ABSTRACT
Breast cancer is the most common cancer diagnosed in women worldwide. Early-stage breast cancer is curable in ~70-80% of patients, while advanced metastatic breast cancer is considered incurable with current therapies. Breast cancer is a highly heterogeneous disease categorized into three main subtypes based on key markers orientating specific treatment strategies for each subtype. The complexity of breast carcinogenesis is often associated with epigenetic modification regulating different signaling pathways, involved in breast tumor initiation and progression, particularly by the methylation of arginine residues. Protein arginine methyltransferases (PRMT1-9) have emerged, through their ability to methylate histones and non-histone substrates, as essential regulators of cancers. Here, we present an updated overview of the mechanisms by which PRMT1 and PRMT5, two major members of the PRMT family, control important signaling pathways impacting breast tumorigenesis, highlighting them as putative therapeutic targets.
Subject(s)
Breast Neoplasms , Protein-Arginine N-Methyltransferases , Repressor Proteins , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Repressor Proteins/metabolism , Repressor Proteins/genetics , Signal Transduction , Methylation , Epigenesis, Genetic , Animals , Gene Expression Regulation, NeoplasticABSTRACT
PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization.
Subject(s)
Protein-Arginine N-Methyltransferases , Radiation Tolerance , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Animals , Humans , Radiation Tolerance/drug effects , Radiation Tolerance/genetics , Cell Line, Tumor , Mice , DNA Repair , Cell Proliferation/radiation effects , Xenograft Model Antitumor Assays , DNA Breaks, Double-Stranded/radiation effects , Mice, NudeABSTRACT
Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.
Subject(s)
Protein Multimerization , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/chemistry , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Substrate Specificity , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Repressor Proteins/genetics , MethylationABSTRACT
OBJECTIVES: Obesity related glomerulopathy (ORG) is induced by obesity, but the pathogenesis remains unclear. This study aims to investigate the expression of early growth response protein 3 (EGR3) in the renal cortex tissues of ORG patients and high-fat diet-induced obese mice, and to further explore the molecular mechanism of EGR3 in inhibiting palmitic acid (PA) induced human podocyte inflammatory damage. METHODS: Renal cortex tissues were collected from ORG patients (n=6) who have been excluded from kidney damage caused by other diseases and confirmed by histopathology, and from obese mice induced by high-fat diet (n=10). Human and mouse podocytes were intervened with 150 µmol/L PA for 48 hours. EGR3 was overexpressed or silenced in human podocytes. Enzyme linked immunosorbent assay (ELISA) was used to detcet the levels of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß). Real-time RT-PCR was used to detect the mRNA expressions of EGR3, podocytes molecular markers nephrosis 1 (NPHS1), nephrosis 2 (NPHS2), podocalyxin (PODXL), and podoplanin (PDPN). RNA-seq was performed to detect differentially expressed genes (DEGs) after human podocytes overexpressing EGR3 and treated with 150 µmol/L PA compared with the control group. Co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS) was used to detect potential interacting proteins of EGR3 and the intersected with the RNA-seq results. Co-IP confirmed the interaction between EGR3 and protein arginine methyltransferases 1 (PRMT1), after silencing EGR3 and PRMT1 inhibitor intervention, the secretion of IL-6 and IL-1ß in PA-induced podocytes was detected. Western blotting was used to detect the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) after overexpression or silencing of EGR3. RESULTS: EGR3 was significantly upregulated in renal cortex tissues of ORG patients and high-fat diet-induced obese mice (both P<0.01). In addition, after treating with 150 µmol/L PA for 48 hours, the expression of EGR3 in human and mouse podocytes was significantly upregulated (both P<0.05). Overexpression or silencing of EGR3 in human podocytes inhibited or promoted the secretion of IL-6 and IL-1ß in the cell culture supernatant after PA intervention, respectively, and upregulated or downregulated the expression of NPHS1, PODXL, NPHS2,and PDPN (all P<0.05). RNA-seq showed a total of 988 DEGs, and Co-IP+LC-MS identified a total of 238 proteins that may interact with EGR3. Co-IP confirmed that PRMT1 was an interacting protein with EGR3. Furthermore, PRMT1 inhibitors could partially reduce PA-induced IL-6 and IL-1ß secretion after EGR3 silencing in human podocytes (both P<0.05). Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3. CONCLUSIONS: EGR3 may reduce ORG podocyte inflammatory damage by inhibiting the PRMT1/p-STAT3 pathway.
