ABSTRACT
NHE5, an isoform of the Na+/H+ exchanger (NHE) protein, is an ion-transporting membrane protein that regulates intracellular pH and is highly expressed in colorectal adenocarcinoma. Therefore, we hypothesized that NHE5 inhibitors can be used as anticancer drugs. However, because NHE1 is ubiquitously expressed in all cells, it is extremely important to demonstrate its selective inhibitory activity against NHE5. We used amiloride, an NHE non-selective inhibitor, as a lead compound and created UTX-143, which has NHE5-selective inhibitory activity, using a structure-activity relationship approach. UTX-143 showed selective cytotoxic effects on cancer cells and reduced the migratory and invasive abilities of cancer cells. These results suggest a new concept wherein drugs exhibit cancer-specific cytotoxic effects through selective inhibition of NHE5 and the possibility of UTX-143 as a lead NHE5-selective inhibitor.
Subject(s)
Amiloride , Sodium , Amiloride/pharmacology , Sodium/metabolism , Sodium-Hydrogen Exchangers/metabolism , Membrane Proteins/metabolism , Hydrogen , Hydrogen-Ion ConcentrationABSTRACT
Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/etiology , Breast Neoplasms/metabolism , Mammary Neoplasms, Experimental/etiology , Mammary Neoplasms, Experimental/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Breast Neoplasms/immunology , Carcinogenesis , Cytokines/metabolism , Female , Humans , Inflammation/etiology , Inflammation/metabolism , Mammary Neoplasms, Experimental/immunology , Mammary Tumor Virus, Mouse , Mice , Mice, Knockout , NF-kappa B/metabolism , Pregnancy , Receptor, ErbB-2/metabolism , Retroviridae Infections , Tumor Microenvironment/immunology , Tumor Virus InfectionsABSTRACT
N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.
Subject(s)
Epithelial-Mesenchymal Transition , Lung Neoplasms , Humans , Epithelial-Mesenchymal Transition/genetics , Transforming Growth Factor beta/metabolism , Methylation , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , RNA, Messenger/genetics , Transcription Factor AP-1/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Transforming Growth Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
ASH2L (Absent-Small-Homeotic-2-Like protein) is a core subunit of the COMPASS (COMplex of Proteins ASsociated with Set1) complex, the most notable writer of the methylation of histone H3 lysine 4 (H3K4). The COMPASS complex regulates active promoters or enhancers for gene expression, and its dysfunction is associated with aberrant development and disease. Here, we demonstrated that ASH2L mediated the cell invasion and migration activity of triple-negative breast cancer cells through the interaction with the COMPASS components and the target genomic regions. Transcriptome analysis indicated a potential correlation between ASH2L and the genes involved in inflammatory/immune responses. Among them, we found that the intrinsic expression of IL1B (interleukin 1 beta), an essential proinflammatory gene, was directly regulated by ASH2L. These results revealed a novel role of ASH2L on the maintenance of breast cancer malignancy possibly through H3K4 methylation of the target inflammatory/immune responsive genes.
Subject(s)
Histones , Triple Negative Breast Neoplasms , Humans , Histones/metabolism , Methylation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Lysine/metabolism , Triple Negative Breast Neoplasms/genetics , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Epigenesis, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Polycomb repressive complex-1 (PRC1) induces transcriptional repression by regulating monoubiquitination of lysine 119 of histone H2A (H2AK119) and as such is involved in a number of biological and pathological processes including cancer development. Previously we demonstrated that PRC2, which catalyzes the methylation of histone H3K27, has an essential function in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung and pancreatic cancer cell lines. Since the cooperative activities of PRC1 and PRC2 are thought to be important for transcriptional repression in EMT program, we investigated the role of KDM2B, a member of PRC1 complex, on TGF-ß-induced EMT in this study. Knockdown of KDM2B inhibited TGF-ß-induced morphological conversion of the cells and enhanced cell migration and invasion potentials as well as the expression changes of EMT-related marker genes. Overexpression of KDM2B influenced the expression of several epithelial marker genes such as CDH1, miR200a, and CGN and enhanced the effects of TGF-ß. Mechanistic investigations revealed that KDM2B specifically recognized the regulatory regions of CDH1, miR200a, and CGN genes and induced histone H2AK119 monoubiquitination as a component of PRC1 complex, thereby mediating the subsequent EZH2 recruitment and histone H3K27 methylation process required for gene repression. Studies using KDM2B mutants confirmed that its DNA recognition property but not its histone H3 demethylase activity was indispensable for its function during EMT. This study demonstrated the significance of the regulation of histone H2A ubiquitination in EMT process and provided the possibility to develop novel therapeutic strategies for the treatment of cancer metastasis.
