ABSTRACT
Although the MAPK/MEK/ERK pathway is prevalently activated in colorectal cancer (CRC), MEK/ERK inhibitors show limited efficiency in clinic. As a downstream target of MAPK, ELK4 is thought to work primarily by forming a complex with SRF. Whether ELK4 can serve as a potential therapeutic target is unclear and the transcriptional regulatory mechanism has not been systemically analyzed. Here, it is shown that ELK4 promotes CRC tumorigenesis. Integrated genomics- and proteomics-based approaches identified SP1 and SP3, instead of SRF, as cooperative functional partners of ELK4 at genome-wide level in CRC. Serum-induced phosphorylation of ELK4 by MAPKs facilitated its interaction with SP1/SP3. The pathological neoangiogenic factor LRG1 is identified as a direct target of the ELK4-SP1/SP3 complex. Furthermore, targeting the ELK4-SP1/SP3 complex by combination treatment with MEK/ERK inhibitor and the relatively specific SP1 inhibitor mithramycin A (MMA) elicited a synergistic antitumor effect on CRC. Clinically, ELK4 is a marker of poor prognosis in CRC. A 9-gene prognostic model based on the ELK4-SP1/3 complex-regulated gene set showed robust prognostic accuracy. The results demonstrate that ELK4 cooperates with SP1 and SP3 to transcriptionally regulate LRG1 to promote CRC tumorigenesis in an SRF-independent manner, identifying the ELK4-SP1/SP3 complex as a potential target for rational combination therapy.
Subject(s)
Colorectal Neoplasms , Gene Expression Regulation , Humans , Promoter Regions, Genetic , Colorectal Neoplasms/genetics , Carcinogenesis/genetics , Mitogen-Activated Protein Kinase Kinases/genetics , ets-Domain Protein Elk-4/genetics , GlycoproteinsABSTRACT
BACKGROUND: Transfer RNA-derived fragments (tRFs) are a new class of small non-coding RNAs. Recent studies suggest that tRFs participate in some pathological processes. However, the biological functions and mechanisms of tRFs in non-small cell lung cancer (NSCLC) are largely unknown. METHODS: Differentially expressed tRFs were identified by tRF and tiRNA sequencing using 9 pairs of pre- and post-operation plasma from patients with NSCLC. Quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to determine the levels of tRF in tissues, plasma, and cells. Gain- and loss-of-function experiments were implemented to investigate the oncogenic effects of tRF on NSCLC cells in vitro and in vivo. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation (RIP), Western blot, co-immunoprecipitation (Co-IP) assays, and rescue experiments were performed to explore the regulatory mechanisms of tRF in NSCLC. RESULTS: AS-tDR-007333 was an uncharacterized tRF and significantly up-regulated in NSCLC tissues, plasma, and cells. Clinically, AS-tDR-007333 overexpression could distinguish NSCLC patients from healthy controls and associated with poorer prognosis of NSCLC patients. Functionally, overexpression of AS-tDR-007333 enhanced proliferation and migration of NSCLC cells, whereas knockdown of AS-tDR-007333 resulted in opposite effects. Mechanistically, AS-tDR-007333 promoted the malignancy of NSCLC cells by activating MED29 through two distinct mechanisms. First, AS-tDR-007333 bound to and interacted with HSPB1, which activated MED29 expression by enhancing H3K4me1 and H3K27ac in MED29 promoter. Second, AS-tDR-007333 stimulated the expression of transcription factor ELK4, which bound to MED29 promoter and increased its transcription. Therapeutically, inhibition of AS-tDR-007333 suppressed NSCLC cell growth in vivo. CONCLUSIONS: Our study identifies a new oncogenic tRF and uncovers a novel mechanism that AS-tDR-007333 promotes NSCLC malignancy through the HSPB1-MED29 and ELK4-MED29 axes. AS-tDR-007333 is a potential diagnostic or prognostic marker and therapeutic target for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/genetics , Gene Expression Regulation , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/genetics , Molecular Chaperones , RNA, Transfer/genetics , RNA, Transfer/metabolism , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolismABSTRACT
The blood-brain barrier (BBB) plays a pivotal role in the maintenance and regulation of the neural microenvironment. The BBB breakdown is a pathological change in early Alzheimer's disease (AD). RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are involved in the regulation of BBB permeability. Our study demonstrates the role of TRA2A/LINC00662/ELK4 axis in regulating BBB permeability in AD microenvironment. In Aß1-42-incubated microvascular endothelial cells (ECs) of the BBB model in vitro, TRA2A and LINC00662 were enriched. TRA2A increased the stability of LINC00662 by binding with it. The knockdown of either TRA2A or LINC00662 decreased BBB permeability due to increased expression of tight junction-related proteins. ELK4 was less expressed in the BBB model in AD microenvironment in vitro. LINC00662 mediated the degradation of ELK4 mRNA by SMD pathway. Downregulation of ELK4 increased BBB permeability by increasing the tight junction-related protein expression.TRA2A/LINC00662/ELK4 axis plays a crucial role in the regulation of BBB permeability in AD microenvironment, which may provide a novel target for the therapy of AD.
