RESUMEN
The MYB protein is a pivotal player in the cellular transcriptional network, influencing major important processes such as cell proliferation, differentiation, and apoptosis. Because of its role in oncogenesis, MYB is now a compelling target for therapeutic interventions in cancer research. This review summarizes its molecular functions and current therapeutic approaches aiming to inhibit its oncogenic activity.
Asunto(s)
Carcinoma Adenoide Quístico , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Regulación de la Expresión Génica , Carcinoma Adenoide Quístico/metabolismoRESUMEN
Aim: Efficient and readily available anticancer drugs are sought as treatment options. For this reason, chromene derivatives were prepared using the one-pot reaction and tested for their anticancer and anti-angiogenic properties. Methods: 2-Amino-3-cyano-4-(aryl)-7-methoxy-4H-chromene compounds (2A-R) were repurposed or newly synthesized via a three-component reaction of 3-methoxyphenol, various aryl aldehydes, and malononitrile. We performed assays to study the inhibition of tumor cell growth [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromid (MTT) assay], effects on microtubules (immunofluorescence), cell cycle (flow-activated cell sorting analysis), angiogenesis (zebrafish model), and MYB activity (luciferase reporter assay). Fluorescence microscopy was applied for localization studies via copper-catalyzed azide-alkyne click reaction of an alkyne-tagged drug derivative. Results: Compounds 2A-C and 2F exhibited robust antiproliferative activities against several human cancer cell lines (50% inhibitory concentrations in the low nanomolar range) and showed potent MYB inhibition. The alkyne derivative 3 was localized in the cytoplasm after only 10 min of incubation. Substantial microtubule disruption and G2/M cell-cycle arrest were observed, where compound 2F stood out as a promising microtubule-disrupting agent. The study of anti-angiogenic properties showed that 2A was the only candidate with a high potential to inhibit blood vessel formation in vivo. Conclusion: The close interplay of various mechanisms, including cell-cycle arrest, MYB inhibition, and anti-angiogenic activity, led to identifying promising multimodal anticancer drug candidates.
RESUMEN
Studies on the role of transcription factor MYB in acute myeloid leukemia (AML) have identified MYB as a key regulator of a transcriptional program for self-renewal of AML cells. Recent work summarized here has now highlighted the CCAAT-box/enhancer binding protein beta (C/EBPß) as an essential factor and potential therapeutic target that cooperates with MYB and coactivator p300 in the maintenance of the leukemic cells.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT , Leucemia Mieloide Aguda , Proteínas Oncogénicas v-myb , Humanos , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas v-myb/genética , Proteínas Oncogénicas v-myb/metabolismoRESUMEN
Based on the promising c-Myb inhibitor 1b, a series of 2-amino-4-aryl-4H-naphtho[1,2-b]pyran-3-carbonitriles (1a, 2a-q, 3a-g) were repurposed or newly synthesized via a three-component reaction of 1-naphthol, and various aryl aldehydes and malononitrile and screened for their c-Myb inhibitory activities. 1b also served as a lead compound for seven new naphthopyran derivatives (3a-f), which were cytotoxic with nanomolar IC50 values, to inhibit the polymerization of tubulin, and to destabilize microtubules in living cells. Especially, the alkyne 3f, originally made for intracellular localization studies using click chemistry, showed an overall high activity in all assays performed. A strong G2/M cell cycle arrest was detected, which resulted in a distinct increase in sub-G1 cells through the induction of effector caspases 3 and 7. Inhibition of angiogenesis was confirmed in vitro and in vivo. In summary, 3f was found to be a pleiotropic compound with high selectivity for cancer cells, combining c-Myb inhibitory, microtubule destabilizing, and antiangiogenic effects.
RESUMEN
The transcription factor MYB is expressed predominantly in hematopoietic progenitor cells, where it plays an essential role in the development of most lineages of the hematopoietic system. In the myeloid lineage, MYB is known to cooperate with members of the CCAAT box/enhancer binding protein (C/EBP) family of transcription factors. MYB and C/EBPs interact with the co-activator p300 or its paralog CREB-binding protein (CBP), to form a transcriptional module involved in myeloid-specific gene expression. Recent work has demonstrated that MYB is involved in the development of human leukemia, especially in acute T-cell leukemia (T-ALL) and acute myeloid leukemia (AML). Chemical entities that inhibit the transcriptional activity of the MYB-C/EBPß-p300 transcription module may therefore be of use as potential anti-tumour drugs. In searching for small molecule inhibitors, studies from our group over the last 10 years have identified natural products belonging to different structural classes, including various sesquiterpene lactones, a steroid lactone, quinone methide triterpenes and naphthoquinones that interfere with the activity of this transcriptional module in different ways. This review gives a comprehensive overview on the various classes of inhibitors and the inhibitory mechanisms by which they affect the MYB-C/EBPß-p300 transcriptional module as a potential anti-tumor target. We also focus on the current knowledge on structure-activity relationships underlying these biological effects and on the potential of these compounds for further development.
