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The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs. Cancer stem cells (CSCs) are associated with chemoresistance and poor prognosis in multiple cancer types. Here, the authors investigate the role of secreted Wnt ligands in pancreatic and breast CSCs and identify E2F1/4-GCN5-pRb/RBL2 as a regulatory axis underlying Wnt secretion.

Multiciliated cells (MCCs) play a crucial role in various physiological processes, including cerebrospinal fluid flow, mucus clearance, and reproductive transport, by coordinating ciliary movement. Their differentiation is regulated by the Notch signaling pathway, along with its downstream targets, Gemc1 and Mcidas transcription factors. This study focuses on Zmynd10, a dynein axonemal assembly factor, to investigate its molecular mechanisms that regulate polycilia differentiation. By constructing a model of Zmynd10-specific knockdown in mouse ependymal cells (mEPCs), we found that Zmynd10 knockdown resulted in a decrease in ciliary density and significantly downregulated the mRNA and protein expression of E2f4 and Deup1. Further experiments demonstrated that E2f4 knockdown inhibited Deup1 expression and reduced cilia numbers, while Zmynd10 regulated the E2f4-Deup1 axis by activating the E2f4 promoter. This study reveals for the first time that Zmynd10 drives centriole amplification through the transcriptional regulation of the E2f4-Deup1 pathway, providing new insights into the molecular mechanisms underlying multicilia differentiation.

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G1-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers.

Esophageal cancer (EC) is driven by complex dysregulated molecular networks, and ferroptosis—an iron‐dependent, non‐apoptotic form of regulated cell death—has emerged as a critical modulator of tumorigenesis. However, the functional contribution and mechanistic basis of GPR176 in ferroptosis regulation during EC progression remain largely unexplored. Here, we integrated computational and experimental approaches to delineate the role of GPR176 and its upstream regulator E2F4 in EC ferroptosis. Bioinformatic analysis revealed consistent upregulation of both GPR176 and E2F4 in EC tissues, which was further confirmed by molecular validation. Functional assays demonstrated that GPR176 overexpression conferred resistance to ferroptosis in EC cells, as reflected by reduced malondialdehyde, intracellular Fe2+, and lipid reactive oxygen species (ROS) accumulation, alongside altered expression of core ferroptosis mediators. This protective effect was associated with the suppression of mitophagy, as indicated by alterations in mitochondrial function and autophagy‐related markers. Mechanistically, we demonstrated that E2F4 directly binds to the GPR176 promoter and transcriptionally activates its expression. Rescue experiments further validated that GPR176 overexpression abrogated the enhanced mitophagy and ferroptosis induced by E2F4 depletion. Collectively, our findings define an E2F4/GPR176/mitophagy axis that acts to suppress ferroptosis in EC, highlighting this pathway as a novel therapeutic target for inducing ferroptosis in EC intervention.

Background Epithelial ovarian cancer (EOC) is the malignant tumor of the female reproductive system with the highest fatality rate. Tolerance of chemotherapeutic drugs like cisplatin (DDP) occurring in very early stage is one of the important factors of the poor prognosis of epithelial ovarian cancer. Here we aim to study the dysregulation of a particular long noncoding RNA, lncRNA GAS5, and its role in EOC progression. Methods The low expression of lncRNA GAS5 in EOC tissues and OC cell lines was determined by microarray analyses and Real-Time qPCR. Flow cytometer assays were used to detect cell cycle and apoptosis of OC cells. CCK8 assay were performed to investigate the DDP sensitivity of OC cells. Western blot was carried out to detect cell growth markers, apoptotic markers, PARP1, E2F4, MAPK pathway protein expression and other protein expression in OC cell lines. The binding of GAS5 and E2F4 were proved by RNA pull-down and RIP assay. The effect of E2F4 on PARP1 were determined by CHIP-qPCR assay and luciferase reporter assay. The effect of lncRNA GAS5 on OC cells was assessed in vitro and in vivo. Results By microarray (3 EOC tissues νs. 3 normal ovary tissues) and RT- qPCR (53 EOC tissues νs. 10 normal ovary tissues) we identified lncRNA GAS5 to be dramatically low expressed in EOC samples and correlated with prognosis. Compared with sensitive cell lines, GAS5 was also low expressed in DDP resistant OC cell lines, and over-expression of GAS5 significantly enhanced the sensitivity of OC cells to DDP in vivo and in vitro. Meanwhile the over-expression of GAS5 also caused OC cells G0/G1 arrest and apoptosis increase. Mechanistically, GAS5 might regulate PARP1 expression by recruiting the transcription factor E2F4 to its promoter, and then affect the MAPK pathway activity. Due to the 5’TOP structure, GAS5 could be regulated by transcription inhibitor rapamycin in OC cells. Conclusion Here we explored the specific mechanisms of EOC cisplatin resistance and tumor progress due to lncRNA-GAS5, presented the GAS5-E2F4-PARP1-MAPK axis and its role in OC drug-sensitivity and progression for the first time, and the results may provide experimental basis for clinical application.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells. E2F transcription factors are regulators of cell division and cell fate decisions. Here the authors show that E2F4 is important for proliferation and survival of mouse ESCs, independent of the RB family, and that E2F4 interacts with chromatin regulators associated with gene activation.