Subject(s)
Blood-Brain Barrier/metabolism , Cellular Microenvironment/genetics , Gene Expression Regulation , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/metabolism , ets-Domain Protein Elk-4/genetics , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Biomarkers , Endothelial Cells/metabolism , Gene Knockdown Techniques , Humans , Peptide Fragments/metabolism , Permeability , Promoter Regions, Genetic , Protein Binding , RNA Interference , RNA Stability , RNA, Long Noncoding/genetics , Tight Junction Proteins/genetics , Tight Junction Proteins/metabolismABSTRACT
Aberrant expression of laminin-332 promotes tumour growth and metastasis in multiple cancers. However, the dysregulated expression and mechanism of action of LAMB3, which encodes the ß3 subunit of laminin-332, and the mechanism underlying dysregulated LAMB3 expression in CRC remain obscure. Here, we show that LAMB3 is overexpressed in CRC and that this overexpression is correlated with tumour metastasis and poor prognosis. Overexpression of LAMB3 promoted cell proliferation and cell migration in vitro and tumour growth and metastasis in vivo, while knockdown of LAMB3 elicited opposing effects. LAMB3 inhibited the tumour suppressive function of FOXO3/4 by activating AKT in CRC. Both the BET inhibitor JQ1 and the MEK inhibitor U0126 decreased the mRNA level of LAMB3 in multiple CRC cells. Mechanistically, ELK4 cooperated with BRD2 to regulate the transcription of LAMB3 in CRC by directly binding to the ETS binding motifs in the LAMB3 promoter. ELK4 was as acetylated at K125, which enhanced the interaction between ELK4 and BRD2. JQ1 disrupted the interaction between ELK4 and BRD2, resulting in decreased binding of BRD2 to the LAMB3 promoter and downregulation of LAMB3 transcription. Both ELK4 and BRD2 expression was associated with LAMB3 expression in CRC. LAMB3 expression was also negatively correlated with FOXO3/4 in CRC. Our study reveals the pro-tumorigenic role of LAMB3 through the AKT-FOXO3/4 axis and the transcriptional mechanism of LAMB3 in CRC, demonstrating that LAMB3 is a potential therapeutic target that can be targeted by BET inhibitors and MEK inhibitors.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Cycle Proteins/metabolism , Colorectal Neoplasms/metabolism , Forkhead Box Protein O3/metabolism , Forkhead Transcription Factors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transcription Factors/metabolism , ets-Domain Protein Elk-4/metabolism , Acetylation , Animals , Cell Adhesion Molecules/genetics , Cell Cycle Proteins/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Female , Forkhead Box Protein O3/genetics , Forkhead Transcription Factors/genetics , Humans , Male , Mice , Proto-Oncogene Proteins c-akt/genetics , Transcription Factors/genetics , ets-Domain Protein Elk-4/genetics , KalininABSTRACT
BACKGROUND: Both hormone-sensitive and castration- and enzalutamide-resistant prostate cancers (PCa) depend on the ternary complex factor (TCF) protein ELK1 to serve as a tethering protein for the androgen receptor (AR) to activate a critical set of growth genes. The two sites in ELK1 required for AR binding are conserved in other members of the TCF subfamily, ELK3 and ELK4. Here we examine the potential utility of the three proteins as prognosticators of disease recurrence in PCa. METHODS: Transcriptional activity assays; Retrospective analysis of PCa recurrence using data on 501 patients in The Cancer Genome Atlas (TCGA) database; Unpaired Wilcoxon rank-sum test and multiple comparison correction using the Holm's method; Spearman's correlations; Kaplan-Meier methods; Univariable and multivariable Cox regression analyses; LASSO-based penalized Cox regression models; Time-dependent area under the receiver operating characteristic (ROC) curve. RESULTS: ELK4 but not ELK3 was coactivated by AR similar to ELK1. Tumor expression of neither ELK3 nor ELK4 was associated with disease-free survival (DFS). ELK1 was associated with higher clinical T-stage, pathology T-stage, Gleason score, prognostic grade, and positive lymph node status. ELK1 was a negative prognosticator of DFS, independent of ELK3, ELK4, clinical T-stage, pathology T-stage, prognostic grade, lymph node status, age, and race. Inclusion of ELK1 increased the abilities of the Oncotype DX and Prolaris gene panels to predict disease recurrence, correctly predicting disease recurrence in a unique subset of patients. CONCLUSIONS: ELK1 is a strong, independent prognosticator of disease recurrence in PCa, underscoring its unique role in PCa growth. Inclusion of ELK1 may enhance the utility of currently used prognosticators for clinical decision making in prostate cancer.
