RESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , MicroARNs/genética , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Quimiocina CXCL12/genética , Humanos , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Receptores CXCR/genética , Receptores CXCR4/genéticaRESUMEN
Analogues of hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signal through the nuclear vitamin D receptor (VDR). They have potential in combination therapies with other anticancer agents such as histone deacetylase inhibitors (HDACi's). Here, we characterize the ZG series of hybrid compounds that combine HDACi within the backbone of a VDR agonist. All display improved solubility, with ZG-126 being the most robustly bifunctional molecule in multiple cell lines. ZG-126 is well tolerated and strongly induces VDR target gene expression in vivo at therapeutic doses. Its antitumor efficacy is superior to 1,25D and the HDACi SAHA, separately or together, in mouse models of melanoma and triple-negative breast cancer (TNBC). Notably, ZG-126 treatment reduces metastases almost 4-fold in an aggressive TNBC model. ZG-126 also reduces total macrophage infiltration and the proportion of immunosuppressive M2-polarized macrophages in TNBC tumors by 2-fold. ZG-126 thus represents a bifunctional and efficacious anticancer agent with improved physicochemical properties.
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Antineoplásicos , Inhibidores de Histona Desacetilasas , Receptores de Calcitriol , Animales , Femenino , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/uso terapéutico , Ratones Endogámicos C57BL , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Relación Estructura-Actividad , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Eudistomin Y class compounds are a series of ß-carbolines which was originally isolated from a marine turnicate or ascidian near the South Korea Sea. These compounds contain bromo-substituted groups, which is one of the typical characters of marine natural products. We report herein the chemical synthesis and biological evaluation of seven new ß-carboline-based metabolites, Eudistomins Y1-Y7, and their hydroxyl-methylated phenyl derivatives. Using bromo-substituted tryptamines and bromo-substituted phenylglyoxals as the key intermediates, Eudistomins Y1-Y7 and their derivatives were synthesized via the acid-catalyzed Pictet-Spengler reaction and fully characterized by 1H- and 13C-NMR and mass spectroscopy. Biological studies revealed that all of the compounds showed moderate growth inhibitory activity against breast carcinoma cell line MDA-231 with IC50 of 15-63 µM and the inhibitory activities of hydroxyl-methylated phenyl products were higher than that of the corresponding natural products Eudistomins Y1-Y7.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carbolinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/patología , Carbolinas/síntesis química , Carbolinas/química , Línea Celular Tumoral , Femenino , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
Overexpression of the pro-survival protein heme oxygenase-1 (HO-1) and loss of the pro-apoptotic tumour suppressor PTEN are common events in prostate cancer (PCA). We assessed the occurrence of both HO-1 expression and PTEN deletion in two cohorts of men with localized and castration-resistant prostate cancer (CRPC). The phenotypic cooperation of these markers was examined in preclinical and clinical models. Overall, there was a statistically significant difference in HO-1 epithelial expression between benign, high-grade prostatic intraepithelial neoplasia (HGPIN), localized PCA, and CRPC (p < 0.0001). The highest epithelial HO-1 expression was noted in CRPC (2.00 ± 0.89), followed by benign prostate tissue (1.49 ± 1.03) (p = 0.0003), localized PCA (1.20 ± 0.95), and HGPIN (1.07 ± 0.87) (p < 0.0001). However, the difference between HGPIN and PCA was not statistically significant (p = 0.21). PTEN deletions were observed in 35/55 (63.6%) versus 68/183 (37.1%) cases of CRPC and localized PCA, respectively. Although neither HO-1 overexpression nor PTEN deletions alone in localized PCA showed a statistically significant association with PSA relapse, the combined status of both markers correlated with disease progression (log-rank test, p = 0.01). In a preclinical model, inhibition of HO-1 by shRNA in PTEN-deficient PC3M cell line and their matched cells where PTEN is restored strongly reduced cell growth and invasion in vitro and inhibited tumour growth and lung metastasis formation in mice compared to cells where only HO-1 is inhibited or PTEN is restored. In summary, we provide clinical and experimental evidence for cooperation between epithelial HO-1 expression and PTEN deletions in relation to the PCA patient's outcome. These findings could potentially lead to the discovery of novel therapeutic modalities for advanced PCA.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Hemo-Oxigenasa 1/metabolismo , Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proliferación Celular , Transformación Celular Neoplásica , Progresión de la Enfermedad , Eliminación de Gen , Hemo-Oxigenasa 1/antagonistas & inhibidores , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Orquiectomía , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Pronóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Análisis por Matrices de Proteínas/métodos , Células Tumorales CultivadasRESUMEN
Stemness and chromosomal instability (CIN) are two common contributors to intratumor heterogeneity and therapy relapse in advanced cancer, but their interplays are poorly defined. Here, in anaplastic thyroid cancer (ATC), we show that ALDH+ stem-like cancer cells possess increased CIN-tolerance owing to transcriptional upregulation of the scaffolding protein NEDD9. Thyroid patient tissues and transcriptomic data reveals NEDD9/ALDH1A3 to be co-expressed and co-upregulated in ATC. Compared to bulk ALDH- cells, ALDH+ cells were highly efficient at propagating CIN due to their intrinsic tolerance of both centrosome amplification and micronuclei. ALDH+ cells mitigated the fitness-impairing effects of centrosome amplification by partially inactivating supernumerary centrosomes. Meanwhile, ALDH+ cells also mitigated cell death caused by micronuclei-mediated type 1 interferon secretion by suppressing the expression of the DNA-sensor protein STING. Both mechanisms of CIN-tolerance were lost upon RNAi-mediated NEDD9 silencing. Both in vitro and in vivo, NEDD9-depletion attenuated stemness, CIN, cell/tumor growth, while enhancing paclitaxel effectiveness. Collectively, these findings reveal that ATC progression can involve an ALDH1A3/NEDD9-regulated program linking their stemness to CIN-tolerance that could be leveraged for ATC treatment.
