RESUMEN
NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.
Asunto(s)
Replicación del ADN , Coactivadores de Receptor Nuclear/fisiología , Origen de Réplica , Animales , Células Cultivadas , Senescencia Celular , Células HeLa , Humanos , Ratones , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.
Asunto(s)
Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Dominio Catalítico , Membrana Celular/metabolismo , Glicosilación , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Leupeptinas/farmacología , Nitrilos/farmacología , Inhibidores de Proteasoma/farmacología , Unión Proteica , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Tirosina Quinasa del Receptor AxlRESUMEN
ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC(50): 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC(50): 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC(50): 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC(50): 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.
Asunto(s)
Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/genética , Quinazolinas/farmacología , Secuencia de Aminoácidos , Células HeLa , Humanos , Riñón/citología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neoplasias/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ret/química , Proteínas Proto-Oncogénicas c-ret/metabolismo , Relación Estructura-Actividad , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
PURPOSE: Targeting of KIT and platelet-derived growth factor receptor (PDGFR) tyrosine kinases by imatinib is an effective anticancer strategy. However, mutations of the gatekeeper residue (T670 in KIT and T681 in PDGFRbeta) render the two kinases resistant to imatinib. The aim of this study was to evaluate whether sorafenib (BAY 43-9006), a multitargeted ATP-competitive inhibitor of KIT and PDGFR, was active against imatinib-resistant KIT and PDGFRbeta kinases. EXPERIMENTAL DESIGN: We used in vitro kinase assays and immunoblot with phosphospecific antibodies to determine the activity of sorafenib on KIT and PDGFRbeta kinases. We also exploited reporter luciferase assays to measure the effects of sorafenib on KIT and PDGFRbeta downstream signaling events. The activity of sorafenib on interleukin-3-independent proliferation of Ba/F3 cells expressing oncogenic KIT or its imatinib-resistant T670I mutant was also tested. RESULTS: Sorafenib efficiently inhibited gatekeeper mutants of KIT and PDGFRbeta (IC(50) for KIT T670I, 60 nmol/L; IC(50) for PDGFRbeta T681I, 110 nmol/L). Instead, it was less active against activation loop mutants of the two receptors (IC(50) for KIT D816V, 3.8 micromol/L; IC(50) for PDGFRbeta D850V, 1.17 micromol/L) that are also imatinib-resistant. Sorafenib blocked receptor autophosphorylation and signaling of KIT and PDGFRbeta gatekeeper mutants in intact cells as well as activation of AP1-responsive and cyclin D1 gene promoters, respectively. Finally, the compound inhibited KIT-dependent proliferation of Ba/F3 cells expressing the oncogenic KIT mutant carrying the T670I mutation. CONCLUSIONS: Sorafenib might be a promising anticancer agent for patients carrying KIT and PDGFRbeta gatekeeper mutations.
Asunto(s)
Bencenosulfonatos/farmacología , Mutación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/farmacología , Benzamidas , Unión Competitiva , Proliferación Celular , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Interleucina-3/metabolismo , Ratones , Niacinamida/análogos & derivados , Compuestos de Fenilurea , SorafenibRESUMEN
PURPOSE: We performed this phase III study to compare the irinotecan, leucovorin (LV), and fluorouracil (FU) regimen (FOLFIRI) versus the oxaliplatin, LV, and FU regimen (FOLFOX4) in previously untreated patients with advanced colorectal cancer. PATIENTS AND METHODS: A total of 360 chemotherapy-naive patients were randomly assigned to receive, every 2 weeks, either arm A (FOLFIRI: irinotecan 180 mg/m(2) on day 1 with LV 100 mg/m(2) administered as a 2-hour infusion before FU 400 mg/m(2) administered as an intravenous bolus injection, and FU 600 mg/m(2) as a 22-hour infusion immediately after FU bolus injection on days 1 and 2 [LV5FU2]) or arm B (FOLFOX4: oxaliplatin 85 mg/m(2) on day 1 with LV5FU2 regimen). RESULTS: One hundred sixty-four and 172 patients were assessable in arm A and B, respectively. Overall response rates (ORR) were 31% in arm A (95% CI, 24.6% to 38.3%) and 34% in arm B (95% CI, 27.2% to 41.5%; P = .60). In both arms A and B, median time to progression (TTP; 7 v 7 months, respectively), duration of response (9 v 10 months, respectively), and overall survival (OS; 14 v 15 months, respectively) were similar, without any statistically significant difference. Toxicity was mild in both groups: alopecia and gastrointestinal disturbances were the most common toxicities in arm A; thrombocytopenia and neurosensorial were the most common toxicities in arm B. Grade 3 to 4 toxicities were uncommon in both arms, and no statistical significant difference was observed. CONCLUSION: There is no difference in ORR, TTP, and OS for patients treated with the FOLFIRI or FOLFOX4 regimen. Both therapies seemed effective as first-line treatment in these patients. The difference between these two combination therapies is mainly in the toxicity profile.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Fluorouracilo/administración & dosificación , Humanos , Infusiones Intravenosas , Leucovorina/administración & dosificación , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/administración & dosificación , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
RET/papillary thyroid carcinoma (PTC) oncogenes, generated by recombination of the tyrosine kinase-encoding domain of RET with different heterologous genes, are prevalent in papillary carcinomas of the thyroid. Point mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) familial cancer syndrome and are found in sporadic medullary thyroid carcinomas. Here, we show that ZD6474, a low molecular weight tyrosine kinase inhibitor, blocks the enzymatic activity of RET-derived oncoproteins at a one-half maximal inhibitory concentration of 100 nM. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells-treated ZD6474 lost proliferative autonomy and showed morphological reversion. ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, it blocked anchorage-independent growth of RET/PTC3-transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH-RET/PTC3 cells into nude mice. Thus, targeting RET oncogenes with ZD6474 might offer a potential treatment strategy for carcinomas sustaining oncogenic activation of RET.