Subject(s)
Neoplasm Recurrence, Local/genetics , Prostatic Neoplasms/genetics , ets-Domain Protein Elk-1/genetics , Adult , Aged , Cluster Analysis , Disease-Free Survival , HeLa Cells , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/pathology , Prognosis , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-ets/genetics , Receptors, Androgen/genetics , Retrospective Studies , Transcriptional Activation , ets-Domain Protein Elk-4/geneticsABSTRACT
In mouse thymocyte development, signaling by the TCR through the ERK pathway is required for positive selection of conventional naive T cells. The Ets transcription factor ELK4 (SAP-1), an ERK-regulated cofactor of the SRF transcription factor, plays an important role in positive selection by activating immediate-early genes such as the Egr transcription factor family. The role of ELK4-SRF signaling in development of other T cell types dependent on ERK signaling has been unclear. In this article, we show that ELK4, and its close relative ELK1, act cell autonomously in the thymus to control the generation of innate-like αß CD8+ T cells with memory-like characteristics. Mice lacking ELK4 and ELK1 develop increased numbers of innate-like αß CD8+ T cells, which populate the periphery. These cells develop cell autonomously rather than through expansion of PLZF+ thymocytes and concomitantly increased IL-4 signaling. Their development is associated with reduced TCR-mediated activation of ELK4-SRF target genes and can be partially suppressed by overexpression of the ELK4-SRF target gene EGR2. Consistent with this, partial inhibition of ERK signaling in peripheral CD8+T cells promotes the generation of cells with innate-like characteristics. These data establish that low-level ERK signaling through ELK4 (and ELK1) promotes innate-like αß CD8+ T cell differentiation, tuning conventional versus innate-like development.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , MAP Kinase Signaling System/immunology , Thymus Gland/immunology , ets-Domain Protein Elk-1/immunology , ets-Domain Protein Elk-4/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation/genetics , Immunity, Innate , MAP Kinase Signaling System/genetics , Mice , Mice, Knockout , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Thymus Gland/cytology , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-4/geneticsABSTRACT
Sirtuin 7 (SIRT7), a member of the NAD+-dependent class III histone deacetylases, is involved in the regulation of various cellular processes and in resisting various stresses, such as hypoxia, low glucose levels, and DNA damage. Interestingly, SIRT7 is linked to the control of glycolysis, suggesting a role in glucose metabolism. Given the important roles of SIRT7, it is critical to clarify how SIRT7 activity is potentially regulated. It has been reported that some transcriptional and post-transcriptional regulatory mechanisms are involved. However, little is known how SIRT7 is regulated by the post-translational modifications. Here, we identified ubiquitin-specific peptidase 7 (USP7), a deubiquitinase, as a negative regulator of SIRT7. We showed that USP7 interacts with SIRT7 both in vitro and in vivo, and we further demonstrated that SIRT7 undergoes endogenous Lys-63-linked polyubiquitination, which is removed by USP7. Although the USP7-mediated deubiquitination of SIRT7 had no effect on its stability, the deubiquitination repressed its enzymatic activity. We also showed that USP7 coordinates with SIRT7 to regulate the expression of glucose-6-phosphatase catalytic subunit (G6PC), a gluconeogenic gene. USP7 depletion by RNA interference increased both G6PC expression and SIRT7 enzymatic activity. Moreover, SIRT7 targeted the G6PC promoter through the transcription factor ELK4 but not through forkhead box O1 (FoxO1). In summary, SIRT7 is a USP7 substrate and has a novel role as a regulator of gluconeogenesis. Our study may provide the basis for new clinical approaches to treat metabolic disorders related to glucose metabolism.
