ABSTRACT
Dysregulation of splicing factor expression plays a crucial role in the progression of hepatocellular carcinoma (HCC). Our research found that the expression level of splicing factor ZMAT2 was increased in HCC, promoting the proliferation of HCC cells. RNAseq data indicated that the absence of ZMAT2 induced skipping exon of mRNA, while RIPseq data further revealed the mRNA binding motifs of ZMAT2. A comprehensive analysis of RNAseq and RIPseq data indicateed that ZMAT2 played a crucial role in the maturation process of TRIM28 mRNA. Knocking down of ZMAT2 led to the deletion of 25 bases in exon 11 of TRIM28, ultimately resulting in nonsense-mediated decay (NMD). Our data revealed that ZMAT2 could regulate TRIM28 to reduce the accumulation of ROS in HCC cells, thereby promoting their proliferation. Our research also discovered that ZMAT2 was capable of undergoing phase separation, resulting in the formation of liquid droplet condensates within HCC cells. Additionally, it was found that ZMAT2 was able to form protein-nucleic acid condensates with TRIM28 mRNA. In summary, this study is the first to reveal that ZMAT2 and TRIM28 mRNA form protein-nucleic acid condensates, thereby regulating the splicing of TRIM28 mRNA. The increased expression of ZMAT2 in HCC leads to upregulated TRIM28 expression and reduced ROS accumulation, ultimately accelerating the proliferation of HCC cells.
Subject(s)
Alternative Splicing , Carcinoma, Hepatocellular , Cell Proliferation , Liver Neoplasms , Reactive Oxygen Species , Tripartite Motif-Containing Protein 28 , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Cell Proliferation/genetics , Reactive Oxygen Species/metabolism , Alternative Splicing/genetics , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/geneticsABSTRACT
This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. In vitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an in situ transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.
Subject(s)
CD8-Positive T-Lymphocytes , Membrane Proteins , Prostatic Neoplasms, Castration-Resistant , Tripartite Motif-Containing Protein 28 , Animals , Humans , Male , Mice , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Macrophages/metabolism , Macrophages/immunology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/immunology , Prostatic Neoplasms, Castration-Resistant/pathology , Signal Transduction , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/geneticsABSTRACT
Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.
Subject(s)
RNA Polymerase II , Tripartite Motif-Containing Protein 28 , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , RNA Polymerase II/metabolism , Humans , Kinetics , Transcription Elongation, Genetic , Genes, Immediate-Early , Transcription, Genetic , Signal Transduction , Transcriptional Activation , AnimalsABSTRACT
Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , DNA End-Joining Repair , Signal Transduction , Tripartite Motif-Containing Protein 28 , Tumor Suppressor p53-Binding Protein 1 , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases , Ubiquitination , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Humans , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Phosphorylation , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , HEK293 Cells , Telomere/metabolism , DNA Damage , Chromatin/metabolism , AnimalsABSTRACT
Type I interferons exhibit anti-proliferative and anti-cancer activities, but their detailed regulatory mechanisms in cancer have not been fully elucidated yet. RNA binding proteins are master orchestrators of gene regulation, which are closely related to tumor progression. Here we show that the upregulated RNA binding protein RBM45 correlates with poor prognosis in breast cancer. Depletion of RBM45 suppresses breast cancer progression both in cultured cells and xenograft mouse models. Mechanistically, RBM45 ablation inhibits breast cancer progression through regulating type I interferon signaling, particularly by elevating IFN-ß production. Importantly, RBM45 recruits TRIM28 to IRF7 and stimulates its SUMOylation, thereby repressing IFNB1 transcription. Loss of RBM45 reduced the SUMOylation of IRF7 by reducing the interaction between TRIM28 and IRF7 to promote IFNB1 transcription, leading to the inhibition of breast cancer progression. Taken together, our finding uncovers a vital role of RBM45 in modulating type I interferon signaling and cancer aggressive progression, implicating RBM45 as a potential therapeutic target in breast cancer.
