ABSTRACT
We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/enzymology , Extracellular Signal-Regulated MAP Kinases/metabolism , Histones/metabolism , Serum Response Factor/metabolism , TCF Transcription Factors/metabolism , Transcription, Genetic , Animals , Cell Line , Chromatin/drug effects , Chromatin/genetics , Chromatin Assembly and Disassembly/drug effects , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Enzyme Activation , Mice , Mice, Knockout , Mutation , Phosphorylation , RNA Interference , Serum Response Factor/genetics , Signal Transduction , TCF Transcription Factors/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transcription Initiation Site , Transcription, Genetic/drug effects , Transfection , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolismABSTRACT
The transcription factor SRF (serum response factor) recruits two families of coactivators, the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors), to couple gene transcription to growth factor signaling. Here we investigated the role of the SRF network in the immediate transcriptional response of fibroblasts to serum stimulation. SRF recruited its cofactors in a gene-specific manner, and virtually all MRTF binding was directed by SRF. Much of SRF DNA binding was serum-inducible, reflecting a requirement for MRTF-SRF complex formation in nucleosome displacement. We identified 960 serum-responsive SRF target genes, which were mostly MRTF-controlled, as assessed by MRTF chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) and/or sensitivity to MRTF-linked signals. MRTF activation facilitates RNA polymerase II (Pol II) recruitment or promoter escape according to gene context. MRTF targets encode regulators of the cytoskeleton, transcription, and cell growth, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing. Finally, we show that specific activation of either MRTFs or TCFs can reset the circadian clock.
Subject(s)
Actins/metabolism , Fibroblasts/physiology , Serum/metabolism , Signal Transduction , Transcription, Genetic/genetics , Animals , CLOCK Proteins/genetics , Circadian Clocks/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Serum Response Factor/metabolismABSTRACT
Oncogenic KRAS impairs antitumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of the mechanisms by which oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive COX2 in cancer cells. Oncogenic KRAS potently induced COX2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX2/PGE2 remodeled the tumor microenvironment by inducing proinflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer. Significance: COX2 signaling via prostaglandin E2 is a major mediator of immune evasion driven by oncogenic KRAS that promotes immunotherapy and KRAS-targeted therapy resistance, suggesting effective combination treatments for KRAS-mutant lung cancer.
Subject(s)
CRISPR-Cas Systems , Cyclooxygenase 2 , Drug Resistance, Neoplasm , Immunotherapy , Lung Neoplasms , Proto-Oncogene Proteins p21(ras) , Animals , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/therapy , Lung Neoplasms/drug therapy , Cyclooxygenase 2/metabolism , Cyclooxygenase 2/genetics , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Humans , Drug Resistance, Neoplasm/genetics , Immunotherapy/methods , Dinoprostone/metabolism , Adenocarcinoma of Lung/immunology , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/therapy , Tumor Microenvironment/immunology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Cell Line, Tumor , Mice, Inbred C57BL , FemaleABSTRACT
In order to identify new genes with differential expression in early intestinal tumours, we performed mRNA (messenger ribonucleic acid) expression profiling of 16 human and 63 mouse adenomas. All individuals had germline APC mutations to ensure that tumorigenesis was driven by 'second hits' at APC. Using stringent filtering to identify changes consistent between humans and mice, we identified 60 genes up-regulated and 151 down-regulated in tumours. For 22 selected genes--including known Wnt targets--expression differences were confirmed by qRT-PCR (quantitative reverse transcription polymerase chain reaction). Most, but not all, differences were also present in colorectal carcinomas. In situ analysis showed a complex picture. Expression of up-regulated genes in adenomas was usually uniform/diffuse (e.g. ITGA6) or prominent in the tumour core (e.g. LGR5); in normal tissue, these genes were expressed at crypt bases or the transit amplifying zone. Down-regulated genes were often undetectable in adenomas, but in normal tissue were expressed in mesenchyme (e.g. GREM1/2) or differentiated cells towards crypt tops (e.g. SGK1). In silico analysis of TCF4-binding motifs showed that some of our genes were probably direct Wnt targets. Previous studies, mostly focused on human tumours, showed partial overlap with our 'expression signature', but 37 genes were unique to our study, including TACSTD2, SEMA3F, HOXA9 and IER3 (up-regulated), and TAGLN, GREM1, GREM2, MAB21L2 and RARRES2 (down-regulated). Combined analysis of our and published human data identified additional genes differentially expressed in adenomas, including decreased BMPs (bone morphogenetic proteins) and increased BUB1/BUB1B. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours.
