Cell Cycle Signaling Research Articles

Nitidine chloride inhibits G2/M phase by regulating the p53/14-3-3 Sigma/CDK1 axis for hepatocellular carcinoma treatment

BackgroundLiver cancer had become the sixth most common cancer. Nitidine chloride (NC) has demonstrated promising anti-HCC properties; however, further elucidation of its mechanism of action is necessary. MethodsThe anti-HCC targets of NC were identified through the utilization of multiple databases and ChIPs data analysis. The GO and KEGG analyses to determine the specific pathway affected by NC. The Huh 7 and Hep G2 cells were subjected to a 24-h treatment with NC, followed by evaluating the impact of NC on cell proliferation and cell cycle. The involvement of the p53/14-3-3 Sigma/CDK1 axis in HCC cells was confirmed by qPCR and WB analysis of the corresponding genes and proteins. ResultsThe GO and KEGG analysis showed the targets were related to cell cycle and p53 signaling pathways. In vitro experiments showed that NC significantly inhibited the proliferation of HCC cells and induced G2/M phase arrest. In addition, qPCR and WB experiments showed that the expression of p53 in HCC cells increased after NC intervention, while the expression of 14-3-3 Sigma and CDK1 decreased. ConclusionNC can inhibit the proliferation of HCC cells and induce G2/M cell cycle arrest, potentially by regulating the p53/14-3-3 Sigma/CDK1 axis.

The Correlation of Centromere Protein Q with Diagnosis and Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the major types of liver cancer. Previous studies have shown that the centromere protein family is associated with malignant biological behaviors such as HCC proliferation. As a member of the centromere protein family, centromere protein Q (CENPQ) is closely associated with immunotherapy and immune cell infiltration in various tumors. However, the role and mechanism of CENPQ in HCC remain unclear. Multiple public databases and RT-qPCR were used to study the expression of CENPQ in HCC. Based on TCGA data, the correlation between CENPQ and clinicopathological characteristics and prognosis of HCC patients was analyzed, and its diagnostic value was evaluated. The potential biological functions of CENPQ in HCC were explored by functional enrichment analysis of differentially expressed genes. The distribution of tumor-infiltrating immune cell types was assessed using single-sample GSEA, and immune checkpoint gene expression was analyzed using Spearman correlation. Subsequently, loss-of-function experiments were performed to determine the function of CENPQ on the cell cycle and proliferation of HCC cells in vitro. CENPQ was found highly expressed in HCC and correlated with weight, BMI, age, AFP, T stage, pathologic stage, histologic grade, and prothrombin time (all p < 0.05). ROC and Kaplan-Meier analyses indicated that CENPQ may be potentially used as a diagnostic marker for HCC (AUC = 0.881), and its upregulation is associated with decreased OS (p = 0.002), DSS (p < 0.001), and PFI (p = 0.002). Functional enrichment analysis revealed an association of CENPQ with biological processes such as immune cell infiltration, cell cycle, and hippo-merlin signaling deregulation in HCC. Furthermore, knockdown of CENPQ manifested in HCC cells with G0/1 phase cycle arrest and decreased proliferative capacity. CENPQ expression was higher in HCC tissues than in normal liver tissues. It was significantly associated with poor prognosis, immune cell infiltration, cell cycle, and proliferation. Therefore, CENPQ may become a promising prognostic biomarker for HCC patients.

ACOX2 Serves as a Favorable Indicator Related to Lipid Metabolism and Oxidative Stress for Biochemical Recurrence in Prostate Cancer.

