RESUMO
Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.
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Natural products (NPs) are highly profitable pharmacological tools due to their chemical diversity and ability to modulate biological systems. Accessing new chemical entities while retaining the biological relevance of natural chemotypes is a fundamental goal in the design of novel bioactive compounds. Notably, NPs have played a crucial role in understanding Hedgehog (HH) signalling and its pharmacological modulation in anticancer therapy. However, HH antagonists developed so far have shown several limitations, thus growing interest in the design of second-generation HH inhibitors. Through smart manipulation of the NPs core-scaffold, unprecedented and intriguing architectures have been achieved following different design strategies. This study reports the rational design and synthesis of a first and second generation of anthraquinone-based hybrids by combining the rhein scaffold with variously substituted piperazine nuclei that are structurally similar to the active portion of known SMO antagonists, the main transducer of the HH pathway. A thorough functional and biological investigation identified RH2_2 and RH2_6 rhein-based hybrids as valuable candidates for HH inhibition through SMO antagonism, with the consequent suppression of HH-dependent tumour growth. These findings also corroborated the successful application of the NPs-based hybrid design strategy in the development of novel NP-based SMO antagonists.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Receptor Smoothened/uso terapêutico , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antraquinonas/farmacologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
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
Withanolides constitute a well-known family of plant-based alkaloids characterised by widespread biological properties, including the ability of interfering with Hedgehog (Hh) signalling pathway. Following our interest in natural products and in anticancer compounds, we report here the synthesis of a new class of Hh signalling pathway inhibitors, inspired by withaferin A, the first isolated member of withanolides. The decoration of our scaffolds was rationally supported by in silico studies, while functional evaluation revealed promising candidates, confirming once again the importance of natural products as inspiration source for the discovery of novel bioactive compounds. A stereoselective approach, based on Brown chemistry, allowed the obtainment and the functional evaluation of the enantiopure hit compounds.
Assuntos
Antineoplásicos , Vitanolídeos , Antineoplásicos/farmacologia , Proteínas Hedgehog/farmacologia , Transdução de Sinais , Vitanolídeos/farmacologiaRESUMO
The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Purinas/farmacologia , Receptor Smoothened/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HT29 , Proteínas Hedgehog/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Purinas/química , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismoRESUMO
Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.
Assuntos
Proteínas F-Box/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Ratos , Transdução de Sinais , UbiquitinaçãoRESUMO
The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild "molar tooth sign"), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies.
Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Cerebelo/anormalidades , Anormalidades Craniofaciais/genética , Anormalidades do Olho/genética , Genes Recessivos , Proteínas Hedgehog/metabolismo , Doenças Renais Císticas/genética , Mutação de Sentido Incorreto , Proteínas Repressoras/genética , Retina/anormalidades , Anormalidades Múltiplas/patologia , Doenças do Desenvolvimento Ósseo/patologia , Células Cultivadas , Cerebelo/patologia , Criança , Estudos de Coortes , Anormalidades Craniofaciais/patologia , Anormalidades do Olho/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Doenças Renais Císticas/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Retina/patologia , Análise de Sequência de DNA , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Proteína Gli3 com Dedos de ZincoRESUMO
The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation.-Savoia, M., Cencioni, C., Mori, M., Atlante, S., Zaccagnini, G., Devanna, P., Di Marcotullio, L., Botta, B., Martelli, F., Zeiher, A. M., Pontecorvi, A., Farsetti, A., Spallotta, F., Gaetano, C. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.
Assuntos
Diferenciação Celular/genética , Proteína p300 Associada a E1A/genética , Isocitrato Desidrogenase/genética , Transcrição Gênica/genética , Acetilação , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Fibras Musculares Esqueléticas/fisiologia , Mioblastos/fisiologia , Processamento de Proteína Pós-Traducional/genética , Ativação Transcricional/genéticaRESUMO
Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.
Assuntos
DNA/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Isoflavonas/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glioblastoma/metabolismo , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Receptores Patched , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.
Assuntos
Cromonas/farmacologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Metabolômica , Animais , Proliferação de Células/efeitos dos fármacos , Glioma/diagnóstico , Glicólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
This work aims at elucidating the mechanism and kinetics of hydrolysis of GANT61, the first and most-widely used inhibitor of the Hedgehog (Hh) signalling pathway that targets Glioma-associated oncogene homologue (Gli) proteins, and at confirming the chemical nature of its bioactive form. GANT61 is poorly stable under physiological conditions and rapidly hydrolyses into an aldehyde species (GANT61-A), which is devoid of the biological activity against Hh signalling, and a diamine derivative (GANT61-D), which has shown inhibition of Gli-mediated transcription. Here, we combined chemical synthesis, NMR spectroscopy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61 hydrolysis pathway. Our results show that GANT61-D is the bioactive form of GANT61 in NIH3T3 Shh-Light II cells and SuFu-/- mouse embryonic fibroblasts, and clarify the structural requirements for GANT61-D binding to Gli1. This study paves the way to the design of GANT61 derivatives with improved potency and chemical stability.
Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Hidrólise , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The transcription factor Nanog plays a critical role in the self-renewal of embryonic stem cells as well as in neural stem cells (NSCs). microRNAs (miRNAs) are also involved in stemness regulation. However, the miRNA network downstream of Nanog is still poorly understood. High-throughput screening of miRNA expression profiles in response to modulated levels of Nanog in postnatal NSCs identifies miR-17-92 cluster as a direct target of Nanog. Nanog controls miR-17-92 cluster by binding to the upstream regulatory region and maintaining high levels of transcription in NSCs, whereas Nanog/promoter association and cluster miRNAs expression are lost alongside differentiation. The two miR-17 family members of miR-17-92 cluster, namely miR-17 and miR-20a, target Trp53inp1, a downstream component of p53 pathway. To support a functional role, the presence of miR-17/20a or the loss of Trp53inp1 is required for the Nanog-induced enhancement of self-renewal of NSCs. We unveil an arm of the Nanog/p53 pathway, which regulates stemness in postnatal NSCs, wherein Nanog counteracts p53 signals through miR-17/20a-mediated repression of Trp53inp1.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Ciclo Celular , Proliferação de Células , Células Cultivadas , Cerebelo/citologia , Proteínas de Choque Térmico/genética , Proteínas de Homeodomínio/genética , Camundongos , MicroRNAs/genética , Proteína Homeobox Nanog , Células-Tronco Neurais/citologiaRESUMO
BACKGROUND: Aberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326. METHODS: We evaluated ß-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays. RESULTS: Low levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation. CONCLUSIONS: Our results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal.
Assuntos
Meduloblastoma/genética , MicroRNAs/genética , Proteína GLI1 em Dedos de Zinco/genética , beta-Arrestina 1/genética , Autorrenovação Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de Sinais/genéticaRESUMO
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
Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion and tethering with the ER. The role of MFN2 in mitochondrial transport has however remained elusive. Like MFN2, acetylated microtubules play key roles in mitochondria dynamics. Nevertheless, it is unknown if the α-tubulin acetylation cycle functionally interacts with MFN2. Here, we show that mitochondrial contacts with microtubules are sites of α-tubulin acetylation, which occurs through MFN2-mediated recruitment of α-tubulin acetyltransferase 1 (ATAT1). This activity is critical for MFN2-dependent regulation of mitochondria transport, and axonal degeneration caused by CMT2A MFN2 associated R94W and T105M mutations may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in α-tubulin acetylation and suggest that disruption of this activity plays a role in the onset of MFN2-dependent CMT2A.
RESUMO
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
The developmental protein Numb is a major determinant of binary cell fates. It is also required for the differentiation of cerebellar granule cell progenitors (GCPs) at a stage of development responsive to the morphogenic glycoprotein Hedehog. Hedgehog signalling is crucial for the physiological maintenance and self-renewal of neural stem cells and its deregulation is responsible for their progression towards tumorigenesis. The mechanisms that inhibit this pathway during the differentiation stage are poorly understood. Here, we identify Numb as a Hedgehog-pathway inhibitor that is downregulated in early GCPs and GCP-derived cancer cells. We demonstrate that the Hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination, which suppresses Hedgehog signals, thus arresting growth and promoting cell differentiation. This novel Numb-dependent regulatory loop may limit the extent and duration of Hedgehog signalling during neural-progenitor differentiation, and its subversion may be a relevant event in brain tumorigenesis.
Assuntos
Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Cerebelo/patologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Meduloblastoma/genética , Camundongos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
Acetylated microtubules play key roles in the regulation of mitochondria dynamics. It has however remained unknown if the machinery controlling mitochondria dynamics functionally interacts with the alpha-tubulin acetylation cycle. Mitofusin-2 (MFN2), a large GTPase residing in the mitochondrial outer membrane and mutated in Charcot-Marie-Tooth type 2 disease (CMT2A), is a regulator of mitochondrial fusion, transport and tethering with the endoplasmic reticulum. The role of MFN2 in regulating mitochondrial transport has however remained elusive. Here we show that mitochondrial contacts with microtubules are sites of alpha-tubulin acetylation, which occurs through the MFN2-mediated recruitment of alpha-tubulin acetyltransferase 1 (ATAT1). We discover that this activity is critical for MFN2-dependent regulation of mitochondria transport, and that axonal degeneration caused by CMT2A MFN2 associated mutations, R94W and T105M, may depend on the inability to release ATAT1 at sites of mitochondrial contacts with microtubules. Our findings reveal a function for mitochondria in regulating acetylated alpha-tubulin and suggest that disruption of the tubulin acetylation cycle play a pathogenic role in the onset of MFN2-dependent CMT2A.
RESUMO
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.