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Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Centrosoma/metabolismo , Inestabilidad Cromosómica/genética , ADN/metabolismo , Humanos , Recurrencia Local de Neoplasia/patología , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC.
Asunto(s)
Carcinoma Papilar , Neoplasias de la Tiroides , Humanos , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/metabolismo , Carcinoma Papilar/genética , Carcinoma Papilar/patología , Mutación/genética , Recurrencia Local de Neoplasia , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
Aurora kinases and protein kinase C (PKC) have been shown to be involved in different aspects of cancer progression. To date, no dual Aurora/PKC inhibitor with clinical efficacy and low toxicity is available. Here, we report the identification of compound 2e as a potent small molecule capable of selectively inhibiting Aurora A kinase and PKC isoforms α, ß1, ß2 and θ. Compound 2e demonstrated significant inhibition of the colony forming ability of metastatic breast cancer cells in vitro and metastasis development in vivo. In vitro kinase screening and molecular modeling studies revealed the critical role of the selenium-containing side chains within 2e, where selenium atoms were shown to significantly improve its selectivity and potency by forming additional interactions and modulating the protein dynamics. In comparison to other H-bonding heteroatoms such as sulfur, our studies suggested that these selenium atoms also confer more favorable PK properties.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Compuestos de Selenio/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Enlace de Hidrógeno , Isoenzimas , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Especificidad por Sustrato , Ensayo de Tumor de Célula MadreRESUMEN
Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in specific podocyte proteins. This study addresses mediation of GEC injury, focusing on endoplasmic reticulum (ER) stress. We studied signaling in cultured GECs in the presence or absence of the extracellular matrix (ECM). Adhesion to collagen supports cell survival, but adhesion to plastic (loss of contact with ECM) leads to apoptosis. Compared with collagen-adherent cells, GECs on plastic showed increased protein misfolding in the ER, and an adaptive-protective ER stress response, including increased expression of ER chaperones, increased phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), and a reduction in protein synthesis. Activation of these ER stress pathways counteracted apoptosis. However, tunicamycin (a potent stimulator of ER stress) changed the ER stress response from protective to cytotoxic, as tunicamycin induced the proapoptotic ER stress gene, C/EBP homologous protein-10, and exacerbated apoptosis in GECs adherent to plastic, but not collagen. In GECs adherent to plastic, adaptive ER stress was associated with an increase in polyubiquitinated proteins and "choking" of the proteasome. Furthermore, pharmacological inhibition of the proteasome induced ER stress in GECs. Finally, we show that ER stress (induction of ER chaperones and eIF2α phosphorylation) was evident in experimental FSGS in vivo. Thus interactions of GECs with ECM may regulate protein folding and induction of the ER stress response. FSGS is associated with induction of ER stress. Enhancing protective aspects of the ER stress response may reduce apoptosis and possibly glomerulosclerosis.
Asunto(s)
Apoptosis/fisiología , Retículo Endoplásmico/fisiología , Células Epiteliales/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Glomérulos Renales/patología , Estrés Fisiológico/fisiología , Animales , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/fisiología , Matriz Extracelular/fisiología , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomérulos Renales/fisiopatología , Masculino , Ratones , Ratones Endogámicos , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Tunicamicina/efectos adversos , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.
