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In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive "conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)" (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active "cytomegalovirus enhancer and β-actin promoter", provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active "Rous sarcoma virus long terminal repeat", not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) co-infections with human papillomavirus (HPV) have been observed in oropharyngeal squamous cell carcinoma. Modeling EBV/HPV co-infection in organotypic epithelial raft cultures revealed that HPV16 E7 inhibited EBV productive replication through the facilitated degradation of the retinoblastoma protein pRb/p105. To further understand how pRb is required for EBV productive replication, we generated CRISPR-Cas9 pRb knockout (KO) normal oral keratinocytes (NOKs) in the context of wild-type and mutant K120E p53. EBV replication was examined in organotypic rafts as a physiological correlate for epithelial differentiation. In pRb KO rafts, EBV DNA copy number was statistically decreased compared to vector controls, regardless of p53 context. Loss of pRb did not affect EBV binding or internalization of calcium-treated NOKs or early infection of rafts. Rather, the block in EBV replication correlated with impaired immediate early gene expression. An EBV infection time course in rafts with mutant p53 demonstrated that pRb-positive basal cells were initially infected with delayed replication occurring in differentiated layers. Loss of pRb showed increased S-phase progression makers and elevated activator E2F activity in raft tissues. Complementation with a panel of pRb/E2F binding mutants showed that wild type or pRbÎ"685 mutant capable of E2F binding reduced S-phase marker gene expression, rescued EBV DNA replication, and restored BZLF1 expression in pRb KO rafts. However, pRb KO complemented with pRb661W mutant, unable to bind E2Fs, failed to rescue EBV replication in raft culture. These findings suggest that EBV productive replication in differentiated epithelium requires pRb inhibition of activator E2Fs to restrict S-phase progression. [ABSTRACT FROM AUTHOR]

Objectives: To determine whether 6 months of preoperative apalutamide for intermediate‐risk prostate cancer (IRPCa) reduces the aggregate postoperative radiotherapy risk and to evaluate associations of molecular perturbations with clinical outcomes in this study cohort. Patients and Methods: Between May 2018 and February 2020, eligible patients with IRPCa (Gleason 3 + 4 or 4 + 3 and clinical T2b‐c or prostate‐specific antigen level of 10–20 ng/mL) were treated with apalutamide 240 mg/day for 6 months followed by radical prostatectomy (RP) in this single‐arm, phase II trial. The primary endpoint was presence of any adverse pathological feature at risk of pelvic radiation (pathological T stage after neoadjuvant therapy [yp]T3 or ypN1 or positive surgical margins). Translational studies, including germline and somatic DNA alterations and RNA and protein expression, were performed on post‐apalutamide RP specimens, and assessed for associations with clinical outcomes. Results: A total of 40 patients underwent a RP, and only one patient discontinued apalutamide prior to 6 months. In all, 40% had adverse pathological features at time of RP, and the 3‐year biochemical recurrence (BCR) rate was 15%, with 27.5% being not evaluable. Genomic alterations frequently seen in metastatic PCas, such as androgen receptor (AR), tumour protein p53 (TP53), phosphatase and tensin homologue (PTEN), or BReast CAncer associated gene (BRCA1/2) were underrepresented in this localised cohort. Adverse pathological features and BCR at 3‐years were associated with increased expression of select cell cycle (e.g., E2F targets: adjusted P value [Padj] < 0.001, normalised enrichment score [NES] 2.47) and oxidative phosphorylation (Padj < 0.001, NES 1.62) pathways. Conclusions: Preoperative apalutamide did not reduce the aggregate postoperative radiation risk to the pre‐specified threshold in unselected men with IRPCa. However, transcriptomic analysis identified key dysregulated pathways in tumours associated with adverse pathological outcomes and BCR, which warrant future study. Further investigation of preoperative therapy is underway for men with high‐risk PCa. [ABSTRACT FROM AUTHOR]

The bromodomain and extraterminal domain (BET) family proteins serve as primary readers of acetylated lysine residues and play crucial roles in cell proliferation and differentiation. Dysregulation of BET proteins has been implicated in tumorigenesis, making them important therapeutic targets. BET-bromodomain (BD) inhibitors and BET-targeting degraders have been developed to inhibit BET proteins. In this study, we found that the BET inhibitor MS645 exhibited superior antiproliferative activity than BET degraders including ARV771, AT1, MZ1 and dBET1 in triple-negative breast cancer (TNBC) cells. Treatment with MS645 led to the dissociation of BETs, MED1 and RNA polymerase II from the E2F1–3 promoter, resulting in the suppression of E2F1–3 transcription and subsequent inhibition of cell growth in TNBC. In contrast, while ARV771 displaced BET proteins from chromatin, it did not significantly alter E2F1–3 expression. Mechanistically, ARV771 induced BRD4 depletion at protein level, which markedly increased EGR1 expression. This elevation of EGR1 subsequently recruited septin 2 and septin 9 to E2F1–3 promoters, enhancing E2F1–3 transcription and promoting cell proliferation rate in vitro and in vivo. Our findings provide valuable insights into differential mechanisms of BET inhibition and highlight potential of developing BET-targeting molecules as therapeutic strategies for TNBC.

