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OBJECTIVES: Diversity of laboratory-developed tests (LDTs) using next-generation sequencing (NGS) raises concerns about their accuracy for selection of targeted therapies. A working group developed a pilot study of traceable reference samples to measure NGS LDT performance among a cohort of clinical laboratories. METHODS: Human cell lines were engineered via CRISPR/Cas9 and prepared as formalin-fixed, paraffin-embedded cell pellets ("wet" samples) to assess the entire NGS test cycle. In silico mutagenized NGS sequence files ("dry" samples) were used to assess the bioinformatics component of the NGS test cycle. Single and multinucleotide variants (n = 36) of KRAS and NRAS were tested at 5% or 15% variant allele fraction to determine eligibility for therapy with the EGFR inhibitor panitumumab in the setting of metastatic colorectal cancer. RESULTS: Twenty-one (21/21) laboratories tested wet samples; 19 of 21 analyzed dry samples. Of the laboratories that tested both the wet and dry samples, 7 (37%) of 19 laboratories correctly reported all variants, 3 (16%) of 19 had fewer than five errors, and 9 (47%) of 19 had five or more errors. Most errors were false negatives. CONCLUSIONS: Genetically engineered cell lines and mutagenized sequence files are complementary reference samples for evaluating NGS test performance among clinical laboratories using LDTs. Variable accuracy in detection of genetic variants among some LDTs may identify different patient populations for targeted therapy.
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Neoplasias del Colon , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Proyectos PilotoRESUMEN
PURPOSE: This first-in-human, open-label phase I study evaluated AMG 337, an oral, highly selective small-molecule inhibitor of MET in advanced solid tumors.Patients and Methods: Patients enrolled into dose-escalation cohorts received AMG 337 up to 400 mg once daily or up to 250 mg twice daily, following a modified 3+3+3 design. Dose expansion was conducted in MET-amplified patients at the maximum tolerated dose (MTD). Primary endpoints included assessment of adverse events (AEs), establishment of the MTD, and pharmacokinetics; clinical response was a secondary endpoint. RESULTS: The safety analysis set included 111 patients who received ≥1 dose of AMG 337. Thirteen patients had ≥1 AE qualifying as dose-limiting toxicity. The MTD was determined to be 300 mg once daily; the MTD for twice-daily dosing was not reached. Most frequent treatment-related AEs were headache (63%) and nausea (31%). Grade ≥3 treatment-related AEs occurred in 23 patients (21%), most commonly headache (n = 6) and fatigue (n = 5). Maximum plasma concentration occurred at 3.0 hours following 300-mg once-daily dosing, indicating AMG 337 absorption soon after treatment. Objective response rate was 9.9% (11/111; 95% CI, 5.1%-17.0%) in all patients and 29.6% (8/27; 95% CI, 13.8%-50.2%) in MET-amplified patients; median (range) duration of response was 202 (51-1,430+) days in all patients and 197 (64-1,430+) days in MET-amplified patients. CONCLUSIONS: Oral AMG 337 was tolerated with manageable toxicities, with an MTD and recommended phase II dose of 300 mg once daily. The promising response rate observed in patients with heavily pretreated MET-amplified tumors warrants further investigation.See related commentary by Ma, p. 2375.