Subject(s)
Gene Expression Regulation, Enzymologic , Glucose-6-Phosphatase/metabolism , Promoter Regions, Genetic , Protein Processing, Post-Translational , Sirtuins/metabolism , Ubiquitin Thiolesterase/metabolism , ets-Domain Protein Elk-4/metabolism , Amino Acid Substitution , Cell Line, Tumor , Gene Deletion , Gluconeogenesis , Glucose-6-Phosphatase/antagonists & inhibitors , Glucose-6-Phosphatase/genetics , HEK293 Cells , Humans , Hydrolysis , Lysine/metabolism , Mutation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sirtuins/antagonists & inhibitors , Sirtuins/genetics , Substrate Specificity , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7 , Ubiquitination , ets-Domain Protein Elk-4/geneticsABSTRACT
The adapter protein Dok-4 (downstream of kinase-4) has been reported as both an activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4 through an atypical phosphotyrosine-binding domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. The Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both an interaction-dependent mechanism and a pleckstrin homology domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA, and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms, including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Nuclear Export Signals/genetics , Nuclear Localization Signals/genetics , ets-Domain Protein Elk-4/genetics , Active Transport, Cell Nucleus/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Proliferation/genetics , Dogs , Gene Expression Regulation , HEK293 Cells , Humans , Immunoblotting , Madin Darby Canine Kidney Cells , Mice , Microscopy, Confocal , Protein Binding , RNA Interference , Sequence Homology, Amino Acid , Two-Hybrid System Techniques , ets-Domain Protein Elk-4/metabolismABSTRACT
We aimed to elucidate the roles and regulatory mechanism of miR-3188 in oxidized low-density lipoprotein (ox-LDL)-induced cell injury in THP-1 derived macrophages, thus providing a new insight for the treatment of atherosclerosis (AS). A total of 85 AS patients and 45 healthy controls were enrolled. The levels of miR-3188 and lipoprotein-associated phospholipase A2 (Lp-PLA2) in AS patients and healthy controls were detected. Then ox-LDL was used to treat human THP-1 derived macrophages. The effects of overexpression and suppression of miR-3188 on regulating ox-LDL-induced cell injury in THP-1 derived macrophages were investigated. Additionally, the potential target of miR-3188 was identified, which was verified by luciferase reporter assay. Besides, the relationship between miR-3188 and RhoA/ROCK pathway was explored. miR-3188 was downregulated in AS patients, while the levels of Lp-PLA2 in AS patients were increased. Ox-LDL significantly induced cell injury by decreasing cell viability, inducing cell apoptosis and increasing the production of inflammatory cytokines, including IL-1ß, IL-6, MCP-1 and TNF-α. In addition, miR-3188 was significantly downregulated after ox-LDL treatment. Overexpression of miR-3188 alleviated ox-LDL-induced cell injury, while inhibition of miR-3188 had opposite effects. ETS-domain protein 4 (ELK4) was a target of miR-3188. The effects of miR-3188 inhibition on ox-LDL-induced cell injury were markedly reversed by knockdown of ELK4. Besides, inhibition of miR-3188 enhanced ox-LDL-activated RhoA/ROCK pathway, while knockdown of ELK4 suppressed this pathway. Downregulation of miR-3188 may contribute to AS development via negatively regulating Lp-PLA2, targeting ELK4 and activating RhoA/ROCK pathway. miR-3188 may serve as a target for AS treatment.
