested the possible role of SNHG4 in cell-cell adhesion, and KEGG enrichment analysis revealed that SNHG4 could be associated with the gastric cancer signaling pathway. ELAVL1 and IGF2BP2 have the highest number of SNHG4 target sites, and these proteins are involved in the PI3K-Akt-mTOR and ERK-MAPK signaling pathways. Based on our results, we conclude that SNHG4 may have a function in GC development by regulating tumor-related signaling pathways.

E2F8 exerts cancer-promoting effects by transcriptionally activating RRM2 and E2F8 knockdown synergizes with WEE1 inhibition in suppressing lung adenocarcinoma

Ribonucleotide reductase (RR) is a rate-limiting enzyme that facilitates DNA replication and repair by reducing nucleotide diphosphates (NDPs) to deoxyribonucleotide diphosphates (dNDPs) and is thereby crucial for cell proliferation and cancer development. The E2F family of transcription factors includes key regulators of gene expression involved in cell cycle control. In this study, E2F8 expression was significantly increased in most cancer tissues of lung adenocarcinoma (LUAD) patients and was correlated with the expression of RRM2 through database and clinical samples analysis. The protein expression of E2F8 and RRM2 were positively correlated with tumor–node–metastasis (TNM) pathological stage, and high expression of E2F8 and RRM2 predicted a low 5-year overall survival rate in LUAD patients. Overexpression and knockdown experiments showed that E2F8 was essential for LUAD cell proliferation, DNA synthesis, and cell cycle progression, which were RRM2-dependent. Reporter gene, ChIP-qPCR, and DNA pulldown–Western blot assays indicated that E2F8 activated the transcription of the RRM2 gene by directly binding with the RRM2 promoter in LUAD cells. Previous studies indicated that inhibition of WEE1 kinase can suppress the phosphorylation of CDK1/2 and promote the degradation of RRM2. We further showed here that the combination of E2F8 knockdown with MK-1775, an inhibitor of WEE1 being evaluated in clinical trials, synergistically suppressed proliferation and promoted apoptosis of LUAD cells in vitro and in vivo. Thus, this study reveals a novel role of E2F8 as a proto-oncogenic transcription activator by activating RRM2 expression in LUAD, and targeting both the transcription and degradation mechanisms of RRM2 could produce a synergistic inhibitory effect for LUAD treatment in addition to conventional inhibition of RR enzyme activity.

MyD88 dimerization inhibitor ST2825 targets the aggressiveness of synovial fibroblasts in rheumatoid arthritis patients

BackgroundDimerization of the myeloid differentiation primary response 88 protein (MyD88) plays a pivotal role in the exacerbated response to innate immunity-dependent signaling in rheumatoid arthritis (RA). ST2825 is a highly specific inhibitor of MyD88 dimerization, previously shown to inhibit the pro-inflammatory gene expression in peripheral blood mononuclear cells from RA patients (RA PBMC). In this study, we elucidated the effect of disrupting MyD88 dimerization by ST2825 on the pathological properties of synovial fibroblasts from RA patients (RA SFs).MethodsRA SFs were treated with varying concentrations of ST2825 in the presence or absence of bacterial lipopolysaccharides (LPS) to activate innate immunity-dependent TLR signaling. The DNA content of the RA SFs was quantified by imaging cytometry to investigate the effect of ST2825 on different phases of the cell cycle and apoptosis. RNA-seq was used to assess the global response of the RA SF toward ST2825. The invasiveness of RA SFs in Matrigel matrices was measured in organoid cultures. SFs from osteoarthritis (OA SFs) patients and healthy dermal fibroblasts were used as controls.ResultsST2825 reduced the proliferation of SFs by arresting the cells in the G0/G1 phase of the cell cycle. In support of this finding, transcriptomic analysis by RNA-seq showed that ST2825 may have induced cell cycle arrest by primarily inhibiting the expression of critical cell cycle regulators Cyclin E2 and members of the E2F family transcription factors. Concurrently, ST2825 also downregulated the genes encoding for pain, inflammation, and joint catabolism mediators while upregulating the genes required for the translocation of nuclear proteins into the mitochondria and members of the mitochondrial respiratory complex 1. Finally, we demonstrated that ST2825 inhibited the invasiveness of RA SFs, by showing decreased migration of LPS-treated RA SFs in spheroid cultures.ConclusionsThe pathological properties of the RA SFs, in terms of their aberrant proliferation, increased invasiveness, upregulation of pain and inflammation mediators, and disruption of mitochondrial homeostasis, were attenuated by ST2825 treatment. Taken together with the previously reported anti-inflammatory effects of ST2825 in RA PBMC, this study strongly suggests that targeting MyD88 dimerization could mitigate both systemic and synovial pathologies in a variety of inflammatory arthritic diseases.