Given the heterogeneity of tumors, there is an urgent need for accurate prognostic parameters in prostate cancer (PCa) patients. Lipid metabolism (LM) reprogramming and oxidative stress (OS) play a vital role in the progression of PCa. In this work, we identified five LM-OS-related genes (including ACOX2, PPRAGC1A, PTGS1, PTGS2, and HAO1) associated with the biochemical recurrence (BCR) of PCa. Subsequently, a prognostic signature was established based on these five genes. Kaplan-Meier survival estimates, receiver operating characteristic curves, and relationship analysis between risk score and clinical characters were applied to measure the robustness of the signature in an external cohort. A nomogram of risk score combined with clinical characteristics was constructed for clinical application. Functional enrichment analysis suggested that the underlying mechanism related to the signature included the calcium signaling, lipid transport, and cell cycle signaling pathways. Furthermore, WEE1 inhibitor was identified as a potential agent related to the cell cycle for high-risk patients. The mRNA expression and the prognostic value of the five genes were determined, and ACOX2 was identified as the key gene related to the prognostic signature. The protein expression of ACOX2 was measured in a prostate tissue microarray through an immunohistochemistry assay, confirming the bioinformatics results. By constructing the ACOX2-overexpressing PCa cell lines PC-3 and 22Rv1, the biological function of PCa cells was investigated. The cell viability, colony formation, migration, and invasion ability of PCa cell lines overexpressing ACOX2 were hindered. Decreased cellular lipid content and elevated cellular ROS content were observed in ACOX2-overexpressing PCa cell lines with reduced G2/M phases. In conclusion, this work presents the first prognostic signature specifically focused on LM-OS for PCa. ACOX2 could serve as a favorable indicator for the BCR in PCa. Further experiments are required to identify the potential underlying mechanism.

Comprehensive Analysis of the Prognostic Implications and Biological Function of HDACs in Liver Hepatocellular Carcinoma.

Background: The prognostic significance and biological functions of the histone deacetylases (HDACs) gene family in liver hepatocellular carcinoma (LIHC) have not been fully investigated. Methods: Using Kaplan-Meier and Cox regression analysis, this study determined if HDAC genes were relevant for prognosis in LIHC. A regression model utilizing HDAC genes and the least absolute shrinkage and selection operator (LASSO) was created to foretell LIHC risk. A selective inhibitor of endogenous HDACs, CKD-581, was studied in vitro and in vivo to determine its effects on the development, invasion, migration, and proliferation of LIHC cell lines. Results: Six HDACs were identified as correlating with the prognosis of LIHC. Overall survival (OS) was found to be shorter in individuals with higher risk scores when compared to those with lower risk scores, according to survival study. Natural killer cell infiltration was higher in individuals with lower risk ratings, which was mainly explained by the type II interferon (IFN) response. Limiting the activity of endogenous HDACs caused LIHC cell death by preventing their migration, invasion, and proliferation. In vivo studies confirmed that blocking HDAC expression inhibited tumor growth in mice. Further mechanistic studies showed that inhibition of HDACs expression elevates the protein levels of P21 and P27, and reduces those of cyclins A2, B1, D1 and E1. Conclusions: The risk score prognostic model based on HDAC genes could provide a valuable prognostic biomarker for LIHC. CKD-581 prohibits LIHC progression via inhibiting the cell cycle signaling pathway. CKD-581 holds promise as a therapeutic agent for the clinical management of LIHC.

Bladder Cancer Patients with Elevated SPRR1B Expression Experiencing a Poor Prognosis.

SPRR1B, a member of the small proline-rich protein family, is implicated in various epithelial cancers as a potential oncogene linked to tumour growth and poor survival outcomes. However, its role in urothelial bladder carcinoma (UBC) remains to be fully elucidated. Transcriptional profiling data from The Cancer Genome Atlas grouped UBC samples in accordance with SPRR1B expression. Bioinformatic analysis was conducted to evaluate whether SPRR1B is a prognostic factor and a survival factor in UBC. Gene set enrichment analysis (GSEA) was performed to study immune cells and pathways. Reverse transcription quantitative real-time polymerase chain reaction detected gene expression. Immunohistochemistry assessed protein expression. Spearman correlation test analysed the correlation between SPRR1B and the protein p53. The bioinformatics results indicated that the expression level of SPRR1B in UBC tissues was significantly increased compared with that in normal bladder tissues, correlating with clinical characteristics. A high expression predicted poor prognosis and survival. Univariate Cox statistics showed that a high expression level of SPRR1B was correlated with UBC patients having poor overall survival (OS) (p < 0.05). In addition, on the basis of the multivariate Cox analysis, SPRR1B expression was independently correlated with OS (p = 0.005). GSEA analysis revealed enrichment in the p53, apoptosis, and cell cycle signalling pathways, and an association with B cells, lymphocytes, and natural killer cells. In addition, SPRR1B was found to be associated with immune infiltration based on the analysis of immune cell infiltration. Performing corresponding verification on a small number of tissues collected from bladder cancer patients revealed that the expression of this protein was negatively correlated with the expression of p53. SPRR1B overexpression predicts poor UBC outcomes, suggesting its role as a prognostic marker and therapeutic target. Further research is necessary to elucidate its role in UBC progression.