Abstract Background The early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed. Results In this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription. Conclusions This study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Simple Summary: Addressing RB1-deficient cancers poses substantial challenges. Dysfunctional or deleted RB1 can enhance the proliferation and spread of different cancer types. Scientists strive to grasp the intricate role of RB1 in regulating various biological processes and molecular pathways to devise innovative therapeutic strategies tailored to RB1-deficient cancers. Recent advancements have revealed encouraging tactics for combating these malignancies, heralding a path toward more precise and efficacious treatments. This review underscores the pivotal role of RB1 in cancer research and highlights its potential as a focal point for personalized therapies. The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1′s canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy. [ABSTRACT FROM AUTHOR]

Objective: PDZ‐binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells. Methods: Four OSCC cell lines (HSC‐2, HSC‐3, SAS, and OSC‐19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514. Results: OTS514 decreased the cell survival of OSCC cells dose‐dependently, and administration of OTS514 readily suppressed the HSC‐2‐derived tumor growth in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase‐3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, TP53 knockdown attenuated OTS514‐induced apoptosis. Conclusion: OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular‐targeted therapeutics against OSCC. [ABSTRACT FROM AUTHOR]

Purpose: Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken. Methods: MYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells. Results: The expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis. Conclusion: In conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour. [ABSTRACT FROM AUTHOR]

Simple Summary: RNA polymerase (pol) III synthesizes essential and abundant non-coding RNAs, including all tRNAs. This activity can be constrained by RB, an action that may help to limit cell growth and proliferation. Previous work established that RB binds and represses TFIIIB, a factor required for pol III to transcribe any of its target genes; this explains the apparently universal effect of RB on the pol III transcriptome. We find E2F bound to chromatin in close proximity to many pol III-transcribed genes, where it may recruit RB to influence local chromatin and control the production of specific products. RB recruitment to subsets of pol III-dependent genes provides the potential for more selective regulation than the general dampening of output that can be achieved through interaction with TFIIIB. The regulatory impact of E2F may therefore be strengthened by differentially influencing levels of key non-coding RNAs such as individual tRNAs. In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB. [ABSTRACT FROM AUTHOR]

Objective. E2F transcription factors are associated with tumor development, but their underlying mechanisms in gastric cancer (GC) remain unclear. This study explored whether E2Fs determine the prognosis or immune and therapy responses of GC patients. Methods. E2F regulation patterns from The Cancer Genome Atlas (TCGA) were systematically investigated and E2F patterns were correlated with the characteristics of cellular infiltration in the tumor microenvironment (TME). A principal component analysis was used to construct an E2F scoring model based on prognosis-related differential genes to quantify the E2F regulation of a single tumor. This scoring model was then tested in patient cohorts to predict effects of immunotherapy. Results. Based on the expression profiles of E2F transcription factors in GC, two different regulatory patterns of E2F were identified. TME and survival differences emerged between the two clusters. Lower survival rates in the Cluster2 group were attributed to limited immune function due to stromal activation. The E2F scoring model was then constructed based on the E2F-related prognostic genes. Evidence supported the E2F score as an independent and effective prognostic factor and predictor of immunotherapy response. A gene-set analysis correlated E2F score with the characteristics of immune cell infiltration within the TME. The immunotherapy cohort database showed that patients with a higher E2F score demonstrated better survival and immune responses. Conclusions. This study found that differences in GC prognosis might be related to the E2F patterns in the TME. The E2F scoring system developed in this study has practical value as a predictor of survival and treatment response in GC patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The RB and p53 pathways are two major tumor suppressive pathways. Hence, in almost all cancers, both pathways are disabled. E2F transcription factor is under the control of the RB pathway and fulfills pivotal roles in tumor suppression by triggering the p53 pathway upon loss of RB function. Although this notion has been well established, emerging evidence indicates that each player of the E2F-RB-p53 pathway possesses novel functions and regulatory mechanisms. This review aims to introduce the expanding roles of the E2F-RB-p53 pathway in tumor suppression. The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology. [ABSTRACT FROM AUTHOR]

Cyclin‐dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two‐hybrid screen to identify new atypical cyclin–CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.