Asunto(s)
Productos Biológicos , Leucemia Mieloide Aguda , Triterpenos , Productos Biológicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción , Triterpenos/farmacologíaRESUMEN
Recent studies have disclosed transcription factor MYB as a potential drug target for malignancies that are dependent on deregulated MYB function, including acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often regarded as undruggable, successful targeting of MYB by low-molecular-weight compounds has recently been demonstrated. In an attempt to repurpose known drugs as novel MYB-inhibitory agents, we have screened libraries of approved drugs and drug-like compounds for molecules with MYB-inhibitory potential. Here, we present initial evidence for the MYB-inhibitory activity of the protein kinase inhibitors bosutinib, PD180970 and PD161570, that we identified in a recent screen. We show that these compounds interfere with the activity of the MYB transactivation domain, apparently by disturbing the ability of MYB to cooperate with the coactivator p300. We show that treatment of the AML cell line HL60 with these compounds triggers the up-regulation of the myeloid differentiation marker CD11b and induces cell death. Importantly, we show that these effects are significantly dampened by forced expression of an activated version of MYB, confirming that the ability to suppress MYB function is a relevant activity of these compounds. Overall, our work identifies several protein kinase inhibitors as novel MYB-inhibitory agents and suggests that the inhibition of MYB function may play a role in their pharmacological impact on leukemic cells.
Asunto(s)
Carcinoma Adenoide Quístico , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myb/metabolismo , Factores de Transcripción , Familia-src QuinasasRESUMEN
C/EBPß has recently emerged as a pro-leukemogenic transcription factor that cooperates with oncoprotein MYB to maintain proliferation and differentiation block of AML cells, making C/EBPß an interesting drug target for AML. Here we have studied the inhibitory potential and biological effects of a synthetic analog of the natural product helenalin, a known inhibitor of C/EBPß. The synthetic compound inhibits C/EBPß by covalent binding to cysteine residues in the transactivation domain, thereby causing up-regulation of differentiation-associated genes, cell death and reduced self-renewal potential of AML cells. Suppression of these effects by ectopic expression of C/EBPß or MYB and gene expression profiling validate C/EBPß as a relevant target of the helenalin-mimic and highlight its role as a pro-leukemogenic factor. Overall, our work demonstrates that the synthetic helenalin mimic acts as a covalent inhibitor of C/EBPß and identifies the cysteine residues in the transactivation domain of C/EBPß as ligandable sites. The helenalin mimic can be considered a potential "lead molecule" but needs further development towards more effective C/EBPß inhibitors before being used as a therapeutic agent.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Sesquiterpenos de Guayano/farmacología , Activación Transcripcional/efectos de los fármacos , Células 3T3 , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Ligandos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Células THP-1RESUMEN
Transcription factor MYB is a key regulator of gene expression in hematopoietic cells and has emerged as a novel drug target for acute myeloid leukemia (AML). Studies aiming to identify potential MYB inhibitors have found that the natural compound helenalin acetate (HA) inhibits viability and induces cell death and differentiation of AML cells by disrupting the MYB-induced gene expression program. Interestingly, CCAAT-box/enhancer binding protein ß (C/EBPß), a transcription factor known to cooperate with MYB and the co-activator p300 in myeloid cells, rather than MYB itself, was identified as the primary target of HA. This supports a model in which MYB, C/EBPß, and p300 form the core of a transcriptional module that is essential for maintenance of the proliferative potential of AML cells, highlighting a novel role for C/EBPß as a proleukemogenic factor.