Asunto(s)
Proteínas de Drosophila , Inhibidores Enzimáticos/farmacología , Piperidinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Quinazolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Células 3T3 , Animales , Carcinoma Papilar/genética , Carcinoma Papilar/patología , Carcinoma Papilar/prevención & control , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/efectos de los fármacos , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/prevención & control , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Oncogenic activation of the RET receptor tyrosine kinase is common in different human cancers. We found that the pyrazolo-pyrimidine PP1 inhibited RET-derived oncoproteins with a half maximal inhibitor concentration of 80 nM. Furthermore, RET/PTC3-transformed cells treated with 5 microM of PP1 lost proliferative autonomy and showed morphological reversion. PP1 prevented the growth of two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene. These findings suggest targeting RET oncogenes with PP1 or related compounds as a novel treatment strategy for RET-associated neoplasms.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Proteínas de Drosophila , Inhibidores Enzimáticos/farmacología , Hormona Liberadora de Gonadotropina/análogos & derivados , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Células 3T3/citología , Células 3T3/efectos de los fármacos , Animales , Transformación Celular Neoplásica/metabolismo , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Oncogenes , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/genética , Células Tumorales CultivadasRESUMEN
We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC(50)< or =100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino- acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC(50): 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.
Asunto(s)
Carcinoma Medular/genética , Inhibidores Enzimáticos/farmacología , Mutación Puntual , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Dominio Catalítico , Línea Celular , Resistencia a Medicamentos , Mitógenos/farmacología , Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-ret , Pirazoles/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Transducción de Señal , Valina/genéticaRESUMEN
CONTEXT: Anaplastic thyroid carcinomas (ATC) are among the most aggressive human malignancies and are characterized by high mitotic activity. Minichromosome maintenance proteins (MCM) 2-7 are required to initiate eukaryotic DNA replication, and their overexpression has been associated with dysplasia and malignancy. OBJECTIVE: In an attempt to cast light on the mechanisms governing ATC, we evaluated MCM5 and MCM7 expression in human normal, papillary (PTC), and anaplastic thyroid samples, as well as in primary culture cells and transgenic mouse models. RESULTS: MCM5 and MCM7 expression was high in 65% of ATC and negligible in normal thyroid tissue and papillary thyroid carcinomas. In ATC, high MCM5 and MCM7 expression was paralleled by high levels of MCM2 and MCM6. An analysis of human ATC primary cell cultures and of a transgenic mouse model of ATC confirmed these findings. An increased transcription rate accounted for MCM7 up-regulation, because the activity of the MCM7 promoter was more than 10-fold higher in ATC cells compared with normal thyroid cells. Adoptive overexpression of wild-type p53, but not of its inactive (R248W and R273H) mutants, strongly down-regulated transcription from the MCM7 promoter, suggesting that p53 knock-out contributes to MCM7 up-regulation in ATC. Treatment with small inhibitory duplex RNAs, which decrease MCM7 protein levels, reduced the rate of DNA synthesis in ATC cells. CONCLUSION: MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.
Asunto(s)
Carcinoma Papilar/fisiopatología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Tiroides/fisiopatología , Animales , División Celular/fisiología , Células Cultivadas , ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Mitógenos/metabolismo , ARN Mensajero/análisis , Regulación hacia ArribaRESUMEN
Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.