Subject(s)
Early Growth Response Protein 3 , Obesity , Podocytes , Protein-Arginine N-Methyltransferases , Repressor Proteins , STAT3 Transcription Factor , Animals , Humans , Male , Mice , Diet, High-Fat/adverse effects , Early Growth Response Protein 3/metabolism , Early Growth Response Protein 3/genetics , Inflammation/metabolism , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Interleukin-6/genetics , Kidney Cortex/metabolism , Kidney Cortex/pathology , Kidney Diseases/metabolism , Kidney Diseases/etiology , Kidney Diseases/pathology , Mice, Inbred C57BL , Mice, Obese , Obesity/complications , Obesity/metabolism , Palmitic Acid/pharmacology , Podocytes/metabolism , Podocytes/pathology , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Signal Transduction , STAT3 Transcription Factor/metabolismABSTRACT
Non-alcoholic fatty liver disease (NAFLD) has emerged as the leading cause of chronic liver disease worldwide. Caspase 8 and FADD-like apoptosis regulator (CFLAR) has been identified as a potent factor in mitigating non-alcoholic steatohepatitis (NASH) by inhibiting the N-terminal dimerization of apoptosis signal-regulating kinase 1 (ASK1). While arginine methyltransferase 1 (PRMT1) was previously reported to be associated with increased hepatic glucose production, its involvement in hepatic lipid metabolism remains largely unexplored. The interaction between PRMT1 and CFLAR and the methylation of CFLAR were verified by Co-IP and immunoblotting assays. Recombinant adenoviruses were generated for overexpression or knockdown of PRMT1 in hepatocytes. The role of PRMT1 in NAFLD was investigated in normal and high-fat diet-induced obese mice. In this study, we found a significant upregulation of PRMT1 and downregulation of CFLAR after 48h of fasting, while the latter significantly rebounded after 12h of refeeding. The expression of PRMT1 increased in the livers of mice fed a methionine choline-deficient (MCD) diet and in hepatocytes challenged with oleic acid (OA)/palmitic acid (PA). Overexpression of PRMT1 not only inhibited the expression of genes involved in fatty acid oxidation (FAO) and promoted the expression of genes involved in fatty acid synthesis (FAS), resulting in increased triglyceride accumulation in primary hepatocytes, but also enhanced the gluconeogenesis of primary hepatocytes. Conversely, knockdown of hepatic PRMT1 significantly alleviated MCD diet-induced hepatic lipid metabolism abnormalities and liver injury in vivo, possibly through the upregulation of CFLAR protein levels. Knockdown of PRMT1 suppressed the expression of genes related to FAS and enhanced the expression of genes involved in FAO, causing decreased triglyceride accumulation in OA/PA-treated primary hepatocytes in vitro. Although short-term overexpression of PRMT1 had no significant effect on hepatic triglyceride levels under physiological conditions, it resulted in increased serum triglyceride and fasting blood glucose levels in normal C57BL/6J mice. More importantly, PRMT1 was observed to interact with and methylate CFLAR, ultimately leading to its ubiquitination-mediated protein degradation. This process subsequently triggered the activation of c-Jun N-terminal kinase 1 (JNK1) and lipid deposition in primary hepatocytes. Together, these results suggested that PRMT1-mediated methylation of CFLAR plays a critical role in hepatic lipid metabolism. Targeting PRMT1 for drug design may represent a promising strategy for the treatment of NAFLD.
Subject(s)
Hepatocytes , Lipid Metabolism , Liver , Non-alcoholic Fatty Liver Disease , Protein-Arginine N-Methyltransferases , Animals , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Liver/metabolism , Mice , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/genetics , Hepatocytes/metabolism , Methylation , Male , Diet, High-Fat/adverse effects , Humans , Mice, Inbred C57BL , Apoptosis Regulatory Proteins/metabolism , Apoptosis Regulatory Proteins/geneticsABSTRACT
Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.