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Neoplasias , Inhibidores de Proteínas Quinasas , Humanos , Dosis Máxima Tolerada , Piridonas , Resultado del Tratamiento , TriazolesRESUMEN
Treatment of myeloma has benefited from the introduction of more effective and better tolerated agents, improvements in supportive care, better understanding of disease biology, revision of diagnostic criteria, and new sensitive and specific tools for disease prognostication and management. Assessment of minimal residual disease (MRD) in response to therapy is one of these tools, as longer progression-free survival (PFS) is seen consistently among patients who have achieved MRD negativity. Current therapies lead to unprecedented frequency and depth of response, and next-generation flow and sequencing methods to measure MRD in bone marrow are in use and being developed with sensitivities in the range of 10-5 to 10-6 cells. These technologies may be combined with functional imaging to detect MRD outside of bone marrow. Moreover, immune profiling methods are being developed to better understand the immune environment in myeloma and response to immunomodulatory agents while methods for molecular profiling of myeloma cells and circulating DNA in blood are also emerging. With the continued development and standardization of these methodologies, MRD has high potential for use in gaining new drug approvals in myeloma. The FDA has outlined two pathways by which MRD could be qualified as a surrogate endpoint for clinical studies directed at obtaining accelerated approval for new myeloma drugs. Most importantly, better understanding of MRD should also contribute to better treatment monitoring. Potentially, MRD status could be used as a prognostic factor for making treatment decisions and for informing timing of therapeutic interventions. Clin Cancer Res; 23(15); 3980-93. ©2017 AACR.
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ADN Tumoral Circulante/sangre , Mieloma Múltiple/sangre , Mieloma Múltiple/tratamiento farmacológico , Neoplasia Residual/sangre , Biomarcadores de Tumor/genética , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Supervivencia sin Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mieloma Múltiple/complicaciones , Mieloma Múltiple/genética , Neoplasia Residual/inducido químicamente , Neoplasia Residual/genética , Selección de Paciente , PronósticoRESUMEN
BACKGROUND: MET gene amplification and Met protein overexpression may be associated with a poor prognosis. The MET/Met status is typically determined with fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Targeted proteomics uses mass spectrometry-based selected reaction monitoring (SRM) to accurately quantitate Met expression. FISH, IHC, and SRM analyses were compared to characterize the prognostic value of MET/Met in gastroesophageal adenocarcinoma (GEC). METHODS: Samples from 447 GEC patients were analyzed for MET gene amplification (FISH) and Met protein expression (IHC and SRM). Cox proportional hazards models and Kaplan-Meier estimates were applied to explore relations between Met, overall survival (OS), and clinical/pathological characteristics. Spearman's rank coefficient was used to assess the correlation between parameters. RESULTS: Patients with MET-amplified tumors had worse OS when: the MET/centromere enumeration probe for chromosome 7 FISH ratio was ≥ 2 (hazard ratio [HR], 3.13; 95% confidence interval [CI], 1.84-5.33), the MET gene copy number was ≥5 (HR, 2.51; 95% CI, 1.45-4.34), or ≥ 10% of the cells had ≥15 copies (HR, 4.28; 95% CI, 2.18-8.39). Similar observations were made with Met protein overexpression by IHC (≥1 + intensity in ≥ 25% of the tumor cell membrane: HR, 1.39; 95% CI, 1.04-1.86) or SRM (≥400 amol/µg: HR, 1.76; 95% CI, 1.06-2.90). A significant correlation was observed between MET FISH/Met IHC, MET FISH/Met SRM, and Met IHC/Met SRM; only MET FISH and Met SRM were independent negative prognostic biomarkers in multivariate analyses. CONCLUSIONS: MET amplification and overexpression, assessed by multiple methods, were associated with a worse prognosis in univariate analyses. However, only MET amplification by FISH and Met expression by SRM were independent prognostic biomarkers. Compared with IHC, SRM may provide an added benefit for informed decisions about Met-targeted therapy. Cancer 2017;123:1061-70. © 2016 American Cancer Society.
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Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidad , Amplificación de Genes , Expresión Génica , Proteínas Proto-Oncogénicas c-met/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidad , Biomarcadores de Tumor , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Espectrometría de Masas , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. METHOD: We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. RESULTS: Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. CONCLUSION: Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.
