RESUMEN
Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.
Asunto(s)
Adenosina Trifosfatasas/genética , Indoles/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , Proteínas Nucleares/genética , Factor Nuclear 1 de Respiración/genética , Inhibidores de Proteasoma/administración & dosificación , Pirimidinas/administración & dosificación , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas Nucleares/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Ubiquitina/genética , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Homeostasis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Sistemas CRISPR-Cas , Línea Celular Tumoral , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Humanos , Indoles/química , Indoles/farmacología , Células K562 , Ratones Desnudos , Ratones SCID , Estructura Molecular , Terapia Molecular Dirigida/métodos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ubiquitinadas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antineoplásicos/química , Indoles/química , Proteínas Nucleares/antagonistas & inhibidores , Pirimidinas/química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Xenoinjertos , Humanos , Indoles/farmacocinética , Indoles/farmacología , Ratones Desnudos , Simulación del Acoplamiento Molecular , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-Actividad , Ubiquitina/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
Dysregulation of PI3K/PTEN pathway components, resulting in hyperactivated PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, has been attributed to the absence or attenuation of downregulating signals along the PI3K/PTEN pathway. Thus, PI3K inhibitors have therapeutic potential as single agents and in combination with other therapies for a variety of cancer indications. XL147 (SAR245408) is a potent and highly selective inhibitor of class I PI3Ks (α, ß, γ, and δ). Moreover, broad kinase selectivity profiling of >130 protein kinases revealed that XL147 is highly selective for class I PI3Ks over other kinases. In cellular assays, XL147 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 in multiple tumor cell lines with diverse genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL147 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL147 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of at least 24 hours. Repeat-dose administration of XL147 results in significant tumor growth inhibition in multiple human xenograft models in nude mice. Administration of XL147 in combination with chemotherapeutic agents results in antitumor activity in xenograft models that is enhanced over that observed with the corresponding single agents.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinoxalinas/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinoxalinas/administración & dosificación , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Activation of the PI3K (phosphoinositide 3-kinase) pathway is a frequent occurrence in human tumors and is thought to promote growth, survival, and resistance to diverse therapies. Here, we report pharmacologic characterization of the pyridopyrimidinone derivative XL765 (SAR245409), a potent and highly selective pan inhibitor of class I PI3Ks (α, ß, γ, and δ) with activity against mTOR. Broad kinase selectivity profiling of >130 protein kinases revealed that XL765 is highly selective for class I PI3Ks and mTOR over other kinases. In cellular assays, XL765 inhibits the formation of PIP(3) in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 phosphorylation in multiple tumor cell lines with different genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL765 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL765 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of approximately 24 hours. Repeat dose administration of XL765 results in significant tumor growth inhibition in multiple human xenograft models in nude mice that is associated with antiproliferative, antiangiogenic, and proapoptotic effects.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/genética , Neoplasias/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Quinoxalinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinoxalinas/administración & dosificación , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sulfonamidas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: A limited number of approved therapeutic options are available to metastatic medullary thyroid cancer (MTC) patients, and the response to conventional chemotherapy and/or radiotherapy strategies is inadequate. Sporadic and inherited mutations in the tyrosine kinase RET result in oncogenic activation that is associated with the pathogenesis of MTC. Cabozantinib is a potent inhibitor of MET, RET, and vascular endothelial factor receptor 2 (VEGFR2), as well as other tyrosine kinases that have been implicated in tumor development and progression. The object of this study was to determine the in vitro biochemical and cellular inhibitory profile of cabozantinib against RET, and in vivo antitumor efficacy using a xenograft model of MTC. METHODS: Cabozantinib was evaluated in biochemical and cell-based assays that determined the potency of the compound against wild type and activating mutant forms of RET. Additionally, the pharmacodynamic modulation of RET and MET and in vivo antitumor activity of cabozantinib was examined in a MTC tumor model following subchronic oral administration. RESULTS: In biochemical assays, cabozantinib inhibited multiple forms of oncogenic RET kinase activity, including M918T and Y791F mutants. Additionally, it inhibited proliferation of TT tumor cells that harbor a C634W activating mutation of RET that is most often associated with MEN2A and familial MTC. In these same cells grown as xenograft tumors in nude mice, oral administration of cabozantinib resulted in dose-dependent tumor growth inhibition that correlated with a reduction in circulating plasma calcitonin levels. Moreover, immunohistochemical analyses of tumors revealed that cabozantinib reduced levels of phosphorylated MET and RET, and decreased tumor cellularity, proliferation, and vascularization. CONCLUSIONS: Cabozantinib is a potent inhibitor of RET and prevalent mutationally activated forms of RET known to be associated with MTC, and effectively inhibits the growth of a MTC tumor cell model in vitro and in vivo.
