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1.
J Proteome Res ; 10(7): 3200-11, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21609022

RESUMEN

Colorectal cancer (CRC) is the second leading cause of death from cancer. The MET receptor tyrosine kinase and/or its ligand HGF are frequently amplified or overexpressed in CRC. It is known that tyrosine phosphorylated proteins are involved in progression and metastasis of colorectal cancer; however, little is known about the MET phospho-proteome in CRC. High resolution mass spectrometry was used to characterize immunoaffinity-purified, phosphotyrosine (pY)-containing tryptic peptides of the MET-expressing CRC cell model, DLD1. A total of 266 unambiguously identified pY sites spanning 168 proteins were identified. Quantification of mass spectrometry ion currents identified 161 pY sites, including many not previously linked to MET signaling, that were modulated in abundance by HGF stimulation. Overlay of these data with protein-protein interaction data sets suggested that many of the identified HGF-modulated phospho-proteins may be directly or indirectly associated with MET. Analysis of pY sequence motifs indicated a prevalence of Src family kinase consensus sequences, and reciprocal signaling between Src and MET was confirmed by using selective small molecule inhibitors of these kinases. Therefore, using quantitative phospho-proteomics profiling, kinase modulation by ligand and inhibitors, and data integration, an outline of the MET signaling network was generated for the CRC model.


Asunto(s)
Neoplasias Colorrectales/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismo , Western Blotting , Línea Celular Tumoral , Cromatografía Liquida , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Expresión Génica , Perfilación de la Expresión Génica , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Inmunoprecipitación , Fosfoproteínas/genética , Fosforilación , Fosfotirosina/genética , Unión Proteica/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteoma/genética , Proteínas Proto-Oncogénicas c-met/genética , Transducción de Señal/genética , Espectrometría de Masas en Tándem , Familia-src Quinasas/genética
2.
SLAS Discov ; 26(8): 947-960, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34154424

RESUMEN

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad
4.
Nat Commun ; 11(1): 2396, 2020 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-32409666

RESUMEN

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.


Asunto(s)
Arginina/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Estrés Fisiológico , Animales , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Metilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Sf9
5.
J Cell Biochem ; 107(6): 1168-81, 2009 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-19533669

RESUMEN

Cell-matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand-independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non-neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto-activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with beta1 integrins and is co-localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src-dependent phosphorylation of Met requires cell-matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src-induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF-dependent and Src/integrin-dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells.


Asunto(s)
Transformación Celular Neoplásica , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias Mamarias Animales/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Familia-src Quinasas/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Forma de la Célula , Células Epiteliales/patología , Femenino , Integrinas , Ratones , Fosforilación , Seudópodos
6.
Nat Commun ; 10(1): 5759, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31848333

RESUMEN

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.


Asunto(s)
Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Sondas Moleculares/farmacología , Cristalografía por Rayos X , Metilación de ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/ultraestructura , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Sondas Moleculares/química , Dominios Proteicos , S-Adenosilmetionina/metabolismo
7.
Sci Rep ; 7: 39692, 2017 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-28059079

RESUMEN

Non-small-cell lung carcinoma (NSCLC) accounts for 85% of malignant lung tumors and is the leading cause of cancer deaths. Our group previously identified Tripartite Motif 14 (TRIM14) as a component of a prognostic multigene expression signature for NSCLC. Little is known about the function of TRIM14 protein in normal or disease states. We investigated the functional and prognostic role of TRIM14 in NSCLC using in vitro and in vivo perturbation model systems. Firstly, a pooled RNAi screen identified TRIM14 to effect cell proliferation/survival in NSCLC cells. Secondly, silencing of TRIM14 expression significantly enhanced tumor growth in NSCLC xenograft mouse models, while exogenous TRIM14 expression attenuated tumorigenesis. In addition, differences in apoptotic activity between TRIM14-deficient and control tumors suggests that TRIM14 tumor suppressor activity may depend on cell death signaling pathways. TRIM14-deficient cell lines showed both resistance to hypoxia-induced cell death and attenuation of interferon response via STAT1 signaling. Consistent with these phenotypes, multivariate analyses on published mRNA expression datasets of over 600 primary NSCLCs demonstrated that low TRIM14 mRNA levels are significantly associated with poorer prognosis in early stage NSCLC patients. Our functional data therefore establish a novel tumor suppressive role for TRIM14 in NSCLC progression.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Proteínas Portadoras/inmunología , Inmunidad Innata , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones SCID , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Proteínas de Motivos Tripartitos , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Nat Commun ; 8(1): 1527, 2017 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-29142305