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Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Neoplasias/genética , Formaldehído , Humanos , Neoplasias/patología , Adhesión en Parafina , Reproducibilidad de los Resultados , Fijación del TejidoRESUMEN
BACKGROUND: This first-in-human study evaluated AMG 208, a small-molecule MET inhibitor, in patients with advanced solid tumors. METHODS: Three to nine patients were enrolled into one of seven AMG 208 dose cohorts (25, 50, 100, 150, 200, 300, and 400 mg). Patients received AMG 208 orally on days 1 and days 4-28 once daily. The primary objectives were to evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of AMG 208. RESULTS: Fifty-four patients were enrolled. Six dose-limiting toxicities were observed: grade 3 increased aspartate aminotransferase (200 mg), grade 3 thrombocytopenia (200 mg), grade 4 acute myocardial infarction (300 mg), grade 3 prolonged QT (300 mg), and two cases of grade 3 hypertension (400 mg). The MTD was not reached. The most frequent grade ≥3 treatment-related adverse event was anemia (n = 3) followed by hypertension, prolonged QT, and thrombocytopenia (two patients each). AMG 208 exposure increased linearly with dose; mean plasma half-life estimates were 21.4-68.7 hours. One complete response (prostate cancer) and three partial responses (two in prostate cancer, one in kidney cancer) were observed. CONCLUSIONS: In this study, AMG 208 had manageable toxicities and showed evidence of antitumor activity, particularly in prostate cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridazinas/farmacocinética , Piridazinas/uso terapéutico , Triazoles/farmacocinética , Triazoles/uso terapéutico , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Biomarcadores de Tumor/genética , Relación Dosis-Respuesta a Droga , Fatiga/inducido químicamente , Femenino , Humanos , Hipertensión/inducido químicamente , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Náusea/inducido químicamente , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/efectos adversos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Piridazinas/efectos adversos , Inducción de Remisión , Resultado del Tratamiento , Triazoles/efectos adversosRESUMEN
BACKGROUND: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. METHODS: In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. RESULTS: Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤ 0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. CONCLUSION: Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.
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Aurora Quinasas/antagonistas & inhibidores , Histonas/metabolismo , Especificidad de Órganos/efectos de los fármacos , Ftalazinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto , Animales , Aurora Quinasas/metabolismo , Biopsia con Aguja Fina , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Ratones Desnudos , Fosforilación/efectos de los fármacos , Ftalazinas/sangreRESUMEN
Breast cancer is the most prevalent malignancy affecting women and ranks second in cancer-related deaths, in which death occurs primarily from metastatic disease. Triple-negative breast cancer (TNBC) is a more aggressive and metastatic subtype of breast cancer that is initially responsive to treatment of microtubule-targeting agents (MTA) such as taxanes. Recently, we reported the characterization of AMG 900, an orally bioavailable, potent, and highly selective pan-Aurora kinase inhibitor that is active in multidrug-resistant cell lines. In this report, we investigate the activity of AMG 900 alone and in combination with two distinct classes of MTAs (taxanes and epothilones) in multidrug-resistant TNBC cell lines and xenografts. In TNBC cells, AMG 900 inhibited phosphorylation of histone H3 on Ser(10), a proximal substrate of Aurora-B, and induced polyploidy and apoptosis. Furthermore, AMG 900 potentiated the antiproliferative effects of paclitaxel and ixabepilone at low nanomolar concentrations. In mice, AMG 900 significantly inhibited the growth of MDA-MB-231 (F(11); parental), MDA-MB-231 (F(11)) PTX-r (paclitaxel-resistant variant), and DU4475 xenografts. The combination of AMG 900 with docetaxel enhanced tumor inhibition in MDA-MB-231 (F(11)) xenografts compared with either monotherapy. Notably, combining AMG 900 with ixabepilone resulted in regressions of MDA-MB-231 (F(11)) PTX-r xenografts, in which more than 50% of the tumors failed to regrow 75 days after the cessation of drug treatment. These findings suggest that AMG 900, alone and in combination with MTAs, may be an effective intervention strategy for the treatment of metastatic breast cancer and provide potential therapeutic options for patients with multidrug-resistant tumors.
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Antineoplásicos/farmacología , Aurora Quinasas/antagonistas & inhibidores , Metástasis de la Neoplasia/patología , Ftalazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias de la Mama Triple Negativas/patología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Aurora Quinasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Epotilonas/farmacología , Femenino , Humanos , Neoplasias Mamarias Experimentales , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/tratamiento farmacológico , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Poliploidía , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Moduladores de Tubulina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.