Asunto(s)
Anilidas/farmacología , Carcinoma Medular/tratamiento farmacológico , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Neoplasias de la Tiroides/tratamiento farmacológico , Anilidas/uso terapéutico , Animales , Carcinoma Medular/metabolismo , Carcinoma Medular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Piridinas/uso terapéutico , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Resultado del Tratamiento , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.
Asunto(s)
Diseño de Fármacos , Indoles/síntesis química , Piperidinas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Citocromo P-450 CYP3A/metabolismo , Femenino , Semivida , Humanos , Indoles/farmacocinética , Indoles/uso terapéutico , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Neoplasias/tratamiento farmacológico , Piperidinas/farmacocinética , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
The signaling pathway of the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) is important for cell growth, survival, and motility and is functionally linked to the signaling pathway of VEGF, which is widely recognized as a key effector in angiogenesis and cancer progression. Dysregulation of the MET/VEGF axis is found in a number of human malignancies and has been associated with tumorigenesis. Cabozantinib (XL184) is a small-molecule kinase inhibitor with potent activity toward MET and VEGF receptor 2 (VEGFR2), as well as a number of other receptor tyrosine kinases that have also been implicated in tumor pathobiology, including RET, KIT, AXL, and FLT3. Treatment with cabozantinib inhibited MET and VEGFR2 phosphorylation in vitro and in tumor models in vivo and led to significant reductions in cell invasion in vitro. In mouse models, cabozantinib dramatically altered tumor pathology, resulting in decreased tumor and endothelial cell proliferation coupled with increased apoptosis and dose-dependent inhibition of tumor growth in breast, lung, and glioma tumor models. Importantly, treatment with cabozantinib did not increase lung tumor burden in an experimental model of metastasis, which has been observed with inhibitors of VEGF signaling that do not target MET. Collectively, these data suggest that cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling.
Asunto(s)
Anilidas/uso terapéutico , Procesos de Crecimiento Celular/efectos de los fármacos , Metástasis de la Neoplasia/prevención & control , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Piridinas/uso terapéutico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Anilidas/farmacología , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Biológicos , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridinas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in a wide variety of human malignancies. Vascular endothelial growth factor (VEGF) receptors are expressed on the surface of vascular endothelial cells and cooperate with Met to induce tumor invasion and vascularization. EXEL-2880 (XL880, GSK1363089) is a small-molecule kinase inhibitor that targets members of the HGF and VEGF receptor tyrosine kinase families, with additional inhibitory activity toward KIT, Flt-3, platelet-derived growth factor receptor beta, and Tie-2. Binding of EXEL-2880 to Met and VEGF receptor 2 (KDR) is characterized by a very slow off-rate, consistent with X-ray crystallographic data showing that the inhibitor is deeply bound in the Met kinase active site cleft. EXEL-2880 inhibits cellular HGF-induced Met phosphorylation and VEGF-induced extracellular signal-regulated kinase phosphorylation and prevents both HGF-induced responses of tumor cells and HGF/VEGF-induced responses of endothelial cells. In addition, EXEL-2880 prevents anchorage-independent proliferation of tumor cells under both normoxic and hypoxic conditions. In vivo, these effects produce significant dose-dependent inhibition of tumor burden in an experimental model of lung metastasis. Collectively, these data indicate that EXEL-2880 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of invasion and angiogenesis mediated by HGF and VEGF receptors.
