Prevotella copri exhausts intrinsic indole-3-pyruvic acid in the host to promote breast cancer progression: inactivation of AMPK via UHRF1-mediated negative regulation.

Emerging evidence has revealed the novel role of gut microbiota in the development of cancer. The characteristics of function and composition in the gut microbiota of patients with breast cancer patients has been reported, however the detailed causation between gut microbiota and breast cancer remains uncertain. In the present study, 16S rRNA sequencing revealed that Prevotella, particularly the dominant species Prevotella copri, is significantly enriched and prevalent in gut microbiota of breast cancer patients. Prior-oral administration of P. copri could promote breast cancer growth in specific pathogen-free mice and germ-free mice, accompanied with sharp reduction of indole-3-pyruvic acid (IPyA). Mechanistically, the present of excessive P. copri consumed a large amount of tryptophan (Trp), thus hampering the physiological accumulation of IPyA in the host. Our results revealed that IPyA is an intrinsic anti-cancer reagent in the host at physiological level. Briefly, IPyA directly suppressed the transcription of UHRF1, following by the declined UHRF1 and PP2A C in nucleus, thus inhibiting the phosphorylation of AMPK, which is just opposite to the cancer promoting effect of P. copri. Therefore, the exhaustion of IPyA by excessive P. copri strengthens the UHRF1-mediated negative control to inactivated the energy-controlling AMPK signaling pathway to promote tumor growth, which was indicated by the alternation in pattern of protein expression and DNA methylation. Our findings, for the first time, highlighted P. copri as a risk factor for the progression of breast cancer.

Roles of post-translational modifications of UHRF1 in cancer.

UHRF1 as a member of RING-finger type E3 ubiquitin ligases family, is an epigenetic regulator with five structural domains. It has been involved in the regulation of a series of biological functions, such as DNA replication, DNA methylation, and DNA damage repair. Additionally, aberrant overexpression of UHRF1 has been observed in over ten cancer types, indicating that UHRF1 is a typical oncogene. The overexpression of UHRF1 repressed the transcription of such tumor-suppressor genes as CDKN2A, BRCA1, and CDH1 through DNMT1-mediated DNA methylation. In addition to the upstream transcription factors regulating gene transcription, post-translational modifications (PTMs) also contribute to abnormal overexpression of UHRF1 in cancerous tissues. The types of PTM include phosphorylation, acetylation, methylationand ubiquitination, which regulate protein stability, histone methyltransferase activity, intracellular localization and the interaction with binding partners. Recently, several novel PTM types of UHRF1 have been reported, but the detailed mechanisms remain unclear. This comprehensive review summarized the types of UHRF1 PTMs, as well as their biological functions. A deep understanding of these crucial mechanisms of UHRF1 is pivotal for the development of novel UHRF1-targeted anti-cancer therapeutic strategies in the future.

Unveiling dissociation mechanisms and binding patterns in the UHRF1-DPPA3 complex via multi-replica molecular dynamics simulations.

CONTEXT: Ubiquitin-like with PHD and RING finger domain containing protein 1 (UHRF1) is responsible for preserving the stability of genomic methylation through the recruitment of DNA methyltransferase 1 (DNMT1). However, the interaction between Developmental pluripotency associated 3 (DPPA3) and the pre-PHD-PHD (PPHD) domain of UHRF1 hinders the nuclear localization of UHRF1. This disruption has implications for potential cancer treatment strategies. Drugs that mimic the binding pattern between DPPA3 and PPHD could offer a promising approach to cancer treatment. Our study reveals that DPPA3 undergoes dissociation from the C-terminal through three different modes of helix unfolding. Furthermore, we have identified key residue pairs involved in this dissociation process and potential drug-targeting residues. These findings offer valuable insights into the dissociation mechanism of DPPA3 from PPHD and have the potential to inform the design of novel drugs targeting UHRF1 for cancer therapy. METHODS: To comprehend the dissociation process and binding patterns of PPHD-DPPA3, we employed enhanced sampling techniques, including steered molecular dynamics (SMD) and conventional molecular dynamics (cMD). Additionally, we utilized self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. The Gromacs software was used for performing molecular dynamics simulations, and the AMBER FF14SB force field was applied to the protein.

CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids.