Investigation of iso-propylchaetominine anticancer activity on apoptosis, cell cycle and Wnt signaling pathway in different cancer models

Dysregulation of the Wnt signaling pathway contributes to the development of many cancer types. Natural compounds produced with biotechnological systems have been the focus of research for being a new drug candidate both with unlimited resources and cost-effective production.In this study, it was aimed to reveal the effects of isopropylchaetominine on cytotoxic, cytostatic, apoptotic and Wnt signaling pathways in brain, pancreatic and prostate cancer.The IC50 values of isopropylchaetominine in U-87 MG, PANC1, PC3 and LNCaP cells were calculated as 91.94 μM, 41.68 μM, 54.54 μM and 7.86 μM in 72nd h, respectively. The metabolite arrests the cell cycle in G0/G1 phase in each cancer cells. Iso-propylchaetominine induced a 4.3-fold and 1.9-fold increase in apoptosis in PC3 and PANC1 cells, respectively. The toxicity of isopropylchaetominine in healthy fibroblast cells was assessed using the annexin V method, and no significant apoptotic activity was observed between the groups treated with the active substance and untreated. In U-87 MG, PANC1, PC3, and LNCaP cells under treatment with isopropylchaetominin, the expression levels of DKK3, TLE1, AES, DKK1, FRZB, DAB2, AXIN1/2, PPARD, SFRP4, APC and SOX17 tumor suppressor genes increased significantly. Decreases in expression of Wnt1, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt10, Wnt11, FRZ2, FRZ3, FRZ7, TCF7L1, BCL9, PYGO, CCND2, c-MYC, WISP1 and CTNNB1 oncogenic genes were detected.All these result shows that isopropylchaetominine can present promising new treatment strategy in different cancer types.

Thrombospondin 1 and Reelin act through Vldlr to regulate cardiac growth and repair.

Adult mammalian cardiomyocytes have minimal cell cycle capacity, which leads to poor regeneration after cardiac injury such as myocardial infarction. Many positive regulators of cardiomyocyte cell cycle and cardioprotective signals have been identified, but extracellular signals that suppress cardiomyocyte proliferation are poorly understood. We profiled receptors enriched in postnatal cardiomyocytes, and found that very-low-density-lipoprotein receptor (Vldlr) inhibits neonatal cardiomyocyte cell cycle. Paradoxically, Reelin, the well-known Vldlr ligand, expressed in cardiac Schwann cells and lymphatic endothelial cells, promotes neonatal cardiomyocyte proliferation. Thrombospondin1 (TSP-1), another ligand of Vldlr highly expressed in adult heart, was then found to inhibit cardiomyocyte proliferation through Vldlr, and may contribute to Vldlr's overall repression on proliferation. Mechanistically, Rac1 and subsequent Yap phosphorylation and nucleus translocation mediate the regulation of the cardiomyocyte cell cycle by TSP-1/Reelin-Vldlr signaling. Importantly, Reln mutant neonatal mice displayed impaired cardiomyocyte proliferation and cardiac regeneration after apical resection, while cardiac-specific Thbs1 deletion and cardiomyocyte-specific Vldlr deletion promote cardiomyocyte proliferation and are cardioprotective after myocardial infarction. Our results identified a novel role of Vldlr in consolidating extracellular signals to regulate cardiomyocyte cell cycle activity and survival, and the overall suppressive TSP-1-Vldlr signal may contribute to the poor cardiac repair capacity of adult mammals.

JunB condensation attenuates vascular endothelial damage under hyperglycemic condition.