Survival of living organisms is fully dependent on their maintenance of genome integrity, being permanently threatened by replication stress in proliferating cells. Although the plant DNA damage response (DDR) regulator SOG1 has been demonstrated to cope with replication defects, accumulating evidence points to other pathways functioning independent of SOG1. Here, we report the roles of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in plant response to replication stress. Through a combination of reverse genetics and chromatin immunoprecipitation approaches, we show that E2FA and E2FB share many target genes with SOG1, providing evidence for their involvement in the DDR. Analysis of double- and triple-mutant combinations revealed that E2FB, rather than E2FA, plays the most prominent role in sustaining plant growth in the presence of replication defects, either operating antagonistically or synergistically with SOG1. Conversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Collectively, our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors. This study investigates the respective roles of E2FA and E2FB transcription factors during plant response to replication stress. The results show that E2FA and E2FB function together with SOG1 to control the expression of DDR genes and that E2FB plays a unique role in allowing cell cycle progression from G2/M despite replication stress. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is one of the most prevalent cancers in men, yet its pathogenic pathways remain poorly understood. Transcriptomics and high-throughput sequencing can help uncover cancer diagnostic targets and understand biological circuits. Using prostate adenocarcinoma (PRAD) datasets of various web-based applications (GEPIA, UALCAN, cBioPortal, SR Plot, hTFtarget, Genome Browser, and MetaCore), we found that upregulated dysbindin domain-containing 1 (DBNDD1) expression in primary prostate tumors was strongly correlated with pathways involving the cell cycle, mitotic in KEGG, WIKI, and REACTOME database, and transcription factor-binding sites with the DBNDD1 gene in prostate samples. DBNDD1 gene expression was influenced by sample type, cancer stage, and promoter methylation levels of different cancers, such as PRAD, liver hepatocellular carcinoma (LIHC), and lung adenocarcinoma (LUAD). Regulation of glycogen synthase kinase (GSK)-3β in bipolar disorder and ATP/ITP/GTP/XTP/TTP/CTP/UTP metabolic pathways was closely correlated with the DBNDD1 gene and its co-expressed genes in PCa. DBNDD1 gene expression was positively associated with immune infiltration of B cells, Myeloid-derived suppressor cell (MDSC), M2 macrophages, andneutrophil, whereas negatively correlated with CD8+ T cells, T follicular helper cells, M1 macrophages, and NK cells in PCa. These findings suggest that DBNDD1 may serve as a viable prognostic marker not only for early-stage PCa but also for immunotherapies. [ABSTRACT FROM AUTHOR]

The family of mammalian E2F transcription factors (E2Fs) comprise of 8 members (E2F1-E2F8) classified as activators (E2F1-E2F3) and repressors (E2F4-E2F8) primarily regulating the expression of several genes related to cell proliferation, apoptosis and differentiation, mainly in a cell cycle-dependent manner. E2F activity is frequently controlled via the retinoblastoma protein (pRb), cyclins, p53 and the ubiquitin-proteasome pathway. Additionally, genetic or epigenetic changes result in the deregulation of E2F family genes expression altering S phase entry and apoptosis, an important hallmark for the onset and development of cancer. Although studies reveal E2Fs to be involved in several human malignancies, the mechanisms underlying the role of E2Fs in oral cancer lies nascent and needs further investigations. This review focuses on the role of E2Fs in oral cancer and the etiological factors regulating E2Fs activity, which in turn transcriptionally control the expression of their target genes, thus contributing to cell proliferation, metastasis, and drug/therapy resistance. Further, we will discuss therapeutic strategies for E2Fs, which may prevent oral tumor growth, metastasis, and drug resistance. [ABSTRACT FROM AUTHOR]

Cyclin‐dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two‐hybrid screen to identify new atypical cyclin–CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control. [ABSTRACT FROM AUTHOR]

Mutations that result in the loss of function of pRB were first identified in retinoblastoma and since then have been associated with the propagation of various forms of cancer. pRB is best known for its key role as a transcriptional regulator during cell cycle exit. Beyond the ability of pRB to regulate transcription of cell cycle progression genes, pRB can remodel chromatin to exert several of its other biological roles. In this review, we discuss the diverse functions of pRB in epigenetic regulation including nucleosome mobilization, histone modifications, DNA methylation and non-coding RNAs.

Prostate cancer (PCa) is characterized by a unique dependence on optimal androgen receptor (AR) activity where physiological androgen concentrations induce proliferation but castrate and supraphysiological levels suppress growth. This feature has been exploited in bipolar androgen therapy (BAT) for castrate resistant malignancies. Here, we investigated the role of the tumor suppressor protein p14ARF in maintaining optimal AR activity and the function of the AR itself in regulating p14ARF levels. We used a tumor tissue array of differing stages and grades to define the relationships between these components and identified a strong positive correlation between p14ARF and AR expression. Mechanistic studies utilizing CWR22 xenograft and cell culture models revealed that a decrease in AR reduced p14ARF expression and deregulated E2F factors, which are linked to p14ARF and AR regulation. Chromatin immunoprecipitation studies identified AR binding sites upstream of p14ARF. p14ARF depletion enhanced AR-dependent PSA and TMPRSS2 transcription, hence p14ARF constrains AR activity. However, p14ARF depletion ultimately results in apoptosis. In PCa cells, AR co-ops p14ARF as part of a feedback mechanism to ensure optimal AR activity for maximal prostate cancer cell survival and proliferation.