Asunto(s)
Leucemia Mieloide Aguda , Diferenciación Celular , Regulación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Células Mieloides , Oncogenes , Proteínas Proto-Oncogénicas c-myb/genéticaRESUMEN
A large body of work has shown that MYB acts as a master transcription regulator in hematopoietic cells and has pinpointed MYB as a potential drug target for acute myeloid leukemia (AML). Here, we have examined the MYB-inhibitory potential of the HDAC inhibitor LAQ824, which was identified in a screen for novel MYB inhibitors. We show that nanomolar concentrations of LAQ824 and the related HDAC inhibitors vorinostat and panobinostat interfere with MYB function in two ways, by inducing its degradation and inhibiting its activity. Reporter assays show that the inhibition of MYB activity by LAQ824 involves the MYB transactivation domain and the cooperation of MYB with co-activator p300, a key MYB interaction partner and driver of MYB activity. In AML cells, LAQ824-induced degradation of MYB is accompanied by expression of myeloid differentiation markers and apoptotic and necrotic cell death. The ability of LAQ824 to inhibit MYB activity is supported by the observation that down-regulation of direct MYB target genes MYC and GFI1 occurs without apparent decrease of MYB expression already after 2 h of treatment with LAQ824. Furthermore, ectopic expression of an activated version of MYB In HL60 cells counteracts the induction of myeloid differentiation by LAQ824. Overall, our data identify LAQ824 and related HDAC inhibitors as potent MYB-inhibitory agents that exert dual effects on MYB expression and activity in AML cells.
RESUMEN
Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often considered un-druggable, recent work has demonstrated successful targeting of MYB by low molecular weight compounds. This has fueled the notion that inhibition of MYB has potential as a therapeutic approach against MYB-driven malignancies. Here, we have used a MYB reporter cell line to screen a library of FDA-approved drugs for novel MYB inhibitors. We demonstrate that proteasome inhibitors have significant MYB-inhibitory activity, prompting us to characterize the proteasome inhibitor oprozomib in more detail. Oprozomib was shown to interfere with the ability of the co-activator p300 to stimulate MYB activity and to exert anti-proliferative effects on human AML and ACC cells. Overall, our work demonstrated suppression of oncogenic MYB activity as a novel result of proteasome inhibition.
Asunto(s)
Carcinoma Adenoide Quístico/tratamiento farmacológico , Proteína p300 Asociada a E1A/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myb/genética , Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Terapia Molecular Dirigida , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma/farmacologíaRESUMEN
Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPß. C/EBPß-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPß broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPß, and co-activator p300, and is down-regulated by C/EBPß-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPß-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPß-inhibitory STLs. Overall, our data identify C/EBPß as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPß may have therapeutic potential against AML.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT , Leucemia Mieloide Aguda , Diferenciación CelularRESUMEN
Homeodomain-interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto-phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin-dependent degradation. By comparing the expression of wild-type and kinase-defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co-translational degradation of kinase-defective versus wild-type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37-HSP90 chaperone complex and points to a novel role of CDC37 in the co-translational degradation of a client protein.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Chaperoninas/antagonistas & inhibidores , Chaperoninas/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/genética , Codorniz , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
HIPK2 is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in a broad spectrum of biological processes. We have previously reported that the expression of HIPK2 is auto-regulated by a mechanism that depends on the activity of its kinase domain, leading to decreased expression of kinase-dead versus wild-type HIPK2. We have now explored this mechanism in more detail. Differential expression of wild-type and kinase-dead HIPK2 is dependent on sequences located in the C-terminal part of HIPK2, but is only observed when this part of HIPK2 is translated together with the defective kinase domain. On their own, both the defective kinase domain and the C-terminal amino acid sequences are expressed at normal levels and independently of kinase activity. Insertion of a 2A-ribosomal skipping sequence into the HIPK2 coding sequence revealed that the differential expression of wild-type and kinase-dead HIPK2 is caused by degradation of nascent kinase-dead HIPK2. Because HIPK2 is constitutively active and auto-activates its kinase domain already during its translation we speculate that the regulatory mechanism discovered here serves as a quality control mechanism that leads to degradation of nascent kinase molecules with defective kinase domains. Overall our work provides insight into a novel auto-regulatory mechanism of HIPK2 expression, thereby adding a new layer of control to the regulation of HIPK2.