Endothelial damage is the initial and crucial factor in the occurrence and development of vascular complications in diabetic patients, contributing to morbidity and mortality. Although hyperglycemia has been identified as a damaging effector, the detailed mechanisms remain elusive. In this study, identified by ATAC-seq and RNA-seq, JunB reverses the inhibition of proliferation and the promotion of apoptosis in human umbilical vein endothelial cells treated with high glucose, mainly through the cell cycle and p53 signaling pathways. Furthermore, JunB undergoes phase separation in the nucleus and in vitro, mediated by its intrinsic disordered region and DNA-binding domain. Nuclear localization and condensation behaviors are required for JunB-mediated proliferation and apoptosis. Thus, our study uncovers the roles of JunB and its coacervation in repairing vascular endothelial damage caused by high glucose, elucidating the involvement of phase separation in diabetes and diabetic endothelial dysfunction.

Class IA PI3K isoforms lead to differential signalling downstream of PKB/Akt

Abstract Objectives The catalytic subunits of Class IA PI3K, p110α, p110β, and p110δ, phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3) on the plasma membrane. In cancer, these catalytic subunits are usually found to be altered or amplified. Because pan-PI3K inhibition results in systemic toxicities, finding specific targets for the ubiquitous PI3K isoforms offers considerable potential for enhancing the effectiveness of PI3K-targeted therapy. Methods We aim to delineate the isoform-specific druggable targets of the PI3K by deleting PIK3CA (encoding p110α) and PIK3CB (encoding p110β) by Cre mediated excision and ectopically expressing p110α, p110β, or p110δ with or without myristoylation (Myr) tag in mouse embryonic fibroblasts (MEFs). Myr is a lipidation signal that translocates proteins to plasma membrane permanently. This translocation renders p110s constitutively activated as they remain in close proximity to PIP2 on the membrane. Results Unique and redundant Akt targets are identified downstream of different PI3K isoforms. mTORC1, one of the targets of fully-activated Akt, has been observed to be differentially regulated in MEFs upon expression of p110α or p110β. The varying dependencies on mTORC1 and Rac1 led us to analyse a potential scaffolding function of p110β with Rac1 to mediate phosphorylation and activation of mTOR using platforms for the modeling of biomolecular complexes. We also documented that p110α and p110β support cell cycle kinetics differentially. Conclusions This study suggests differential regulation of protein translation, metabolism, cell cycle, and survival signaling downstream of unique p110 targets, underlying the importance of cancer treatment according to the deregulated p110 isoform.

Metal-Organic Framework-Mediated Synergistic Hypoxia-Activated Chemo-Immunotherapy Induced by High Intensity Focused Ultrasound for Enhanced Cancer Theranostics.

High intensity focused ultrasound (HIFU) has attracted considerable attention as a noninvasive, efficient, and economic therapeutic modality for solid tumors. However, HIFU surgery has its intrinsic limitation in completely ablating tumors, leading to residual tumor tissue. Furthermore, the severely hypoxic environment ensuring after surgery can exacerbate the unrestricted proliferation and metabolism of residual tumor cells, leading to tumor recurrence and metastasis. To address these limitations, a versatile HIFU-specific metal-organic framework nanosystem (called ADMOFs) is developed by coordinating hypoxia-activated prodrug AQ4N, Mn2+, and DOX based on the postoperative response to changes in the tumor microenvironment. ADMOFs loaded with AQ4N/Mn2+ exhibited remarkable tumor-targeting behavior in vivo and enhanced photoacoustic/magnetic resonance imaging effects, enabling more accurate guidance for HIFU surgery. After surgery, the ADMOFs exploited the severely hypoxic tumor environment induced by HIFU, overcoming hypoxia-associated drug resistance, and inducing immunogenic cell death. Finally, it effectively inhibited tumor growth and eliminated lung metastasis. Transcriptome studies revealed that this strategy significantly up-regulated genes involved in apoptosis, cell cycle, and HIF-1 signaling pathway while downregulating genes related to tumor proliferation and metastasis. These findings suggest that combining hypoxia-activated chemo-immunotherapy with HIFU is a promising strategy for enhancing cancer theranostics.

Fucoxanthin Inhibits Development of Sigmoid Colorectal Cancer in a PDX Model With Alterations of Growth, Adhesion, and Cell Cycle Signals.