Asunto(s)
Anilidas/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Experimentales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Quinolinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Dermis/citología , Dermis/efectos de los fármacos , Dermis/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Neovascularización Patológica/prevención & control , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
PURPOSE: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non-small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. EXPERIMENTAL DESIGN: The activity of EGFR inhibitors against WT and mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and T790M mutant EGFR) xenograft tumors. RESULTS: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. CONCLUSIONS: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Azabiciclo/farmacología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/genética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Clorhidrato de Erlotinib , Femenino , Gefitinib , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Ratones SCID , Mutación , Fosforilación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chk1 and Chk2 kinases are critically involved in modulating DNA damage checkpoints. In particular, Chk1, a key activator of the S-phase DNA damage response, may be involved in resistance to genotoxic therapies that target DNA synthesis. We studied the in vitro and in vivo effects of EXEL-9844 (XL844), a potent, orally available, and specific inhibitor of Chk1 and Chk2, in combination with gemcitabine. In clonogenic assays using multiple cell lines in vitro, EXEL-9844 had only minor effects as a single agent but substantially enhanced gemcitabine-induced cell killing. Correspondingly, in PANC-1 cells, EXEL-9844 increased gemcitabine-induced H2AX phosphorylation, blocked Cdc25A phosphorylation, and induced premature mitotic entry. In a PANC-1 xenograft model, EXEL-9844 significantly enhanced gemcitabine antitumor activity but had limited effect as a single agent. Together, these data show that cell cycle checkpoint inhibitors may have significant clinical utility in potentiating the activity of gemcitabine.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Genes cdc/efectos de los fármacos , Fase S/efectos de los fármacos , Animales , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Genes cdc/fisiología , Ratones , Ratones Desnudos , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , GemcitabinaRESUMEN
We have examined the possible mechanisms of resistance to the epidermal growth factor receptor (EGFR) inhibitors in tumor cells with variable levels of EGFR. ZD1839 (Iressa) is a small-molecular-weight, ATP-mimetic that specifically inhibits the EGFR tyrosine kinase. A431 cell growth was markedly inhibited by ZD1839 (IC(50)< or =0.1 microM) whereas the MDA-468 cells were relatively resistant (IC(50)2 microM). Low doses of ZD1839 delayed cell cycle progression and induced apoptosis in A431 cells but not in MDA-468 cells. In both cell lines, 0.1 microM ZD1839 eliminated EGFR phosphorylation. However, the basal activity of the phosphatidylinositol-3 kinase (PI3 K) target Akt was eliminated in A431 but not in MDA-468 cells, implying that their Akt activity is independent of EGFR signals. A431 cells express PTEN/MMAC1/TEP, a phosphatase that can dephosphorylate position D3 of phosphatidylinositol-3,4,5 trisphosphate, the site that recruits the plecstrin-homology domain of Akt to the cell membrane. On the contrary, MDA-468 cells lack the phosphatase and tensin homolog (PTEN), potentially setting Akt activity at a high threshold that is unresponsive to EGFR inhibition alone. Therefore, we reintroduced (PTEN) by retroviral infection in MDA-468 cells. In MDA-468/PTEN but not in vector controls, treatment with ZD1839 inhibited P-Akt levels, induced relocalization of the Forkhead factor FKHRL1 to the cell nucleus, and increased FKHRL1-dependent transcriptional activity. ZD1839 induced a greater degree of apoptosis and cell cycle delay in PTEN-reconstituted than in control cells. These data suggest that loss of PTEN, by permitting a high level of Akt activity independent of receptor tyrosine kinase inputs, can temporally dissociate the inhibition of the EGFR with that of Akt induced by EGFR inhibitors. Thus, in EGFR-expressing tumor cells with concomitant amplification(s) of PI3K-Akt signaling, combined blockade of the EGFR tyrosine kinase and Akt should be considered as a therapeutic approach.