The SGLT2 inhibitor canagliflozin suppresses growth and enhances prostate cancer response to radiotherapy

Radiotherapy is a non-invasive standard treatment for prostate cancer (PC). However, PC develops radio-resistance, highlighting a need for agents to improve radiotherapy response. Canagliflozin, an inhibitor of sodium-glucose co-transporter-2, is approved for use in diabetes and heart failure, but is also shown to inhibit PC growth. However, whether canagliflozin can improve radiotherapy response in PC remains unknown. Here, we show that well-tolerated doses of canagliflozin suppress proliferation and survival of androgen-sensitive and insensitive human PC cells and tumors and sensitize them to radiotherapy. Canagliflozin blocks mitochondrial respiration, promotes AMPK activity, inhibits the MAPK and mTOR-p70S6k/4EBP1 pathways, activates cell cycle checkpoints, and inhibits proliferation in part through HIF-1α suppression. Canagliflozin mediates transcriptional reprogramming of several metabolic and survival pathways known to be regulated by ETS and E2F family transcription factors. Genes downregulated by canagliflozin are associated with poor PC prognosis. This study lays the groundwork for clinical investigation of canagliflozin in PC prevention and treatment in combination with radiotherapy.

E2F family play important roles in tumorigenesis.

Tumors are serious threats to human health. The transcription factors are regarded as the potential targets for tumor treatment. As an important family of transcription factors, E2F family transcription factors (E2Fs) play vital roles in cell proliferation and regulation. However, the expression feature, gene functions, and molecular interactions of E2Fs in tumorigenesis are not clear. In this study, the transcriptome data, mutation data, and protein-protein interaction data of 10 high-incidence tumors in China from the TCGA database were integrated and analyzed to explore the expression, structure, function, mutation, and phylogenetic characteristics of E2Fs. The results showed that E2F1 and E2F7 were regularly upregulated in the tumor samples. Moreover, E2Fs participated in the regulation of the cell cycle, cell aging, and other signaling pathways. As an important regulator, E2F1 interacted with more proteins than other E2Fs. At the same time, the genetic mutation types of E2Fs varied in tumor type and patient sex, of which gene amplification accounts for the largest proportion. Phylogenetic analysis showed that E2Fs were conserved in 41 species, including fruit flies, nematodes, and humans. Meanwhile, E2Fs had a tendency for gene expansion during evolution. In conclusion, this study clarified the expression pattern, mutation characteristics, and evolutionary trend of E2Fs in high-incidence tumors in China, and suggested that E2F family transcription factors could be novel diagnostic markers for tumor diseases. Furthermore, this work can provide a theoretical basis for the development of anti-tumor-targeted drugs.

Abstract 1723: Ferroptosis is a novel therapeutic target for RB1 deficient lethal prostate cancer

