RESUMEN
The miR-34 family of microRNAs suppresses the expression of proteins involved in pluripotency and oncogenesis. miR-34 expression is frequently reduced in cancers; however, the regulation of their expression is not well understood. We used genome-wide miRNA profiling and mechanistic analysis to show that SUMOylation regulates miR-34b/c expression, which impacts the expression of c-Myc and other tested miR-34 targets. We used site-directed mutagenesis and other methods to show that protein kinase B (also known as Akt) phosphorylation of FOXO3a plays an important role in SUMOylation-dependent expression of miR-34b/c. This study reveals how the miR-34-targeted gene expression program is regulated by SUMOylation and shows that SUMOylation need not regulate target proteins through direct modification, but instead can act through the expression of their targeting miRNAs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Sumoilación , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteína Forkhead Box O3/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Enzimas Activadoras de UbiquitinaRESUMEN
E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl small ubiquitin-like modifier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics.
Asunto(s)
Modelos Moleculares , Procesamiento Proteico-Postraduccional , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Sustitución de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/metabolismo , Bases de Datos de Proteínas , Disulfuros/química , Disulfuros/metabolismo , Disulfuros/farmacología , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Ligandos , Mutación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homología Estructural de Proteína , Ubiquitina/química , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Enhancers are fundamental to gene regulation. Post-translational modifications by the small ubiquitin-like modifiers (SUMO) modify chromatin regulation enzymes, including histone acetylases and deacetylases. However, it remains unclear whether SUMOylation regulates enhancer marks, acetylation at the 27th lysine residue of the histone H3 protein (H3K27Ac). To investigate whether SUMOylation regulates H3K27Ac, we performed genome-wide ChIP-seq analyses and discovered that knockdown (KD) of the SUMO activating enzyme catalytic subunit UBA2 reduced H3K27Ac at most enhancers. Bioinformatic analysis revealed that TFAP2C-binding sites are enriched in enhancers whose H3K27Ac was reduced by UBA2 KD. ChIP-seq analysis in combination with molecular biological methods showed that TFAP2C binding to enhancers increased upon UBA2 KD or inhibition of SUMOylation by a small molecule SUMOylation inhibitor. However, this is not due to the SUMOylation of TFAP2C itself. Proteomics analysis of TFAP2C interactome on the chromatin identified histone deacetylation (HDAC) and RNA splicing machineries that contain many SUMOylation targets. TFAP2C KD reduced HDAC1 binding to chromatin and increased H3K27Ac marks at enhancer regions, suggesting that TFAP2C is important in recruiting HDAC machinery. Taken together, our findings provide insights into the regulation of enhancer marks by SUMOylation and TFAP2C and suggest that SUMOylation of proteins in the HDAC machinery regulates their recruitments to enhancers.
RESUMEN
BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy. Although Dexamethasone (Dex) is the most widely used therapeutic drug in MM treatment, patients develop Dex resistance leading to progressive disease, demanding an urgent need to investigate the mechanisms driving Dex resistance and develop new reagents to address this problem. We propose SUMOylation as a potential mechanism regulating Dex resistance and SUMOylation inhibition can enhance Dex sensitivity in MM. METHODS: Using MM cell lines and primary MM samples from relapsing MM patients, we evaluated the effects of knockdown of SUMO E1 (SAE2) or using TAK-981, a novel and specific SUMO E1 inhibitor, on Dex sensitivity. Xenograft mouse models were generated to determine the in vivo anti-MM effects of TAK-981 as a single agent and in combination with Dex. miRNA-seq, RNA-seq and GSEA analysis were utilized for evaluating key factors mediating Dex resistance. Chromatin immunoprecipitation (ChIP) assay was performed to determine the binding occupancy of c-Myc on promoter region of miRs. RESULTS: We observed a significant negative correlation between SUMO E1 (SAE2) expression and Dex sensitivity in primary MM samples. Knockdown of SAE2 or using TAK-981 significantly enhances myeloma sensitivity to Dex in MM cell lines. Moreover, the enhanced anti-MM activity by TAK-981 and Dex combination has been validated using primary relapsing MM patient samples and xenograft mouse models. SUMOylation inhibition increased glucocorticoid receptor (GR) expression via downregulation miR-130b. Using RNA and microRNA sequencing, we identified miR-551b and miR-25 as important