RESUMEN
Head and neck squamous cell carcinoma (HNSCC), a prevalent cancer worldwide, has a high incidence of loco-regional dissemination, frequent recurrence, and lower 5-year survival rates. Current gold standard treatments for advanced HNSCC rely primarily on radiotherapy and chemotherapy but with limited efficacy and significant side effects. In this study, we characterized a novel 5-fluorouracil (5-FU) carrier composed of chitosan solution (CS) and polycaprolactone (PCL) microparticles (MPs) in HNSCC preclinical models. The designed MPs were evaluated for their size, morphology, drug entrapment efficiency (EE%) and in vitro drug release profile. The anti-cancer activity of 5-FU-loaded particles was assessed in HNSCC human cell lines (CAL27 and HSC3) and in a preclinical mouse model (AT84) utilizing cell proliferation and survival, cell motility, and autophagy endpoints. The results demonstrated a 38.57 % in 5-FU entrapment efficiency associated with reduced 5-FU in vitro release up to 96 h post-exposure. Furthermore, CS-decorated PCL MPs were able to promote a significant inhibition of cancer cell proliferation based on the metabolic and colony formation assays, in comparison to controls. In contrast, CS-decorated PCL MPs did not influence the pharmacological efficacy of 5-FU to inhibit in vitro cancer cell migration. Last, cell protein analysis revealed a significant increase of autophagy and cell death evaluated by LC3-II expression and PARP1 cleavage, respectively. In summary, these results support the potential utility of CS-decorated PCL MPs as an effective 5-FU-delivery carrier to improve HNSCC therapeutic management.
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Antimetabolitos Antineoplásicos/farmacología , Quitosano/química , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Nanopartículas/administración & dosificación , Poliésteres/química , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/química , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quitosano/administración & dosificación , Liberación de Fármacos , Fluorouracilo/administración & dosificación , Fluorouracilo/química , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Nanopartículas/química , Tamaño de la Partícula , Poliésteres/administración & dosificaciónRESUMEN
PURPOSE: Prostate cancer metastasis to secondary organs is considered an initial event in the development of hormone refractory disease and remains the major cause of death among prostate cancer patients. In this study, we investigated the role of fascin, a cytoskeleton actin-bundling protein involved in the formation of filopodia and cell migration, in prostate cancer progression. EXPERIMENTAL DESIGN: Fascin protein expression was examined by immunohistochemistry in a cohort of 196 patients with localized prostate cancer and across several stages of disease progression, including hormone refractory disease. Cellular changes were also assessed in vitro and in vivo in DU145 prostate cancer cell line using fascin gene silencing. RESULTS: Fascin epithelial expression was significantly up-regulated in localized and hormone refractory prostate cancer compared with benign prostate tissue (P<0.05). Furthermore, high fascin expression was associated with an increased rate of prostate-specific antigen recurrence following radical prostatectomy (P=0.075), signifying more aggressive clinical course, thus supporting a function for fascin in prostate cancer progression. In cellular models, fascin gene silencing using small interfering RNA in the androgen-independent prostate cancer cell line DU145 decreased cell motility and invasiveness while increasing cell adhesive properties. In addition, fascin small interfering RNA-expressing DU145 cells implanted orthotopically in mouse prostate showed significantly decreased growth (P<0.005) and drastically prevented the formation of lymph node metastases (P<0.001) compared with their matched controls. CONCLUSIONS: Our data show a function of fascin in the regulation of prostate cancer progression and emphasize the importance of fascin as a prognostic marker for aggressive disease and as a potential therapeutic target for advanced androgen independent disease.
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Proteínas Portadoras/fisiología , Proteínas de Microfilamentos/fisiología , Neoplasias de la Próstata/patología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Humanos , Metástasis Linfática , Masculino , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in alpha-actinin-4. This study addresses how FSGS-associated mutant alpha-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant alpha-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an alpha-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2alpha subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing alpha-actinin-4 K256E. Previously, we showed that expression of this alpha-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that alpha-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of alpha-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, alpha-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus alpha-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.
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Actinina/metabolismo , Retículo Endoplásmico/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Actinina/genética , Animales , Apoptosis , Células COS , Chlorocebus aethiops , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Podocitos/metabolismo , Ratas , Factor de Transcripción CHOP/metabolismoRESUMEN
Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Receptor ErbB-2/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Activación Enzimática , Femenino , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/deficiencia , Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Humanos , Ratones , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Receptor ErbB-3/metabolismo , Análisis de Matrices TisularesRESUMEN
With the goal of identifying small molecule modulators of protein-protein interactions, we developed a solid-phase synthesis method, which was then successfully utilized in a library generation of 164 aminoindoline-derived, natural-product-like compounds. This library and several other related intermediates synthesized during this project were then subjected to different biological assays in search of small molecule modulators of focal adhesion kinase (FAK)-mediated signaling pathways. This study included (i) an in vitro, full length FAK inhibition assay, (ii) a cell proliferation assay, and (iii) a wound healing assay. In FAK inhibition assay, eight library members (5-12) and three aminoindoline derivatives (13, 14, and 2) were identified as promising candidates. Compounds 13 and 2 inhibited the FAK activity by 25-45% at 10 microM. These two lead compounds also showed activity in a wound healing assay. To our knowledge, these aminoindoline-derived small molecules belong to a new family of FAK inhibitors.