Subject(s)
Atherosclerosis/pathology , MicroRNAs/physiology , ets-Domain Protein Elk-4/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Apoptosis/physiology , Atherosclerosis/genetics , Case-Control Studies , Cell Survival/physiology , Cytokines/metabolism , Down-Regulation , Female , Gene Knockdown Techniques , Humans , Lipoproteins, LDL/metabolism , Macrophages/pathology , Male , MicroRNAs/genetics , Middle Aged , ets-Domain Protein Elk-4/geneticsABSTRACT
Cyclin-dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression markedly decreases epidermal growth factor (EGF)-induced cell transformation mediated through the downregulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates the ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is overexpressed in melanoma and knocking down the ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for the chemoprevention and therapy against skin cancer.
Subject(s)
Cyclin-Dependent Kinase 2/genetics , Melanoma/genetics , Proto-Oncogene Proteins c-fos/biosynthesis , ets-Domain Protein Elk-4/biosynthesis , Animals , Cell Cycle/genetics , Cell Line , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase 2/biosynthesis , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , Humans , Melanoma/pathology , Mice , Phosphorylation , Transcriptional Activation/genetics , ets-Domain Protein Elk-4/geneticsABSTRACT
Chromosomal rearrangements and fusion genes play important roles in tumor development and progression. Four high-frequency prostate cancer-specific fusion genes were recently reported in Chinese cases. We attempted to confirm one of the fusion genes, USP9Y-TTTY15, by reverse transcription PCR, but detected the presence of the USP9Y-TTTY15 fusion transcript in cancer samples, nonmalignant prostate tissues, and normal tissues from other organs, demonstrating that it is a transcription-induced chimeric RNA, which is commonly produced in normal tissues. In 105 prostate cancer samples and case-matched adjacent nonmalignant tissues, we determined the expression level of USP9Y-TTTY15 and a previously reported transcription-induced chimeric RNA, SLC45A3-ELK4. The expression levels of both chimeric RNAs vary greatly in cancer and normal cells. USP9Y-TTTY15 expression is neither higher in cancer than adjacent normal tissues, nor correlated with features of advanced prostate cancer. Although the expression level of SLC45A3-ELK4 is higher in cancer than normal cells, and a dramatic increase in its expression from normal to cancer cells is correlated with advanced disease, its expression level in cancer samples alone is not correlated with any clinical parameters. These data show that both chimeric RNAs contribute less to prostate carcinogenesis than previously reported.
Subject(s)
Oncogene Proteins, Fusion/genetics , Prostatic Neoplasms/metabolism , RNA, Untranslated/genetics , Aged , Humans , Male , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Minor Histocompatibility Antigens , Monosaccharide Transport Proteins , Neoplasm Staging , Oncogene Proteins, Fusion/metabolism , Prostate/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Untranslated/metabolism , Transcription, Genetic , Transcriptome , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolismABSTRACT
BACKGROUND: Serum response factor (SRF) is a widely expressed transcription factor involved in multiple regulatory programs. It is believed that SRF can toggle between disparate programs of gene expression through association with different cofactors. However, the direct evidence as to how these factors function on a genome-wide level is still lacking. RESULTS: In the present study, I explored the functions of SRF and its representative cofactors, megakaryoblastic leukemia 1/2 (MKL1/2) and ETS-domain protein 4 (ELK4), during fungal infection challenge in macrophages. The knockdown study, combined with gene expression array analysis, revealed that MKL1/2 regulated SRF-dependent genes were related to actin cytoskeleton organization, while ELK4 regulated SRF-dependent genes were related to external stimulus responses. Subsequent chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) suggested that many of these regulations were mediated directly in cis. CONCLUSIONS: I conclude that SRF utilizes MKL1/2 to fulfill steady state cellular functions, including cytoskeletal organization, and utilizes ELK4 to facilitate acute responses to external infection. Together, these findings indicate that SRF, along with its two cofactors, are important players in both cellular homeostasis and stress responses in macrophages.