Abstract Background It is well-established that biochemical recurrence is detrimental to prostate cancer (PCa). In the present study, we explored the mechanisms underlying PCa progression. Methods Five cohorts from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to perform gene set variation analysis (GSVA) between nonrecurrent and recurrent PCa patients. We obtained the intersection of pathway enrichment results and extracted the corresponding gene list. LASSO Cox regression analysis was used to identify recurrence-free survival (RFS)-related significant genes and establish an RFS prediction gene signature and nomogram. MTT and colony formation assays were conducted to validate our findings. Results The E2F signaling pathway was activated in recurrent PCa patients compared to nonrecurrent patients. We established an E2F-related gene signature for RFS prediction based on the four identified E2F-related genes (CDKN2C, CDKN3, RACGAP1, and RRM2) using LASSO Cox regression in the Memorial Sloan Kettering Cancer Center (MSKCC) cohort. The risk score of each patient in MSKCC was calculated based on the expression levels of CDKN2C, CDKN3, RACGAP1, and RRM2. PCa patients with low-risk scores exhibited higher RFS than those with high-risk scores. Receiver operating characteristic (ROC) curve analysis validated the good performance and prognostic accuracy of the E2F-related gene signature, which was validated in the TCGA-prostate adenocarcinoma (TCGA-PRAD) cohort. Compared to patients with low Gleason scores and early T stages, PCa patients with high Gleason scores and advanced T stages had high-risk scores. Moreover, the E2F-related gene signature-based nomogram yielded good performance in RFS prediction. Functional experiments further confirmed these results. Conclusions The E2F signaling pathway is associated with biochemical recurrence in PCa. Our established E2F-related gene signature and nomogram yielded good accuracy in predicting the biochemical recurrence in PCa.

Castration-resistant prostate cancer (CRPC) poses a major clinical challenge with the androgen receptor (AR) remaining to be a critical oncogenic player. Several lines of evidence indicate that AR induces a distinct transcriptional program after androgen deprivation in CRPCs. However, the mechanism triggering AR binding to a distinct set of genomic loci in CRPC and how it promotes CRPC development remain unclear. We demonstrate here that atypical ubiquitination of AR mediated by an E3 ubiquitin ligase TRAF4 plays an important role in this process. TRAF4 is highly expressed in CRPCs and promotes CRPC development. It mediates K27-linked ubiquitination at the C-terminal tail of AR and increases its association with the pioneer factor FOXA1. Consequently, AR binds to a distinct set of genomic loci enriched with FOXA1- and HOXB13-binding motifs to drive different transcriptional programs including an olfactory transduction pathway. Through the surprising upregulation of olfactory receptor gene transcription, TRAF4 increases intracellular cAMP levels and boosts E2F transcription factor activity to promote cell proliferation under androgen deprivation conditions. Altogether, these findings reveal a posttranslational mechanism driving AR-regulated transcriptional reprogramming to provide survival advantages for prostate cancer cells under castration conditions. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression. [ABSTRACT FROM AUTHOR]

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

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

Human embryonic stem cells (hESCs) hold the potential to solve the problem of the shortage of functional hepatocytes in clinical applications and drug development. However, a large number of usable hepatocytes derived from hESCs cannot be effectively obtained due to the limited proliferation capacity. In this study, we found that enhancement of liver transcription factor C/EBPβ during hepatic differentiation could not only significantly promote the expression of hepatic genes, such as albumin, alpha fetoprotein, and alpha-1 antitrypsin, but also dramatically reinforce proliferation-related phenotypes, including increasing the expression of proliferative genes, such as CDC25C, CDC45L, and PCNA, and the activation of cell cycle and DNA replication pathways. In addition, the analysis of CUT&Tag sequencing further revealed that C/EBPβ is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBPβ and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBPβ could significantly inhibit the expression of the aforementioned proliferative genes. RNA transcriptome analysis and GSEA enrichment indicated that the E2F family was enriched, and the expression of E2F2 was changed with the overexpression or knockdown of C/EBPβ. Moreover, the results of CUT&Tag sequencing showed that C/EBPβ also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBPβ and E2F2 proteins, and this interaction between these two proteins was also verified in the LO2 cell line, a hepatic progenitor cell line. Thus, our results demonstrated that C/EBPβ first initiated E2F2 expression and then coupled with E2F2 to regulate the expression of proliferative genes in hepatocytes during the differentiation of hESCs. Therefore, our findings open a new avenue to provide an in vitro efficient approach to generate proliferative hepatocytes to potentially meet the demands for use in cell-based therapeutics as well as for pharmaceutical and toxicological studies. [ABSTRACT FROM AUTHOR]

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells. [ABSTRACT FROM AUTHOR]

Background: It is well-established that biochemical recurrence is detrimental to prostate cancer (PCa). In the present study, we explored the mechanisms underlying PCa progression. Methods: Five cohorts from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to perform gene set variation analysis (GSVA) between nonrecurrent and recurrent PCa patients. We obtained the intersection of pathway enrichment results and extracted the corresponding gene list. LASSO Cox regression analysis was used to identify recurrence-free survival (RFS)-related significant genes and establish an RFS prediction gene signature and nomogram. MTT and colony formation assays were conducted to validate our findings. Results: The E2F signaling pathway was activated in recurrent PCa patients compared to nonrecurrent patients. We established an E2F-related gene signature for RFS prediction based on the four identified E2F-related genes (CDKN2C, CDKN3, RACGAP1, and RRM2) using LASSO Cox regression in the Memorial Sloan Kettering Cancer Center (MSKCC) cohort. The risk score of each patient in MSKCC was calculated based on the expression levels of CDKN2C, CDKN3, RACGAP1, and RRM2. PCa patients with low-risk scores exhibited higher RFS than those with high-risk scores. Receiver operating characteristic (ROC) curve analysis validated the good performance and prognostic accuracy of the E2F-related gene signature, which was validated in the TCGA-prostate adenocarcinoma (TCGA-PRAD) cohort. Compared to patients with low Gleason scores and early T stages, PCa patients with high Gleason scores and advanced T stages had high-risk scores. Moreover, the E2F-related gene signature-based nomogram yielded good performance in RFS prediction. Functional experiments further confirmed these results. Conclusions: The E2F signaling pathway is associated with biochemical recurrence in PCa. Our established E2F-related gene signature and nomogram yielded good accuracy in predicting the biochemical recurrence in PCa. [ABSTRACT FROM AUTHOR]