Hematopoiesis is a continuous process of blood cell production driven by hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Proliferation and differentiation of HSPCs are regulated by complex transcriptional networks. In order to identify transcription factors with key roles in HSPC-mediated hematopoietic reconstitution, we developed an efficient and robust CRISPR/Cas9-based in vivo genetic screen. Using this experimental system, we identified the TFDP1 transcription factor to be essential for HSPC proliferation and post-transplant hematopoiesis. We further discovered that E2F4, an E2F transcription factor, serves as a binding partner of TFDP1 and is required for HSPC proliferation. Deletion of TFDP1 caused downregulation of genes associated with the cell cycle, with around 50% of these genes being identified as direct targets of TFDP1 and E2F4. Thus, our study expands the transcriptional network governing hematopoietic development through an in vivo CRISPR/Cas9-based genetic screen and identifies TFDP1/E2F4 as positive regulators of cell cycle genes in HSPCs.

Aberrant expression of long noncoding RNA GAS5 in bladder cancer (BC) cells was identified in recent studies. However, the regulatory functions and underlying molecular mechanisms of GAS5 in BC development remain unclear. Here, we confirmed that there was a negative correlation between GAS5 level and bladder tumor clinical stage. Functionally, overexpression of GAS5 reduced cell viability and induced cell apoptosis in T24 and EJ bladder cancer cells. Mechanistically, GAS5 effectively repressed EZH2 transcription by directly interacting with E2F4 and recruiting E2F4 to EZH2 promoter. We previously reported that miR-101 induced the apoptosis of BC cells by inhibiting the expression of EZH2. Interestingly, the present study showed that downregulation of EZH2 by GAS5 resulted in overexpression of miR-101 in T24 and EJ cells. Furthermore, the level of GAS5 was increased under the treatment of Gambogic acid (GA), a promising natural anti-cancer compound, whereas knockdown of GAS5 suppressed the inhibitory effect of GA on cell viability and abolished GA-induced apoptosis in T24 and EJ cells. Taken together, our findings demonstrated a tumor-suppressor role of GAS5 by inhibiting EZH2 on transcriptional level, and additionally provided a novel therapeutic strategy for treating human bladder cancer.

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

SUMOylation plays a crucial role in numerous cellular biological and pathophysiological processes associated with human disease; however, the mechanisms regulating the genes involved in SUMOylation remain unclear. In the present study, E2F transcription factor 4 (E2F4) was identified as an E2F member related to hepatocellular carcinoma (HCC) progression by public database analysis. It was found that E2F4 promoted the proliferation and invasiveness of HCC cells via SUMOylation using Soft agar and Transwell migration assays. Mechanistically, it was demonstrated that E2F4 upregulated the transcript and protein expression levels of baculoviral IAP repeat containing 5, cell division cycle associated 8 and DNA topoisomerase II α using western blotting. Furthermore, the interaction between E2F4 with lin-9 DREAM multi-vulva class B core complex component (LIN9) was explored by co-immunoprecipitation, immunofluorescence co-localization and bimolecular fluorescence complementation assays. Moreover, it was demonstrated that E2F4 promoted the progression of HCC cells via LIN9. Rescue experiments revealed that LIN9 facilitated the SUMOylation and proliferation of HCC cells, which was prevented by knocking down E2F4 expression. In conclusion, the findings of the present study indicated that E2F4 plays a major role in the proliferation of HCC cells and may be a potential therapeutic target in the future.