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The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.
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Exosomas , Sarcoma , Neoplasias de los Tejidos Blandos , Humanos , Exosomas/patología , Estudios Prospectivos , Biomarcadores de Tumor , Sarcoma/patología , Biopsia Líquida , Neoplasias de los Tejidos Blandos/diagnósticoRESUMEN
BACKGROUND: Prostate cancer (PCa) is characterized by clinical and biological heterogeneity and has differential outcomes and mortality rates. Therefore, it is necessary to identify molecular alterations to define new therapeutic strategies based on the risk of progression. In this study, the prognostic relevance of the insulin-like growth factor (IGF) system was examined in molecular subtypes defined by TMPRSS2-ERG (T2E) gene fusion within a series of patients with primary localized PCa. METHODS: A cohort of 270 formalin-fixed and paraffin-embedded (FFPE) primary PCa samples from patients with more than 5 years' follow-up was collected. IGF-1R, IGF-1, IGFBP-3 and INSR expression was analyzed using quantitative RT-PCR. The T2E status and immunohistochemical ERG findings were considered in the analyses. The association with both biochemical and clinical progression-free survival (BPFS and PFS, respectively) was evaluated for the different molecular subtypes using the Kaplan-Meier proportional risk log-rank test and the Cox proportional hazards model. RESULTS: An association between IGF-1R overexpression and better BPFS was found in T2E-negative patients (35.3% BPFS, p-value = 0.016). Multivariate analysis demonstrated that IGF-1R expression constitutes an independent variable in T2E-negative patients [HR: 0.41. CI 95% (0.2-0.82), p = 0.013]. These data were confirmed using immunohistochemistry of ERG as subrogate of T2E. High IGF-1 expression correlated with prolonged BPFS and PFS independent of the T2E status. CONCLUSIONS: IGF-1R, a reported target of T2E, constitutes an independent factor for good prognosis in T2E-negative PCa. Quantitative evaluation of IGF-1/IGF-1R expression combined with molecular assessment of T2E status or ERG protein expression represents a useful marker for tumor progression in localized PCa.
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Proteínas de Fusión Oncogénica , Neoplasias de la Próstata/metabolismo , Receptores de Somatomedina/metabolismo , Serina Endopeptidasas/genética , Anciano , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/mortalidad , Receptor IGF Tipo 1 , Receptores de Somatomedina/análisis , Receptores de Somatomedina/genética , Regulador Transcripcional ERG/genéticaRESUMEN
BACKGROUND: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. RESULTS: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. CONCLUSIONS: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.
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Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Biopelículas/crecimiento & desarrollo , Xanthomonas/fisiología , Adhesión Bacteriana , Mutación , Hojas de la Planta/microbiología , Proteoma/análisis , Xanthomonas/genética , Xanthomonas/metabolismoRESUMEN
Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.
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Neoplasias de la Mama/genética , Ciclina D1/genética , Regulación Neoplásica de la Expresión Génica , Receptores Androgénicos/metabolismo , Elementos de Respuesta , Andrógenos/farmacología , Sitios de Unión , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Receptor Nuclear Huérfano DAX-1/metabolismo , Dihidrotestosterona/farmacología , Femenino , Humanos , Regiones Promotoras GenéticasRESUMEN
Bone and soft tissue sarcomas (STSs) represent a group of heterogeneous rare malignant tumors of mesenchymal origin, with a poor prognosis. Due to their low incidence, only a few studies have been reported addressing circulating tumor cells (CTCs) in sarcoma, despite the well-documented relevance for applications of liquid biopsy in precision medicine. In the present review, the most recent data relative to the detection and isolation of viable and intact CTCs in these tumors will be reviewed, and the heterogeneity in CTCs will be discussed. The relevance of epithelial-mesenchymal plasticity and stemness in defining the phenotypic and functional properties of these rare cells in sarcoma will be highlighted. Of note, the existence of dynamic epithelial-mesenchymal transition (EMT)-related processes in sarcoma tumors has only recently been related to their clinical aggressiveness. Also, the presence of epithelial cell adhesion molecule (EpCAM)-positive CTC in sarcoma has been weakly correlated with poor outcome and disease progression, thus proving the existence of both epithelial and mesenchymal CTC in sarcoma. The advancement in technologies for capturing and enumerating all diverse CTCs phenotype originating from these mesenchymal tumors are presented, and results provide a promising basis for clinical application of CTC detection in sarcoma.
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Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.
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Soft tissue sarcomas (STS) are heterogeneous rare malignancies comprising ~1% of all solid cancers in adults and including more than 70 histological and molecular subtypes with different pathological and clinical development characteristics. Over the last two decades, the increased knowledge of the new molecular and genomic mechanisms of different STS histotypes allowed for a reclassification of these tumors and consequently to the development of novel chemotherapeutic agents. Generally, surgery, in combination with radiotherapy only in selected cases of localized disease, represents the most common treatment of primary STS, whereas the principal treatment modality for locally advanced or metastatic disease is first-line chemotherapy. The principal treatment for the preponderance of STS patients is usually an anthracycline (epirubicin and doxorubicin) in monotherapy or in combination with other drug novel chemotherapeutic agents. However, survival for treated patients with metastatic disease is poor, and a 2-years survival rate is about 30%. In this scenario, Pharmacogenomics (PGx) biomarkers that can predict drug response play an important role in the improvement of molecular diagnostics in clinical routines and contribute to elucidating the genetic basis for the differences in treatment efficacy and toxicity among STS patients. This review focuses on recent insight in the PGx biomarkers that have been described to modulate responsiveness and toxicity parameters of conventional and new chemotherapeutics drugs in several STS histotypes.