KEY MESSAGE: Two peanut LEC1-type genes exhibit partial functional redundancy. AhNFYB10 could complement almost all the defective phenotypes of lec1-2 in terms of embryonic morphology, while AhNF-YB1 could partially affect these phenotypes. LEAFY COTYLEDON1 (LEC1) is a member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two LEC1-type genes from Arachis hypogeae were identified and designated as AhNF-YB1 and AhNF-YB10; these genes belong to subgenome A and subgenome B, respectively. The functions of AhNF-YB1 and AhNF-YB10 were investigated by complementation analysis of their defective phenotypes of the Arabidopsis lec1-2 mutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, AhNF-YB10 complemented almost all the defective phenotypes of lec1-2 in terms of embryonic morphology and hypocotyl length, while AhNF-YB1 had only a partial effect. In addition, 30-40% of the seeds of the AhNF-YB1 transformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing AhNF-YB10-overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content.

YTHDF1 promotes gallbladder cancer progression via post-transcriptional regulation of the m6A/UHRF1 axis.

Gallbladder cancer is a rare but fatal malignancy. However, the mechanisms underlying gallbladder carcinogenesis and its progression are poorly understood. The function of m6A modification and its regulators was still unclear for gallbladder cancer. The current study seeks to investigate the function of YTH m6A RNA-binding protein 1 (YTHDF1) in gallbladder cancer. Transcriptomic analysis and immunochemical staining of YTHDF1 in gallbladder cancer tissues revealed its upregulation compared to paracancerous tissues. Moreover, YTHDF1 promotes the proliferation assays, Transwell migration assays, and Transwell invasion assays of gallbladder cancer cells in vitro. And it also increased tumour growth in xenograft mouse model and metastases in tail vein injection model in vivo. In vitro, UHRF1 knockdown partly reversed the effects of YTHDF1 overexpression. Mechanistically, dual-luciferase assays proved that YTHDF1 promotes UHRF1 expression via direct binding to the mRNA 3'-UTR in a m6A-dependent manner. Overexpression of YTHDF1 enhanced UHRF1 mRNA stability, as demonstrated by mRNA stability assays, and Co-IP studies confirmed a direct interaction between YTHDF1 and PABPC1. Collectively, these findings provide new insights into the progression of gallbladder cancer as well as a novel post-transcriptional mechanism of YTHDF1 via stabilizing target mRNA.

UHRF1 poly-auto-ubiquitination induced by the anti-cancer drug, thymoquinone, is involved in the DNA repair machinery recruitment.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates atherosclerosis in mice via regulating the SMAD7/YAP1 axis.

BACKGROUND: Endothelial cell injury and dysfunction lead to cholesterol and lipid accumulation and atherosclerotic plaque formation in the arterial wall during atherosclerosis (AS) progression, Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), a DNA methylation regulator, was strongly upregulated in atherosclerotic plaque lesions in mice. This study aimed to investigate the precise biological functions and regulatory mechanisms of UHRF1 on endothelial dysfunction during AS development. METHODS: UHRF1 levels in the atherosclerotic plaque tissues and normal arterial intima from AS patients were tested with Western blot analysis and immunohistochemistry assays. Human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce an injury model and then transfected with short hairpin RNA targeting UHRF1 (sh-UHRF1). Cell proliferation, migration, apoptosis, the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the protein levels adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured. Moreover, co-immunoprecipitation assay was used to determine the interactions between UHRF1 and DNA methyltransferases 1 (DNMT1), As well as mothers against DPP homolog 7 (SMAD7) and yes-associated protein 1 (YAP1). SMAD7 promoter methylation was examined with methylation-specific PCR. In addition, we established an AS mouse model to determine the in vivo effects of UHRF1 on AS progression. RESULTS: UHRF1 was upregulated in atherosclerotic plaque tissues and ox-LDL-treated HUVECs. UHRF1 knockdown mitigated ox-LDL-induced proliferation and migration inhibition, apoptosis and the production of TNF-α, IL-6, VCAM-1, and ICAM-1 in HUVECs. Mechanistically, UHRF1 promoted DNMT1-mediated SMAD7 promoter methylation and inhibited its expression. SMAD7 knockdown abolished the protective effects of UHRF1 knockdown on ox-LDL-induced HUVEC injury. Moreover, SMAD7 interacted with YAP1 and inhibited YAP1 expression by promoting YAP1 protein ubiquitination-independent degradation in HUVECs. YAP1 overexpression abrogated SMAD7 overexpression-mediated protective effects on ox-LDL-induced HUVEC injury. Finally, UHRF1 knockdown alleviated atherosclerotic plaque deposition and arterial lesions in AS mice. CONCLUSION: UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates AS progression in mice by regulating the SMAD7/YAP1 axis.