Abstract Background: Although the development of next-generation antiandrogens has significantly extended the survival time of patients with metastatic castration-resistant prostate cancer (mCRPC), drug resistance inevitably developed over time. Therefore, the identification of novel therapeutic strategies targeting mCRPC is urgent. RB1 deficiency is a common genetic event and contributes to the development of therapy resistance and poor prognosis in prostate cancer. However, effective therapies against RB1-deficient lethal prostate cancer remain elusive. Here we determined how RB1 regulates the sensitivity of prostate cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, and whether we can exploit ferroptosis for the treatment of RB1 deficient lethal prostate cancer. Methods: To determine how RB1 regulates ferroptosis in prostate cancer, we generated RB1 stable knockdown prostate cancer cell lines using lentivirus-delivered shRNAs. We compared cellular sensitivity to ferroptosis and associated lipid peroxidation between control and RB1 stable knockdown prostate cancer cells. Using western blotting, qPCR, and ChIP analyses, we examined the regulation of ferroptosis by RB1. We also evaluated the therapeutic efficacy of ferroptosis inducers in cell-derived xenograft, patient-derived xenograft, and genetically engineered mouse models. Results: Our studies revealed that RB1 deficiency sensitizes prostate cancer cells to ferroptosis. Mechanistically, we found that the E2F family of transcription factors directly binds and activates the ACSL4 promoter and that RB1 inhibits ferroptosis by suppressing E2F-mediated ACSL4 transcriptional activation. More importantly, our preclinical studies demonstrated that induction of ferroptosis by JKE-1674, a recently discovered ferroptosis inducer, significantly blocks RB1 deficient prostate tumor growth and metastasis, and improves the overall survival of mice in the absence of obvious toxicity. Conclusions: Our findings uncover an RB1/E2F/ACSL4 molecular axis that governs ferroptosis and also suggest a new approach to the treatment of RB1-deficient lethal prostate cancer. Citation Format: Mu-En Wang, Jiaqi Chen, Yi Lu, Jinjin Wy, Jianhong Ou, John Asara, Andrew Armstrong, Qianben Wang, Lei Li, Yuzhuo Wang, Jiaoti Huang, Ming Chen. Ferroptosis is a novel therapeutic target for RB1 deficient lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1723.

Abstract 4756: NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC)

Abstract Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4756.

Decursin inhibits EGFR-ERK1/2 signaling axis in advanced human prostate carcinoma cells.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

Integrative Analysis of Genetic and Epigenetic Alterations in the CBX7 Gene Reveals Its Tumor-Suppressive Function by Regulating the Cell Cycle in Human Breast Cancer

CBX7 is a member of the chromobox gene family, which plays an important role in epigenetic transcriptional regulation. In this study, we found that compared to normal mammary tissues, mRNA levels of CBX7 are consistently significantly downregulated in breast cancers (BCs) across different datasets. Integrative multiomics analysis revealed the genetic and epigenetic mechanisms for the loss of CBX7 expression in BCs. Lower expression levels of CBX7 are significantly associated with shorter overall, disease-free, and distant metastasis-free survival of patients with BC. These prognostic impacts of CBX7 are independent of estrogen receptor status and PAM50 molecular subtypes. Coexpression analysis identified 207 genes consistently coexpressed with CBX7 (157 negatively and 50 positively). Gene Ontology, KEGG, and Reactome enrichment analysis revealed that cell cycle‑, DNA replication‑, and mitosis-related pathways are significantly overrepresented within the set of CBX7 negatively coexpressed genes, suggesting that CBX7 functions as a suppressor of the cell cycle. Moreover, transcription factor enrichment analysis detected the E2F family of transcription factors significantly associated with CBX7 negatively coexpressed genes, consistent with E2F function regulating the cell cycle. Furthermore, we found that loss of CBX7 expression significantly increases genomic instability and tumor mutation burden. Our findings indicate that CBX7 acts as a tumor suppressor in BC through its potential role in the negative regulation of cell proliferation and the maintenance of genome integrity.

MEKs/ERKs-mediated FBXO1/E2Fs interaction interference modulates G1/S cell cycle transition and cancer cell proliferation.

E2F 1, 2, and 3a, (refer to as E2Fs) are a subfamily of E2F transcription factor family that play essential roles in cell-cycle progression, DNA replication, DNA repair, apoptosis, and differentiation. Although the transcriptional regulation of E2Fs has focused on pocket protein retinoblastoma protein complex, recent studies indicate that post-translational modification and stability regulation of E2Fs play key roles in diverse cellular processes. In this study, we found that FBXO1, a component of S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) complex, is an E2Fs binding partner. Furthermore, FBXO1 to E2Fs binding induced K48 ubiquitination and subsequent proteasomal degradation of E2Fs. Binding domain analysis indicated that the Arg (R)/Ile (I) and R/Val (V) motifs, which are located in the dimerization domain of E2Fs, of E2F 1 and 3a and E2F2, respectively, acted as degron motifs (DMs) for FBXO1. Notably, RI/AA or RV/AA mutation in the DMs reduced FBXO1-mediated ubiquitination and prolonged the half-lives of E2Fs. Importantly, the stabilities of E2Fs were affected by phosphorylation of threonine residues located near RI and RV residues of DMs. Phosphorylation prediction database analysis and specific inhibitor analysis revealed that MEK/ERK signaling molecules play key roles in FBXO1/E2Fs' interaction and modulate E2F protein turnover. Moreover, both elevated E2Fs protein levels by knockdown of FBXO1 and decreased E2Fs protein levels by sh-E2F3a delayed G1/S cell cycle transition, resulting in inhibition of cancer cell proliferation. These results demonstrated that FBXO1-E2Fs axis-mediated precise E2Fs stability regulation plays a key role in cell proliferation via G1/S cell cycle transition.

Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy.

The E2F family of transcription factors play a crucial role in the development of various cancers. However, E2F members lack targetable binding pockets and are typically considered "undruggable". Unlike canonical small-molecule therapeutics, molecular glues mediate new E3 ligase-protein interactions to induce selective proteasomal degradation, which represents an attractive option to overcome these limitations. Human proteome microarray was utilized to identify a natural product-derived molecular glue for targeting E2F2 degradation. Co-IP analysis with stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics was carried out to further explore the E3 ligase for E2F2 degradation. In this study, we identified a molecular glue bufalin, which significantly promoted E2F2 degradation. Unexpectedly, E2F2 underwent ubiquitination and proteasomal degradation via a previously undisclosed atypical E3 ligase, zinc finger protein 91 (ZFP91). In particular, we observed that bufalin markedly promoted E2F2-ZFP91 complex formation, thereby leading to E2F2 polyubiquitination via K48-linked ubiquitin chains for degradation. E2F2 degradation subsequently caused transcriptional suppression of multiple oncogenes including c-Myc, CCNE1, CCNE2, MCM5 and CDK1, and inhibited hepatocellular carcinoma growth invitro and invivo. Collectively, our findings open up a new direction for transcription factors degradation by targeting atypical E3 ligase ZFP91. Meanwhile, the chemical knockdown strategy with molecular glue may promote innovative transcription factor degrader development in cancer therapy. This work was financially supported by the NationalKeyResearchandDevelopment Projectof China (2022YFC3501601), National Natural Sciences Foundation of China (81973505, 82174008, 82030114), and China Postdoctoral Science Foundation (2019M650396), the Fundamental Research Funds for the Central Universities.

Decoding the Immune Landscape of Pathogenic T Cells in Acute Graft-Versus-Host Disease By Single-Cell RNA-Seq

