RESUMO
Autophagy is a lysosome-dependent degradation process. During autophagy, cytoplasmic components are sequestered and catabolized to supply nutrition and energy under starvation conditions. Recent work has demonstrated that many cargos can be specifically recognized and then eliminated via the core mechanism of autophagy which is termed as selective autophagy. The cargo recognition program provides the basis for the specific degradation of selective autophagy; thus, the exploration of the interaction between the cargo and the receptor is the key for revealing the underlying mechanism. Also, receptor protein complexes are required in various selective autophagy subtypes which process and guide the cargo to the core mechanism. Ubiquitination and phosphorylation are the main methods to modulate the affinity of the receptor toward cargo. Although many key processes of selective autophagy subtypes have been discovered and intensively studied, the precise ways in which the mechanisms of cargo recognition function remain mostly elusive. A fuller mechanistic understanding of selective autophagy will be important for efforts to promote disease treatment and drug development.
Assuntos
Autofagia , Lisossomos , Proteínas de Transporte , Citosol , UbiquitinaçãoRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Meduloblastoma/fisiopatologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Ciclo Celular/fisiologia , Senescência Celular/fisiologia , Cerebelo/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Instabilidade Genômica , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc , Metástase Neoplásica/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genéticaRESUMO
BRAFV600E is a common finding in glioma (about 10-60% depending on histopathologic subclassification). BRAFV600E monotherapy shows modest preclinical efficacy against BRAFV600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAFV600E inhibition in glioma. Furthermore, we investigate BRAFV600E/MEK combination therapy that overcomes intrinsic resistance to BRAFV600E inhibitor and also prevents BRAFV600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAFV600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAFV600E/MEK combination therapy and the effect on secondary malignancies. BRAFV600E inhibition leads to recovery of ERK phosphorylation. Combined BRAFV600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAFV600E glioma showed reduced tumor growth when treated with a combination of BRAFV600E and MEK inhibitor compared to BRAFV600E inhibition alone. Additional benefit of BRAFV600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAFV600E inhibitor. Combined BRAFV600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.
Assuntos
Antineoplásicos/administração & dosagem , Glioma/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Benzamidas/administração & dosagem , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Difenilamina/administração & dosagem , Difenilamina/análogos & derivados , Modelos Animais de Doenças , Feminino , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Indóis/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Glioma/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Glioma/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Roscovitina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.
Assuntos
Glioma/tratamento farmacológico , Glioma/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática , Receptores ErbB/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organoplatínicos/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Especificidade por Substrato , Serina-Treonina Quinases TOR , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismoRESUMO
Gliomas represent the most common primary brain tumor and among the most aggressive of cancers. Patients with glioma typically relapse within a year of initial diagnosis. Recurrent glioma is associated with acquired therapeutic resistance. Although neurosurgical resection, radiation and chemotherapy provide clear benefit, survival remains disappointing. It is, therefore, critical that we identify effective medical therapies and appropriate tumor biomarkers in patients at initial presentation, to promote durable responses in glioma. Pathways linking receptor tyrosine kinases, PI3 kinase, Akt, and mTOR feature prominently in this disease and represent therapeutic targets. Small molecules that inhibit one or more of these kinases are now being introduced into the clinic and may have some activity. Disappointingly, however, preclinical studies demonstrate these agents to be primarily cytostatic rather than cytotoxic to glioma cells. Here, we detail activation of the EGFR-PI3K-Akt-mTOR signaling network in glioma, review class I PI3K inhibitors, discuss roles for Akt, PKC and mTOR, and the importance of biomarkers. We further delineate attempts to target both single and multiple components within the EGFR-PI3K-Akt-mTOR axes. Lastly, we discuss the need to combine targeted therapies with cytotoxic chemotherapy, radiation and with inhibitors of survival signaling to improve outcomes in glioma.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Receptor EphB3RESUMO
Dysregulation of kinase-based signal transduction networks contributes to multiple aspects of malignancy. Chemical genetic approaches interrogate perturbed signaling in the immediate context of small molecule inhibitor treatment. In recent years, such approaches have identified new kinase targets, clarified the impact of poly-specific inhibition using agents for which at least one primary target is known, and have identified targets for which combinatorial inhibition leads to improved efficacy. Elucidation of the mechanisms through which specific small molecule drug-like agents impact crucial cancer pathways should yield important and clinically translatable insights into the use of similar agents in patients.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/genética , Sequência de Aminoácidos , Animais , Genes abl , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Modelos Biológicos , Neoplasias/etiologia , Proteínas Quinases/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacosRESUMO
In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.
