RESUMEN
Compounds that inhibit glutathione peroxidase 4 (GPX4) hold promise as cancer therapeutics in their ability to induce a form of nonapoptotic cell death called ferroptosis. Our research identified 24, a structural analog of the potent GPX4 inhibitor RSL3, that has much better plasma stability (t1/2 > 5 h in mouse plasma). The bioavailability of 24 provided efficacious plasma drug concentrations with IP dosing, thus enabling in vivo studies to assess tolerability and efficacy. An efficacy study in mouse using a GPX4-sensitive tumor model found that doses of 24 up to 50 mg/kg were tolerated for 20 days but had no effect on tumor growth, although partial target engagement was observed in tumor homogenate.
Asunto(s)
Ferroptosis , Neoplasias , Ratones , Animales , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Disponibilidad BiológicaRESUMEN
S-Palmitoylation is a reversible post-translational lipid modification that regulates protein trafficking and signaling. The enzymatic depalmitoylation of proteins is inhibited by the beta-lactones Palmostatin M and B, which have been found to target several serine hydrolases. In efforts to better understand the mechanism of action of Palmostatin M, we describe herein the synthesis, chemical proteomic analysis, and functional characterization of analogs of this compound. We identify Palmostatin M analogs that maintain inhibitory activity in N-Ras depalmitoylation assays while displaying complementary reactivity across the serine hydrolase class as measured by activity-based protein profiling. Active Palmostatin M analogs inhibit the recently characterized ABHD17 subfamily of depalmitoylating enzymes, while sparing other candidate depalmitoylases such as LYPLA1 and LYPLA2. These findings improve our understanding of the structure-activity relationship of Palmostatin M and refine the set of serine hydrolase targets relevant to the compound's effects on N-Ras palmitoylation dynamics.
Asunto(s)
Lactonas/análisis , Propiolactona/análogos & derivados , Proteómica , Sulfonas/análisis , Proteínas ras/metabolismo , Humanos , Lactonas/metabolismo , Lactonas/farmacología , Estructura Molecular , Propiolactona/análisis , Propiolactona/metabolismo , Propiolactona/farmacología , Sulfonas/metabolismo , Sulfonas/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/químicaRESUMEN
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
Asunto(s)
Membrana Celular/metabolismo , Hidrolasas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas ras/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/patología , Lipoilación , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura MolecularRESUMEN
Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Hematológicas/genética , Hematopoyesis/genética , Lipoilación/genética , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Sustitución de Aminoácidos , Animales , Ácido Aspártico/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Glicina/genética , Neoplasias Hematológicas/metabolismo , Células Madre Hematopoyéticas/fisiología , Redes y Vías Metabólicas/genética , Ratones , Ratones Transgénicos , Ácido Palmítico/metabolismoRESUMEN
The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias/metabolismo , Proteínas ras/metabolismo , Células A549 , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células HCT116 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Pelados , Ratones SCID , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/patología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Leucemia Mieloide Aguda/mortalidad , Análisis de SupervivenciaRESUMEN
Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
Asunto(s)
Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Neoplasias Colorrectales/genética , Difenilamina/análogos & derivados , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Evolución Clonal , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Difenilamina/farmacología , Difenilamina/uso terapéutico , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutación , RetroviridaeRESUMEN
Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-Ras(G60_A66dup) and K-Ras(E62_A66dup) proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Hepatocitos/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Mutagénesis Insercional , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Preescolar , Humanos , Ratones , Mutación , Secuencias Repetidas en Tándem , Ensayo de Tumor de Célula MadreRESUMEN
Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.
Asunto(s)
Alelos , Proteína Adaptadora GRB10/genética , Impresión Genómica , Neurofibromatosis 1/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fibroblastos/metabolismo , Proteína Adaptadora GRB10/metabolismo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes de Neurofibromatosis 1 , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas p21(ras) , Radiación , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.