Introduction Graft-versus-host disease (GvHD) is the major cause of non-relapse mortality in allogeneic hematopoietic cell transplantation (alloHCT), and about 40-60% cases would progress into steroid refractory (SR) aGvHD resulting in dismal outcomes. The pathophysiology of aGvHD is complex, and donor derived T cells are identified as the culprits of aGvHD. However, the relationships among distinct T cell subsets and molecular mechanisms underlying aGvHD remain incompletely understood. Methods We employed a single-cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing (scTCR-seq) to profile 49288 T cells in peripheral blood isolated from alloHCT recipients developed into aGvHD (n=9) or not (as control, n=5). Then, samples from SR-aGvHD patients were further investigated to explore the molecular mechanism of pathogenic T cell subsets in SR-aGvHD. Results A total of thirteen unique T cell subsets with distinct molecular properties were identified, including four clusters of CD8+ T cells (CD8+ TN, CD8+ TCM, CD8+ TEM and CD8+ TEFF), five clusters of CD4+ T cells (CD4+ TN, CD4+ TCM, Th1, Th17 and Treg), NK cell cluster and three clusters of other T cells (IFN-T, γδT and MAIT). The composition shifted towards more-developed CD8+ TCM, CD8+ TEM, CD4+ TCM and Th1 cells in aGvHD group due to rapid cell cycling. The expansion of CD8+ TEM subpopulation was confirmed by multiparameter flow cytometry. The CD8+ TEFF and Th1 were considered to be pathogenic in aGvHD as they exhibited aGvHD-related cytokine/granzyme expression and signaling pathway activation. Upregulated genes in aGvHD-derived-Th1 cells were related to chemotaxis (CCL3, CXCR6), cytokine production (type I interferon, IL-23) and JAK-STAT signaling activation. CD8+ TEFF cells showed activity of chemotaxis (CCL3, CXCR6), granzyme and cytokine expression (GZMB, GZMA, TNF-α, IL-6, IL-10, IL-12) and MAPK/MTOR/NF-kappaB activation. The different composition of cytokine expression and activated signaling pathways suggested distinct functions of each subset. Furthermore, CD8+ TEM represented four traits related to a range of T-cell differentiation stages, spanning rapid cell cycling (transcription factor E2F family), chemotaxis (CCL3, CXCR6), cytotoxicity (GZMA, GZMB, and GZMK) and exhaustion (PDCD1, LAG3, HAVCR2 and TIMD4), suggesting CD8+ TEM as a transient stage which might further develop into CD8+ TEFF for aGvHD pathogenesis (Figure 1). To elucidate the molecular mechanism of SR-aGvHD, we dissected the composition and functional heterogeneity of T-cell subsets between SR- and steroid sensitive (SS)-aGvHD. CD8+ TEM expanded to be the largest subset which resulted in the increase of CD8+ TEFF proportion in SR-aGvHD. CD8+ TEM and CD8+ TEFF showed enrichment of GvHD pathogenic scores and up-regulated expression of inflammation transcription factors STAT1 and STAT3 in SR-aGvHD. Of note, the expression of steroid receptor NR3C1 was downregulated in CD8+ TEM subset, as well as the steroid target gene ANXA1, compared with counterparts. Furthermore, both CD8+ TEM and CD8+ TEFF cells exhibited enrichment of JAK-STAT, MAPK, NF-kappaB, and PI3K-AKT signaling activation (Figure 2). Conclusions The effector T-cell subsets CD8+ TEFF and Th1 were co-activated and undertook distinct expression profiles and signaling pathways, which suggests their different roles in aGvHD pathogenesis. CD8+ TEM, characterized by proliferative, cytotoxicity, chemotaxis and exhaustion capacities, provided functional repertoire for CD8+ TEFF. Reduced NR3C1 expression and activation of by-pass survival pathways of CD8+ TEM might provide persistent differentiation origin of CD8+ TEFF to mediate resistance to steroid therapy. Collectively, our data described the immune landscape of aGvHD and provided novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Expanded Interplay Network between NF-κB p65 (RelA) and E2F1 Transcription Factors: Roles in Physiology and Pathology

Simple SummaryMembers of the NF-kappaB (κB) and the E2F transcription factor (TF) families regulate the expression of numerous genes controlling several biological processes, including cell proliferation and viability, metabolic pathways, pro-inflammatory, immune and stress-like responses, maintaining cell homeostasis, and physiology. While the NF-κB RelA/p65 and E2F1 TFs functions are quite distinct, they regulate the transcription of several common genes, but their interplay in target-gene promoters can either activate, blunt, or repress gene expression in different cell types affecting several processes, with physiological consequences. Using available genomics data, we showed that the RelA/p65 TF subunit binds to distal active enhancers, while the E2F1 TF binds to gene promoters. Further, RelA/p65 may attract and recruit E2F1 in gene promoters, acting potentially as a recruiting TF to properly control the transcription of target genes, a potential mechanism allowing for concerted actions of RelA/p65 and E2F1 in response to mitogenic, inflammatory, or genotoxic stimuli.Transcription Factors (TFs) are the main regulators of gene expression, controlling among others cell homeostasis, identity, and fate. TFs may either act synergistically or antagonistically on nearby regulatory elements and their interplay may activate or repress gene expression. The family of NF-κB TFs is among the most important TFs in the regulation of inflammation, immunity, and stress-like responses, while they also control cell growth and survival, and are involved in inflammatory diseases and cancer. The family of E2F TFs are major regulators of cell cycle progression in most cell types. Several studies have suggested the interplay between these two TFs in the regulation of numerous genes controlling several biological processes. In the present study, we compared the genomic binding landscape of NF-κB RelA/p65 subunit and E2F1 TFs, based on high throughput ChIP-seq and RNA-seq data in different cell types. We confirmed that RelA/p65 has a binding profile with a high preference for distal enhancers bearing active chromatin marks which is distinct to that of E2F1, which mostly generates promoter-specific binding. Moreover, the RelA/p65 subunit and E2F1 cistromes have limited overlap and tend to bind chromatin that is in an active state even prior to immunogenic stimulation. Finally, we found that a fraction of the E2F1 cistrome is recruited by NF-κΒ near pro-inflammatory genes following LPS stimulation in immune cell types.