Subject(s)
Epithelial-Mesenchymal Transition/drug effects , F-Box Proteins/metabolism , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Lung Neoplasms/pathology , Pancreatic Neoplasms/pathology , Transforming Growth Factor beta/pharmacology , Antigens, CD/metabolism , Cadherins/metabolism , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/metabolism , F-Box Proteins/genetics , Gene Expression Regulation, Neoplastic , Histones/genetics , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolismABSTRACT
N6-Methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the role of m6A methyltransferase METTL3 in TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung cancer cell lines. The expression of METTL3 and m6A RNA modification were increased during TGF-ß-induced EMT of A549 and LC2/ad lung cancer cells. Knockdown of METTL3 inhibited TGF-ß-induced morphological conversion of the cells, enhanced cell migration potential and the expression changes of EMT-related marker genes such as CDH1/E-cadherin, FN1/Fibronectin and VIM/Vimentin. Mechanistic investigations revealed that METTL3 knockdown decreased the m6A modification, total mRNA level and mRNA stability of JUNB, one of the important transcriptional regulators of EMT. Over-expression of JUNB partially rescued the inhibitory effects of METTL3 knockdown in the EMT phenotypes. This study demonstrates that m6A methyltransferase METTL3 is indispensable for TGF-ß-induced EMT of lung cancer cells through the regulation of JUNB.
Subject(s)
Epithelial-Mesenchymal Transition/drug effects , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Methyltransferases/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/pharmacology , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Lung Neoplasms/genetics , Methyltransferases/genetics , Phenotype , RNA Stability/drug effects , Transcription Factors/geneticsABSTRACT
Long noncoding RNAs (lncRNAs) are important regulatory molecules in various biological and pathological processes, including cancer development. We have previously shown that the MEG3 lncRNA plays an essential role in transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) of human lung cancer cells. In this study, we investigated the function of another lncRNA, MEG8, which shares the DLK1-DIO3 locus with MEG3, in the regulation of EMT. MEG8 lncRNA expression was immediately induced during TGF-ß-mediated EMT of A549 and LC2/ad lung cancer and Panc1 pancreatic cancer cell lines. MEG8 overexpression specifically suppressed the expression of microRNA-34a and microRNA-203 genes, resulting in up-regulation of SNAIL family transcriptional repressor 1 (SNAI1) and SNAI2 transcription factors, which repressed expression of cadherin 1 (CDH1)/E-cadherin. Mechanistic investigations revealed that MEG8 associates with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) protein and induces its recruitment to the regulatory regions of the two microRNA genes for histone H3 methylation and transcriptional repression. Interestingly, expression of both MEG8 and MEG3, but not each individually, could induce EMT-related cell morphological changes and increased cell motility in the absence of TGF-ß by activating the gene expression program required for EMT. MEG8 knockdown indicated that endogenous MEG8 lncRNA is indispensable for TGF-ß-induced EMT in A549 lung cancer and Panc1 pancreatic cancer cells. Our findings indicate that MEG8 lncRNA significantly contributes to epigenetic EMT induction and increase our understanding of the lncRNA-mediated regulatory mechanisms involved in malignant progression of cancer.
