RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.
Asunto(s)
Carcinoma Ductal Pancreático , Inmunoconjugados , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Microambiente Tumoral , Antígenos CD , Moléculas de Adhesión Celular , Proteínas Ligadas a GPIRESUMEN
Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.
Asunto(s)
Antimitóticos , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Tirosina Quinasa del Receptor Axl , Antimitóticos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Resistencia a Antineoplásicos , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite their outstanding antitumour activity in mice, the limited supply from the natural sources has prevented drug discovery/development based on intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) on a >10 g scale with >99.8% purity under GMP conditions. Interestingly, E7130 not only is a novel microtubule dynamics inhibitor but can also increase intratumoural CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. According to these unique effects, E7130 significantly augment the effect of antitumour treatments in mouse models and is currently in a clinical trial. Overall, our work demonstrates that a total synthesis can address the issue of limited material supply in drug discovery/development even for the cases of complex natural products.
Asunto(s)
Antineoplásicos Fitogénicos/síntesis química , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma de Células Escamosas/tratamiento farmacológico , Éteres Cíclicos/síntesis química , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Macrólidos/síntesis química , Moduladores de Tubulina/síntesis química , Actinas/genética , Actinas/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Productos Biológicos/síntesis química , Productos Biológicos/farmacología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Cetuximab/farmacología , Descubrimiento de Drogas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Éteres Cíclicos/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Macrólidos/farmacología , Ratones , Ratones Endogámicos BALB C , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Análisis de Supervivencia , Moduladores de Tubulina/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most of preclinical cancer studies use xenograft models established from human cell lines or patient-derived cancer cells subcutaneously implanted into the flank of immunocompromised mice. These models are often assumed to represent the original diseases and are valuable tools, at least to some extent, for understanding both the basic biology of cancer and for proof-of-concept studies of molecularly targeted therapies. However, analyzing the cellular behavior of individual components within xenografts, including tumor cells, stromal cells, immune cells, and blood vessels, is challenging. In particular, it has been difficult and urgently required to trace the whole process of heterogeneous tumor microenvironment formation mediated by various components described above. Here we demonstrate a method for monitoring this process using a window device system that we have recently developed and a subcutaneous xenograft model that accurately recapitulates the histology of human lung adenocarcinoma. Use of our imaging window device and a multiphoton laser scanning microscope provides a powerful tool for investigating tumor heterogeneity and responses to drug treatments in an in vivo live imaging system.
Asunto(s)
Adenocarcinoma/patología , Neoplasias Pulmonares/patología , Imagen Molecular/instrumentación , Animales , Femenino , Humanos , Inyecciones Subcutáneas , Ratones , Ratones Desnudos , Ratones SCID , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immune surveillance is a critical component of the antitumor response in vivo, yet the specific components of the immune system involved in this regulatory response remain unclear. In this study, we demonstrate that autoantibodies can mitigate tumor growth in vitro and in vivo We generated two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from monkey-induced pluripotent stem cells (iPSC) carrying a homozygous haplotype of major histocompatibility complex (MHC, Mafa in Macaca fascicularis). To establish a monkey cancer model, we transplanted these cells into monkeys carrying the matched Mafa haplotype in one of the chromosomes. Neither Mafa-homozygous cancer cell line grew in monkeys carrying the matched Mafa haplotype heterozygously. We detected in the plasma of these monkeys an IgG autoantibody against GRP94, a heat shock protein. Injection of the plasma prevented growth of the tumor cells in immunodeficient mice, whereas plasma IgG depleted of GRP94 IgG exhibited reduced killing activity against cancer cells in vitro These results indicate that humoral immunity, including autoantibodies against GRP94, plays a role in cancer immune surveillance. Cancer Res; 77(21); 6001-10. ©2017 AACR.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Células Madre Pluripotentes Inducidas/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Proteínas de la Membrana/inmunología , Neoplasias/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/metabolismo , Carcinoma Embrionario/genética , Carcinoma Embrionario/inmunología , Carcinoma Embrionario/patología , Línea Celular Tumoral , Células Cultivadas , Femenino , Glioblastoma/genética , Glioblastoma/inmunología , Glioblastoma/patología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Haplotipos , Homocigoto , Células Madre Pluripotentes Inducidas/metabolismo , Macaca fascicularis , Complejo Mayor de Histocompatibilidad/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Trasplante de Neoplasias/métodos , Neoplasias/genética , Neoplasias/patologíaRESUMEN
We previously reported that eribulin mesylate (eribulin), a tubulin-binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin-induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post-erlotinib treatment H1650 (PE-H1650) xenograft model. Furthermore, infiltrating CD11b-positive immune cells were significantly increased in multiple eribulin-treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.