A bioinformatics analysis for diagnostic roles of the E2F family in esophageal cancer.

Esophageal cancer (EC) is the eighth most commonly occurring cancer worldwide and the sixth leading cause of cancer-related deaths. The therapeutic effect of EC patients is not ideal, and new biomarkers are needed to guide diagnosis and prognosis of EC patients. E2F family transcription factors are among the most important links in the cell cycle regulatory network. E2Fs dysregulation not only promotes the early stages of tumor development but also the progression of benign tumors to malignant tumors. E2F is expected to be a new biomarker. The prognostic significance of the E2F family in EC requires further research. We analyzed The Cancer Genome Atlas (TCGA), Gene Expression Profiling Interactive Analysis (GEPIA), and GeneMANIA databases to obtain RNA-sequencing data and clinical data. The clinical data included age, gender, race, stage, type, status, etc. The prognosis outcome included overall survival (OS) and progression-free interval (PFI). Subsequently, we conducted further research on gene expressions, enrichment analysis, interaction network, and prognostic values by R software, containing ggplot2, ComplexHeatmap, DESeq2, pROC R package, based on t-test, Wilcoxon rank sum test, Spearman rank correlation analysis, log-rank test and COX model. We found that mRNA transcription levels of E2F1, E2F3-8 were more highly expressed in esophageal carcinoma (ESCA) tissues than in normal tissues. E2F8 expression was correlated with tumor stage [Pr(>F)=0.00856]. E2F-related genes played a role in development and differentiation, and were prevalent in the endoplasmic reticulum lumen, Golgi lumen, and lipoprotein particle, catalyzing translation activities and lipid metabolism. Each gene was found to be related to each other to some degree. The GeneMANIA network analysis revealed links between E2Fs and other genes. We compared the correlations between 24 kinds of tumor-infiltrating immune cells and E2Fs. E2F1 (AUC =0.945, CI: 0.890-1.000) and E2F7 (AUC =0.958, CI: 0.920-0.996) exhibited higher predictive power accuracy. However, only E2F7 was closely related to OS [HR =1.91 (1.16-3.16), P=0.011]. We discover that E2F7 is a prognostic biomarker. E2F family may take part in the development of EC through lipid metabolism pathways, which is helpful to predict the prognosis of EC patients and guide accurate diagnosis and treatment.

PP2A-B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle-related genes.

The retinoblastoma (RB) tumour suppressor protein regulates cell proliferation, motility, differentiation and apoptosis. The phosphorylation state of RB is modulated by kinases and phosphatases, and RB exhibits phosphorylation-sensitive interactions with E2F family transcription factors. Here, we characterize RB dephosphorylation by protein phosphatase 2A (PP2A). The growth factor-inducible immediate early response (IER) proteins IER2 and IER5 possess an adapter-like function in which IER proteins bind to both PP2A and its target proteins and enhance PP2A activity towards the proteins. IER2 interacts with RB and facilitates dephosphorylation of RB at T821/T826 by PP2A. In IER2 knockdown cells, elevated phosphorylation of RB resulted in reduced binding of RB to the promoters and derepression of cyclin D1 and p21. IER5 binds to both RB and RB-like 1 (p107/RBL1), enhances dephosphorylation of these proteins by PP2A and represses the expression of various cell cycle-related genes. However, IER2-regulated dephosphorylation at T821/T826 is not necessary for the repression function of RB in cell mobility-related gene expression. Our data identify PP2A adapter proteins as critical regulators of RB family proteins and suggest that the phosphorylation status of RB differentially affects gene expression.