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Técnicas Químicas Combinatorias/métodos , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Indoles/química , Indoles/farmacología , Transducción de Señal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/química , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.
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Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Células Epiteliales/enzimología , Células Epiteliales/patología , Glomérulos Renales/enzimología , Glomérulos Renales/inmunología , MAP Quinasa Quinasa Quinasa 5/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Citotoxicidad Inmunológica , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/patología , Activación Enzimática , Células Epiteliales/inmunología , Genes Dominantes , Glomerulonefritis Membranosa/enzimología , Glomerulonefritis Membranosa/inmunología , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Glomérulos Renales/patología , L-Lactato Deshidrogenasa/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Regulación hacia Arriba/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Patients with triple-negative breast cancer (TNBC) often have a poor prognosis largely due to lack of effective targeted therapy. Using a library of seleno-purines coupled to a high-throughput biochemical enzymatic assays we identified a potent pharmacological enhancer of autophagy (referred herein as SLLN-15) that selectively activated cytostatic macroautophagy/autophagy in TNBC preclinical models. SLLN-15 induced a dose-dependent anti-proliferative activity in the TNBC cell lines MDA-MB-231 and BT-20 via induction of autophagy and autophagic flux. This induction was associated with a selective inhibition of AKT-MTOR signaling. Conversely, rapamycin, a known autophagy inducer and MTOR inhibitor, was unable to duplicate SLLN-15's effect on TNBC cells. Inhibition of autophagy by siRNA-mediated targeting of the autophagy regulators, BECN1, ATG5 and ATG7 or using 3-methyladenine (3-MA), significantly protected against SLLN-15-induced inhibition of cell viability, further supporting that SLLN-15-induced inhibition of cancer cell proliferation was autophagy-dependent. SLLN-15-induced autophagy in TNBC cells was also associated with decreased AURKA expression, decreased AKT phosphorylation and subsequent blockage of the AKT-MTOR pathway. In vivo, oral SLLN-15 revealed a potent anticancer and anti-metastatic activity in mice bearing TNBC. Altogether, this study describes a novel regulator of mammalian autophagy, with potential utility as an experimental therapeutic for TNBCs. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; AURKA: aurora kinase A; AURKB: aurora kinase B; BECN1: beclin 1; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; ERBB2: erb-b2 receptor tyrosine kinase 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase 1; PI: propidium iodide; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.
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Autofagia , Citostáticos/farmacología , Progresión de la Enfermedad , Purinas/farmacología , Selenio/farmacología , Neoplasias de la Mama Triple Negativas/patología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Aurora Quinasa A/metabolismo , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Ratones Endogámicos BALB C , Ratones SCID , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/química , Selenio/química , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/ultraestructuraRESUMEN
TRAF2, a RING finger adaptor protein, plays an important function in tumor necrosis factor (TNF)- and TNF-like weak inducer of apoptosis (TWEAK)-dependent signaling, in particular during inflammatory and immune responses. We identified a functional interaction of TRAF2 with focal adhesion (FA) signaling involving the focal adhesion kinase (FAK) in the regulation of cell susceptibility to anoikis. Comparison of TRAF2-proficient (TRAF2+/+) versus TRAF2-deficient (TRAF2-/-), and FAK-proficient (FAK+/+) versus FAK-deficient (FAK-/-) mouse embryonic fibroblasts and their matched reconstituted cells demonstrated that TRAF2 interacts physically with the N-terminal portion of FAK and colocalizes to cell membrane protrusions. This interaction was found to be critical for promoting resistance to cell anoikis. Similar results were confirmed in the human breast cancer cell line MDA-MB-231, where TRAF2 and FAK downregulation promoted cell susceptibility to anoikis. In human breast cancer tissues, genomic analysis of The Cancer Genome Atlas database revealed coamplification of TRAF2 and FAK in breast cancer tissues with a predictive value for shorter survival, further supporting a potential role of TRAF2-FAK cooperative signaling in cancer progression.
Asunto(s)
Neoplasias de la Mama/genética , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Animales , Anoicis , Sitios de Unión , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/química , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Transducción de Señal , Análisis de SupervivenciaRESUMEN
The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.