Asunto(s)
Furanos/administración & dosificación , Cetonas/administración & dosificación , Neoplasias/tratamiento farmacológico , Microambiente Tumoral/efectos de los fármacos , Remodelación Vascular/efectos de los fármacos , Animales , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Femenino , Células HCT116 , Humanos , Ratones , Neoplasias/patología , Polietilenglicoles/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells (CSCs), drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance. Although there has been substantial progress in CSC research into many solid tumour types, an understanding of the biology of CSCs in lung cancer remains elusive, mainly because of their heterogeneous origins and high plasticity. Here, we demonstrate that engineered lung cancer cells derived from normal human airway basal epithelial cells possessed CSC-like characteristics in terms of multilineage differentiation potential and strong tumour-initiating ability. Moreover, we established an in vitro 3D culture system that allowed the in vivo differentiation process of the CSC-like cells to be recapitulated. This engineered CSC model provides valuable opportunities for studying the biology of CSCs and for exploring and evaluating novel therapeutic approaches and targets in lung CSCs.
Asunto(s)
Ingeniería Celular/métodos , Pulmón/citología , Células Madre Neoplásicas/fisiología , Mucosa Respiratoria/citología , Animales , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Femenino , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Extremely low infectivity has hampered direct (cell-free) infection studies of human T-cell leukemia virus type I (HTLV-I). In order to break through this barrier, we examined the susceptibility of many kinds of cells to HTLV-I and found a feline kidney cell line, 8C, that is highly susceptible to HTLV-I and produced remarkable amounts of infectious progeny viruses. Tax1 protein encoded by HTLV-I is known as a transcription activator for viral and cellular genes. We found that the 8C cells expressing the Tax1 protein (8C/TaxWT cells) can produce more progeny viruses than 8C cells when the cells were exposed to cell-free HTLV-I. A large number of syncytia were also induced in these cells. Here, we propose 8C/TaxWT cells as a useful tool to study the cell-free HTLV-I infection.
Asunto(s)
Expresión Génica , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Replicación Viral , Animales , Gatos , Línea Celular , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Virología/métodos , Cultivo de Virus/métodosRESUMEN
Oncogenic stimuli such as H-Ras induce oncogene-induced senescence (OIS) in fibroblasts to protect against transformation. Here we found that a population of the human diploid fibroblasts can escape from OIS induced by H-RasV12. We designated these OIS-escaped cells as OISEC (OIS-escaped cells). OISEC lost the expression of p16 which plays an important role for cell cycle arrest for induction of senescence, but OISEC preserved the p16 expression machinery and exhibited senescence by the treatment with hydrogen peroxide (H(2)O(2)) as stress-induced premature senescence (SIPS). OISEC did not possess anchorage-independent growth potential, and functional disruption of p53 and Rb by SV40 early region encoding large T and small t antigens, induced the aneuploidy phenotype and colony-forming potential of OISEC together with the exhibition of in vivo tumor formation. Finally, we also found that the distinctive feature of OISEC is expression of transcription factors, Oct3/4, SOX2, and Nanog which is closely related to stem-like cell features. This study highlights the presence of a cell population which escaped from OIS, and this OISEC may transform into malignant cancer cells by the additional hits of several genes in vivo.
Asunto(s)
Transformación Celular Neoplásica/patología , Senescencia Celular , Fibroblastos/patología , Genes ras , Animales , Transformación Celular Neoplásica/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Diploidia , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Estrés Fisiológico/genéticaRESUMEN
It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets.
Asunto(s)
Transformación Celular Viral/fisiología , Retroviridae/fisiología , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/virología , Adenocarcinoma del Pulmón , Adulto , Animales , Diferenciación Celular/fisiología , Transformación Celular Viral/genética , Quinasa 4 Dependiente de la Ciclina/biosíntesis , Quinasa 4 Dependiente de la Ciclina/genética , Transición Epitelial-Mesenquimal , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/virología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fenotipo , Retroviridae/genética , Telomerasa/biosíntesis , Telomerasa/genética , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Adulto JovenRESUMEN
The small GTPase Rho regulates cell morphogenesis through remodeling of the actin cytoskeleton. While Rho is overexpressed in many clinical cancers, the role of Rho signaling in oncogenesis remains unknown. mDia1 is a Rho effector producing straight actin filaments. Here we transduced mouse embryonic fibroblasts from mDia1-deficient mice with temperature-sensitive v-Src and examined the involvement and mechanism of the Rho-mDia1 pathway in Src-induced oncogenesis. We showed that in v-Src-transduced mDia1-deficient cells, formation of actin filaments is suppressed, and v-Src in the perinuclear region does not move to focal adhesions upon a temperature shift. Consequently, membrane translocation of v-Src, v-Src-induced morphological transformation, and podosome formation are all suppressed in mDia1-deficient cells with impaired tyrosine phosphorylation. mDia1-deficient cells show reduced transformation in vitro as examined by focus formation and colony formation in soft agar and exhibit suppressed tumorigenesis and invasion when implanted in nude mice in vivo. Given overexpression of c-Src in various cancers, these findings suggest that Rho-mDia1 signaling facilitates malignant transformation and invasion by manipulating the actin cytoskeleton and targeting Src to the cell periphery.