Density-dependent regulation of cell growth is presumed to be caused by cell-cell contact, but the underlying molecular mechanism is not yet clearly defined. Here, we report that receptor-type protein tyrosine phosphatase-kappa (R-PTP-κ) is an important regulator of cell contact-dependent growth inhibition. R-PTP-κ expression increased in proportion to cell density. siRNA-mediated R-PTP-κ downregulation led to the loss of cell contact-mediated growth inhibition, whereas its upregulation reduced anchorage-independent cell growth in soft agar as well as tumor growth in nude mice. Expression profiling and luciferase reporter system-mediated signaling pathway analysis revealed that R-PTP-κ induced under cell contact conditions distinctly suppressed E2F activity. Among the structural domains of R-PTP-κ, the cytoplasmic domain containing the tandemly repeated PTP motif acts as a potent downregulator of the E2F pathway. Specifically, R-PTP-κ suppressed CDK2 activity through the induction of p21Cip1/WAF-1 and p27Kip1, resulting in cell cycle arrest at the G1 phase. In transcriptome-based public datasets generated from four different tumor types, R-PTP-κ expression was negatively correlated with the expression pattern and prognostic value of two known E2F1 target genes (CCNE1 and CDC25A). Therefore, our results indicate that the R-PTP-κ-E2F axis plays a crucial role in cell growth-inhibitory signaling arising from cell-cell contact conditions. [ABSTRACT FROM AUTHOR]

ZhaoHui Xu,1,2,&ast; Hui Qu,1,2,&ast; YanYing Ren,1 ZeZhong Gong,1,2 Hyok Ju Ri,1,2 Fan Zhang,1,&ast; Shuai Shao,1 XiaoLiang Chen,1 Xin Chen1 1Department of Hernia and Colorectal Surgery, The Second Hospital of Dalian Medical University, Dalian, People’s Republic of China; 2Graduate School of Dalian Medical University, Dalian, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Xin Chen, Tel +86 17709872266, Email chenxincjz@gmail.comBackground: The E2 factor (E2F) family of transcription factors is dysregulated in numerous cancer types and may play a vital role in the development of various malignancies. However, to our knowledge, the specific function of each of the E2Fs and their relation to the disease prognosis of colorectal cancer (CRC) patients remain unknown.Materials and Methods: We used different publicly available databases and tools, such as ONCOMINE, GEPIA2, UALCAN, cBioPortal, Kaplan–Meier plotter, Metascape, and TIMER analysis, to do an in silico exploration of the potential roles of E2Fs in CRC.Results: In our analyses, we found a downregulation of E2F2 expression and an upregulation of E2F1 and E2F3-8 expression in CRC tissues compared to normal controls. These findings were consistent with our subgroup analysis using the different clinicopathological features of CRC patients. Furthermore, overexpression of E2F3 and E2F4 were significantly correlated with worse overall survival (OS) in colon cancer patients. Meanwhile, low levels of E2F2 resulted in a shorter OS in rectal cancer patients. The E2F family members had varying degrees of genetic alterations with the highest alteration rate observed in E2F1 (23%). Interestingly, a moderate positive express correlation had been found in the following E2F family members: E2F1 with E2F4, E2F2 with E2F7, E2F2 with E2F8, and E2F7 with E2F8. In addition, spearman analysis revealed that E2Fs have a strong positive correlation with the critical oncogenes in CRC patients. Lastly, the expression of E2Fs was significantly associated with the infiltration of six immune cells.Conclusion: In this study, we found that E2F2, E2F3, and E2F4 have the potential to be novel prognostic biomarkers in CRC. The role of these E2F family members in disease pathology may be related to their functions in cell cycle regulation, therapeutic resistance, immune cell infiltration, and epithelia-to-mesenchymal transition. Further studies are required to validate our results; however, our findings may help provide a foundation for broadening our understanding of CRC pathology.Keywords: colorectal cancer, E2F, biomarkers, prognosis, bioinformatics

• Spt-Ada-Gcn5-Acetyltransferase (SAGA) and Transcription Factor IID (TFIID) differentially regulate the transcriptional activation of the P i transporter gene PHO84 in response to nutrient stress. • Target of Rapamycin (TOR) differentially regulates transcription activation of ribosomal protein (RP) genes and non-RP genes in response to nutrient stress. • Upon cellular stress, transcription factors like p53, CHOP, ATF6α, E2Fs, APC, (TGF)-β, MYC, and SMAD have been implicated in regulation of transcription of several target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. • Cellular level of arrested/paused RNA Polymerase II (RNA Pol II) during transcription in response to genotoxic stress is maintained via ubiquitin–proteasome system (UPS) • Upon metabolic stress, long noncoding RNAs (lncRNA) regulate transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans -regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans -regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress. [ABSTRACT FROM AUTHOR]