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Eliminación de Gen , Monoéster Fosfórico Hidrolasas/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas , División Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Treonina/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Proteínas de Ciclo Celular/genética , Fraccionamiento Celular , Línea Celular , Núcleo Celular/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Protein tyrosine kinases are tightly regulated enzymes that play an important role in the control of most fundamental cellular processes, including cell proliferation, differentiation, metabolism, migration, and survival. These signaling proteins are the frequent target of oncogenic mutations or other genetic alterations leading to dysregulated tyrosine kinase activity, cellular transformation, and subsequent tumor progression. Many of the known dominant oncogenes encode aberrant protein tyrosine kinases and are causally associated with a significant fraction of human neoplasms, including breast carcinoma. The epidermal growth factor receptor and HER2/neu are two transmembrane tyrosine kinases that are members of the HER (erbB) signaling network. Aberrant signaling by this network is present in a cohort of breast carcinomas. Structure/function studies of these kinases have led to the identification of molecular approaches aimed at disabling signaling by this transforming network. Trastuzumab, a monoclonal antibody that binds the ectodomain of HER2, was recently shown to induce regression of HER2-overexpressing breast cancers, confirming the role of HER2 in tumor maintenance and progression. A rational therapeutic approach that builds on these results with trastuzumab and expands the targeting of the HER network will be presented.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Femenino , Gefitinib , Humanos , Quinazolinas/administración & dosificación , Receptor ErbB-2/metabolismo , TrastuzumabRESUMEN
We have examined whether inhibition of phosphatidylinositol-3 kinase (PI3K) and its target, the serine/threonine kinase Akt, play a role in the antitumor effect of the HER2 antibody Herceptin. Herceptin inhibited colony formation, down-regulated cyclin D1, and increased p27 protein levels in the HER2 gene-amplified BT-474 and SKBR-3 human breast cancer cells. These effects were temporally associated with the inhibition of PI3K activity in vitro as well as Akt function as measured by steady-state levels of phospho-Ser473 Akt and kinase activity against glycogen synthase kinase (GSK)-3beta. These responses were not observed in MDA-361 and MDA-453 cells, which do not exhibit HER2 gene amplification and are relatively resistant to Herceptin. Treatment of BT-474 cells with Herceptin inhibited the constitutive tyrosine phosphorylation of HER3 and disrupted the basal association of HER3 with HER2 and of HER3 with p85alpha potentially explaining the inhibition of PI3K. Treatment with either Herceptin or the PI3K inhibitor LY294002 increased the levels of p27 in the nucleus>cytosol, thus increasing the ratio of p27:Cdk2 in the nucleus and inhibiting Cdk2 activity and cell proliferation. Antisense p27 oligonucleotides abrogated the increase in p27 induced by Herceptin and prevented the antibody-mediated reduction in S phase. Transduction of BT-474 cells with an adenovirus-encoding active (myristoylated) Akt (Myr-Akt), but not with a beta-galactosidase control adenovirus, prevented the Herceptin- or LY294002-induced down-regulation of cyclin D1 and of phosphorylated GSK-3beta and prevented the accumulation of p27 in the nucleus and cytosol. In addition, Myr-Akt prevented Herceptin-induced inhibition of the cell proliferation of BT-474 cells and Herceptin-induced apoptosis of SKBR-3 cells. These data suggest that (a) changes in cell cycle- and apoptosis-regulatory molecules after HER2 blockade with Herceptin result, at least in part, from the inhibition of Akt; and (b) disabling PI3K and Akt is required for the antitumor effect of HER2 inhibitors.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Amplificación de Genes , Genes erbB-2 , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Trastuzumab , Células Tumorales CultivadasRESUMEN
The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this "gene-environment interaction" paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA beta-polymerase (beta-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene-environment interaction. The beta-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in beta-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N'-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in beta-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in beta-pol null cells eliminates the effect. In contrast, beta-pol/AAG double null cells are slightly more mutable than the beta-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.