Subject(s)
Gene Expression Regulation , Macrophages/metabolism , Serum Response Factor/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Transcription, Genetic , ets-Domain Protein Elk-4/metabolism , Animals , Binding Sites , Cell Line , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Macrophages/drug effects , Male , Mice , Nucleotide Motifs , Position-Specific Scoring Matrices , Promoter Regions, Genetic , Protein Binding , Protein Transport , Reproducibility of Results , Trans-Activators/genetics , Transcription Factors/genetics , Zymosan/pharmacology , ets-Domain Protein Elk-4/geneticsABSTRACT
Analysis of 4,405 variants in 89,050 European subjects from 41 case-control studies identified three independent association signals for estrogen-receptor-positive tumors at 11q13. The strongest signal maps to a transcriptional enhancer element in which the G allele of the best candidate causative variant rs554219 increases risk of breast cancer, reduces both binding of ELK4 transcription factor and luciferase activity in reporter assays, and may be associated with low cyclin D1 protein levels in tumors. Another candidate variant, rs78540526, lies in the same enhancer element. Risk association signal 2, rs75915166, creates a GATA3 binding site within a silencer element. Chromatin conformation studies demonstrate that these enhancer and silencer elements interact with each other and with their likely target gene, CCND1.
Subject(s)
Breast Neoplasms/genetics , Chromosomes, Human, Pair 11/genetics , Cyclin D1/genetics , Enhancer Elements, Genetic/genetics , Polymorphism, Single Nucleotide/genetics , Binding Sites , Case-Control Studies , Cell Line, Tumor , Chromatin/chemistry , Chromatin/genetics , Chromatin Immunoprecipitation , Cyclin D1/metabolism , Electrophoretic Mobility Shift Assay , Female , GATA3 Transcription Factor/antagonists & inhibitors , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Gene Expression Regulation, Neoplastic , Humans , Luciferases/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Silencer Elements, Transcriptional/genetics , ets-Domain Protein Elk-4/antagonists & inhibitors , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolismABSTRACT
Using a series of detailed experiments, Zhang and colleagues establish that the prostate cancer RNA chimera SLC45A3-ELK4 is generated by cis-splicing between the 2 adjacent genes and does not involve DNA rearrangements or trans-splicing. The chimera expression is induced by androgen treatment likely by overcoming the read-through block imposed by the intergenic CCCTC insulators bound by CCCTC-binding factor repressor protein. The chimeric transcript, but not wild-type ELK4, is shown to augment prostate cancer cell proliferation.
Subject(s)
Cell Proliferation , Membrane Transport Proteins/genetics , Prostatic Neoplasms/genetics , RNA Splicing , Recombinant Fusion Proteins/genetics , ets-Domain Protein Elk-4/genetics , Animals , Humans , Male , Monosaccharide Transport ProteinsABSTRACT
UNLABELLED: Gene fusion is a common event in cancer. The fusion RNA and protein products often play causal roles in tumorigenesis and therefore represent ideal diagnostic and therapeutic targets. Formerly, fusion chimeric products in cancer were thought to be produced solely by chromosomal translocation. Here, we show that a chimeric SLC45A3-ELK4 RNA is generated in the absence of chromosomal rearrangement. We showed that it is not a product of RNA trans-splicing, but formed by cis-splicing of adjacent genes/read-through. The binding of CCCTC-binding factor (CTCF) to the insulator sequences inversely correlates with the expression of the chimera transcript. The SLC45A3-ELK4 fusion, but not wild-type, ELK4 plays important roles in regulating cell growth in both androgen-dependent and -independent prostate cancer cells. The level of the chimeric transcript correlates with disease progression, with the highest levels in prostate cancer metastases. Our results suggest that gene fusions can arise from cis-splicing of adjacent genes without corresponding DNA changes. SIGNIFICANCE: With the absence of corresponding DNA rearrangement, chimeric fusion SLC45A3-ELK4 transcript in prostate cancer cells is generated by cis-splicing of adjacent genes/gene read-through instead of trans-splicing. SLC45A3-ELK4 controls prostate cancer cell proliferation, and the chimera level correlates with prostate cancer disease progression.