Subject(s)
Arginine , Ferroptosis , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2 , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Animals , Arginine/metabolism , Arginine/analogs & derivatives , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Mice , Cell Line, Tumor , Kelch-Like ECH-Associated Protein 1/metabolism , Kelch-Like ECH-Associated Protein 1/genetics , Feedback, Physiological , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Sequestosome-1 Protein/metabolism , Sequestosome-1 Protein/genetics , Mice, Nude , Signal Transduction , Phase Separation , RNA-Binding ProteinsABSTRACT
Protein arginine methyltransferases (PRMTs) in mammals play a role in various signaling pathways, such as virus infection, inflammasome responses, and cancer growth. While some PRMTs have been found to regulate interferon production in mammals, the mechanism in chickens remains to be fully understood. This study focused on investigating the function of chicken PRMTs. Our findings indicate that chicken PRMTs act as inhibitors of interferon production in response to dsRNA or MDA5 stimulation. Each PRMT is involved in different stages of interferon induction through the MDA5-MAVS-TBK1 pathway. Furthermore, we observed the colocalization of multiple PRMTs with the viral protein VP3 of infectious bursal disease virus (IBDV). Among the chicken PRMTs studied, PRMT3 was found to be widely expressed in various organs and its expression was upregulated during IBDV infection. Notably, PRMT3 supported IBDV replication, as demonstrated by ectopic expression and inhibition studies using SGC-707. Silencing of PRMT3 led to enhanced interferon production and inhibition of IBDV replication. This study provides novel insights into the role of chicken PRMTs, particularly PRMT3, in promoting IBDV replication by suppressing interferon signaling.
Subject(s)
Avian Proteins , Chickens , Infectious bursal disease virus , Poultry Diseases , Protein-Arginine N-Methyltransferases , Virus Replication , Animals , Infectious bursal disease virus/physiology , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Avian Proteins/genetics , Avian Proteins/metabolism , Poultry Diseases/virology , Birnaviridae Infections/veterinary , Birnaviridae Infections/virologyABSTRACT
Abnormal epigenetic modifications are involved in the regulation of Warburg effect in tumor cells. Protein arginine methyltransferases (PRMTs) mediate arginine methylation and have critical functions in cellular responses. PRMTs are deregulated in a variety of cancers, but their precise roles in Warburg effect in cancer is largely unknown. Experiments from the current study showed that PRMT1 was highly expressed under conditions of glucose sufficiency. PRMT1 induced an increase in the PKM2/PKM1 ratio through upregulation of PTBP1, in turn, promoting aerobic glycolysis in non-small cell lung cancer (NSCLC). The PRMT1 level in p53-deficient and p53-mutated NSCLC remained relatively unchanged while the expression was reduced in p53 wild-type NSCLC under conditions of glucose insufficiency. Notably, p53 activation under glucose-deficient conditions could suppress USP7 and further accelerate the polyubiquitin-dependent degradation of PRMT1. Melatonin, a hormone that inhibits glucose intake, markedly suppressed cell proliferation of p53 wild-type NSCLC, while a combination of melatonin and the USP7 inhibitor P5091 enhanced the anticancer activity in p53-deficient NSCLC. Our collective findings support a role of PRMT1 in the regulation of Warburg effect in NSCLC. Moreover, combination treatment with melatonin and the USP7 inhibitor showed good efficacy, providing a rationale for the development of PRMT1-based therapy to improve p53-deficient NSCLC outcomes.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Membrane Proteins , Protein-Arginine N-Methyltransferases , Thyroid Hormone-Binding Proteins , Thyroid Hormones , Tumor Suppressor Protein p53 , Warburg Effect, Oncologic , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Warburg Effect, Oncologic/drug effects , Tumor Suppressor Protein p53/metabolism , Thyroid Hormones/metabolism , Cell Line, Tumor , Membrane Proteins/metabolism , Membrane Proteins/genetics , Cell Proliferation/drug effects , Carrier Proteins/metabolism , Carrier Proteins/genetics , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitin-Specific Peptidase 7/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Animals , Glycolysis/drug effects , Mice, Nude , Glucose/metabolism , Mice , Gene Expression Regulation, Neoplastic , A549 Cells , Polypyrimidine Tract-Binding ProteinABSTRACT
Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.