miRs mediating Dex resistance in MM. Overexpression of miR-551b and miR-25 caused resistance to Dex, however, knockdown of miR-551b and miR-25 significantly enhanced Dex sensitivity in MM. SAE2 knockdown or TAK-981 treatment downregulated the expression of miR-551b and miR-25, leading to induction of miR targets ZFP36, ULK1 and p27, resulting in apoptosis and autophagy. We demonstrated c-Myc as a major transcriptional activator of miR-130b, miR-551b and miR-25 and SUMOylation inhibition downregulates these miRs level by decreasing c-Myc level. CONCLUSION: Our study proves SUMOylation plays a crucial role in Dex resistance in MM and SUMOylation inhibition appears to be an attractive strategy to advance to the clinic for MM patients.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dexametasona/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Sumoilación/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Dexametasona/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Mieloma Múltiple/patología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase 6 (HDAC6), a member of the HDAC family whose major substrate is α-tubulin, has become a target for drug development to treat cancer due to its major contribution in oncogenic cell transformation. Overexpression of HDAC6 correlates with tumorigenesis and improved survival; therefore, HDAC6 may be used as a marker for prognosis. Previous work demonstrated that in multiple myeloma cells, inhibition of HDAC6 results in apoptosis. Furthermore, HDAC6 is required for the activation of heat-shock factor 1 (HSF1), an activator of heat-shock protein encoding genes (HSPs) and CYLD, a cylindromatosis tumor suppressor gene. HDAC6 contributes to cancer metastasis since its upregulation increases cell motility in breast cancer MCF-7 cells and its interaction with cortactin regulates motility. HDAC6 also affects transcription and translation by regulating the heat-shock protein 90 (Hsp90) and stress granules (SGs), respectively. This review will discuss the role of HDAC6 in the pathogenesis and treatment of cancer.
Asunto(s)
Histona Desacetilasas/fisiología , Neoplasias/enzimología , Histona Desacetilasa 6 , HumanosRESUMEN
Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.
Asunto(s)
Sumoilación , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Regulación Alostérica , Sitio Alostérico , Animales , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones SCID , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sumoilación/efectos de los fármacos , Trasplante Heterólogo , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Central to discovering novel approaches to treating leukemias and lymphomas is a clear understanding of the signaling networks which lead to unchecked cell cycle progression, proliferation, and survival. Cyclic-adenosine monophosphate (cAMP) responsive element-binding protein (CREB) represents a critical integrator of numerous signals from cytoplasmic kinase cascades, and is directly involved in controlling the transcription of genes critical for normal cellular proliferation and survival. Several lines of evidence implicate CREB as a proto-oncogene, as a number of translocations involving CREB and dysregulation of expression are both associated with oncogenesis. Thus, CREB represents a potential therapeutic target in leukemia. Here, we review CREB function and regulation in normal and aberrant hematopoiesis.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Neoplasias Hematológicas/fisiopatología , Hematopoyesis/fisiología , Transducción de Señal/fisiología , Animales , Supervivencia Celular/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Hematopoyesis/genética , Humanos , Fosforilación , Proto-Oncogenes Mas , Transducción de Señal/genéticaRESUMEN
Over the past decade, histone deacetylase inhibitors have increasingly been used to treat various malignancies. Tubacin (tubulin acetylation inducer) is a small molecule that inhibits histone deacetylase 6 (HDAC6) and induces acetylation of α-tubulin. We observed a higher antiproliferative effect of tubacin in acute lymphoblastic leukemia (ALL) cells than in normal hematopoietic cells. Treatment with tubacin led to the induction of apoptotic pathways in both pre-B and T cell ALL cells at a 50% inhibitory concentration (IC(50)) of low micromolar concentrations. Acetylation of α-tubulin increases within the first 30 min following treatment of ALL cells with tubacin. We also observed an accumulation of polyubiquitinated proteins and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, the signaling pathways activated by tubacin appear to be distinct from those observed in multiple myeloma. In this article, we demonstrate that tubacin enhances the effects of chemotherapy to treat primary ALL cells in vitro and in vivo. These results suggest that targeting HDAC6 alone or in combination with chemotherapy could provide a novel approach to treat ALL.