Fucoxanthin (Fx), a dietary marine xanthophyll, exerts potent anticancer effects in various colorectal cancer (CRC) animal models. However, therapeutic effects of Fx in human cancer tissues remain unclear. A patient-derived xenograft (PDX) mouse model transplanted with cancer tissues from patients is widely accepted as the best preclinical model for evaluating the anticancer potential of drug candidates. Herein, we investigated the anticancer effects of Fx in PDX mice transplanted with cancer tissues derived from a patient with CRC (CRC-PDX) using LC-MS/MS- and western blot-based proteome analysis. The tumor in the patient with CRC was a primary adenocarcinoma (T3N0M0, stage II) showing mutations of certain genes that were tumor protein p53 (TP53), AT-rich interaction domain 1A (ARID1A), neuroblastoma RAS viral oncogene homolog (NRAS), and PMS1 homolog 2 (PMS2). Administration of Fx significantly suppressed the tumor growth (0.6-fold) and tended to induce differentiation in CRC-PDX mice. Fx up-regulated glycanated-decorin (Gc-DCN) expression, and down-regulated Kinetochore-associated protein DSN1 homolog (DSN1), phospho(p) focal adhesion kinase (pFAK)(Tyr397), pPaxillin(Tyr31), and c-MYC involved in growth, adhesion, and/or cell cycle, in the tumors of CRC-PDX mice than in control mice. Alterations in the five proteins were consistent with those in human CRC HT-29 and HCT116 cells treated with fucoxanthinol (FxOH, a major metabolite of Fx). Fx suppresses development of human-like CRC tissues, especially through growth, adhesion, and cell cycle signals.

MaTPIP: A deep-learning architecture with eXplainable AI for sequence-driven, feature mixed protein-protein interaction prediction

Background and ObjectiveProtein-protein interaction (PPI) is a vital process in all living cells, controlling essential cell functions such as cell cycle regulation, signal transduction, and metabolic processes with broad applications that include antibody therapeutics, vaccines, and drug discovery. The problem of sequence-based PPI prediction has been a long-standing issue in computational biology. MethodsWe introduce MaTPIP, a cutting-edge deep-learning framework for predicting PPI. MaTPIP stands out due to its innovative design, fusing pre-trained Protein Language Model (PLM)-based features with manually curated protein sequence attributes, emphasizing the part-whole relationship by incorporating two-dimensional granular part (amino-acid) level features and one-dimensional whole-level (protein) features. What sets MaTPIP apart is its ability to integrate these features across three different input terminals seamlessly. MatPIP also includes a distinctive configuration of Convolutional Neural Network (CNN) with Transformer components for concurrent utilization of CNN and sequential characteristics in each iteration and a one-dimensional to two-dimensional converter followed by a unified embedding. The statistical significance of this classifier is validated using McNemar's test. ResultsMaTPIP outperformed the existing methods on both the Human PPI benchmark and cross-species PPI testing datasets, demonstrating its immense generalization capability for PPI prediction. We used seven diverse datasets with varying PPI target class distributions. Notably, within the novel PPI scenario, the most challenging category for Human PPI Benchmark, MaTPIP improves the existing state-of-the-art score from 74.1% to 78.6% (measured in Area under ROC Curve), from 23.2% to 32.8% (in average precision) and from 4.9% to 9.5% (in precision at 3% recall) for 50%, 10% and 0.3% target class distributions, respectively. In cross-species PPI evaluation, hybrid MaTPIP establishes a new benchmark score (measured in Area Under precision-recall curve) of 81.1% from the previous 60.9% for Mouse, 80.9% from 56.2% for Fly, 78.1% from 55.9% for Worm, 59.9% from 41.7% for Yeast, and 66.2% from 58.8% for E.coli. Our eXplainable AI-based assessment reveals an average contribution of different feature families per prediction on these datasets. ConclusionsMaTPIP mixes manually curated features with the feature extracted from the pre-trained PLM to predict sequence-based protein-protein association. Furthermore, MaTPIP demonstrates strong generalization capabilities for cross-species PPI predictions.