Assuntos
Células Epiteliais/metabolismo , Miosina Tipo II/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Western Blotting , Linhagem Celular , Polaridade Celular , Células Epiteliais/citologia , Humanos , Miosina Tipo II/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Feminino , Glioblastoma/patologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Purinas/farmacologia , Purinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have shown previously that blockade of epidermal growth factor receptor (EGFR) cooperates with a pan-selective inhibitor of phosphoinositide-3-kinase (PI3K) in EGFR-driven glioma. In this communication, we tested EGFR-driven glioma differing in PTEN status, treating with the EGFR inhibitor erlotinib and a novel dual inhibitor of PI3Kalpha and mTOR (PI-103). Erlotinib blocked proliferation only in PTEN(wt) cells expressing EGFR. Although erlotinib monotherapy showed little effect in PTEN(mt) glioma, PI-103 greatly augmented the antiproliferative efficacy of erlotinib in this setting. To address the importance of PI3K blockade, we showed in PTEN(mt) glioma that combining PI-103 and erlotinib was superior to either monotherapy or to therapy combining erlotinib with either rapamycin (an inhibitor of mTOR) or PIK-90 (an inhibitor of PI3Kalpha). These experiments show that a dual inhibitor of PI3Kalpha and mTOR augments the activity of EGFR blockade, offering a mechanistic rationale for targeting EGFR, PI3Kalpha, and mTOR in the treatment of EGFR-driven, PTEN-mutant glioma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Furanos/farmacologia , Glioma/tratamento farmacológico , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Cloridrato de Erlotinib , Furanos/administração & dosagem , Glioma/genética , Glioma/patologia , Humanos , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Quinazolinas/administração & dosagem , Serina-Treonina Quinases TOR , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Glioblastoma is the most common and aggressive adult brain cancer. Tumors show frequent dysregulation of the PI3K-mTOR pathway. Although a number of small molecules target the PI3K-AKT-mTOR axis, their preclinical and clinical efficacy has been limited. Reasons for treatment failure include poor penetration of agents into the brain and observations that blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Clinical trials using allosteric mTOR inhibitors (rapamycin and rapalogs) to treat patients with glioblastoma have also been unsuccessful or uncertain, in part, because rapamycin inefficiently blocks the mTORC1 target 4EBP1 and feeds back to activate PI3K-AKT signaling. Inhibitors of the mTOR kinase (TORKi) such as TAK-228/MLN0128 interact orthosterically with the ATP- and substrate-binding pocket of mTOR kinase, efficiently block 4EBP1 in vitro, and are currently being investigated in the clinical trials. Preclinical studies suggest that TORKi have poor residence times of mTOR kinase, and our data suggest that this poor pharmacology translates into disappointing efficacy in glioblastoma xenografts. RapaLink-1, a TORKi linked to rapamycin, represents a drug with improved pharmacology against 4EBP1. In this review, we clarify the importance of 4EBP1 as a biomarker for the efficacy of PI3K-AKT-mTOR inhibitors in glioblastoma. We also review mechanistic data by which RapaLink-1 blocks p-4EBP1 and discuss future clinical strategies for 4EBP1 inhibition in glioblastoma. Clin Cancer Res; 24(1); 14-21. ©2017 AACR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Biomarcadores Tumorais , Glioblastoma/metabolismo , Fosfoproteínas/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Amplification of epidermal growth factor receptor (EGFR) and its active mutant EGFRvIII occurs frequently in glioblastoma (GBM). While EGFR and EGFRvIII play critical roles in pathogenesis, targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs) or antibodies has only shown limited efficacy in patients. Here we discuss signaling pathways mediated by EGFR/EGFRvIII, current therapeutics, and novel strategies to target EGFR/EGFRvIII-amplified GBM.
Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB/fisiologia , Glioblastoma/terapia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Neoplasias Encefálicas/genética , Receptores ErbB/antagonistas & inibidores , Glioblastoma/genética , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
: Amplification of the EGFR gene and its truncation mutant EGFRvIII are hallmarks of glioblastoma. Although coexpression of EGFR and EGFRvIII confers a growth advantage, how EGFR and EGFRvIII influence the tumor microenvironment remains incompletely understood. Here, we show that EGFR and EGFRvIII cooperate to induce macrophage infiltration via upregulation of the chemokine CCL2. EGFRvIII was significantly enriched in glioblastoma patient samples with high CCL2, and knockout of CCL2 in tumors coexpressing EGFR and EGFRvIII led to decreased infiltration of macrophages. KRAS was a critical signaling intermediate for EGFR- and EGFRvIII-induced expression of CCL2. Our results illustrate how EGFR and EGFRvIII direct the microenvironment in glioblastoma. SIGNIFICANCE: Full-length EGFR and truncated EGFRvIII work through KRAS to upregulate the chemokine CCL2 and drive macrophage infiltration in glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Quimiocina CCL2/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Microglia/metabolismo , Transplante de Neoplasias , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Microambiente Tumoral , Regulação para CimaRESUMO
BACKGROUND: Our understanding of protein kinase inhibition in the treatment of cancer is clearly limited by the lack of inhibitors that selectively block a single kinase implicated in neoplastic transformation. One approach to developing specific inhibitors is to engineer in protein kinases silent mutations that allow selective inhibition while retaining kinase activity. Because it is implicated in a large number of malignancies, EGFR provides an attractive target for such selective kinase inhibition. RESULTS: We generated an inhibitor-sensitized allele of the transforming receptor tyrosine kinase v-erbB. Transformation of immortalized rodent fibroblasts by sensitized versions of v-erbB (v-erbB-as1) was blocked by 1-napthyl PP1 (NaPP1), a cell-permeable ATP-competitive inhibitor. NaPP1 also reversed morphological transformation by v-erbB-as1. Signaling through MAP kinase and PI(3) kinase was initially blocked by inhibitor treatment and then recovered to levels comparable to those in nontransformed cells. Surprisingly, NaPP1-treated v-erbB-as1 cells failed to re-enter the cell cycle, showed decreased levels of D- and A-type cyclins, and showed increased levels of p27. To extend this result, we showed that NaPP1 treatment of v-Src-as1 cells also led to cell cycle arrest. Arrested cells could be rescued with a conditional allele of Raf or by transduction of a constitutive allele of cyclin D1. CONCLUSIONS: These data suggest that mammalian cells can become dependent on aberrant oncogenic signaling; this dependency renders them incapable of returning to a normal, proliferative phenotype.
Assuntos
Transformação Celular Neoplásica , Genes erbB-1/genética , Proteínas Oncogênicas v-erbB/genética , Proteínas Oncogênicas v-erbB/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Células 3T3 , Alelos , Animais , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Tamanho Celular , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/metabolismo , Citoesqueleto/metabolismo , Inibidores Enzimáticos/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Imuno-Histoquímica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Naftalenos/metabolismo , Proteína Oncogênica pp60(v-src)/genética , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Amplification and mutation of the epidermal growth factor receptor (EGFR) is common in astrocytoma. The most frequently occurring mutation (DeltaEGFR, EGFRvIII) deletes exons 2-7 from this receptor tyrosine kinase (RTK), and signals constitutively in the absence of ligand. DeltaEGFR is not found in normal tissue, and therefore represents an attractive therapeutic target. Here, we show that a small interfering RNA (siRNA) directed against the unique exon 1/exon 8 junction sequence of DeltaEGFR efficiently suppressed expression of DeltaEGFR in rodent fibroblasts and in two human glioblastoma cell lines. SiRNA-mediated depletion of DeltaEGFR led to reduction in the levels of phosphorylated Akt in glioma cells, was associated with increased apoptosis, and induced partial arrest at the G2M phase of the cell cycle. Inhibitors of PI3 kinase cooperated with siRNA treatment, leading to further increases in both cell cycle blockade and apoptosis. Importantly, cell cycle blockade could be reversed, and apoptosis rescued using a conditional allele of Akt, implicating Akt as a primary target of combination therapy. This study demonstrates the therapeutic potential of siRNA to impact DeltaEGFR as a glioma-specific target, and offers a mechanistic rationale for combining siRNA and small molecule inhibitor therapies against distinct components in the EGFR signaling pathway.