Subject(s)
Cell Movement , Enhancer of Zeste Homolog 2 Protein , Glioblastoma , Neoplasm Invasiveness , Protein Stability , Protein-Arginine N-Methyltransferases , Ubiquitination , Animals , Female , Humans , Male , Mice , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/metabolism , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Glioblastoma/metabolism , Glioblastoma/genetics , Mice, Inbred BALB C , Mice, Nude , Nuclear Proteins , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Proteolysis , TNF Receptor-Associated Factor 6/metabolism , TNF Receptor-Associated Factor 6/geneticsABSTRACT
Gastric cancer is a most malignancy in digestive tract worldwide. This study aimed to investigate the roles of protein arginine methyltransferase 6 (PRMT6) in gastric cancer. Immunohistochemistry was performed to detect PRMT6 expression in gastric tumors. Real-time transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to detected mRNA levels. Protein expression was determined using western blot. Gastric cancer cells were co-cultured with CD8+ T cells. Colony formation assay was performed to detect cell proliferation. Flow cytometry was performed to determine CD8+ T cell function and tumor cell apoptosis. PRMT6 was overexpressed in gastric tumors. High level of PRMT6 predicted poor outcomes of gastric cancer patients and inhibition of CD8+ T cell infiltration. PRMT6 promoted proliferation of CD8+ T cells and enhanced its tumor killing ability. Moreover, PRMT6 upregulated annexin A1 (ANXA1) and promoted ANXA1 protein stability. ANXA1 overexpression suppressed the proliferation of CD8+ T cells and promoted tumor cell survival. PRMT6 functions as an oncogene in gastric cancer. PRMT6-mediated protein stability inhibits the infiltration of CD8+ T cells, resulting in immune evasion of gastric cancer. The PRMT6-ANXA1 may be a promising strategy for gastric cancer.
Subject(s)
Annexin A1 , CD8-Positive T-Lymphocytes , Cell Proliferation , Gene Expression Regulation, Neoplastic , Protein-Arginine N-Methyltransferases , Stomach Neoplasms , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Stomach Neoplasms/immunology , Stomach Neoplasms/metabolism , Humans , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Annexin A1/genetics , Annexin A1/metabolism , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Up-Regulation , Apoptosis , Tumor Escape/genetics , Male , Immune Evasion , Female , Nuclear ProteinsABSTRACT
The dynamic regulation of mitochondria through fission and fusion is essential for maintaining cellular homeostasis. In this study, we discovered a role of coactivator-associated arginine methyltransferase 1 (CARM1) in mitochondrial dynamics. CARM1 methylates specific residues (R403 and R634) on dynamin-related protein 1 (DRP1). Methylated DRP1 interacts with mitochondrial fission factor (Mff) and forms self-assembly on the outer mitochondrial membrane, thereby triggering fission, reducing oxygen consumption, and increasing reactive oxygen species (ROS) production. This sets in motion a feedback loop that facilitates the translocation of CARM1 from the nucleus to the cytoplasm, enhancing DRP1 methylation and ROS production through mitochondrial fragmentation. Consequently, ROS reinforces the CARM1-DRP1-ROS axis, resulting in cellular senescence. Depletion of CARM1 or DRP1 impedes cellular senescence by reducing ROS accumulation. The uncovering of the above-described mechanism fills a missing piece in the vicious cycle of ROS-induced senescence and contributes to a better understanding of the aging process.
Subject(s)
Cellular Senescence , Cytoplasm , Dynamins , Mitochondrial Dynamics , Protein-Arginine N-Methyltransferases , Reactive Oxygen Species , Dynamins/metabolism , Dynamins/genetics , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Reactive Oxygen Species/metabolism , Methylation , Cytoplasm/metabolism , Mitochondria/metabolism , Mitochondria/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Membrane ProteinsABSTRACT
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.
Subject(s)
Gene Products, tax , Human T-lymphotropic virus 1 , Protein-Arginine N-Methyltransferases , Humans , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Gene Products, tax/genetics , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Cyclin D2/genetics , Cyclin D2/metabolism , Transcriptional Activation , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/metabolism , NF-kappa B/metabolism , NF-kappa B/genetics , Histones/metabolism , Histones/genetics , Epigenesis, Genetic , Jurkat CellsABSTRACT
Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.