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Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Tienopiridinas/síntesis química , Animales , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Humanos , Janus Quinasa 1/química , Janus Quinasa 2/química , Leucocitos Mononucleares/efectos de los fármacos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Porcinos , Tienopiridinas/química , Tienopiridinas/farmacologíaRESUMEN
HSC homing, quiescence, and self-renewal depend on the bone marrow HSC niche. A large proportion of solid tumor metastases are bone metastases, known to usurp HSC homing pathways to establish footholds in the bone marrow. However, it is not clear whether tumors target the HSC niche during metastasis. Here we have shown in a mouse model of metastasis that human prostate cancer (PCa) cells directly compete with HSCs for occupancy of the mouse HSC niche. Importantly, increasing the niche size promoted metastasis, whereas decreasing the niche size compromised dissemination. Furthermore, disseminated PCa cells could be mobilized out of the niche and back into the circulation using HSC mobilization protocols. Finally, once in the niche, tumor cells reduced HSC numbers by driving their terminal differentiation. These data provide what we believe to be the first evidence that the HSC niche serves as a direct target for PCa during dissemination and plays a central role in bone metastases. Our work may lead to better understanding of the molecular events involved in bone metastases and new therapeutic avenues for an incurable disease.
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Neoplasias de la Médula Ósea/secundario , Células Madre Hematopoyéticas/patología , Neoplasias de la Próstata , Animales , Neoplasias de la Médula Ósea/patología , Trasplante de Médula Ósea , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Biológicos , Trasplante de Neoplasias , Osteoblastos/patología , Neoplasias de la Próstata/patología , Donantes de Tejidos , Trasplante HeterólogoRESUMEN
Our recent studies have shown that annexin II, expressed on the cell surface of osteoblasts, plays an important role in the adhesion of hematopoietic stem cells (HSCs) to the endosteal niche. Similarly, prostate cancer (PCa) cells express the annexin II receptor and seem to use the stem cell niche for homing to the bone marrow. The role of the niche is thought to be the induction and sustenance of HSC dormancy. If metastatic PCa cells occupy a similar or the same ecological niche as HSCs, then it is likely that the initial role of the HSC niche will be to induce dormancy in metastatic cells. In this study, we demonstrate that the binding of PCa to annexin II induces the expression of the growth arrest-specific 6 (GAS6) receptors AXL, Sky, and Mer, which, in the hematopoietic system, induce dormancy. In addition, GAS6 produced by osteoblasts prevents PCa proliferation and protects PCa from chemotherapy-induced apoptosis. Our results suggest that the activation of GAS6 receptors on PCa in the bone marrow environment may play a critical role as a molecular switch, establishing metastatic tumor cell dormancy.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Nicho de Células Madre/metabolismo , Animales , Anexina A2/metabolismo , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Ratones , Osteoblastos/metabolismo , Neoplasias de la Próstata/patología , Proteínas Tirosina Quinasas Receptoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicho de Células Madre/citología , Análisis de Matrices TisularesRESUMEN
Monocyte chemoattractant protein 1 (CCL2) is a recently identified prominent regulator of prostate cancer growth and metastasis. The purpose of this study was to investigate the mechanistic role of CCL2 in prostate cancer growth in bone. The present study found that CCL2 was up-regulated in osteoblasts (3-fold by PC-3 and 2-fold by VCaP conditioned medium) and endothelial cells (2-fold by PC-3 and VCaP conditioned medium). Parathyroid hormone-related protein (PTHrP) treatment of osteoblastic cells up-regulated CCL2 and was blocked by a PTHrP antagonist, suggesting that prostate cancer-derived PTHrP plays an important role in elevation of osteoblast-derived CCL2. CCL2 indirectly increased blood vessel formation in endothelial cells through vascular endothelial growth factor-A, which was up-regulated 2-fold with administration of CCL2 in prostate cancer cells. In vivo, anti-CCL2 treatment suppressed tumor growth in bone. The decreased tumor burden was associated with decreased bone resorption (serum TRAP5b levels were decreased by 50-60% in anti-CCL2-treated animals from VCaP or PC-3 cell osseous lesions) and microvessel density was decreased by 70% in anti-CCL2-treated animals with bone lesions from VCaP cells. These data suggest that a destructive cascade is driven by tumor cell-derived, PTHrP-mediated induction of CCL2, which facilitates tumor growth via enhanced osteoclastic and endothelial cell activity in bone marrow. Taken together, CCL2 mediates the interaction between tumor-derived factors and host-derived chemokines acting in cooperation to promote skeletal metastasis.