Assuntos
Divisão Celular/fisiologia , Receptores ErbB/genética , Fase G2/fisiologia , Interferência de RNA/fisiologia , Astrocitoma , Divisão Celular/genética , Linhagem Celular Tumoral , Primers do DNA , Receptores ErbB/deficiência , Fase G2/genética , Glioma , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Whether the apparent efficacy of a specific kinase inhibitor is attributable solely to inhibition of its primary target, or to combined inhibition of additional unidentified kinases, is a critical issue in cancer therapy. We used a chemical genetic approach to generate a selective inhibitor of v-erbB [a transforming allele of epidermal growth factor receptor (EGFR)] and interrogated inhibition in known downstream signaling pathways. On the basis of this analysis, we hypothesized that dual inhibition of v-erbB and phosphatidylinositol 3' (PI3) kinases could show improved potency. We, therefore, used two different cell lines to examine the effects of v-erbB or EGFR inhibitors, in combination with PI3 kinase inhibitors, in mouse models for EGFR-driven cancers. When treated with NaPP1, v-erbB-as1-transformed fibroblasts showed cell-cycle arrest and decreased activity of Akt kinase. Inhibitors of v-erbB-as1 and of PI3 kinase showed enhanced efficacy in treating established 3T3:v-erbB-as1 tumor allografts. We extended these results to the human glioma cell line U87:MG transduced with DeltaEGFR, a tumor-derived activated allele, treating tumor-bearing mice with vehicle, the EGFR inhibitor ZD1839, LY294002, or ZD1839 plus LY294002. In human glioma xenografts, inhibition of EGFR cooperated similarly with inhibition of PI3 kinase. Our experiments provide a preclinical mechanistic basis for combining biologically based therapies directed against two targets within a complex signaling cascade.
Assuntos
Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Gefitinibe , Glioma/tratamento farmacológico , Glioma/enzimologia , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Naftalenos/farmacologia , Proteínas Oncogênicas v-erbB/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The gene that encodes the epidermal growth factor receptor (EGFR) is frequently overexpressed or mutated in human cancers, including glioblastoma. However, the efficacy of EGFR-targeted small-molecule inhibitors or monoclonal antibodies in glioblastomas that also have mutation or deletion of the gene encoding phosphatase and tensin homolog (PTEN) has been modest. We found that EGFR signaling was blocked by a small molecule (G5-7) that selectively inhibited Janus kinase 2 (JAK2)-mediated phosphorylation and activation of EGFR and STAT3 (signal transducer and activator of transcription 3) by binding to JAK2, thereby decreasing the activity of downstream signaling by mTOR (mammalian target of rapamycin) and inducing cell cycle arrest. G5-7 inhibited the proliferation of PTEN-deficient glioblastoma cell lines harboring a constitutively active variant of EGFR (U87MG/EGFRvIII) and human glioblastoma explant neurosphere cultures, but the drug only weakly inhibited the proliferation of either glioblastoma cell lines that were wild type for EGFR and stably transfected with PTEN (U87MG/PTEN) or normal neural progenitor cells and astrocytes. Additionally, G5-7 reduced vascular endothelial growth factor (VEGF) secretion and endothelial cell migration and induced apoptosis in glioblastoma xenografts, thereby suppressing glioblastoma growth in vivo. Furthermore, G5-7 was more potent than EGFR or JAK2 inhibitors that interfere with either ligand or adenosine 5'-triphosphate (ATP) binding at impeding glioblastoma cell proliferation, demonstrating that this allosteric JAK2 inhibitor may be an effective clinical strategy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glioma/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Deleção de Genes , Glioma/enzimologia , Glioma/genética , Glioma/patologia , Xenoenxertos , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/química , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genéticaRESUMO
EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document coexpression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Alelos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Transplante de Neoplasias , Fosforilação , Transdução de SinaisRESUMO
Amplification of the gene encoding the epidermal growth factor receptor (EGFR) occurs commonly in glioblastoma (GBM), leading to activation of downstream kinases, including phosphatidylinositol 3'-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). A serine-threonine kinase, mTOR controls cell growth by regulating mRNA translation, metabolism, and autophagy; acting as both a downstream effector and upstream regulator of PI3K. These signaling functions are distributed between at least two distinct complexes, mTORC1 and mTORC2 with respect to pathway specificity. We have investigated mTOR signaling in glioma cells with the allosteric mTORC1 inhibitor rapamycin, the mTORC1/2 inhibitor Ku-0063794, a dual PI3K/mTORC1/2 kinase inhibitor PI-103, and siRNA against raptor, rictor, or mTOR, and evaluated the value of mTOR inhibitors for the treatment of glioblastoma.