Wnt/ß-catenin signaling regulates amino acid metabolism through the suppression of CEBPA and FOXA1 in liver cancer cells.

Deregulation of the Wnt/ß-catenin pathway is associated with the development of human cancer including colorectal and liver cancer. Although we previously showed that histidine ammonia lyase (HAL) was transcriptionally reduced by the ß-catenin/TCF complex in liver cancer cells, the mechanism(s) of its down-regulation by the complex remain to be clarified. In this study, we search for the transcription factor(s) regulating HAL, and identify CEBPA and FOXA1, two factors whose expression is suppressed by the knockdown of ß-catenin or TCF7L2. In addition, RNA-seq analysis coupled with genome-wide mapping of CEBPA- and FOXA1-binding regions reveals that these two factors also increase the expression of arginase 1 (ARG1) that catalyzes the hydrolysis of arginine. Metabolome analysis discloses that activated Wnt signaling augments intracellular concentrations of histidine and arginine, and that the signal also increases the level of lactic acid suggesting the induction of the Warburg effect in liver cancer cells. Further analysis reveals that the levels of metabolites of the urea cycle and genes coding its related enzymes are also modulated by the Wnt signaling. These findings shed light on the altered cellular metabolism in the liver by the Wnt/ß-catenin pathway through the suppression of liver-enriched transcription factors including CEBPA and FOXA1.

Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

Inhibition of UHRF1 Improves Motor Function in Mice with Spinal Cord Injury.

Spinal-cord injury (SCI) is a severe condition that can lead to limb paralysis and motor dysfunction, and its pathogenesis is not fully understood. The objective of this study was to characterize the differential gene expression and molecular mechanisms in the spinal cord of mice three days after spinal cord injury. By analyzing RNA sequencing data, we identified differentially expressed genes and discovered that the immune system and various metabolic processes play crucial roles in SCI. Additionally, we identified UHRF1 as a key gene that plays a significant role in SCI and found that SCI can be improved by suppressing UHRF1. These findings provide important insights into the molecular mechanisms of SCI and identify potential therapeutic targets that could greatly contribute to the development of new treatment strategies for SCI.

Alterations of UHRF family Expression and was regulated by High Risk Type HPV16 in Uterine Cervical Cancer.

The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1), and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. In this study, we analyzed the UHRF family expression in cervical cancers, and it's regulation by human papillomavirus (HPV). Western blotting was performed to analyze protein expression in cervical cancer cell lines. Immunohistochemical analysis were used to investigate the expression of UHRF family and MIB-1 in cervical cancer tissues. Transfection were done for analyze the relationship between UHRF family and HPVs. We showed that NIRF expression was decreased and ICBP90 expression was increased in cervical cancers compared to normal counterparts. Western blotting also showed that NIRF expression was quite low levels, but ICBP90 was high in human cervical cancer cell lines. Interestingly, ICBP90 was up regulated by high risk type HPV16 E6 and E7, but not low-risk type HPV11. On the other hand, NIRF was down regulated by high risk type HPV16 E6 but not by E7. Low risk type HPV11 E6 did not affect the NIRF expression at all. We propose that ICBP90 overexpression, and reduced NIRF expression, found in cervical cancers, is an important event of a cervical carcinogenesis, and especially ICBP90 may offer a proliferating marker and therapeutic target for treating uterine cervical cancers.

MOF-mediated acetylation of UHRF1 enhances UHRF1 E3 ligase activity to facilitate DNA methylation maintenance.

The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (males absent on the first) acetylates UHRF1 at K670 in the pre-RING linker region, whereas HDAC1 deacetylates UHRF1 at the same site. We also identify that K667 and K668 can also be acetylated by MOF when K670 is mutated. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity. Abolishing UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs DNMT1 recruitment and DNA methylation. Taken together, these findings identify MOF as an acetyltransferase for UHRF1 and define a mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

Structural basis for specific DNA sequence recognition by the transcription factor NFIL3.