Asunto(s)
Proteínas Portadoras/genética , Fosforilación/genética , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Secuencia de Aminoácidos/genética , Proteínas Portadoras/química , Regulación de la Expresión Génica/genética , Células HeLa , Células Hep G2 , Humanos , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Here, we present the initial characterization of 2-amino-4-(3,4,5-trimethoxyphenyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile (Bcr-TMP), a nanomolar-active MYB-inhibitory compound identified in a screen for novel MYB inhibitors. Bcr-TMP affects MYB function in a dual manner by inducing its degradation and suppressing its transactivation potential by disrupting its cooperation with co-activator p300. Bcr-TMP also interferes with the p300-dependent stimulation of C/EBPß, a transcription factor co-operating with MYB in myeloid cells, indicating that Bcr-TMP is a p300-inhibitor. Bcr-TMP reduces the viability of AML cell lines at nanomolar concentrations and induces cell-death and expression of myeloid differentiation markers. It also down-regulates the expression of MYB target genes and exerts stronger anti-proliferative effects on MYB-addicted primary murine AML cells and patient-derived ACC cells than on their non-oncogenic counterparts. Surprisingly, we observed that Bcr-TMP also has microtubule-disrupting activity, pointing to a possible link between MYB-activity and microtubule stability. Overall, Bcr-TMP is a highly potent multifunctional MYB-inhibitory agent that warrants further investigation of its therapeutic potential and mechanism(s) of action.
RESUMEN
The transcription factor B-MYB is an important regulator of cell cycle-related processes that is activated by step-wise phosphorylation of multiple sites by cyclin-dependent kinases (CDKs) and conformational changes induced by the peptidylprolyl cis/trans isomerase Pin1. Here, we show that a conserved amino acid sequence around Ser-577 in the C-terminal part of B-MYB is able to interact with the B-MYB DNA-binding domain. Phosphorylation of Ser-577 disrupts this interaction and is regulated by the interplay of CDKs and the phosphatase CDC14B. Deletion of sequences surrounding Ser-577 hyperactivates the transactivation potential of B-MYB, decreases its proteolytic stability, and causes cell cycle defects. Overall, we show for the first time that B-MYB can undergo an intramolecular interaction that is controlled by the phosphorylation state of Ser-577.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fosfoserina/metabolismo , Serina/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinasas Ciclina-Dependientes/metabolismo , ADN/genética , ADN/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Células HEK293 , Humanos , Fosforilación , Unión Proteica/genética , Dominios Proteicos/genética , Proteolisis , Serina/genética , Transactivadores/genética , Activación Transcripcional/genéticaRESUMEN
B-MYB, a highly conserved member of the MYB transcription factor family, is expressed ubiquitously in proliferating cells and plays key roles in important cell cycle-related processes, such as control of G2/M-phase transcription, cytokinesis, G1/S-phase progression and DNA-damage reponse. Deregulation of B-MYB function is characteristic of several types of tumor cells, underlining its oncogenic potential. To gain a better understanding of the functions of B-MYB we have employed affinity purification coupled to mass spectrometry to discover novel B-MYB interacting proteins. Here we have identified the zinc-finger proteins ZMYM2 and ZMYM4 as novel B-MYB binding proteins. ZMYM4 is a poorly studied protein whose initial characterization reported here shows that it is highly SUMOylated and that its interaction with B-MYB is stimulated upon induction of DNA damage. Unlike knockdown of B-MYB, which causes G2/M arrest and defective cytokinesis in HEK293 cells, knockdown of ZMYM2 or ZMYM4 have no obvious effects on the cell cycle of these cells. By contrast, knockdown of ZMYM2 strongly impaired the G1/S-phase progression of HepG2 cells, suggesting that ZMYM2, like B-MYB, is required for entry into S-phase in these cells. Overall, our work identifies two novel B-MYB binding partners with possible functions in the DNA-damage response and the G1/S-transition.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Fase G1 , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fase S , Sumoilación , Transactivadores/genética , Factores de Transcripción/genética , Dedos de ZincRESUMEN
The master transcriptional regulator MYB is a key oncogenic driver in several human neoplasms, particularly in acute myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). MYB is therefore an attractive target for drug development in MYB-activated malignancies. Here, we employed a MYB-reporter cell line and identified the polyether ionophores monensin, salinomycin, and nigericin as novel inhibitors of MYB activity. As a proof of principle, we show that monensin affects the expression of a significant number of MYB-regulated genes in AML cells and causes down-regulation of MYB expression, loss of cell viability, and induction of differentiation and apoptosis. Furthermore, monensin significantly inhibits proliferation of primary murine AML cells but not of normal hematopoietic progenitors, reflecting a high MYB-dependence of leukemic cells and underscoring the efficacy of monensin in MYB-activated malignancies. Importantly, monensin also suppressed the viability and non-adherent growth of adenoid cystic carcinoma (ACC) cells expressing MYB-NFIB fusion oncoproteins. Our data show that a single compound with significant MYB-inhibitory activity is effective against malignant cells from two distinct MYB-driven human neoplasms. Hence, monensin and related compounds are promising molecular scaffolds for development of novel MYB inhibitors.
