RESUMO
The Wnt signaling pathway, known for regulating genes critical to normal embryonic development and tissue homeostasis, is dysregulated in many types of cancer. Previously, we identified that the anthelmintic drug niclosamide inhibited Wnt signaling by promoting internalization of Wnt receptor Frizzled 1 and degradation of Wnt signaling pathway proteins, Dishevelled 2 and ß-catenin, contributing to suppression of colorectal cancer growth in vitro and in vivo Here, we provide evidence that niclosamide-mediated inhibition of Wnt signaling is mediated through autophagosomes induced by niclosamide. Specifically, niclosamide promotes the co-localization of Frizzled 1 or ß-catenin with LC3, an autophagosome marker. Niclosamide inhibition of Wnt signaling is attenuated in autophagosome-deficient ATG5-/- MEF cells or cells expressing shRNA targeting Beclin1, a critical constituent of autophagosome. Treatment with the autophagosome inhibitor 3MA blocks niclosamide-mediated Frizzled 1 degradation. The sensitivity of colorectal cancer cells to growth inhibition by niclosamide is correlated with autophagosome formation induced by niclosamide. Niclosamide inhibits mTORC1 and ULK1 activities and induces LC3B expression in niclosamide-sensitive cell lines, but not in the niclosamide-resistant cell lines tested. Interestingly, niclosamide is a less effective inhibitor of Wnt-responsive genes (ß-catenin, c-Myc, and Survivin) in the niclosamide-resistant cells than in the niclosamide-sensitive cells, suggesting that deficient autophagy induction by niclosamide compromises the effect of niclosamide on Wnt signaling. Our findings provide a mechanistic understanding of the role of autophagosomes in the inhibition of Wnt signaling by niclosamide and may provide biomarkers to assist selection of patients whose tumors are likely to respond to niclosamide.
Assuntos
Autofagia/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Niclosamida/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Células HCT116 , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Dysregulation of the Wnt signaling pathway is an underlying mechanism in multiple diseases, particularly in cancer. Until recently, identifying agents that target this pathway has been difficult and as a result, no approved drugs exist that specifically target this pathway. We reported previously that the anthelmintic drug Niclosamide inhibits the Wnt/ß-catenin signaling pathway and suppresses colorectal cancer cell growth in vitro and in vivo. In an effort to build on this finding, we sought to discover new Wnt/ß-catenin inhibitors that expanded the chemotype structural diversity. Here, we asked a specific SAR question unresolved in previous SAR studies of Niclosamide's inhibition of Wnt/ß-catenin signaling to identify a new structural class of Wnt/ß-catenin signaling inhibitors based on a triazole motif. Similar to Niclosamide, we found that the new triazole derivatives internalized Frizzled-1 GFP receptors, inhibited Wnt/ß-catenin signaling in the TOPflash assay and reduced Wnt/ß-catenin target gene levels in CRC cells harboring mutations in the Wnt pathway. Moreover, in pilot SAR studies, we found the Wnt/ß-catenin SAR trends in the anilide region were generally similar between the two chemical classes of inhibitors. Overall, these studies demonstrate the ability to use the SAR of the Niclosamide salicylanilide chemical class to expand the structural diversity of Wnt/ß-catenin inhibitors.