The bromodomain and extraterminal domain (BET) family proteins serve as primary readers of acetylated lysine residues and play crucial roles in cell proliferation and differentiation. Dysregulation of BET proteins has been implicated in tumorigenesis, making them important therapeutic targets. BET-bromodomain (BD) inhibitors and BET-targeting degraders have been developed to inhibit BET proteins. In this study, we found that the BET inhibitor MS645 exhibited superior antiproliferative activity than BET degraders including ARV771, AT1, MZ1 and dBET1 in triple-negative breast cancer (TNBC) cells. Treatment with MS645 led to the dissociation of BETs, MED1 and RNA polymerase II from the E2F1–3 promoter, resulting in the suppression of E2F1–3 transcription and subsequent inhibition of cell growth in TNBC. In contrast, while ARV771 displaced BET proteins from chromatin, it did not significantly alter E2F1–3 expression. Mechanistically, ARV771 induced BRD4 depletion at protein level, which markedly increased EGR1 expression. This elevation of EGR1 subsequently recruited septin 2 and septin 9 to E2F1–3 promoters, enhancing E2F1–3 transcription and promoting cell proliferation rate in vitro and in vivo. Our findings provide valuable insights into differential mechanisms of BET inhibition and highlight potential of developing BET-targeting molecules as therapeutic strategies for TNBC. [Display omitted] [ABSTRACT FROM AUTHOR]

The E2F transcription factor family, whose members are encoded by the E2F1-E2F8 genes, plays pivotal roles in the cell cycle, apoptosis, metabolism, stemness, metastasis, aging, angiogenesis, tumor promotion or suppression, and other biological processes. The activity of E2Fs is regulated at multiple levels, with posttranslational modifications being an important regulatory mechanism. There are numerous types of posttranslational modifications, among which phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, and poly(ADP-ribosyl)ation are the most commonly studied in the context of the E2F family. Posttranslational modifications of E2F family proteins regulate their biological activity, stability, localization, and interactions with other biomolecules, affecting cell proliferation, apoptosis, DNA damage, etc., and thereby playing roles in physiological and pathological processes. Notably, these modifications do not always act alone but rather form an interactive regulatory network. Currently, several drugs targeting posttranslational modifications are being studied or clinically applied, in which the proteolysis-targeting chimera and molecular glue can target E2Fs. This review aims to summarize the roles and regulatory mechanisms of different PTMs of E2F family members in the physiological state and in cancer and to briefly discuss their clinical significance and potential therapeutic use. [Display omitted] • Different posttranslational modifications of E2Fs mediated by various enzymes. • Roles and regulatory mechanisms of posttranslational modifications on E2Fs. • Crosstalk of various posttranslational modifications of E2Fs. • Potential value of E2F posttranslational modifications in cancer therapy. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Among the various molecular mechanisms implicated in osteosarcomagenesis, the RB-E2F pathway is of particular importance as virtually all cases of osteosarcoma display alterations in the RB-E2F pathway. In this study, we examined the transcription factor E2F family members that are associated with increased malignancy in Rb1-null osteosarcoma tumors. Using genetically engineered mouse models of osteosarcoma, we found that loss of activator E2Fs, E2F1 and E2F3, significantly delays tumor progression and increases the overall survival of the p53/Rb1-deficient osteosarcoma mouse model. We also studied the role of helicase, lymphoid specific (HELLS), a chromatin remodeling protein identified as a critical downstream effector of the RB-E2F signaling pathway in various cancers. In this study, we confirmed that the RB-E2F pathway directly regulates HELLS gene expression. We also found that HELLS mRNA is upregulated and its protein overexpressed in osteosarcoma. Using loss-of-function assays to study the role of HELLS in human osteosarcoma, we observed that HELLS has no effect on tumor proliferation and migration. Further, we pioneered the study of Hells in developmental tumor models by generating Hells conditional knockout osteosarcoma mouse models to examine the role of HELLS in osteosarcoma tumor development. We found that loss of Hells in osteosarcoma has no effect in tumor initiation and overall survival of mice. This suggests that while HELLS may serve as a biomarker for tumorigenesis and for RB-E2F pathway status, it is unlikely to serve as a relevant target for therapeutics in osteosarcoma.