Asunto(s)
Carcinoma Adenoide Quístico/metabolismo , Regulación hacia Abajo , Leucemia Mieloide Aguda/metabolismo , Monensina/farmacología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Animales , Carcinoma Adenoide Quístico/dietoterapia , Carcinoma Adenoide Quístico/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Nigericina/farmacología , Proteolisis , Proteínas Proto-Oncogénicas c-myb/genética , Piranos/farmacología , Células THP-1RESUMEN
PDCD4, the protein encoded by the tumor suppressor gene PDCD4 (programmed cell death 4) has been implicated in the control of cellular transcription and translation by modulating the activity of specific transcription factors and suppressing the translation of mRNAs with structured 5'-UTRs. Most studies of human PDCD4 have employed tumor cell lines, possibly resulting in a biased picture of its role in normal cells. Here, we have studied the function of PDCD4 in a telomerase-immortalized human epithelial cell line. We show for the first time that PDCD4 is required for the G1/S-transition, demonstrating its crucial role in the cell cycle. Inhibition of p53-dependent activation of p21WAF1/CIP1 overrides the requirement for PDCD4 for the G1/S-transition, suggesting that PDCD4 counteracts basal p53 activity to prevent activation of the G1/S checkpoint by p53. Transcriptome and ribosome profiling data show that silencing of PDCD4 changes the expression levels and translation of many mRNAs, providing an unbiased view of the cellular processes that are affected by PDCD4 in an epithelial cell line. Our data identify PDCD4 as a key regulator of cell cycle- and DNA-related functions that are inhibited when it is silenced, suggesting that decreased expression of PDCD4 might contribute to tumor development by compromising genomic integrity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Células Epiteliales/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Telomerasa/genética , Transcriptoma , Regiones no Traducidas 5' , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Transformada , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Transducción de Señal , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The oncogenic transcription factor B-Myb is an essential regulator of late cell cycle genes whose activation by phosphorylation is still poorly understood. We describe a stepwise phosphorylation mechanism of B-Myb, which involves sequential phosphorylations mediated by cyclin-dependent kinase (Cdk) and Polo-like kinase 1 (Plk1) and Pin1-facilitated peptidyl-prolyl cis/trans isomerization. Our data suggest a model in which initial Cdk-dependent phosphorylation of B-Myb enables subsequent Pin1 binding and Pin1-induced conformational changes of B-Myb. This, in turn, initiates further phosphorylation of Cdk-phosphosites, enabling Plk1 docking and subsequent Plk1-mediated phosphorylation of B-Myb to finally allow B-Myb to stimulate transcription of late cell cycle genes. Our observations reveal novel mechanistic hierarchies of B-Myb phosphorylation and activation and uncover regulatory principles that might also apply to other Myb family members. Strikingly, overexpression of B-Myb and of factors mediating its activation strongly correlates with adverse prognoses for tumor patients, emphasizing B-Myb's role in tumorigenesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Carcinogénesis/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Células Hep G2 , Humanos , Mitosis/efectos de los fármacos , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/química , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Nocodazol/farmacología , Isomerasa de Peptidilprolil/genética , Fosforilación/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Roscovitina/farmacología , Timidina/farmacología , Transactivadores/química , Transcripción Genética/efectos de los fármacos , Quinasa Tipo Polo 1RESUMEN
The transcription factor MYB plays key roles in hematopoietic cells and has been implicated the development of leukemia. MYB has therefore emerged as an attractive target for drug development. Recent work has suggested that targeting MYB by small-molecule inhibitors is feasible and that inhibition of MYB has potential as a therapeutic approach against acute myeloid leukemia. To facilitate the identification of small-molecule MYB inhibitors we have re-designed and improved a previously established cell-based screening assay and have employed it to screen a natural product library for potential inhibitors. Our work shows that teniposide and etoposide, chemotherapeutic agents causing DNA-damage by inhibiting topoisomerase II, potently inhibit MYB activity and induce degradation of MYB in AML cell lines. MYB inhibition is suppressed by caffeine, suggesting that MYB is inhibited indirectly via DNA-damage signalling. Importantly, ectopic expression of an activated version of MYB in pro-myelocytic NB4 cells diminished the anti-proliferative effects of teniposide, suggesting that podophyllotoxins disrupt the proliferation of leukemia cells not simply by inducing general DNA-damage but that their anti-proliferative effects are boosted by inhibition of MYB. Teniposide and etoposide therefore act like double-edged swords that might be particularly effective to inhibit tumor cells with deregulated MYB.