Temporally multiplexed imaging of dynamic signaling networks in living cells

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group &amp;gt; ND group &amp;gt; HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

Casein Kinase II Inhibitor Sensitizes the Anti-Tumor Effect of EZH2 Inhibitor By Suppressing FOSL1 Expression in Acute Myeloid Leukemia

Background More effective combination therapy is emerging needed owing to the high mortality and poor outcome in acute myeloid leukemia (AML). The genomic amplification and overexpression of Enhancer of zeste homolog 2 (EZH2) occur in AML patients, and high expression of the Casein kinase 2 (CK2) is associated with poor survival in AML patients. Both EZH2 inhibitor and CK2 inhibitors are reported to inhibit cell proliferation and induce apoptosis in AML, however, the anti-leukemia effect of EZH2 inhibitor in combination with CK2 inhibitor has not been reported. The present study aims to investigate the effect of the combination of the EZH2 inhibitor DZNeP with CK2 inhibitor CX4945 in AML and possible underlying mechanisms. Methods A total of 40 newly diagnosed AML patients and 40 bone marrow control from healthy donors were obtained from our institute with the approval of the Ethics Committee. Cell Counting Kit-8 assay (CCK8), Cell cycle assay, and apoptosis assays were applied in THP-1 AML cells treated with control, DZNeP, CX4945, and CX4945+DZNeP. Global transcriptome analysis by RNA-seq was performed in U937 cells treated with 2μM DZNeP; 12.5μM CX4945 or vehicle for 48 hours. FOSL1 mRNA level was examined by qPCR; R2 genomics analysis and visualization application were used for comparison of the expression difference and the Kaplan-Meier overall survival analysis was conducted by the Gene Expression Profiling Interactive Analysis (GEPIA) platform. Results DZNeP only has little effect on cell proliferation arrest in THP-1 cells, but it significantly sensitized the effect of CX4945, and CalcuSyn analysis showed the synergistic effect of the CX4945+DZNeP (Fig.1A). The treatment of CX4945+DZNeP combination induced the G0/G1 phase arrest in THP-1 cells versus single-drug control (Fig. 1B). The combination also significantly increased the % of the apoptotic cells in THP-1 cells as well as the AML-5b primary cells from the patient compared to single-drug controls (p&amp;lt;0.0001) (Fig.1C). These data indicate the synergistic anti-tumor effect of CX4945+DZNep combination in AML. To understand the molecular mechanism underlying the synergy, the global transcriptome was identified in U937 cells. The significantly different expression genes (DEGs) were identified upon CX4945 treatment (∣log2FC∣&amp;gt;1.5, FDR&amp;lt;0.05), or DZNeP treatment (∣log2FC∣&amp;gt;1.0, FDR&amp;lt;0.05) versus vehicle-control; and 1908 overlapped DEGs are identified upon either drug treatment, in which 596 were intersected (429 genes changed in the same direction but 167 changed in the opposite (Fig. 1D). KEGG enrichment analyses showed the overlapped DEGs upon the drug treatment mainly involved in cell cycle, DNA replication, and MAPK signaling pathway. (Fig.1E). It is worth noting that FOSL1 is dramatically downregulated in U937 cells upon treatment of either DZNeP or CX4945 (Fig1.D); and qPCR data showed that the mRNA level of FOSL1 is significantly downregulated by CX4945+DZNeP, DZNeP or CX4945 compared to the vehicle control (p&amp;lt;0.0001) in THP-1 cells (Fig. 1F). These data indicated that the combination exerts its anti-tumor effect by down-regulation of FOSL1 expression in AML. FOSL1 is a key component of the AP-1 transcriptional complex, and overexpression of FOSL1 is associated with invasion, migration, proliferation, and stemness in human solid cancers. We also examined the FOSL1 mRNA level in 40 newly diagnosed patients with AML versus 40 normal bone marrow controls. Results showed the mRNA level of FOSL1 is significantly higher in AML patients than that of normal controls (p&amp;lt;0.001) (Fig1.G). Metadata analysis showed that FOSL1 high expression is associated with short overall survival (p=0.033) (Fig.1H). These results not only suggested the oncogenic role of FOSL1 in AML but also further supported the CX4945+DZNeP combination-induced anti-tumor effect mediated by suppressing FOSL1 expression in AML. The mechanism model is summarized in Fig. 1E. Conclusion The combination of EZH2 inhibitor DZNeP and CK2 inhibitor CX4945 exerts a potent synergistic effect on proliferation arrest, cell cycle arrest, and apoptosis in AML by suppressing FOSL1 expression. Our results reveal the oncogenic role of FOSL1 in AML. Our data also provide experimental hints for the potential clinical trial of the novel combination in AML patients.