Subject(s)
Adenoma/genetics , Gene Targeting , Genes, APC/physiology , Intestinal Neoplasms/genetics , Mutation/genetics , Wnt Proteins/physiology , Adenoma/metabolism , Animals , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Intestinal Neoplasms/metabolism , Mice , Oligonucleotide Array Sequence AnalysisABSTRACT
The Elongator complex has been implicated in several cellular processes, including gene expression and tRNA modification. We investigated the biological importance of the Elp3 gene in Drosophila melanogaster. Deletion of Elp3 results in larval lethality at the pupal stage. During early development, larval growth is dramatically impaired, with progression to the third instar delayed for â¼24 hr, and pupariation occurring only at day 14 after egg laying. Melanotic nodules appear after 4 days. Microarray analysis shows that stress response genes are induced and ecdysone-induced transcription factors are severely repressed in the mutant. Interestingly, the phenotypes of Elp3 flies are similar to those of flies lacking the domino gene, encoding a SWI/SNF-like ATP-dependent chromatin-remodeling enzyme. Indeed, the gene expression profiles of these mutants are also remarkably similar. Together, these data demonstrate that Drosophila Elp3 is essential for viability, normal development, and hematopoiesis and suggest a functional overlap with the chromatin remodeler Domino.
Subject(s)
Drosophila melanogaster/growth & development , Drosophila melanogaster/genetics , Genes, Lethal , Histone Acetyltransferases/metabolism , Nerve Tissue Proteins/metabolism , Animals , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly , Drosophila Proteins/genetics , Ecdysone/physiology , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation, Developmental , Histone Acetyltransferases/genetics , Larva/genetics , Larva/metabolism , Linear Models , Nerve Tissue Proteins/genetics , Oligonucleotide Array Sequence Analysis , Pupa/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that "pseudohypoxic" stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors.
Subject(s)
Fumarate Hydratase/deficiency , Fumarate Hydratase/genetics , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Animals , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Proliferation , Cells, Cultured , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Glycolysis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Leiomyomatosis/genetics , Leiomyomatosis/metabolism , Leiomyomatosis/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth/metabolism , Muscle, Smooth/pathology , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Spectral KaryotypingABSTRACT
Homozygosity for the G allele of rs6983267 at 8q24 increases colorectal cancer (CRC) risk approximately 1.5 fold. We report here that the risk allele G shows copy number increase during CRC development. Our computer algorithm, Enhancer Element Locator (EEL), identified an enhancer element that contains rs6983267. The element drove expression of a reporter gene in a pattern that is consistent with regulation by the key CRC pathway Wnt. rs6983267 affects a binding site for the Wnt-regulated transcription factor TCF4, with the risk allele G showing stronger binding in vitro and in vivo. Genome-wide ChIP assay revealed the element as the strongest TCF4 binding site within 1 Mb of MYC. An unambiguous correlation between rs6983267 genotype and MYC expression was not detected, and additional work is required to scrutinize all possible targets of the enhancer. Our work provides evidence that the common CRC predisposition associated with 8q24 arises from enhanced responsiveness to Wnt signaling.
Subject(s)
Chromosomes, Human, Pair 8/genetics , Colorectal Neoplasms/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide/genetics , Signal Transduction , Wnt Proteins/metabolism , Animals , Base Sequence , Binding Sites , Conserved Sequence , Embryo, Mammalian/metabolism , Enhancer Elements, Genetic/genetics , Gene Dosage , Genome-Wide Association Study , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Organ Specificity , Protein Binding , Proto-Oncogene Proteins c-myc/metabolism , Reproducibility of Results , TCF Transcription Factors/metabolism , Transcription Factor 7-Like 2 Protein , beta Catenin/metabolismABSTRACT
Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia.
Subject(s)
Leukemia, Myeloid, Acute/genetics , MicroRNAs/genetics , Analysis of Variance , Base Sequence , Chromosome Aberrations , Chromosomes, Human, Pair 14 , DNA Probes , Gene Expression Profiling , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Oligonucleotides/chemistry , Polymerase Chain ReactionABSTRACT
We recently completed the total synthesis of spiruchostatin A, a depsipeptide natural product with close structural similarities to FK228, a histone deacetylase (HDAC) inhibitor (HDI) currently being evaluated in clinical trials for cancer. Here we report a detailed characterisation of the in vitro activity of spiruchostatin A. Spiruchostatin A was a potent (sub-nM) inhibitor of class I HDAC activity in vitro and acted as a prodrug, requiring reduction for activity. Spiruchostatin A was a potent (low nM) inhibitor of the growth of various cancer cell lines. Spiruchostatin A-induced acetylation of specific lysine residues within histones H3 and H4, and increased the expression of p21(cip1/waf1), but did not induce acetylation of alpha-tubulin. Spiruchostatin A also induced cell cycle arrest, differentiation and cell death in MCF7 breast cancer cells. Like FK228, spiruchostatin A was both an inducer and substrate of the ABCB1 drug efflux pump. Whereas spiruchostatin A and FK228-induced protracted histone acetylation, hydroxamate HDI-induced short-lived histone acetylation. Using a subset of HDI-target genes identified by microarray analysis, we demonstrated that these differences in kinetics of histone acetylation between HDI correlated with differences in the kinetics of induction or repression of specific target genes. Our results demonstrate that spiruchostatin A is a potent inhibitor of class I HDACs and anti-cancer agent. Differences in the kinetics of action of HDI may be important for the clinical application of these compounds.