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

Before a cell divides into two daughter cells, it typically doubles not only its DNA, but also its mass. Numerous studies in cells ranging from yeast to mammals have shown that cellular growth, stimulated by nutrients and/or growth factor signaling, is a prerequisite for cell cycle progression in most types of cells. The textbook view of growth‐regulated cell cycles is that growth signaling activates the transcription of G1 Cyclin genes to induce cell proliferation, and also stimulates anabolic metabolism and cell growth in parallel. However, genetic knockout tests in model organisms indicate that this is not the whole story, and new studies show that additional, "smarter" mechanisms help to coordinate the cell cycle with growth itself. Here we summarize recent advances in this field, and discuss current models in which growth signaling regulates cell proliferation by targeting core cell cycle regulators via non‐transcriptional mechanisms. [ABSTRACT FROM AUTHOR]

Simple Summary: E2F1 and E2F2 are highly expressed in many cancer types, but their contribution to malignancy is not well understood. Here we aimed to define the impact of E2F1/E2F2 deregulation in prostate cancer. We show that inhibition of E2F sensitizes prostate cancer cells to drug-induced replication stress and cell death. We found that E2F target genes involved in nucleotide biosynthesis contribute to maintaining genome stability in prostate cancer cells, but their enzymatic activity is insufficient to prevent replication stress after E2F1/E2F2 depletion. Instead, E2F1/E2F2 hinder premature CDK1 activation during S phase, which is key to ensure genome stability and viability of prostate cancer cells. From a therapeutic perspective, inhibiting E2F activity provokes catastrophic levels of replication stress and blunts xenograft growth in combination with drugs targeting nucleotide biosynthesis or DNA repair. Our results highlight the suitability of targeting E2F for the treatment of prostate cancer. E2F1/E2F2 expression correlates with malignancy in prostate cancer (PCa), but its functional significance remains unresolved. To define the mechanisms governed by E2F in PCa, we analyzed the contribution of E2F target genes to the control of genome integrity, and the impact of modulating E2F activity on PCa progression. We show that silencing or inhibiting E2F1/E2F2 induces DNA damage during S phase and potentiates 5-FU-induced replication stress and cellular toxicity. Inhibition of E2F downregulates the expression of E2F targets involved in nucleotide biosynthesis (TK1, DCK, TYMS), whose expression is upregulated by 5-FU. However, their enzymatic products failed to rescue DNA damage of E2F1/E2F2 knockdown cells, suggesting additional mechanisms for E2F function. Interestingly, targeting E2F1/E2F2 in PCa cells reduced WEE1 expression and resulted in premature CDK1 activation during S phase. Inhibition of CDK1/CDK2 prevented DNA damage induced by E2F loss, suggesting that E2F1/E2F2 safeguard genome integrity by restraining CDK1/CDK2 activity. Importantly, combined inhibition of E2F and ATR boosted replication stress and dramatically reduced tumorigenic capacity of PCa cells in xenografts. Collectively, inhibition of E2F in combination with drugs targeting nucleotide biosynthesis or DNA repair is a promising strategy to provoke catastrophic levels of replication stress that could be applied to PCa treatment. [ABSTRACT FROM AUTHOR]

In general, ensuring safety is the top priority of a new modality. Although oncolytic virus armed with an immune stimulatory transgene (OVI) showed some promise, the strategic concept of simultaneously achieving maximum effectiveness and minimizing side effects has not been fully explored. We generated a variety of survivin-responsive "conditionally replicating adenoviruses that can target and treat cancer cells with multiple factors (m-CRAs)" (Surv.m-CRAs) armed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene downstream of various promoters using our m-CRA platform technology. We carefully analyzed both therapeutic and adverse effects of them in the in vivo syngeneic Syrian hamster cancer models. Surprisingly, an intratumor injection of a conventional OVI, which expresses the GM-CSF gene under the constitutively and strongly active "cytomegalovirus enhancer and β-actin promoter", provoked systemic and lethal GM-CSF circulation and shortened overall survival (OS). In contrast, a new conceptual type of OVI, which expressed GM-CSF under the cancer-predominant and mildly active E2F promoter or the moderately active "Rous sarcoma virus long terminal repeat", not only abolished lethal adverse events but also prolonged OS and systemic anti-cancer immunity. Our study revealed a novel concept that optimal expression levels of an immune stimulatory transgene regulated by a suitable upstream promoter is crucial for achieving high safety and maximal therapeutic effects simultaneously in OVI therapy. These results pave the way for successful development of the next-generation OVI and alert researchers about possible problems with ongoing clinical trials., Competing Interests: Declaration of competing interest All authors have read the journal's policy on disclosure of potential conflicts of interest. The authors declare no competing conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Prostate cancer (PCa) is one of the most prevalent cancers in men, yet its pathogenic pathways remain poorly understood. Transcriptomics and high-throughput sequencing can help uncover cancer diagnostic targets and understand biological circuits. Using prostate adenocarcinoma (PRAD) datasets of various web-based applications (GEPIA, UALCAN, cBioPortal, SR Plot, hTFtarget, Genome Browser, and MetaCore), we found that upregulated dysbindin domain-containing 1 (DBNDD1) expression in primary prostate tumors was strongly correlated with pathways involving the cell cycle, mitotic in KEGG, WIKI, and REACTOME database, and transcription factor-binding sites with the DBNDD1 gene in prostate samples. DBNDD1 gene expression was influenced by sample type, cancer stage, and promoter methylation levels of different cancers, such as PRAD, liver hepatocellular carcinoma (LIHC), and lung adenocarcinoma (LUAD). Regulation of glycogen synthase kinase (GSK)-3β in bipolar disorder and ATP/ITP/GTP/XTP/TTP/CTP/UTP metabolic pathways was closely correlated with the DBNDD1 gene and its co-expressed genes in PCa. DBNDD1 gene expression was positively associated with immune infiltration of B cells, Myeloid-derived suppressor cell (MDSC), M2 macrophages, andneutrophil, whereas negatively correlated with CD8+ T cells, T follicular helper cells, M1 macrophages, and NK cells in PCa. These findings suggest that DBNDD1 may serve as a viable prognostic marker not only for early-stage PCa but also for immunotherapies.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB’s mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB’s cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