Comprehensive clinicopathological significance and putative transcriptional mechanisms of Forkhead box M1 factor in hepatocellular carcinoma

BackgroundThe Forkhead box M1 factor (FOXM1) is a crucial activator for cancer cell proliferation. While FOXM1 has been shown to promote hepatocellular carcinoma (HCC) progression, its transcriptional mechanisms remain incompletely understood.MethodsWe performed an in-house tissue microarray on 313 HCC and 37 non-HCC tissue samples, followed by immunohistochemical staining. Gene chips and high throughput sequencing data were used to assess FOXM1 expression and prognosis. To identify candidate targets of FOXM1, we comprehensively reanalyzed 41 chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sets. We predicted FOXM1 transcriptional targets in HCC by intersecting candidate FOXM1 targets with HCC overexpressed genes and FOXM1 correlation genes. Enrichment analysis was employed to address the potential mechanisms of FOXM1 underlying HCC. Finally, single-cell RNA sequencing analysis was performed to confirm the transcriptional activity of FOXM1 on its predicted targets.ResultsThis study, based on 4235 HCC tissue samples and 3461 non-HCC tissue samples, confirmed the upregulation of FOXM1 in HCC at mRNA and protein levels (standardized mean difference = 1.70 [1.42, 1.98]), making it the largest multi-centered study to do so. Among HCC patients, FOXM1 was increased in Asian and advanced subgroups, and high expression of FOXM1 had a strong ability to differentiate HCC tissue from non-HCC tissue (area under the curve = 0.94, sensitivity = 88.72%, specificity = 87.24%). FOXM1 was also shown to be an independent exposure risk factor for HCC, with a pooled hazard ratio of 2.00 [1.77, 2.26]. The predicted transcriptional targets of FOXM1 in HCC were predominantly enriched in nuclear division, chromosomal region, and catalytic activity acting on DNA. A gene cluster encoding nine transcriptional factors was predicted to be positively regulated by FOXM1, promoting the cell cycle signaling pathway in HCC. Finally, the transcriptional activity of FOXM1 and its targets was supported by single-cell analysis of HCC cells.ConclusionsThis study not only confirmed the upregulation of FOXM1 in HCC but also identified it as an independent risk factor. Moreover, our findings enriched our understanding of the complex transcriptional mechanisms underlying HCC pathogenesis, with FOXM1 potentially promoting HCC progression by activating other transcription factors within the cell cycle pathway.

The Wnt/β-catenin pathway is involved in 2,5-hexanedione-induced ovarian granulosa cell cycle arrest

N-Hexane causes significant ovarian toxicity, and its main active metabolite 2,5-hexanedione (2,5-HD) can induce ovarian injury through mechanisms such as inducing apoptosis in ovarian granulosa cells (GCs); however, the specific mechanism has not been fully elucidated. In this study, we investigated the effects on the cell cycle of rat ovarian GCs exposed in vitro to different concentrations of 2,5-HD (0 mM, 20 mM, 40 mM, and 60 mM) and further explored the mechanism by mRNA and miRNA microarray analyses. The flow cytometry results sindicated that compared with control cells, in ovarian GCs, there was significant cell cycle arrest after 2,5-HD treatment. Cell cycle- and apoptosis- related gene (Cdk2, Ccnd1, Bax, Bcl-2, Caspase3, and Caspase9) expression was altered. The mRNA and miRNA microarray results suggested that 5678 mRNAs and 32 miRNAs were differentially expressed in the 2,5-HD-treated group. A total of 262 target mRNAs were obtained by miRNA and mRNA coexpression analysis, forming 368 miRNA–mRNA coexpression relationship pairs with 27 miRNAs. GO and KEGG analyses showed that differentially expressed genes were significantly enriched in the cell cycle and Wnt signaling pathways. Furthermore, significant changes in the expression of Wnt signaling pathway and cell cycle- related genes (Fzd1, Lrp6, Tcf3, Tcf4, Fzd6, Lrp5, β-catenin, Lef1, GSK3β, and Dvl3) after 2,5-HD treatment were confirmed by qRT–PCR and Western blotting. Ther results of dual-luciferase assays indicated decreased β-catenin/TCF transcriptional activity after 2,5-HD treatment. In addition, Wnt pathway-related miRNAs (rno-miR-145–5p, rno-miR-143–3p, rno-miR-214–3p, rno-miR-138–5p, and rno-miR-199a-3p) were changed significantly after 2,5-HD treatment. In summary, 2,5-HD induced cell cycle arrest in ovarian GCs, and the Wnt/β-catenin signaling pathway may play a very critical role in this process. Alterations in the expression of miRNAs such as rno-miR-145–5p may have significant implications.