Assuntos
Niclosamida/farmacologia , Triazóis/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Niclosamida/síntese química , Niclosamida/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
The Wnt signaling pathway is critical for normal tissue development and is an underlying mechanism of disease when dysregulated. Previously, we reported that the drug Niclosamide inhibits Wnt/ß-catenin signaling by decreasing the cytosolic levels of Dishevelled and ß-catenin, and inhibits the growth of colon cancers both in vitro and in vivo. Since the discovery of Niclosamide's anthelmintic activity, a growing body of literature indicates that Niclosamide is a multifunctional drug. In an effort to identify derivatives of Niclosamide with improved pharmacokinetic properties that maintain the multifunctional drug activity of Niclosamide for clinical evaluation, we designed DK419, a derivative containing a 1H-benzo[d]imidazole-4-carboxamide substructure, using the structure-activity relationships (SAR) of the Niclosamide salicylanilide chemotype. Similar to Niclosamide, we found DK419 inhibited Wnt/ß-catenin signaling, altered cellular oxygen consumption rate and induced production of pAMPK. Moreover, we found DK419 inhibited the growth of CRC tumor cells in vitro, had good plasma exposure when dosed orally, and inhibited the growth of patient derived CRC240 tumor explants in mice dosed orally. DK419, a derivative of Niclosamide with multifunctional activity and improved pharmacokinetic properties, is a promising agent to treat colorectal cancer, Wnt-related diseases and other diseases in which Niclosamide has demonstrated functional activity.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Benzimidazóis/química , Benzimidazóis/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Desenho de Fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Células HEK293 , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Camundongos , Camundongos SCID , Niclosamida/análogos & derivados , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Consumo de Oxigênio/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
Abnormal Wnt activity is a major mechanism responsible for many diseases, including cancer. Previously, we reported that the anthelmintic drug Niclosamide (NIC) inhibits Wnt/ß-catenin signaling and suppresses colon cancer cell growth. Although the pharmacokinetic properties of NIC are appropriate for use as an anthelmintic agent, its low solubility, low bioavailability and low systemic exposure limit its usefulness in treating systemic diseases. To overcome these limitations, we conjugated NIC to recombinant chimeric polypeptides (CPs), and the CP-NIC conjugate spontaneously self-assembled into sub-100 nm near-monodisperse nanoparticles. CP-NIC nanoparticles delivered intravenously act as a pro-drug of NIC to dramatically increase exposure of NIC compared to dosing with free NIC. CP-NIC improved anti-tumor activity compared to NIC in a xenograft model of human colon cancer. Because NIC has multiple biological activities, CP-NIC could be used for treatment of multiple diseases, including cancer, bacterial and viral infection, type II diabetes, NASH and NAFLD.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Nanopartículas , Niclosamida/farmacologia , Peptídeos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Diabetes Mellitus Tipo 2 , Humanos , Masculino , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Wnt signaling pathway plays a key role in organ and tissue homeostasis, and when dysregulated, can become a major underlying mechanism of disease, particularly cancer. We reported previously that the anthelmintic drug Niclosamide inhibits Wnt/ß-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. To define Niclosamide's mechanism of Wnt/ß-catenin inhibition, and to improve its selectivity and pharmacokinetic properties as an anticancer treatment, we designed a novel class of benzimidazole inhibitors of Wnt/ß-catenin signaling based on SAR studies of the Niclosamide salicylanilide chemotype. Niclosamide has multiple biological activities. To address selectivity in our design, we interrogated a protonophore SAR model and used the principle of conformational restriction to identify novel Wnt/ß-catenin inhibitors with less effect on ATP cellular homeostasis. These studies led to the identification of 4-chloro-2-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) phenol (4) and related derivatives with greater selectivity for Wnt/ß-catenin signaling inhibition vs. differential effects on cellular ATP homeostasis. This is the first report that the Wnt signaling inhibitory activity of Niclosamide can be translated into a new chemical class and to show that its effects on ATP homeostasis can be separated from its inhibitory effects on Wnt signaling. These compounds could be useful tools to elucidate the mechanism of Niclosamide's inhibition of Wnt signaling, and aid the discovery of inhibitors with improved pharmacologic properties to treat cancer and diseases in which Niclosamide has important biological activity.
Assuntos
Trifosfato de Adenosina/metabolismo , Benzimidazóis/farmacologia , Niclosamida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Benzimidazóis/química , Linhagem Celular Tumoral , Células HEK293 , Homeostase , Humanos , Niclosamida/química , Relação Estrutura-AtividadeRESUMO
The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/ß-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/ß-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4'-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide's low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.
Assuntos
Niclosamida/uso terapêutico , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. METHODS: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. RESULTS: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Perexilina/farmacologia , Receptor ErbB-3/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neurregulinas/metabolismo , Neurregulinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Smoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.
Assuntos
Carcinogênese/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Proteínas Nucleares/fisiologia , Proteínas Inibidoras de STAT Ativados/fisiologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Cílios/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Oncogenes , Ligação Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Receptor Smoothened , Ubiquitina-Proteína Ligases , Proteína Gli2 com Dedos de ZincoRESUMO
Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.
Assuntos
Brônquios/metabolismo , Brônquios/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Mucinas/metabolismo , Sequência de Bases , Brônquios/citologia , Brônquios/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Microscopia Confocal , Substrato Quinase C Rico em Alanina Miristoilada , Transporte Proteico , Pirimidinonas/farmacologia , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
INTRODUCTION: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. METHODS: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. RESULTS: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. CONCLUSION: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.