Asunto(s)
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Genes ras , Células Madre Hematopoyéticas/citología , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas/genética , Inhibidores de Proteínas Quinasas/química , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Células Madre/citología , TransgenesRESUMEN
Hedgehog (Hh) pathway activation and Gli-dependent transcription play critical roles in embryonic patterning, tissue homeostasis, and tumorigenesis. By conducting a genome-scale cDNA overexpression screen, we have identified the Rho GAP family member Arhgap36 as a positive regulator of the Hh pathway in vitro and in vivo. Arhgap36 acts in a Smoothened (Smo)-independent manner to inhibit Gli repressor formation and to promote the activation of full-length Gli proteins. Arhgap36 concurrently induces the accumulation of Gli proteins in the primary cilium, and its ability to induce Gli-dependent transcription requires kinesin family member 3a and intraflagellar transport protein 88, proteins that are essential for ciliogenesis. Arhgap36 also functionally and biochemically interacts with Suppressor of Fused. Transcriptional profiling further reveals that Arhgap36 is overexpressed in murine medulloblastomas that acquire resistance to chemical Smo inhibitors and that ARHGAP36 isoforms capable of Gli activation are up-regulated in a subset of human medulloblastomas. Our findings reveal a new mechanism of Gli transcription factor activation and implicate ARHGAP36 dysregulation in the onset and/or progression of GLI-dependent cancers.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Meduloblastoma/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Cilios/genética , Cilios/metabolismo , Proteínas Activadoras de GTPasa/genética , Perfilación de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones Noqueados , Células 3T3 NIH , Proteínas Nucleares/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Factores de Transcripción/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteína con Dedos de Zinc GLI1RESUMEN
UNLABELLED: Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic Nras(G12D) in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm that is exacerbated in homozygous mutant animals. Here, we show that increased Nras(G12D) gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of Nras(G12D/G12D) hematopoietic cells. Modulating Nras(G12D) dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MAP-ERK kinase (MEK) inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras-mutant cells nor inhibited myeloid transformation by exogenous Nras(G12D). Importantly, NRAS expression increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers. SIGNIFICANCE: Understanding the mechanisms of Ras -induced transformation and adaptive cellular responses is fundamental. The observation that oncogenic Nras lacks tumor suppressor activity, whereas increased dosage strongly modulates cell growth and alters sensitivity to MEK inhibition, suggests new therapeutic opportunities in cancer.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Genes ras/genética , Sistema Hematopoyético/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Genes Supresores de Tumor , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Progenitoras Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Transducción de SeñalRESUMEN
The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.
Asunto(s)
Citoplasma/enzimología , Dineínas Citoplasmáticas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Quinazolinonas/química , Quinazolinonas/farmacología , Animales , Bovinos , Cilios/efectos de los fármacos , Cilios/metabolismo , Cilios/patología , Dineínas Citoplasmáticas/metabolismo , Proteínas Hedgehog/metabolismo , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Peso Molecular , Movimiento/efectos de los fármacos , Células 3T3 NIH , Peroxisomas/efectos de los fármacos , Peroxisomas/fisiología , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
Controlling cell fate is essential for embryonic development, tissue regeneration, and the prevention of human disease. With each cell in the human body sharing a common genome, achieving the appropriate spectrum of stem cells and their differentiated lineages requires the selective activation of developmental signaling pathways, the expression of specific target genes, and the maintenance of these cellular states through epigenetic mechanisms. Small molecules that target these regulatory processes are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming. Pharmacological modulators of cell fate could also help remediate human diseases caused by dysregulated cell proliferation or differentiation, heralding a new era in molecular therapeutics.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medicina Regenerativa , Humanos , Bibliotecas de Moléculas PequeñasRESUMEN
Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Animales , Química Farmacéutica/métodos , Diseño de Fármacos , Epistasis Genética , Fibroblastos/metabolismo , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Neuronas/metabolismo , Fenotipo , Unión ProteicaRESUMEN
Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.
Asunto(s)
Movimiento Celular/fisiología , Células Germinativas/fisiología , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Alcaloides de Veratrum/farmacología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/fisiología , Células Germinativas/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Smoothened , Pez Cebra/fisiologíaRESUMEN
Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).
Asunto(s)
Proteínas Hedgehog/metabolismo , Compuestos Heterocíclicos con 2 Anillos/farmacología , Microtúbulos/metabolismo , Tiazoles/farmacología , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Compuestos Heterocíclicos con 2 Anillos/química , Ratones , Microtúbulos/efectos de los fármacos , Células 3T3 NIH , Piridinas/química , Transducción de Señal , Tiazoles/químicaRESUMEN
Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.