Subject(s)
Cell Proliferation , Membrane Transport Proteins/genetics , Prostatic Neoplasms/genetics , RNA Splicing , Recombinant Fusion Proteins/genetics , ets-Domain Protein Elk-4/genetics , Animals , Base Sequence , Blotting, Southern , CCCTC-Binding Factor , Cell Line , Cell Line, Tumor , DNA, Neoplasm/genetics , Gene Expression Profiling , HCT116 Cells , HEK293 Cells , Humans , Male , Metribolone/pharmacology , Molecular Sequence Data , Monosaccharide Transport Proteins , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms/pathology , RNA Interference , RNA, Neoplasm/genetics , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/drug effectsABSTRACT
ETS gene rearrangements are frequently found in prostate cancer. Several studies have assessed the rearrangement status of the most commonly found ETS rearranged gene ERG, and the less frequent genes, ETV-1, ETV-4, ETV-5, and ELK-4 in primary prostate cancer. However, frequency in metastatic disease is not well investigated. Recently, we have assessed the ERG rearrangement status in both primary and corresponding lymph node metastases and observed that ERG rearrangement in primary prostate cancer transfers into lymph node metastases, suggesting it to be a clonal expansion event during prostate cancer progression. As a continuation, we investigated in this study whether this observation is valid for the less frequent ETS rearranged genes. Using dual-color break-apart fluorescent in situ hybridization assays, we evaluated the status of all less frequent ETS gene rearrangements for the first time on tissue microarrays constructed from a large cohort of 86 patients with prostate cancer and composed of primary and corresponding lymph node metastases, as well as in a second cohort composed of 43 distant metastases. ETV-1, ETV-4, ETV-5, and ELK-4 rearrangements were found in 8 (10%) of 81, 5 (6%) of 85, 1 (1%) of 85, and 2 (2%) of 86 of primary prostate cancer, respectively, and in 6 (8%) of 73, 4 (6%) of 72, 1 (1%) of 75, and 1 (1%) of 78 of corresponding lymph node metastases, respectively. ETV-1 and ETV-5 rearrangements were not found in the distant metastases cases, whereas ETV-4 and ELK-4 rearrangements were found in 1 (4%) of 25 and 1 (4%) of 24, respectively. Our findings suggest that rearrangement of the less frequent ETS genes is a clonal event during prostate cancer progression.
Subject(s)
Adenocarcinoma/genetics , Adenovirus E1A Proteins/genetics , DNA-Binding Proteins/genetics , Gene Rearrangement , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Transcription Factors/genetics , ets-Domain Protein Elk-4/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Adenovirus E1A Proteins/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/secondary , Brain Neoplasms/genetics , Brain Neoplasms/secondary , Clone Cells , DNA-Binding Proteins/metabolism , Disease Progression , Humans , In Situ Hybridization, Fluorescence , Male , Prognosis , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ets , Transcription Factors/metabolism , ets-Domain Protein Elk-4/metabolismABSTRACT
Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.
Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Glioblastoma/prevention & control , Proto-Oncogene Proteins c-bcl-2/metabolism , ets-Domain Protein Elk-4/metabolism , Adult , Animals , Base Sequence , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Brain Neoplasms/prevention & control , Cell Line, Tumor , Chromatin Immunoprecipitation , Down-Regulation , Electrophoretic Mobility Shift Assay , Glioblastoma/metabolism , Humans , Luciferases/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Sequence Data , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Grading , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Transfection , bcl-X Protein/genetics , bcl-X Protein/metabolism , ets-Domain Protein Elk-4/antagonists & inhibitors , ets-Domain Protein Elk-4/geneticsABSTRACT
The ETS family of transcription factors plays important roles in both normal and neoplastic cells for different biological processes such as proliferation, differentiation, development, transformation, apoptosis, migration, invasion and angiogenesis. The 27 ETS factors are probably a part of complex regulatory networks including interactions among family members. In human prostate cancer, rearrangements have been found in several genes of the ETS family resulting in chimeric oncoproteins. In a previous study we found that the ETS family prototype, Ets-1 affects biological properties of PC3 prostate cancer cells. In a first effort to understand the cooperative interactions between different ETS factors in prostate cancer, in the present study we examined the expression pattern of all 27 ETS members using quantitative RT-PCR (qRT-PCR) in the androgen-sensitive VCaP and LNCaP, and the androgen-insensitive PC3 and DU-145 prostate cancer cell lines as well as in human prostate cancer tissue samples. We further investigated whether the ETS family prototype, Ets-1, regulates other ETS family members by examining the effect of Ets-1 blockade in PC3 cells on their expression. We found an expression specificity of various ETS family members in the prostate cancer cell lines which might reflect their different biological properties. In human prostate samples only 3 among the 27 ETS family members (Ehf, Elk-4 and Ets-2) showed significant expression differences between normal and cancerous prostate glands. We finally demonstrate that the family prototype, Ets-1, regulates the family members Elf-1, Elf-2, Elk-1, Etv-5 and Spi-1 in PC3 prostate cancer cells. Chimeric oncoproteins containing ETS family members arising due to frequent translocations in prostate cancer are probably part of a regulatory network involving other ETS family members as well.