Subject(s)
Inflammation , Leukemia, Myeloid, Acute , Mice, Knockout , NF-kappa B , Protein-Arginine N-Methyltransferases , Signal Transduction , Animals , Female , Humans , Male , Mice , Cell Line, Tumor , Inflammation/metabolism , Inflammation/genetics , Intracellular Signaling Peptides and Proteins , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice, Inbred C57BL , NF-kappa B/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/geneticsABSTRACT
Microglia-mediated inflammatory response is one key cause of many central nervous system diseases, like Alzheimer's disease. We hypothesized that a novel C15orf39 (MAPK1 substrate) plays a critical role in the microglial inflammatory response. To confirm this hypothesis, we used lipopolysaccharide (LPS)-and interferon-gamma (IFN-γ)-induced human microglia HMC3 cells as a representative indicator of the microglial in vitro inflammatory response. We found that C15orf39 was down-regulated when interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) expression increased in LPS/IFN-γ-stimulated HMC3 cells. Once C15orf39 was overexpressed, IL-6 and TNFα expression were reduced in LPS/IFN-γ-stimulated HMC3 cells. In contrast, C15orf39 knockdown promoted IL-6 and TNFα expression in LPS/IFN-γ-stimulated HMC3 cells. These results suggest that C15orf39 is a suppressive factor in the microglial inflammatory response. Mechanistically, C15orf39 interacts with the cytoplasmic protein arginine methyltransferase 2 (PRMT2). Thus, we termed C15orf39 a PRMT2 interaction protein (PRMT2 IP). Furthermore, the interaction of C15orf39 and PRMT2 suppressed the activation of NF-κB signaling via the PRMT2-IκBα signaling axis, which then led to a reduction in transcription of the inflammatory factors IL6 and TNF-α. Under inflammatory conditions, NF-κBp65 was found to be activated and to suppress C15orf39 promoter activation, after which it canceled the suppressive effect of the C15orf39-PRMT2-IκBα signaling axis on IL-6 and TNFα transcriptional expression. In conclusion, our findings demonstrate that in a steady condition, the interaction of C15orf39 and PRMT2 stabilizes IκBα to inhibit IL-6 and TNFα expression by suppressing NF-κB signaling, which reversely suppresses C15orf39 transcription to enhance IL-6 and TNFα expression in the microglial inflammatory condition. Our study provides a clue as to the role of C15orf39 in microglia-mediated inflammation, suggesting the potential therapeutic efficacy of C15orf39 in some central nervous system diseases.
Subject(s)
Inflammation , Interleukin-6 , Lipopolysaccharides , Microglia , Protein-Arginine N-Methyltransferases , Tumor Necrosis Factor-alpha , Humans , Cell Line , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Interleukin-6/metabolism , Interleukin-6/genetics , Lipopolysaccharides/pharmacology , Microglia/metabolism , Microglia/drug effects , NF-kappa B/metabolism , Open Reading Frames , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Chromosomes, Human, Pair 15ABSTRACT
DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.
Subject(s)
DNA Replication , Immunity, Innate , Protein-Arginine N-Methyltransferases , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Signal Transduction , Arginine/metabolism , Arginine/analogs & derivatives , Stress, Physiological , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA Damage , Transcription Factors/metabolism , Transcription Factors/geneticsABSTRACT
Cancer cells are often addicted to serine synthesis to support growth. How serine synthesis is regulated in cancer is not well understood. We recently demonstrated protein arginine methyltransferase 1 (PRMT1) is upregulated in hepatocellular carcinoma (HCC) to methylate and activate phosphoglycerate dehydrogenase (PHGDH), thereby promoting serine synthesis. However, the mechanisms underlying PRMT1 upregulation and regulation of PRMT1-PHGDH axis remain unclear. Here, we show the E3 ubiquitin ligase F-box-only protein 7 (FBXO7) inhibits serine synthesis in HCC by binding PRMT1, inducing lysine 37 ubiquitination, and promoting proteosomal degradation of PRMT1. FBXO7-mediated PRMT1 downregulation cripples PHGDH arginine methylation and activation, resulting in impaired serine synthesis, accumulation of reactive oxygen species (ROS), and inhibition of HCC cell growth. Notably, FBXO7 is significantly downregulated in human HCC tissues, and inversely associated with PRMT1 protein and PHGDH methylation level. Overall, our study provides mechanistic insights into the regulation of cancer serine synthesis by FBXO7-PRMT1-PHGDH axis, and will facilitate the development of serine-targeting strategies for cancer therapy.