Subject(s)
Epithelial-Mesenchymal Transition , Lung Neoplasms/genetics , Lung Neoplasms/physiopathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/physiopathology , RNA, Long Noncoding/genetics , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Epigenesis, Genetic , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Pancreatic Neoplasms/metabolism , RNA, Long Noncoding/metabolism , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolism , Transforming Growth Factor beta1ABSTRACT
Histone methylation is implicated in a number of biological and pathological processes, including cancer development. In this study, we investigated the molecular mechanism for the recruitment of Polycomb repressive complex-2 (PRC2) and its accessory component, JARID2, to chromatin, which regulates methylation of lysine 27 of histone H3 (H3K27), during epithelial-mesenchymal transition (EMT) of cancer cells. The expression of MEG3 long noncoding RNA (lncRNA), which could interact with JARID2, was clearly increased during transforming growth factor-ß (TGF-ß)-induced EMT of human lung cancer cell lines. Knockdown of MEG3 inhibited TGF-ß-mediated changes in cell morphology and cell motility characteristic of EMT and counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1, ZEB family, and the microRNA-200 family. Overexpression of MEG3 influenced the expression of these genes and enhanced the effects of TGF-ß in their expressions. Chromatin immunoprecipitation (ChIP) revealed that MEG3 regulated the recruitment of JARID2 and EZH2 and histone H3 methylation on the regulatory regions of CDH1 and microRNA-200 family genes for transcriptional repression. RNA immunoprecipitation and chromatin isolation by RNA purification assays indicated that MEG3 could associate with JARID2 and the regulatory regions of target genes to recruit the complex. This study demonstrated a crucial role of MEG3 lncRNA in the epigenetic regulation of the EMT process in lung cancer cells.
Subject(s)
Epigenesis, Genetic/genetics , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Antigens, CD , Apoptosis , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Cell Proliferation , Chromatin/genetics , Fluorescent Antibody Technique , Histones/genetics , Humans , Immunoprecipitation , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Tumor Cells, CulturedABSTRACT
Histone methylation is associated with various biological and pathological processes including cancer development. KDM6A is a candidate tumor suppressor gene that encodes a histone H3 lysine 27 (H3K27) demethylase. In this study, we discovered that ectopic expression of KDM6A antagonized TGF-ß-induced epithelial-mesenchymal transition (EMT) and cell migration of lung cancer cell lines through its demethylase activity. KDM6A counteracted TGF-ß-dependent changes in the expression of EMT-related genes such as CDH1/E-cadherin, FN1/Fibronectin, ZEB family and microRNA-200 family. Mechanistic investigations revealed that KDM6A inhibited the recruitment of EZH2 histone H3K27 methyltransferase and H3K27 methylation on the regulatory regions of the target genes such as CDH1 and microRNA-200 family. Knockdown of KDM6A did not proceed EMT by itself, but influenced the expression of specific target genes critical for EMT, suggesting that endogenous KDM6A was involved in EMT-inducing transcriptional program. This study demonstrated a novel regulatory role of KDM6A histone demethylase in the epigenetic control of EMT process in lung cancer cells.
Subject(s)
Epigenesis, Genetic , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Histone Demethylases/genetics , Transforming Growth Factor beta/genetics , A549 Cells , Animals , Antigens, CD , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Fibronectins/genetics , Fibronectins/metabolism , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Histone Demethylases/metabolism , Humans , Lentivirus/genetics , Lentivirus/metabolism , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Transforming Growth Factor beta/metabolism , Zinc Finger E-box-Binding Homeobox 1/genetics , Zinc Finger E-box-Binding Homeobox 1/metabolismABSTRACT
Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells(-/-) (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.
Subject(s)
Cytosine/metabolism , DNA Helicases/genetics , DNA Methylation , DNA/metabolism , Gene Expression Regulation, Developmental , Animals , Cell Nucleus/genetics , Embryonic Stem Cells , Epigenesis, Genetic , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Knockout Techniques , Histones/metabolism , Lamin Type B/metabolism , Mice , Molecular Sequence Data , Mutation , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic AcidABSTRACT
Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh(-/-) (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells.
Subject(s)
Adenosine Triphosphate/metabolism , Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA Methylation , Nucleosomes/metabolism , Repetitive Sequences, Nucleic Acid , Binding Sites , Cells, Cultured , HumansABSTRACT
DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh(-/-) mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh(-/-) MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh(-/-) iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh(-/-) iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity.