Subject(s)
Prostatic Neoplasms/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Proto-Oncogene Proteins c-ets/biosynthesis , Aged , Androgens/metabolism , Cell Line, Tumor , Gene Expression Profiling , Gene Regulatory Networks , Humans , Male , Middle Aged , Oncogene Proteins, Fusion/genetics , Prostatic Neoplasms/genetics , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-ets-2/genetics , Proto-Oncogene Protein c-ets-2/metabolism , Proto-Oncogene Proteins c-ets/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Translocation, Genetic , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolismABSTRACT
The ability of transforming growth factor ß (TGFß) to induce epithelial-mesenchymal transition (EMT) is mediated by SMAD-dependent and SMAD-independent pathways such as the activation of Rho GTPase signalling. Upon activation, GTP-bound Rho stimulates the ROCK kinases, which in turn phosphorylate numerous substrates including the LIM kinases (LIMK). The net result of ROCK activation is increased actin-myosin contractile force generation, with a contribution from LIMK-induced actin filament stabilisation. In this study, we made use of siRNA-mediated knockdown and selective inhibitors to determine the contributions of ROCK and LIMK to TGFß-induced responses. We find that both ROCK and LIMK are required for TGFß stimulation of serum-response factor (SRF) transcriptional activity and actin stress fibre formation during EMT. In contrast, although LIMK inhibition had little effect on cell motility in scratch wound and Transwell migration assays, ROCK inhibition actually promoted TGFß-induced cell motility by helping individual cells to break free from the epithelial sheet. Furthermore, we demonstrate that selective inhibition of LIMK, but not ROCK, effectively blocked TGFß driven invasion through a layer of matrigel extracellular matrix protein. These results indicate that the roles of LIMK and ROCK in the Rho signalling pathway downstream of TGFß are not identical and suggest that LIMK represents an attractive therapeutic target in TGFß driven organ fibrosis and metastatic cancer spread.
Subject(s)
Cell Movement , Lim Kinases/metabolism , Transcription, Genetic , Transforming Growth Factor beta/metabolism , rho-Associated Kinases/metabolism , Actins/metabolism , Animals , Blotting, Western , Epithelial-Mesenchymal Transition/physiology , Extracellular Matrix Proteins/physiology , Fluorescent Antibody Technique , Gene Knockdown Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Phosphorylation , Polymerase Chain Reaction , RNA, Small Interfering , Signal Transduction , Transforming Growth Factor beta/pharmacology , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
ETS-like transcription factor 4 (ELK4) (a.k.a. serum response factor accessory protein 1) belongs to the ternary complex factor (TCF) subfamily of E twenty-six (ETS) domain transcription factors. Compared to the other TCF subfamily members, ELK1 and ELK3 (NET), there is limited information of the mechanisms regulating the ELK4 activity. Here, we show that the ELK4 can be covalently modified (SUMOylated) by small ubiquitin-related modifier (SUMO) 1 protein, an important regulator of signaling and transcription. SUMOylation of ELK4 was reversed by SUMO-specific proteases (SENP) 1 and 2 and stimulated by SUMO E3 ligase PIAS3. Conserved lysine residue 167 that is located in the NET inhibitory domain of ELK4 was identified as the main site of SUMO-1 conjugation. Interestingly, mutation of the K167 disrupting the SUMOylation markedly enhanced the transcriptional activity of the ELK4, but weakened its repressive function on c-fos promoter. In conclusion, our results suggest that covalent modification by SUMO-1 can regulate the activity of ELK4, contributing to the transcriptional repression by the ELK4.