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Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/fisiología , Neoplasias de la Próstata/patología , Animales , Neoplasias Óseas/prevención & control , División Celular/efectos de los fármacos , Línea Celular Tumoral , Quimiocina CCL2/genética , Endotelio Vascular/patología , Humanos , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Osteoblastoma/patología , Proteína Relacionada con la Hormona Paratiroidea/antagonistas & inhibidores , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Trasplante HeterólogoRESUMEN
Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.
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Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular , Transportador de Glucosa de Tipo 1/genética , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Indoles/farmacología , Maleimidas/farmacología , Mutación , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Factores de Tiempo , Transfección , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Nearly 85% of the men who will die of prostate cancer (PCa) have skeletal metastases present. The ability of PCa cells to interact with the microenvironment determines the success of the tumor cell to form metastatic lesions. The ability to bind to human bone marrow endothelial (HBME) cells and undergo transendothelial cell migration are key steps in allowing the PCa cell to extravasate from the bone microvasculature and invade the bone stroma. We have previously demonstrated that monoctyte chemoattractant protein 1 (MCP-1; CCL2) is expressed by HBME cells and promotes PCa proliferation and migration. In the current study, we demonstrate that the CCL2 stimulation of PCa cells activates the small GTPase, Rac through the actin-associated protein PCNT1. Activation of Rac GTPase is accompanied by morphologic changes and the ability of the cells to undergo diapedesis through HBME cells. These data suggest a role for HBME-secreted CCL2 in promoting PCa cell extravasation into the bone microenvironment.
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Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/farmacología , Células Endoteliales/enzimología , Células Endoteliales/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Proteínas de Unión al GTP rac/metabolismo , Actinas/metabolismo , Neoplasias Óseas/secundario , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/genética , Transporte de Proteínas/efectos de los fármacosRESUMEN
One of the most life-threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short-term and long-term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest that annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche.
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Anexina A2/fisiología , Fenómenos Fisiológicos Celulares , Metástasis de la Neoplasia/patología , Neoplasias de la Próstata/patología , Receptores de Péptidos/fisiología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Técnicas de Cocultivo , Células Endoteliales , Humanos , Masculino , OsteoblastosRESUMEN
We developed a sensitive real-time polymerase chain reaction (QPCR) assay that allows us to track early lodging/homing events in vivo. We used this technology to develop a metastasis assay of human prostate cancer (PCa) growth in severe combined immunodeficient mice. For this purpose, marked human PCa cell lines were implanted subcutaneously or in the prostate (orthotopically) of severe combined immunodeficient mice as models of primary tumors. Mice were then sacrificed at various time points, and distant tissues were investigated for the presence of metastatic cells. At 3 weeks, a number of tissues were recovered and evaluated by QPCR for the presence of metastatic cells. The data demonstrate that several PCa cell lines are able to spread from the primary lesion and take up residence in distant sites. If the primary tumors were resected at 3 weeks, in several cases, metastatic lesions were identified over the course of 9 months. We propose that this new model may be particularly useful in exploring the molecular events in early metastasis, identifying the metastatic niche, and studying issues pertaining to dormancy.