RESUMEN

Many compounds with potentially reactive chemical motifs and poor physicochemical properties are published as selective modulators of biomolecules without sufficient validation and then propagated in the scientific literature as useful chemical probes. Several histone acetyltransferase (HAT) inhibitors with these liabilities are now routinely used to probe epigenetic pathways. We profile the most commonly used HAT inhibitors and confirm that the majority of them are nonselective interference compounds. Most (15 out of 23, 65%) of the inhibitors are flagged by ALARM NMR, an industry-developed counter-screen for promiscuous compounds. Biochemical counter-screens confirm that most of these compounds are either thiol-reactive or aggregators. Selectivity panels show many of these compounds modulate unrelated targets in vitro, while several also demonstrate nonspecific effects in cell assays. These data demonstrate the usefulness of performing counter-screens for bioassay promiscuity and assay interference, and raise caution about the utility of many widely used, but insufficiently validated, compounds employed in chemical biology.


Asunto(s)
Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Histona Acetiltransferasas/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Células HEK293 , Histona Acetiltransferasas/antagonistas & inhibidores , Humanos , Células MCF-7 , Estructura Molecular , Compuestos de Sulfhidrilo/química
9.
PLoS One ; 9(1): e86103, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24465899

RESUMEN

KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC.


Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Neoplasias Colorrectales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Proteína Activadora de GTPasa p120/metabolismo , Secuencia de Bases , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fibras de Estrés/metabolismo , Proteína Activadora de GTPasa p120/genética , Proteínas ras/genética
10.
PLoS One ; 7(10): e46677, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23056397

RESUMEN

Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote in vivo tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth in vivo, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth in vivo. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine in vitro, and in vivo when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.


Asunto(s)
Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Lipocalinas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Resistencia a Antineoplásicos/genética , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Neoplasias Pancreáticas/genética , Gemcitabina
11.
Ther Adv Med Oncol ; 3(1 Suppl): S7-S19, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22128289

RESUMEN

c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor, activates a wide range of different cellular signaling pathways, including those involved in proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. This paper provides an overview of the c-MET signaling pathway, including its role in the development of cancers, and provides a rationale for targeting the pathway as a possible treatment option.

12.
Neoplasia ; 11(12): 1292-300, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20019837

RESUMEN

Complete resection of early-stage non-small cell lung cancer (NSCLC) is potentially curative, yet approximately 50% of patients are at risk for developing metastatic recurrence. Met, the receptor for hepatocyte growth factor (HGF) is a receptor tyrosine kinase with demonstrated roles in regulating cellular proliferation, motility, morphogenesis, and apoptosis. Met receptor and its ligand, HGF, are commonly overexpressed in NSCLC, and their overexpression has been associated with poor prognosis, which could potentially involve a paracrine and/or autocrine activation loop. However, there is as yet no direct evidence that HGF-Met signaling directly promotes metastasis in NSCLC cells. Using retroviral transduction, we overexpressed the human c-met and hgf complementary DNA, alone or in combination in the NCI-H460 human large cell carcinoma cell line. The HGF/Met co-overexpressing (H460-HGF/Met) cells demonstrated enhanced tumorigenicity in xenograft SCID mice. When these cells are implanted orthotopically into the lungs of nude rats, only the H460-HGF/Met cells showed higher spontaneous metastases to distant organs including bone, brain, and kidney. These results provide evidence that autocrine overactivation of the Met- HGF loop enhances systemic metastases in NSCLC. Targeted interference of this loop may potentially be an effective adjuvant therapy to improve survival of early-stage NSCLC patients.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundario , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones SCID , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-met/genética , Ratas , Ratas Desnudas , Receptores de Factores de Crecimiento/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Trasplante Heterólogo
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