Assuntos
Anticorpos/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Receptor ErbB-2/imunologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Transplante Heterólogo , Ubiquitinação , VacinaçãoRESUMO
BACKGROUND: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. METHODS: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. RESULTS: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). CONCLUSIONS: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. TRIAL REGISTRY: ClinicalTrials.gov NCT00952692.
Assuntos
Imunoterapia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Idoso , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Demografia , Epitopos/imunologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Lapatinib , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , VacinaçãoRESUMO
UNLABELLED: Hepatic accumulation of myofibroblastic hepatic stellate cells (MF-HSCs) is pivotal in the pathogenesis of cirrhosis. Two events are necessary for MF-HSCs to accumulate in damaged livers: transition of resident, quiescent hepatic stellate cells (Q-HSCs) to MF-HSCs and expansion of MF-HSC numbers through increased proliferation and/or reduced apoptosis. In this study, we identified two novel mediators of MF-HSC accumulation: Ras-related C3 botulinum toxin substrate 1 (Rac1) and Hedgehog (Hh). It is unclear whether Rac1 and Hh interact to regulate the accumulation of MF-HSCs. We evaluated the hypothesis that Rac1 promotes activation of the Hh pathway, thereby stimulating signals that promote transition of Q-HSCs into MF-HSCs and enhance the viability of MF-HSCs. Using both in vitro and in vivo model systems, Rac1 activity was manipulated through adenoviral vector-mediated delivery of constitutively active or dominant-negative rac1. Rac1-transgenic mice with targeted myofibroblast expression of a mutated human rac1 transgene that produces constitutively active Rac1 were also examined. Results in all models demonstrated that activating Rac1 in HSC enhanced Hh signaling, promoted acquisition/maintenance of the MF-HSC phenotype, increased MF-HSC viability, and exacerbated fibrogenesis. Conversely, inhibiting Rac1 with dominant-negative rac1 reversed these effects in all systems examined. Pharmacologic manipulation of Hh signaling demonstrated that profibrogenic actions of Rac1 were mediated by its ability to activate Hh pathway-dependent mechanisms that stimulated myofibroblastic transition of HSCs and enhanced MF-HSC viability. CONCLUSION: These findings demonstrate that interactions between Rac1 and the Hh pathway control the size of MF-HSC populations and have important implications for the pathogenesis of cirrhosis.
Assuntos
Fibroblastos/patologia , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Fenótipo , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a beta-arrestin2-GFP reporter, we identified four FDA-approved drugs, halcinonide, fluticasone, clobetasol, and fluocinonide, as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo, promote Smo internalization, activate Gli, and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide, fluticasone, clobetasol, and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses.
Assuntos
Glucocorticoides/farmacologia , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Medicina Regenerativa/métodos , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Androstadienos/farmacologia , Arrestinas , Western Blotting , Linhagem Celular , Proliferação de Células , Clobetasol/farmacologia , Fluocinonida/farmacologia , Fluticasona , Proteínas de Fluorescência Verde , Halcinonida/farmacologia , Humanos , Estrutura Molecular , Receptor Smoothened , Células-Tronco/metabolismo , beta-ArrestinasRESUMO
The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.
Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Terapia Genética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/genética , Adenoviridae/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Proliferação de Células , Terapia Combinada , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Lapatinib , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and stem cell related tissue-regenerative effects of Wnt signaling. Dysregulated Wnt signaling is associated with many cancers. Currently, there are no drug candidates or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated beta-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide-mediated internalization, the Frizzled1 receptor colocalizes in vesicles containing transferrin and agonist-activated beta(2)-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting upstream signaling molecules (i.e., Frizzled and Dishevelled) and moreover may provide a valuable means of studying the physiological consequences of Wnt signaling.
Assuntos
Receptores Frizzled/metabolismo , Niclosamida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Animais , Antinematódeos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Ratos , beta Catenina/metabolismoRESUMO
beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate beta-arrestin but not G protein-dependent signaling, and are abrogated by beta-arrestin-2 small interfering RNA. These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.