Subject(s)
CpG Islands/genetics , DNA Helicases/deficiency , DNA Methylation/genetics , Embryo, Mammalian/cytology , Fibroblasts/metabolism , Histones/metabolism , Lysine/metabolism , Animals , Biomarkers/metabolism , Cell Lineage , DNA Helicases/metabolism , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic , Fibroblasts/cytology , HeLa Cells , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Knockout , Neurons/cytology , Protein Binding , Signal Transduction , Transcription Factors/metabolismABSTRACT
Genetic studies have shown that aberrant activation of p53 signaling leads to embryonic lethality. Maintenance of a fine balance of the p53 protein level is critical for normal development. Previously, we have reported that Jmjd5, a member of the Jumonji C (JmjC) family, regulates embryonic cell proliferation through the control of Cdkn1a expression. Since Cdkn1a is the representative p53-regulated gene, we have examined whether the expression of other p53 target genes is coincidentally upregulated with Cdkn1a in Jmjd5-deficient embryos. The expression of a subset of p53-regulated genes was increased in both Jmjd5 hypomorphic mouse embryonic fibroblasts (MEFs) and Jmjd5-deficient embryos at embryonic day 8.25 without the induced expression of Trp53. Intercrossing of Jmjd5-deficient mice with Trp53 knockout mice showed that the growth defect of Jmjd5 mutant cells was significantly recovered under a Trp53 null genetic background. Chromatin immunoprecipitation analysis in Jmjd5 hypomorphic MEFs indicated the increased recruitment of p53 at several p53 target gene loci, such as Cdkn1a, Pmaip1, and Mdm2. These results suggest that Jmjd5 is involved in the transcriptional regulation of a subset of p53-regulated genes, possibly through the control of p53 recruitment at the gene loci. In Jmjd5-deficient embryos, the enhanced recruitment of p53 might result in the abnormal activation of p53 signaling leading to embryonic lethality.
Subject(s)
Embryonic Development , Jumonji Domain-Containing Histone Demethylases/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Animals , Cell Proliferation , Embryo, Mammalian/cytology , Embryonic Development/genetics , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Genetic Loci , Humans , Jumonji Domain-Containing Histone Demethylases/deficiency , Jumonji Domain-Containing Histone Demethylases/genetics , Mice , Phenotype , Signal Transduction/genetics , Up-Regulation/geneticsABSTRACT
Covalent modifications of histones play an important role in chromatin architecture and dynamics. In particular, histone lysine methylation is important for transcriptional control during diverse biological processes. The nuclear protein Jmjd5 (also called Kdm8) is a histone lysine demethylase that contains a JmjC domain in the C-terminal region. In this study, we have generated Jmjd5-deficient mice (Jmjd5(Δ)(/)(Δ)) to investigate the in vivo function of Jmjd5. Jmjd5(Δ)(/)(Δ) embryos showed severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos (Jmjd5(neo/neo)) also showed the growth defect. Quantitative PCR analysis of various cell cycle regulators indicated that only Cdkn1a expression was upregulated in Jmjd5(neo/neo) MEFs and Jmjd5(Δ)(/)(Δ) embryos. A knockdown assay with Cdkn1a-specific small interfering RNAs revealed that the growth defect of Jmjd5(neo/neo) MEFs was significantly rescued. In addition, a genetic study using Jmjd5(Δ)(/)(Δ); Cdkn1a(Δ)(/)(Δ) double-knockout mice showed that the growth retardation of Jmjd5(Δ)(/)(Δ) embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in Jmjd5(neo/neo) MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates embryonic cell proliferation through the epigenetic control of Cdkn1a expression.
Subject(s)
Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Animals , Cells, Cultured , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Gene Expression Regulation , Histones/genetics , Humans , In Situ Hybridization , Jumonji Domain-Containing Histone Demethylases/genetics , Lysine/metabolism , Mice , Mice, Knockout , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Histone methylation is involved in various biological and pathological processes including cancer development. In this study, we found that EED, a component of Polycomb repressive complex-2 (PRC2) that catalyzes methylation of lysine 27 of histone H3 (H3K27), was involved in epithelial-mesenchymal transition (EMT) of cancer cells induced by Transforming Growth Factor-beta (TGF-ß). The expression of EED was increased during TGF-ß-induced EMT and knockdown of EED inhibited TGF-ß-induced morphological conversion of the cells associated with EMT. EED knockdown antagonized TGF-ß-dependent expression changes of EMT-related genes such as CDH1, ZEB1, ZEB2 and microRNA-200 (miR-200) family. Chromatin immunoprecipitation assays showed that EED was implicated in TGF-ß-induced transcriptional repression of CDH1 and miR-200 family genes through the regulation of histone H3 methylation and EZH2 occupancies on their regulatory regions. Our study demonstrated a novel role of EED, which regulates PRC2 activity and histone methylation during TGF-ß-induced EMT of cancer cells.