The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system, and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/ß demonstrate a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain result in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis.

SCUBE3 Exerts a Tumor-Promoting Effect in Tongue Squamous Cell Carcinoma by Promoting CEBPA Binding to the CCL2 Promoter.

Tongue squamous cell carcinoma (TSCC) is the main pathologic subtype of oral cancer, and the current therapeutic effect is far from satisfactory. The signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) has been shown to be a tumor-promoting factor in several malignancies. However, little is known about the role of SCUBE3 in TSCC. In this study, we identified that SCUBE3 was highly expressed in TSCC. Clinically, high expression of SCUBE3 was positively associated with tumor stage and T stage of TSCC. Functionally, SCUBE3 silence remarkably restrained cell proliferation, migration, and invasion, induced apoptosis as well as cell cycle arrest in G2-phase, and weakened the tumorigenicity of TSCC cells in vivo. Mechanistically, SCUBE3 promoted the direct binding of CCAAT enhancer binding protein alpha (CEBPA) to C-C motif chemokine ligand 2 (CCL2) promoter in TSCC cells. Interestingly, CCL2 overexpression partially reversed the inhibitory effect of SCUBE3 deficiency on TSCC cell viability and migration. Moreover, STAT3 signaling contributed to CCL2-mediated phenotypes in TSCC cells. IMPLICATIONS: Our data revealed a tumor-promoting role for SCUBE3 in TSCC via the CEBPA/CCL2/STAT3 axis, which provided new insight into novel potential therapeutic target for TSCC.

Cebp1 and Cebpß transcriptional axis controls eosinophilopoiesis in zebrafish.

Eosinophils are a group of granulocytes well known for their capacity to protect the host from parasites and regulate immune function. Diverse biological roles for eosinophils have been increasingly identified, but the developmental pattern and regulation of the eosinophil lineage remain largely unknown. Herein, we utilize the zebrafish model to analyze eosinophilic cell differentiation, distribution, and regulation. By identifying eslec as an eosinophil lineage-specific marker, we establish a Tg(eslec:eGFP) reporter line, which specifically labeled cells of the eosinophil lineage from early life through adulthood. Spatial-temporal analysis of eslec+ cells demonstrates their organ distribution from larval stage to adulthood. By single-cell RNA-Seq analysis, we decipher the eosinophil lineage cells from lineage-committed progenitors to mature eosinophils. Through further genetic analysis, we demonstrate the role of Cebp1 in balancing neutrophil and eosinophil lineages, and a Cebp1-Cebpß transcriptional axis that regulates the commitment and differentiation of the eosinophil lineage. Cross-species functional comparisons reveals that zebrafish Cebp1 is the functional orthologue of human C/EBPεP27 in suppressing eosinophilopoiesis. Our study characterizes eosinophil development in multiple dimensions including spatial-temporal patterns, expression profiles, and genetic regulators, providing for a better understanding of eosinophilopoiesis.

Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma.

BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.

The dopamine receptor D1 inhibitor, SKF83566, suppresses GBM stemness and invasion through the DRD1-c-Myc-UHRF1 interactions.

BACKGROUND: Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. METHODS: GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. RESULTS: Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. CONCLUSIONS: DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

Circ ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 increases the stability of G9a and ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 messenger RNA through recruiting eukaryotic translation initiation factor 4A3, transcriptionally inhibiting PDZ and homeobox protein domain protein 1, and promotes the metastasis of hepatocellular carcinoma.

BACKGROUND AND AIM: Circular ubiquitin-like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1-G9a-ubiquitin-like, containing PHD and ring finger domains 1 (UHRF1) mRNA-eukaryotic translation initiation factor 4A3 (EIF4A3)-PDZ and LIM domain 1 (PDLIM1) network in HCC. METHODS: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial-mesenchymal transition (EMT)-related proteins, and Hippo-Yap pathway components was determined by quantitative polymerase chain reaction (Q-PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit-8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull-down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co-immunoprecipitation was performed to examine the binding between UHRF1 and G9a. RESULTS: Circular ubiquitin-like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription. CONCLUSION: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo-Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1-G9a-UHRF1 mRNA-EIF4A3-PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment.