RESUMO
Myotonic dystrophy 2 (DM2) is a genetic multi-systemic disease primarily affecting skeletal muscle. It is caused by CCTGn expansion in intron 1 of the CNBP gene, which encodes a zinc finger protein. DM2 disease has been successfully modeled in Drosophila melanogaster, allowing the identification and validation of new pathogenic mechanisms and potential therapeutic strategies. Here, we describe the principal tools used in Drosophila to study and dissect molecular pathways related to muscular dystrophies and summarize the main findings in DM2 pathogenesis based on DM2 Drosophila models. We also illustrate how Drosophila may be successfully used to generate a tractable animal model to identify novel genes able to affect and/or modify the pathogenic pathway and to discover new potential drugs.
Assuntos
Proteínas de Drosophila , Distrofia Miotônica , Animais , Drosophila melanogaster/genética , Distrofia Miotônica/genética , Drosophila , Íntrons/genética , Músculo Esquelético , Proteínas de Ligação a RNA , Proteínas de Drosophila/genéticaRESUMO
AIMS: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model. MATERIALS AND METHODS: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)-dependent MB-prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1-depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA-Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. RESULTS: We found that monoallelic Nbn deletion increases SmoA1-dependent MB incidence. In addition to a defective DDR, Nbn+/- GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH-driven tumour allograft. CONCLUSIONS: Our study indicates that Nbn is haploinsufficient for SHH-MB development whereas full NbnKO is epistatic on SHH-driven MB development, thus revealing a gene dosage-dependent effect of Nbn inactivation on SHH-MB development.
Assuntos
Proteínas de Ciclo Celular , Neoplasias Cerebelares , Proteínas de Ligação a DNA , Meduloblastoma , Animais , Proteínas de Ciclo Celular/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/genética , Dosagem de Genes , Genes Essenciais , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos TransgênicosRESUMO
Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC-202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC-202 treatment abrogates GLI activation and HH target gene expression in both SMOi-sensitive and -resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second-line therapy may improve the treatment options for HH-associated malignancies with SMOi resistance.
Assuntos
Benzamidas/farmacologia , Carcinoma Basocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteínas Hedgehog/antagonistas & inibidores , Histona Desacetilases/química , Receptor Smoothened/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Animais , Apoptose , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Proliferação de Células , Proteínas Hedgehog/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais , Receptor Smoothened/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Hedgehog signaling is a key regulator of development and stem cell fate and its aberrant activation is a leading cause of a number of tumors. Activating germline or somatic mutations of genes encoding Hh pathway components are found in Basal Cell Carcinoma (BCC) and Medulloblastoma (MB). Ligand-dependent Hedgehog hyperactivation, due to autocrine or paracrine mechanisms, is also observed in a large number of malignancies of the breast, colon, skin, bladder, pancreas and other tissues. The key tumorigenic role of Hedgehog has prompted effort aimed at identifying inhibitors of this signaling. To date, only the antagonists of the membrane transducer Smo have been approved for therapy or are under clinical trials in patients with BCC and MB linked to Ptch or Smo mutations. Despite the good initial response, patients treated with Smo antagonists have eventually developed resistance due to the occurrence of compensating mechanisms. Furthermore, Smo antagonists are not effective in tumors where the Hedgehog hyperactivation is due to mutations of pathway components downstream of Smo, or in case of non-canonical, Smo-independent activation of the Gli transcription factors. For all these reasons, the research of Hh inhibitors acting downstream of Smo is becoming an area of intensive investigation. In this review we illustrate the progresses made in the identification of effective Hedgehog inhibitors and their application in cancer, with a special emphasis on the newly identified downstream inhibitors. We describe in detail the Gli inhibitors and illustrate their mode of action and applications in experimental and/or clinical settings.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Hedgehog/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Humanos , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Meduloblastoma/metabolismo , Camundongos , Dados de Sequência Molecular , Proteína Homeobox Nanog , Células-Tronco Neoplásicas/metabolismo , Neurônios/citologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Alinhamento de Sequência , Células-Tronco/citologia , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína GLI1 em Dedos de ZincoRESUMO
The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteômica , Fatores de Transcrição/genética , Transferases , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3É, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3É+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.
Assuntos
Progressão da Doença , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptor Notch3 , Receptores CXCR4 , Timócitos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Receptor Notch3/genética , Receptor Notch3/metabolismo , Timócitos/metabolismo , Timócitos/citologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Camundongos , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Humanos , Camundongos Transgênicos , Transdução de Sinais , Diferenciação Celular/genéticaRESUMO
A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy. Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation. Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.
Assuntos
Neoplasias Colorretais , Fatores de Iniciação de Peptídeos , Poliaminas , Proteínas Proto-Oncogênicas c-myc , Animais , Camundongos , Apoptose , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Ornitina Descarboxilase/farmacologia , Poliaminas/metabolismo , Humanos , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Regulation of gene expression in response to mitogenic stimuli is a critical aspect underlying many forms of human cancers. The AP-1 complex mediates the transcriptional response to mitogens, and its deregulation causes developmental defects and tumors. We report that the coactivator CRTC1 cyclic AMP response element-binding protein (CREB)-regulated transcription coactivator 1 is a potent and indispensable modulator of AP-1 function. After exposure of cells to the AP-1 agonist 12-O-tetradecanoylphorbol-13-acetate (TPA), CRTC1 is recruited to AP-1 target gene promoters and associates with c-Jun and c-Fos to activate transcription. CRTC1 consistently synergizes with the proto-oncogene c-Jun to promote cellular growth, whereas AP-1-dependent proliferation is abrogated in CRTC1-deficient cells. Remarkably, we demonstrate that CRTC1-Maml2 oncoprotein, which causes mucoepidermoid carcinomas, binds and activates both c-Jun and c-Fos. Consequently, ablation of AP-1 function disrupts the cellular transformation and proliferation mediated by this oncogene. Together, these data illustrate a novel mechanism required to couple mitogenic signals to the AP-1 gene regulatory program.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica , Fator de Transcrição AP-1/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Transativadores , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Biguanides are a family of antidiabetic drugs with documented anticancer properties in preclinical and clinical settings. Despite intensive investigation, how they exert their therapeutic effects is still debated. Many studies support the hypothesis that biguanides inhibit mitochondrial complex I, inducing energy stress and activating compensatory responses mediated by energy sensors. However, a major concern related to this "complex" model is that the therapeutic concentrations of biguanides found in the blood and tissues are much lower than the doses required to inhibit complex I, suggesting the involvement of additional mechanisms. This comprehensive review illustrates the current knowledge of pharmacokinetics, receptors, sensors, intracellular alterations, and the mechanism of action of biguanides in diabetes and cancer. The conditions of usage and variables affecting the response to these drugs, the effect on the immune system and microbiota, as well as the results from the most relevant clinical trials in cancer are also discussed.
RESUMO
We synthesized new aroyl diheterocyclic pyrrole (ARDHEP) 15 that exhibited the hallmarks of ferroptosis. Compound 15 strongly inhibited U-87 MG, OVCAR-3, and MCF-7 cancer cells, induced an increase of cleaved PARP, but was not toxic for normal human primary T lymphocytes at 0.1 µM. Analysis of the levels of lactoperoxidase, malondialdehyde, lactic acid, total glutathione, and ATP suggested that the in vivo inhibition of cancer cell proliferation by 15 went through stimulation of oxidative stress injury and Fe2+ accumulation. Quantitative polymerase chain reaction analysis of the mRNA expression in U-87 MG and SKOV-3 tumor tissues from 15-treated mice showed the presence of Ptgs2/Nfe2l2/Sat1/Akr1c1/Gpx4 genes correlated with ferroptosis in both groups. Immunofluorescence staining revealed significantly lower expressions of proteins Ki67, CD31, and ferroptosis negative regulation proteins glutathione peroxidase 4 (GPX4) and FTH1. Compound 15 was found to be metabolically stable when incubated with human liver microsomes.