Asunto(s)
Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica , Neoplasias Experimentales/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Animales , Proteínas Portadoras/genética , Proliferación Celular , Células Cultivadas , Citoesqueleto/metabolismo , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Forminas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Microscopía Fluorescente , Microscopía por Video , Invasividad Neoplásica , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteína Oncogénica pp60(v-src)/genética , TransfecciónRESUMEN
LL5beta has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)-bound microtubule plus ends to the cell cortex. In this study, we show that LL5beta and its homologue LL5alpha (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins alpha3beta1 and alpha6beta4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin alpha3 at the basal cell cortex. Activation of integrin alpha3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin-integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Laminina/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Proteínas Portadoras/genética , Adhesión Celular , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Femenino , Humanos , Integrina alfa3/genética , Integrina alfa3/metabolismo , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Laminina/genética , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Proteínas del Tejido Nervioso/genética , Unión Proteica/fisiología , ARN Interferente Pequeño/genética , Receptores de Laminina/genética , Receptores de Laminina/metabolismoRESUMEN
MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localized with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.
Asunto(s)
Citoplasma/enzimología , Fosfatasas de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Activación Transcripcional , Transporte Activo de Núcleo Celular , Animales , Células COS , Núcleo Celular/enzimología , Chlorocebus aethiops , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/farmacología , Humanos , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387-MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v-Src-transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H-RasV12-transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony-forming abilities of v-Src-transformed NIH3T3 cells. Expression of a phosphorylation-mimic mutant MgcRacGAP-S387D enhanced colony formation of v-Src-transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387-MgcRacGAP in v-Src-transformed NIH3T3 cells, suggesting the v-Src-induced pathological positive feedback mechanisms towards Rac1 activation using pS387-MgcRacGAP. These results indicated the difference in the mechanisms between v-Src- and H-RasV12-induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v-Src-induced cell transformation.
Asunto(s)
Transformación Celular Neoplásica , Proteínas Activadoras de GTPasa/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aminoquinolinas/farmacología , Animales , Aurora Quinasa B , Aurora Quinasas , Western Blotting , Ensayo de Unidades Formadoras de Colonias , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Ratones , Células 3T3 NIH , Proteína Oncogénica pp60(v-src)/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Pirimidinas/farmacología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Astrocitos/enzimología , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Indoles/farmacología , Luciferasas de Luciérnaga/genética , Luciferasas de Renilla/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
BACKGROUND: A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways. RESULTS: We established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents. CONCLUSION: Normal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.
Asunto(s)
Astrocitos/enzimología , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioma/tratamiento farmacológico , Glioma/enzimología , Proteínas Supresoras de Tumor/genética , Células 3T3 , Animales , Antineoplásicos/uso terapéutico , Astrocitos/citología , Astrocitos/efectos de los fármacos , División Celular , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Humanos , Ratones , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temozolomida , Transfección , Trasplante Heterólogo , Proteínas Supresoras de Tumor/metabolismo , Ácido Valproico/farmacologíaRESUMEN
The Src family of tyrosine kinases play pivotal roles in regulating cellular functions characteristic of multicellular animals, including cell-cell interactions, cell-substrate adhesion, and cell migration. To investigate the functional alteration of Src kinases during evolution from a unicellular ancestor to multicellular animals, we characterized Src orthologs from the unicellular choanoflagellate Monosiga ovata and the primitive multicellular sponge Ephydatia fluviatilis. Here, we show that the src gene family and its C-terminal Src kinase (Csk)-mediated regulatory system already were established in the unicellular M. ovata and that unicellular Src has unique features relative to multicellular Src: It can be phosphorylated by Csk at the negative regulatory site but still exhibits substantial activity even in the phosphorylated form. Analyses of chimera molecules between M. ovata and E. fluviatilis Src orthologs reveal that structural alterations in the kinase domain are responsible for the unstable negative regulation of M. ovata Src. When expressed in vertebrate fibroblasts, M. ovata Src can induce cell transformation irrespective of the presence of Csk. These findings suggest that a structure of Src required for the stable Csk-mediated negative regulation still is immature in the unicellular M. ovata and that the development of stable negative regulation of Src may correlate with the evolution of multicellularity in animals.