Subject(s)
Epithelial-Mesenchymal Transition/physiology , Neoplasms/pathology , Neoplasms/physiopathology , Polycomb Repressive Complex 2/physiology , Transforming Growth Factor beta/physiology , Antigens, CD , Cadherins/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , HT29 Cells , Histones/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , MicroRNAs/genetics , Neoplasms/genetics , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/genetics , Promoter Regions, Genetic , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger E-box Binding Homeobox 2 , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
Retroviral insertional mutagenesis in mice is considered a powerful forward genetic strategy to identify disease genes involved in cancer. Our high-throughput screens led to frequent identification of the genes encoding the enzymes engaged in histone lysine methylation. Histone methylation can positively or negatively impact on gene transcription, and then fulfill important roles in developmental control and cell-fate decisions. A tremendous amount of progress has accelerated the characterization of histone methylations and the enzymes that regulate them. Deregulation of these histone methyl-modifying enzymes has been increasingly recognized as a hallmark of cancer in the last few years. However, in most cases, we have only limited understanding for the molecular mechanisms by which these enzymes contribute to cancer development and progression. In this review, we summarize the current knowledge regarding some of the best-validated examples of histone lysine methyltransferases and demethylases associated with oncogenesis and discuss their potential mechanisms of action.
Subject(s)
Histone Demethylases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Animals , DNA Methylation , Disease Progression , Histone Demethylases/genetics , Histone-Lysine N-Methyltransferase/genetics , Humans , Mutagenesis, Insertional , Neoplasms/enzymology , Neoplasms/geneticsABSTRACT
PLU1 is a candidate oncogene that encodes H3K4 (Lys(4) of histone H3) demethylase. In the present study, we found that ectopic expression of PLU1 enhanced the invasive potential of the weakly invasive cells dependent on its demethylase activity. PLU1 was shown to repress the expression of the KAT5 gene through its H3K4 demethylation on the promoter. The regulation of KAT5 by PLU1 was suggested to be responsible for PLU1-induced cell invasion. First, knockdown of KAT5 similarly increased the invasive potential of the cells. Secondly, knockdown of PLU1 in the highly invasive cancer cells increased KAT5 expression and reduced the invasive activity. Thirdly, simultaneous knockdown of KAT5 partially relieved the suppression of cell invasion imposed by PLU1 knockdown. Finally, we found that CD82, which was transcriptionally regulated by KAT5, might be a candidate effector of cell invasion promoted by PLU1. The present study demonstrated a functional contribution of PLU1 overexpression with concomitant epigenetic dysregulation in cancer progression.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/physiology , Histone Acetyltransferases/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Neoplasm Invasiveness/genetics , Animals , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA-Binding Proteins/genetics , Histone Acetyltransferases/genetics , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Kangai-1 Protein/genetics , Kangai-1 Protein/metabolism , Lysine Acetyltransferase 5 , Mice , Promoter Regions, GeneticABSTRACT
[This corrects the article DOI: 10.1016/j.bbrep.2021.101072.].
ABSTRACT
Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-ß1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-ß1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-ß1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-ß1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-ß1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-ß1-induced cell invasion into collagen gel. Thus, TGF-ß1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.
ABSTRACT
Utx is a candidate tumor suppressor gene that encodes histone H3 lysine 27 (H3K27) demethylase. In this study, we found that ectopic expression of Utx enhanced the expression of retinoblastoma tumor suppressor gene Rb and its related gene Rbl2. This activation was dependent on the demethylase activity of Utx, and was suggested to contribute to the decreased cell proliferation induced by Utx. A chromatin immunoprecipitation assay showed that over-expressed Utx was associated with the promoter regions of Rb and Rbl2 resulting in the removal of repressive H3K27 tri-methylation and the increase in active H3K4 tri-methylation. Furthermore, siRNA-mediated knockdown of Utx revealed the recruitment of endogenous Utx protein on the promoters of Rb and Rbl2 genes. These results indicate that Rb and Rbl2 are downstream target genes of Utx and may play important roles in Utx-mediated cell growth control.