Subject(s)
Carcinoma, Hepatocellular , F-Box Proteins , Liver Neoplasms , Phosphoglycerate Dehydrogenase , Protein-Arginine N-Methyltransferases , Serine , Ubiquitination , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Humans , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , F-Box Proteins/metabolism , F-Box Proteins/genetics , Serine/metabolism , Serine/biosynthesis , Phosphoglycerate Dehydrogenase/metabolism , Phosphoglycerate Dehydrogenase/genetics , Cell Line, Tumor , Animals , Repressor Proteins/metabolism , Repressor Proteins/genetics , Mice , Cell Proliferation , Methylation , Gene Expression Regulation, Neoplastic , Mice, Nude , Male , HEK293 Cells , Female , Hep G2 CellsABSTRACT
Gastric cancer (GC) is a common form of cancer and the leading cause of cancer-related deaths worldwide. Chemotherapy is the primary treatment for patients with unresectable or partially resectable GC. However, its adverse effects and chemoresistance greatly restrict its applicability and efficacy. Although HER2-targeted therapy and immunotherapy have been successfully used for GC treatment, their beneficial population is limited. To expand the range of cancer treatments, drug repurposing has emerged as a promising strategy. In this study, we evaluated the potential of Metformin, an oral anti-hyperglycemic agent, to suppress GC progression both in vivo and in vitro. Functional investigations showed that Metformin significantly inhibits GC proliferation and migration. Furthermore, we discovered that Metformin bound and disrupted STAT1 phosphorylation, inhibiting PRMT1 expression and consequently GC progression. In conclusion, our study not only provides further evidence for the anti-GC role of Metformin but also identifies the direct target mediating the tumor-inhibitory effects of Metformin in GC.
Subject(s)
Metformin , Mice, Nude , Protein-Arginine N-Methyltransferases , Repressor Proteins , STAT1 Transcription Factor , Stomach Neoplasms , Animals , Humans , Male , Mice , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Progression , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Metformin/therapeutic use , Mice, Inbred BALB C , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/antagonists & inhibitors , STAT1 Transcription Factor/metabolism , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Stomach Neoplasms/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.
Subject(s)
Astrocytoma , Brain Neoplasms , Isocitrate Dehydrogenase , Promoter Regions, Genetic , Protein-Arginine N-Methyltransferases , Tumor Suppressor Proteins , Adult , Aged , Female , Humans , Male , Middle Aged , Astrocytoma/genetics , Astrocytoma/pathology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Disease Progression , DNA Methylation , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Gene Expression Regulation, Neoplastic , Isocitrate Dehydrogenase/genetics , Mutation , Neoplasm Grading , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Retrospective Studies , Temozolomide/therapeutic use , Temozolomide/pharmacology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that deposits asymmetrical dimethylation marks on both histone and nonhistone substrates. The regulatory role of CARM1 in transcription was first identified in estrogen receptor positive (ER+) breast cancer. Since then, the mechanism of CARM1 in activating ER-target genes has been further interrogated. CARM1 is expressed at the highest level in ER negative (ER-) breast cancer and higher expression correlates with poor prognosis, suggesting an oncogenic role of CARM1. Indeed, in ER- breast cancer, CARM1 can promote proliferation and metastasis at least partly through methylation of proteins and activation of oncogenes. In this review, we summarize the mechanisms of transcriptional activation by CARM1 in breast cancer. The methyltransferase activity of CARM1 is important for many of its functions; here, we also highlight the nonenzymatic roles of CARM1. We also cover the biological processes regulated by CARM1 that are often deregulated in cancer and the ways to harness CARM1 in cancer treatment.
Subject(s)
Breast Neoplasms , Protein-Arginine N-Methyltransferases , Humans , Protein-Arginine N-Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Transcriptional Activation , AnimalsABSTRACT
Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.