Assuntos
Neoplasias Ovarianas , Moduladores de Tubulina , Humanos , Animais , Feminino , Camundongos , Tubulina (Proteína) , Pirróis/farmacologia , Apoptose , Linhagem Celular TumoralRESUMO
Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. Here, we show that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.
Assuntos
Proteínas de Drosophila/metabolismo , Atividade Motora/genética , Atividade Motora/fisiologia , Poliaminas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Regulação para Baixo/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Biossíntese de Proteínas , Putrescina/farmacologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Espermidina/farmacologiaRESUMO
Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lisina/metabolismo , Camundongos Nus , Fases de Leitura Aberta/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/química , Peptídeos/metabolismo , Poliaminas/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/química , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Extensive molecular characterization of human colorectal cancer (CRC) via Next Generation Sequencing (NGS) indicated that genetic or epigenetic dysregulation of a relevant, but limited, number of molecular pathways typically occurs in this tumor. The molecular picture of the disease is significantly complicated by the frequent occurrence of individually rare genetic aberrations, which expand tumor heterogeneity. Inter- and intratumor molecular heterogeneity is very likely responsible for the remarkable individual variability in the response to conventional and target-driven first-line therapies, in metastatic CRC (mCRC) patients, whose median overall survival remains unsatisfactory. Implementation of an extensive molecular characterization of mCRC in the clinical routine does not yet appear feasible on a large scale, while multigene panel sequencing of most commonly mutated oncogene/oncosuppressor hotspots is more easily achievable. Here, we report that clinical multigene panel sequencing performed for anti-EGFR therapy predictive purposes in 639 formalin-fixed paraffin-embedded (FFPE) mCRC specimens revealed previously unknown pairwise mutation associations and a high proportion of cases carrying actionable gene mutations. Most importantly, a simple principal component analysis directed the delineation of a new molecular stratification of mCRC patients in eight groups characterized by non-random, specific mutational association patterns (MAPs), aggregating samples with similar biology. These data were validated on a The Cancer Genome Atlas (TCGA) CRC dataset. The proposed stratification may provide great opportunities to direct more informed therapeutic decisions in the majority of mCRC cases.
RESUMO
The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Correpressoras/efeitos dos fármacos , Proteínas Hedgehog/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Neoplasias/tratamento farmacológico , Fenformin/uso terapêutico , Animais , Antineoplásicos/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Camundongos , Fenformin/farmacologiaRESUMO
Hedgehog (Hh) signaling is a critical developmental regulator and its aberrant activation,due to somatic or germline mutations of genes encoding pathway components, causes Basal CellCarcinoma (BCC) and medulloblastoma (MB). A growing effort has been devoted at theidentification of druggable vulnerabilities of the Hedgehog signaling, leading to the identificationof various compounds with variable efficacy and/or safety. Emerging evidence shows that anaberrant polyamine metabolism is a hallmark of Hh-dependent tumors and that itspharmacological inhibition elicits relevant therapeutic effects in clinical or preclinical models ofBCC and MB. We discuss here the current knowledge of polyamine metabolism, its role in cancerand the available targeting strategies. We review the literature about the connection betweenpolyamines and the Hedgehog signaling, and the potential therapeutic benefit of targetingpolyamine metabolism in two malignancies where Hh pathways play a well-established role: BCCand MB.
Assuntos
Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/terapia , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/terapia , Terapia de Alvo Molecular , Poliaminas/metabolismo , Animais , Carcinogênese/metabolismo , HumanosRESUMO
The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDAapproved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HHGLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HHregulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogenactivated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogenactivated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HHregulated transcription factor. Phosphorylation occurred at multiple residues in the Cterminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 1433. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.
Assuntos
Proteínas Hedgehog/genética , MAP Quinase Quinase Quinase 1/genética , Meduloblastoma/genética , Proteína GLI1 em Dedos de Zinco/genética , Anilidas/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Piridinas/administração & dosagem , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/ß-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.
Assuntos
Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , beta-Arrestina 2/metabolismo , Motivos de Aminoácidos , Animais , Carcinogênese , Feminino , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/enzimologia , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , beta-Arrestina 2/genéticaRESUMO
SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.