Common and specific proteins and pathways in heart and cerebral ischemia

ObjectiveTo understand the similarities and differences between acute ischemic stroke and acute myocardial infarction (AMI) to help in the development of specific or common treatment strategies. MethodsUsing an aptamer-based proteomic array, we measured and compared 1310 circulating proteins in the blood of 40 patients with AIS, 9 patients with AMI, and 31 healthy controls. Pathway enrichment analysis was performed using GSEA and g:profiler. ResultsNinety-four proteins were differentially expressed in AIS, and 284 were differentially expressed in AMI. Of these, 8 were specific to cerebral ischemia, and 197 were specific to myocardial infarction. Forty-two proteins were altered in both ischemia processes. Most altered pathways in AIS could be classified as immune response, cell cycle processing, molecular transport, or signaling. Pathways altered in AMI were mostly related to lipid metabolism and transport, highlighting cholesterol metabolic processes and estrogen signaling. In both types of ischemia, we found pathways related to metabolism, specifically purine metabolism, and signaling processes, such as TNF signaling or MAPK1/3. ConclusionsThe present study revealed proteins and pathways that were specifically altered in cerebral ischemia, in cardiac ischemia, or in both diseases, providing information on the similarities and differences of ischemic conditions. The role of common and specific proteins and pathways should be explored in detail to find possible therapeutic targets.

Navigating the Gene Co-Expression Network and Drug Repurposing Opportunities for Brain Disorders Associated with Neurocognitive Impairment.

Neurocognitive impairment refers to a spectrum of disorders characterized by a decline in cognitive functions such as memory, attention, and problem-solving, which are often linked to structural or functional abnormalities in the brain. While its exact etiology remains elusive, genetic factors play a pivotal role in disease onset and progression. This study aimed to identify highly correlated gene clusters (modules) and key hub genes shared across neurocognition-impairing diseases, including Alzheimer's disease (AD), Parkinson's disease with dementia (PDD), HIV-associated neurocognitive disorders (HAND), and glioma. Herein, the microarray datasets AD (GSE5281), HAND (GSE35864), glioma (GSE15824), and PD (GSE7621) were used to perform Weighted Gene Co-expression Network Analysis (WGCNA) to identify highly preserved modules across the studied brain diseases. Through gene set enrichment analysis, the shared modules were found to point towards processes including neuronal transcriptional dysregulation, neuroinflammation, protein aggregation, and mitochondrial dysfunction, hallmarks of many neurocognitive disorders. These modules were used in constructing protein-protein interaction networks to identify hub genes shared across the diseases of interest. These hub genes were found to play pivotal roles in processes including protein homeostasis, cell cycle regulation, energy metabolism, and signaling, all associated with brain and CNS diseases, and were explored for their drug repurposing experiments. Drug repurposing based on gene signatures highlighted drugs including Dorzolamide and Oxybuprocaine, which were found to modulate the expression of the hub genes in play and may have therapeutic implications in neurocognitive disorders. While both drugs have traditionally been used for other medical purposes, our study underscores the potential of a combined WGCNA and drug repurposing strategy for searching for new avenues in the simultaneous treatment of different diseases that have similarities in gene co-expression networks.