Asunto(s)
Evolución Molecular , Familia-src Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Adhesión Celular , Clonación Molecular , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosforilación , Poríferos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Saccharomyces cerevisiae , Familia-src Quinasas/genéticaRESUMEN
Human diploid fibroblasts (HDF) immortalized by hTERT and simian virus 40 (SV40) early region (ER) exhibit a limited degree of transformation upon the expression of activated H-RAS (H-RAS V12) compared with rat embryonic fibroblasts (REF) immortalized by SV40 ER. Here, we identified FRA1 as a determinant for this difference in RAS-induced transformation. FRA1 was not induced by H-RAS V12 in the immortalized HDF, in contrast to its marked accumulation in the immortalized REF. Ectopic expression of FRA1 significantly enhanced anchorage-independent growth of various HDF expressing hTERT, SV40 ER, and H-RAS V12. More importantly, FRA1 could induce anchorage-independent growth as well as nude mice tumor formation of the immortalized HDF in the absence of H-RAS V12. The results of an in vitro kinase assay clearly showed that the RAS-induced extracellular signal-regulated kinase (ERK) activation, which is responsible for FRA1 induction, was markedly attenuated in the HDF compared with that in the REF, despite no obvious differences in the phosphorylation status of ERK between the species. Our results strongly suggest that HDF negatively regulate the mitogen-activated protein kinase kinase (MEK)/ERK pathway more efficiently than REF, and consequently express less malignant phenotypes in response to H-RAS V12.
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Genes ras , Proteínas Proto-Oncogénicas c-fos/fisiología , Animales , Western Blotting , Transformación Celular Neoplásica , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
Oncogenic protein provokes cell cycle arrest termed premature senescence. In this process Ras has been known to induce cyclin-dependent kinase inhibitor (CKI) p16(INK4A) in primary fibroblasts. Here, we present a novel finding that human chimeric oncoprotein SYT-SSX1 induces CKI p21(WAF1/CIP1) (p21) for suppression of cell growth. In human synovial sarcoma cell lines, the expression levels of p21 were high and the transcriptional activity of the p21 gene promoter was significantly elevated. The transient expression of SYT-SSX1-induced activation of the p21 gene promoter in human diploid fibroblasts. The N-terminus deletion form of SYT-SSX1, which failed to bind to hBRM one of the chromatin remodeling factors, preserved the p21 induction ability. This effect of SYT-SSX1 was similar in extent in both wild-type and p53-deficient HCT116 cell lines. Furthermore, the introduction of mutation in Sp1/Sp3 binding sites of the p21 gene promoter abolished the SYT-SSX1-induced transcriptional activity of its promoter. In SW13 cells, the stable expression of SYT-SSX1 suppressed cell growth in culture. These results suggest that SYT-SSX1 is able to induce p21 in a manner independent on hBRM and p53 but dependent on Sp1/Sp3.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Sarcoma Sinovial/genética , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Immunoblotting , Luciferasas/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Sarcoma Sinovial/patologíaRESUMEN
Ras-induced transformation is characterized not only by uncontrolled proliferation but also by drastic morphological changes accompanied by the disruption of the actin cytoskeleton. Previously, we reported that human fibroblasts are more resistant than rodent fibroblasts to Ras-induced transformation. To explore the molecular basis for the difference in susceptibility to Ras-induced transformation, we investigated the effect of activated H-Ras on the actin cytoskeleton in human diploid fibroblasts and in rat embryo fibroblasts, both of which are immortalized by SV40 early region. We demonstrate here that Ras-induced morphological changes, decreased expression of tropomyosin isoforms, and suppression of the ROCK/LIMK/Cofilin pathway observed in the rat fibroblasts were not detected in the human fibroblasts even with high expression levels of Ras. We also show that activation of the MEK/ERK pathway sufficed to induce all of these alterations in the rat fibroblasts, whereas the human fibroblasts were refractory to these MEK/ERK-mediated changes. In addition to morphological changes, we demonstrated that the expression of activated Ras induced an invasive phenotype in the rat, but not in the human fibroblasts. These studies provide evidence for the existence of human-specific mechanisms that resist Ras/MEK/ERK-mediated transformation.