Gliomas portray a large and heterogeneous group of CNS tumors, encompassing a wide range of low- to high-grade tumors, as defined by histological and molecular characteristics. The identification of signature mutations and other molecular abnormalities has largely impacted tumor classification, diagnosis, and therapy. Transcription factors (TFs) are master regulators of gene expression programs, which ultimately shape cell fate and homeostasis. A variety of TFs have been detected to be aberrantly expressed in brain tumors, being highly implicated in critical pathological aspects and progression of gliomas. Herein, we describe a selection of oncogenic (GLI-1/2/3, E2F1–8, STAT3, and HIF-1/2) and tumor suppressor (NFI-A/B, TBXT, MYT1, and MYT1L) TFs that are deregulated in gliomas and are subsequently associated with tumor development, progression, and migratory potential. We further discuss the current targeting options against these TFs, including chemical (Bortezomib) and natural (Plumbagin) compounds, small molecules, and inhibitors, and address their potential implications in glioma therapy. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) hold the potential to solve the problem of the shortage of functional hepatocytes in clinical applications and drug development. However, a large number of usable hepatocytes derived from hESCs cannot be effectively obtained due to the limited proliferation capacity. In this study, we found that enhancement of liver transcription factor C/EBPβ during hepatic differentiation could not only significantly promote the expression of hepatic genes, such as albumin, alpha fetoprotein, and alpha-1 antitrypsin, but also dramatically reinforce proliferation-related phenotypes, including increasing the expression of proliferative genes, such as CDC25C, CDC45L, and PCNA, and the activation of cell cycle and DNA replication pathways. In addition, the analysis of CUT&Tag sequencing further revealed that C/EBPβ is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBPβ and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBPβ could significantly inhibit the expression of the aforementioned proliferative genes. RNA transcriptome analysis and GSEA enrichment indicated that the E2F family was enriched, and the expression of E2F2 was changed with the overexpression or knockdown of C/EBPβ. Moreover, the results of CUT&Tag sequencing showed that C/EBPβ also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBPβ and E2F2 proteins, and this interaction between these two proteins was also verified in the LO2 cell line, a hepatic progenitor cell line. Thus, our results demonstrated that C/EBPβ first initiated E2F2 expression and then coupled with E2F2 to regulate the expression of proliferative genes in hepatocytes during the differentiation of hESCs. Therefore, our findings open a new avenue to provide an in vitro efficient approach to generate proliferative hepatocytes to potentially meet the demands for use in cell-based therapeutics as well as for pharmaceutical and toxicological studies.

Density-dependent regulation of cell growth is presumed to be caused by cell-cell contact, but the underlying molecular mechanism is not yet clearly defined. Here, we report that receptor-type protein tyrosine phosphatase-kappa (R-PTP-κ) is an important regulator of cell contact-dependent growth inhibition. R-PTP-κ expression increased in proportion to cell density. siRNA-mediated R-PTP-κ downregulation led to the loss of cell contact-mediated growth inhibition, whereas its upregulation reduced anchorage-independent cell growth in soft agar as well as tumor growth in nude mice. Expression profiling and luciferase reporter system-mediated signaling pathway analysis revealed that R-PTP-κ induced under cell contact conditions distinctly suppressed E2F activity. Among the structural domains of R-PTP-κ, the cytoplasmic domain containing the tandemly repeated PTP motif acts as a potent downregulator of the E2F pathway. Specifically, R-PTP-κ suppressed CDK2 activity through the induction of p21Cip1/WAF-1 and p27Kip1, resulting in cell cycle arrest at the G1 phase. In transcriptome-based public datasets generated from four different tumor types, R-PTP-κ expression was negatively correlated with the expression pattern and prognostic value of two known E2F1 target genes (CCNE1 and CDC25A). Therefore, our results indicate that the R-PTP-κ-E2F axis plays a crucial role in cell growth-inhibitory signaling arising from cell-cell contact conditions.

Perfectly orchestrated periodic gene expression during cell cycle progression is essential for maintaining genome integrity and ensuring that cell proliferation can be stopped by environmental signals. Genetic and proteomic studies during the past two decades revealed remarkable evolutionary conservation of the key mechanisms that control cell cycle–regulated gene expression, including multisubunit DNA-binding DREAM complexes. DREAM complexes containing a retinoblastoma family member, an E2F transcription factor and its dimerization partner, and five proteins related to products of Caenorhabditis elegans multivulva (Muv) class B genes lin-9, lin-37, lin-52, lin-53, and lin-54 (comprising the MuvB core) have been described in diverse organisms, from worms to humans. This review summarizes the current knowledge of the structure, function, and regulation of DREAM complexes in different organisms, as well as the role of DREAM in human disease. [ABSTRACT FROM AUTHOR]