Asunto(s)
Benzamidas/química , Niacinamida/análogos & derivados , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Piridinas/química , Bibliotecas de Moléculas Pequeñas/química , Animales , Benzamidas/metabolismo , Benzamidas/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Mutación , Células 3T3 NIH , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/toxicidad , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/toxicidad , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Piridinas/metabolismo , Piridinas/toxicidad , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/toxicidad , TransfecciónRESUMEN
Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.
Asunto(s)
Proteínas de Drosophila , Inhibidores Enzimáticos/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Neoplasias de la Tiroides/enzimología , Células 3T3 , Animales , Carcinoma Papilar/enzimología , Línea Celular Transformada , Activación Enzimática , Reordenamiento Génico , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Invasividad Neoplásica , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Tiroides/patología , Transfección , Células Tumorales CultivadasRESUMEN
CONTEXT: Mutations of the RET receptor tyrosine kinase are associated to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of several kinases. HSP90 is specifically inhibited by 17-allyl-amino-17-demethoxygeldanamycin (17-AAG). OBJECTIVE: Our aim was to investigate whether RET protein half-life depends on HSP90 and to dissect the molecular pathway responsible for the degradation of RET upon HSP90 inhibition by 17-AAG. DESIGN: 17-AAG effects were studied in RAT1 fibroblasts exogenously expressing MEN2-associated RET mutants and human MTC-derived cell lines. RESULTS: 17-AAG induced a 26S proteasome-dependent degradation of wild-type RET and MEN2-associated RET mutants. The compound hampered HSP90/RET interaction and stabilized RET binding to HSP70, leading to the recruitment of the HSP70-associated E3 ligase C-terminus of Hsc70-interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RET-dependent transcriptional activation of gene promoters. In human MTC cells carrying oncogenic RET mutants, HSP90 inhibition induced receptor degradation and signaling hindrance leading to cell cycle arrest. CONCLUSION: RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Benzoquinonas/farmacología , Western Blotting , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Humanos , Lactamas Macrocíclicas/farmacología , Macrólidos/farmacología , Fosforilación/genética , Proteínas Proto-Oncogénicas c-ret/genética , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND: Medullary and papillary thyroid carcinomas are often associated with oncogenic activation of the RET tyrosine kinase. We evaluated whether the biaryl urea BAY 43-9006, which is known to inhibit several other tyrosine kinases, blocks RET kinase function and oncogenic activity. METHODS: We examined BAY 43-9006 activity against oncogenic RET in vitro and in cellular RET signaling in oncogenic RET-transfected NIH3T3 fibroblasts by using immunocomplex kinase assays and immunoblotting with phospho-specific antibodies. The effects of BAY 43-9006 on proliferation of human TPC1 and TT thyroid carcinoma cells, which harbor spontaneous oncogenic RET alleles, and on RAT1 fibroblasts transformed with oncogenic RET mutants, including mutants that are resistant to other chemotherapeutic agents, were determined using growth curves and flow cytometry. Growth of TT cell-derived xenograft tumors in athymic mice treated orally with BAY 43-9006 or with vehicle was measured. All statistical tests were two-sided. RESULTS: BAY 43-9006 inhibited oncogenic RET kinase activity at half-maximal inhibitory concentrations (IC50s) of 50 nM or less in NIH3T3 cells. It also arrested the growth of NIH3T3 and RAT1 fibroblasts transformed by oncogenic RET and of thyroid carcinoma cells that harbor spontaneous oncogenic RET alleles. Moreover, BAY 43-9006 inhibited the growth of cells carrying RET V804L (IC50 = 110 nM, 95% confidence interval [CI] = 88 to 133 nM) or RET V804M (IC50 = 147 nM, 95% CI = 123 nM to 170 nM), both mutants that are resistant to anilinoquinazolines and pyrazolopyrimidines. After 3 weeks of oral treatment with BAY 43-9006 (60 mg/kg/day), the volume of TT cell xenografts (n = 7) was reduced from 72.5 to 44 mm3 (difference = 28.5 mm3, 95% CI = 7 mm3 to 50 mm3), whereas in vehicle-treated mice (n = 7), mean tumor volume increased to 408 mm3 (difference = 320 mm3, 95% CI = 180 mm3 to 460 mm3; untreated versus treated, P =.02). This inhibition paralleled a decrease in RET phosphorylation. CONCLUSIONS: BAY 43-9006 is a powerful inhibitor of the RET kinase. Its potential as a therapeutic tool for RET-positive thyroid tumors, including those expressing V804 mutations merits study.