Assuntos
Apoptose/fisiologia , Arrestinas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/fisiologia , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Arrestinas/genética , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína de Morte Celular Associada a bcl/genética , beta-Arrestina 2 , beta-ArrestinasRESUMO
Deleted in colorectal cancer (DCC) and neogenin are receptors of netrins, a family of guidance cues that promote axon outgrowth and guide growth cones in developing nervous system. The intracellular mechanisms of netrins, however, remain elusive. In this paper, we show that both DCC and neogenin become tyrosine phosphorylated in cortical neurons in response to netrin-1. Using a site-specific antiphosphor DCC antibody, we show that Y1420 phosphorylation is increased in netrin-1-stimulated neurons and that tyrosine-phosphorylated DCC is located in growth cones. In addition, we show that tyrosine-phosphorylated DCC selectively interacts with the Src family kinases Fyn and Lck, but not Src, c-Abl, Grb2, SHIP1, Shc, or tensin, suggesting a role of Fyn or Lck in netrin-1-DCC signaling. Of interest to note is that tyrosine-phosphorylated neogenin and uncoordinated 5 H2 (Unc5H2) not only bind to the Src homology 2 (SH2) domains of Fyn and SHP2, but also interact with the SH2 domain of SHIP1, suggesting a differential signaling between DCC and neogenin/Unc5H2. Furthermore, we demonstrate that inhibition of Src family kinase activity attenuated netrin-1-induced neurite outgrowth. Together, these results suggest a role of Src family kinases and tyrosine phosphorylation of netrin-1 receptors in regulating netrin-1 function.
Assuntos
Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Galinhas , Receptor DCC , Feminino , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Receptores de Netrina , Netrina-1 , Neurônios/citologia , Neurônios/fisiologia , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Tirosina/genéticaRESUMO
The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.
Assuntos
Arrestinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Adrenérgicos beta 2/fisiologia , Quinases de Receptores Adrenérgicos beta/metabolismo , Animais , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Quinase 2 de Receptor Acoplado a Proteína G , Quinase 3 de Receptor Acoplado a Proteína G , Quinase 5 de Receptor Acoplado a Proteína G , Quinases de Receptores Acoplados a Proteína G , Genes Reporter , Humanos , Isoproterenol/farmacologia , Rim , Cinética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , beta-ArrestinasRESUMO
Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.
Assuntos
Fatores de Crescimento Neural/fisiologia , Neuritos/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Proteínas Supressoras de Tumor/fisiologia , Fosfolipases Tipo C/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Receptor DCC , Humanos , Camundongos , Netrina-1 , Neurônios/citologia , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Chemotaxis is a cellular response that directs cell migration toward a chemical gradient and is fundamental to a variety of cellular processes. The receptors for most known chemokines belong to the seven transmembrane-spanning superfamily and signal through members of the G(alphai) family. Beta-arrestins, in addition to regulating desensitization, have emerged as potential mediators of G-protein-independent signaling pathways and have been implicated in several chemotactic pathways. Here, we report a system wherein chemotaxis is stimulated in a beta-arrestin 2-dependent and apparently G-protein-independent manner. Human embryonic kidney 293 cells with stable expression of the angiotensin II (Ang II) receptor type 1A (AT(1A)R) undergo chemotaxis in response to Ang II. An Ang II peptide analog S(1)I(4)I(8) Ang II that is unable to activate G-protein-mediated responses induces chemotaxis in these cells that is unaffected by pertussis toxin-mediated suppression of G(alphai). Suppression of beta-arrestin 2 expression using small interfering RNA (siRNA) essentially eliminated AT(1A)R-mediated chemotaxis induced by either Ang II or the S(1)I(4)I(8) Ang II peptide but had no effect on epidermal growth factor (EGF)-induced chemotaxis. It also abolished chemotaxis induced by lysophosphatidic acid (LPA), which was completely sensitive to pertussis toxin. In contrast, reduction of G(alphaq/11) through siRNA and inhibition of protein kinase C, extracellular signal-regulated kinases 1 and 2, or phosphatidylinositol-3-kinase did not diminish AT(1A)R-mediated chemotaxis. Inhibiting p38 mitogen-activated protein kinase decreased AT(1A)R-mediated chemotaxis and eliminated EGF-mediated chemotaxis, suggesting that p38 plays a role in chemotaxis that is not specific to the AT(1A)R in this system. These data suggest that beta-arrestin 2 can mediate chemotaxis through mechanisms which may be G-protein-independent (Ang II receptors) or -dependent (LPA receptors).