Subject(s)
Breast Neoplasms , Disease Progression , Gene Expression Regulation, Neoplastic , Interferon Regulatory Factor-7 , RNA-Binding Proteins , Sumoylation , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Animals , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Interferon Regulatory Factor-7/genetics , Interferon Regulatory Factor-7/metabolism , Mice , Transcription, Genetic , Cell Line, Tumor , Interferon-beta/metabolism , Interferon-beta/genetics , Signal Transduction , Mice, Nude , Cell Proliferation , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , Xenograft Model Antitumor Assays , Mice, Inbred BALB CABSTRACT
TIF1ß/KAP1/TRIM28, a chromatin modulator, both represses and activates the transcription of genes in normal and malignant cells. Analyses of datasets on leukemia patients revealed that the expression level of TIF1ß was increased in patients with chronic myeloid leukemia at the blast crisis and acute myeloid leukemia. We generated a BCR::ABL1 conditional knock-in (KI) mouse model, which developed aggressive myeloid leukemia, and demonstrated that the deletion of the Tif1ß gene inhibited the progression of myeloid leukemia and showed longer survival than that in BCR::ABL1 KI mice, suggesting that Tif1ß drove the progression of BCR::ABL1-induced leukemia. In addition, the deletion of Tif1ß sensitized BCR::ABL1 KI leukemic cells to dasatinib. The deletion of Tif1ß decreased the expression levels of TIF1ß-target genes and chromatin accessibility peaks enriched with the Fosl1-binding motif in BCR::ABL1 KI stem cells. TIF1ß directly bound to the promoters of proliferation genes, such as FOSL1, in human BCR::ABL1 cells, in which TIF1ß and FOSL1 bound to adjacent regions of chromatin. Since the expression of Fosl1 was critical for the enhanced growth of BCR::ABL1 KI cells, Tif1ß and Fosl1 interacted to activate the leukemic transcriptional program in and cellular function of BCR::ABL1 KI stem cells and drove the progression of myeloid leukemia.
Subject(s)
Fusion Proteins, bcr-abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Animals , Mice , Humans , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Gene Expression Regulation, Leukemic , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , Transcription, GeneticABSTRACT
BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.
Subject(s)
N-Myc Proto-Oncogene Protein , Neuroblastoma , Tripartite Motif-Containing Protein 28 , Animals , Humans , Mice , Adenosine/analogs & derivatives , Adenosine/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Mice, Nude , N-Myc Proto-Oncogene Protein/genetics , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/genetics , Neuroblastoma/metabolism , Neuroblastoma/pathology , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/geneticsABSTRACT
Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.
Subject(s)
Mesothelioma, Malignant , Pleural Neoplasms , Tripartite Motif-Containing Protein 28 , Humans , Tripartite Motif-Containing Protein 28/metabolism , Tripartite Motif-Containing Protein 28/genetics , Prognosis , Mesothelioma, Malignant/metabolism , Mesothelioma, Malignant/genetics , Pleural Neoplasms/genetics , Pleural Neoplasms/metabolism , Male , Female , Cell Line, Tumor , Mesothelioma/genetics , Mesothelioma/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Middle Aged , Lung Neoplasms/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathologyABSTRACT
The maturation process of natural killer (NK) cells, which is regulated by multiple transcription factors, determines their functionality, but few checkpoints specifically targeting this process have been thoroughly studied. Here we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) leads to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibit undermined activation, cytotoxicity and IFN-γ production upon stimulation, as well as weakened responses to IL-15 for NK cell maturation, in vitro. In vivo, NK-specific gp96-deficient mice show increased tumor growth. Mechanistically, we identify Eomes as the downstream transcription factor, with gp96 binding to Trim28 to prevent Trim28-mediated ubiquitination and degradation of Eomes. Our study thus suggests the gp96-Trim28-Eomes axis to be an important regulator for NK cell maturation and cancer surveillance in mice.