Asunto(s)
Neoplasias Óseas/secundario , Modelos Animales de Enfermedad , Neoplasias de la Próstata/patología , Animales , Neoplasias Óseas/genética , ADN/genética , ADN/metabolismo , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Células Neoplásicas Circulantes/patología , Hormona Paratiroidea/metabolismo , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Several reports have recently documented that CXCR7/RDC1 functions as a chemokine receptor for SDF-1/CXCL12, which regulates a spectrum of normal and pathological processes. In this study, the role of CXCR7/RDC1 in prostate cancer (PCa) was explored. Staining of high density tissue microarrays demonstrates that the levels of CXCR7/RDC1 expression increase as the tumors become more aggressive. In vitro and in vivo studies with PCa cell lines suggest that alterations in CXCR7/RDC1 expression are associated with enhanced adhesive and invasive activities in addition to a survival advantage. In addition, it was observed that CXCR7/RDC1 levels are regulated by CXCR4. Among the potential downstream targets of CXCR7/RDC1 are CD44 and cadherin-11, which are likely to contribute to the invasiveness of PCa cells. CXCR7/RDC1 also regulates the expression of the proangiogenic factors interleukin-8 or vascular endothelial growth factor, which are likely to participate in the regulation of tumor angiogenesis. Finally, we found that signaling by CXCR7/RDC1 activates AKT pathways. Together, these data demonstrate a role for CXCR7/RDC1 in PCa metastasis and progression and suggest potential targets for therapeutic intervention.
Asunto(s)
Quimiocina CXCL12/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores CXCR/fisiología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Masculino , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Unión ProteicaRESUMEN
Tumor-associated macrophages (TAMs) have been implicated in promoting tumor growth and development. Here we present evidence that demonstrates that co-inoculation of male athymic nude mice with PC-3 prostate cancer cells and U937 promonocytic cells enhances tumor growth and increases tumor angiogenesis. Male athymic nude mice were co-inoculated with PC-3 and U937 cells (control or IL-4 stimulated) and tumor growth was monitored over time. Immunohistochemical analysis of tumor specimens was performed for proliferation markers (e.g., Ki67) and the effects of IL-4 stimulation on U937 cells were analyzed for chemokine expression. The presence of U937 cells increased the rate of tumor growth in vivo and stimulated increased microvascular density within the tumor bed. Stimulation of U937 cells with IL-4 resulted in a significant increase in several pro-angiogenic and pro-tumor chemokines (e.g., CCL2). Co-inoculation increases prostate cancer growth via upregulation of chemokines that induce angiogenesis within the tumor.
Asunto(s)
Neovascularización Patológica , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Animales , Línea Celular Tumoral , Quimiocinas/metabolismo , Técnicas de Cocultivo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-4/farmacología , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Upregulation of Bcl-2 family members is a well-established mechanism in the development of androgen-independent prostate cancer. Inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1 may delay the transition to androgen-independent growth. EXPERIMENTAL DESIGN: We have established a prostate cancer model with VCaP prostate cancer cells in vivo to study the transition to androgen independence. Here, we investigated the efficacy of AT-101 (R-(-)-gossypol acetic acid; a pan small molecule inhibitor of Bcl-2, Bcl-x(L), and Mcl-1) in combination with surgical castration to delay the onset of androgen-independent growth in vivo. RESULTS: AT-101 (15 mg/kg, per os (p.o.) 5 days/week) in combination with surgical castration delayed the onset of androgen-independent prostate cancer growth in vivo. In addition, we demonstrate the induction of caspase-9-and caspase-3-dependent induction of apoptosis following AT-101 treatment in vitro which was accompanied by an AT-101-induced downregulation of Bcl-2 and Mcl-1 mRNA and protein expression. CONCLUSIONS: We conclude that AT-101 in combination with surgical castration delays the onset of androgen-independent prostate cancer in vivo by disrupting the antiapoptotic activity of Bcl-2 upregulation during the transition to androgen independence. Further studies are needed to define the mechanism of action by which AT-101 attenuates the expression of Bcl-2 and Mcl-1 and to characterize the potential for AT-101 in combination with hormone therapy.