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This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.
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[This corrects the article DOI: 10.3389/fonc.2020.00489.].
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Breast cancer development and progression is regulated by growth factors and steroid hormones. Although the majority of human breast cancers expresses androgen receptor (AR), the role of androgens in breast tumorigenesis remains largely unexplored. Here we demonstrate that an AR ligand, 5-alpha-dihydrotestosterone (DHT), inhibits MCF-7 breast cancer cell growth induced by insulin like growth factor 1 (IGF-I). Our results show that DHT induces association of AR with IRS-1, the major IGF-1 receptor signaling molecule. The AR/IRS-1 complex translocates to the nucleus and is recruited to gene promoters containing androgen responsive elements causing an increase of AR transcriptional activity. Moreover, IRS-1 knockdown suggests that IRS-1/AR interaction decreases the ubiquitin/proteasome dependent degradation of AR, increasing its stability. Taken together, these data indicate that nuclear IRS-1 is a novel AR regulator required to sustain AR activity and demonstrate, for the first time in breast cancer cells, the existence of a functional interplay between the IGF system and AR. This interplay may represent the molecular basis of mechanisms through which androgens exert their inhibitory role on the proliferation of breast cancer cells.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Receptores Androgénicos/biosíntesis , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Dihidrotestosterona/metabolismo , Femenino , Humanos , Inmunoprecipitación , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Receptores Androgénicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson-Gilford progeria, a severe LMNA-linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C-HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C-HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Histona Desacetilasa 2/metabolismo , Lamina Tipo A/metabolismo , Estrés Oxidativo , Progeria/metabolismo , Progeria/patología , Adolescente , Anciano , Células Cultivadas , Niño , Preescolar , Daño del ADN , Reparación del ADN , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Masculino , Mutación/genética , Progeria/genética , Unión Proteica , Especificidad por SustratoRESUMEN
BACKGROUND: Insulin receptor substrate 1 (IRS-1), a cytoplasmic protein transmitting signals from the insulin and insulin-like growth factor 1 receptors, has been implicated in breast cancer. Previously, it was reported that IRS-1 can be translocated to the nucleus and modulate oestrogen receptor alpha (ERalpha) activity in vitro. However, the expression of nuclear IRS-1 in breast cancer biopsy specimens has never been examined. AIMS: To assess whether nuclear IRS-1 is present in breast cancer and non-cancer mammary epithelium, and whether it correlates with other markers, especially ERalpha. Parallel studies were carried out for the expression of cytoplasmatic IRS-1. METHODS: IRS-1 and ERalpha expression was assessed by immunohistochemical analysis. Data were evaluated using Pearson's correlation, linear regression and receiver operating characteristic analysis. RESULTS: Median nuclear IRS-1 expression was found to be low in normal mammary epithelial cells (1.6%) and high in benign tumours (20.5%), ductal grade 2 carcinoma (11.0%) and lobular carcinoma (approximately 30%). Median ERalpha expression in normal epithelium, benign tumours, ductal cancer grade 2 and 3, and lobular cancer grade 2 and 3 were 10.5, 20.5, 65.0, 0.0, 80 and 15%, respectively. Nuclear IRS-1 and ERalpha positively correlated in ductal cancer (p<0.001) and benign tumours (p<0.01), but were not associated in lobular cancer and normal mammary epithelium. In ductal carcinoma, both nuclear IRS-1 and ERalpha negatively correlated with tumour grade, size, mitotic index and lymph node involvement. Cytoplasmic IRS-1 was expressed in all specimens and positively correlated with ERalpha in ductal cancer. CONCLUSIONS: A positive association between nuclear IRS-1 and ERalpha is a characteristic for ductal breast cancer and marks a more differentiated, non-metastatic phenotype.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Citoplasma/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Proteínas Sustrato del Receptor de Insulina , Glándulas Mamarias Humanas/metabolismo , Microscopía Confocal , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismoRESUMEN
PURPOSE: Recent in vitro studies suggested that the autocrine leptin loop might contribute to breast cancer development by enhancing cell growth and survival. To evaluate whether the leptin system could become a target in breast cancer therapy, we examined the expression of leptin and its receptor (ObR) in primary and metastatic breast cancer and noncancer mammary epithelium. We also studied whether the expression of leptin/ObR in breast cancer can be induced by obesity-related stimuli, such as elevated levels of insulin, insulin-like growth factor-I (IGF-I), estradiol, or hypoxic conditions. EXPERIMENTAL DESIGN: The expression of leptin and ObR was examined by immunohistochemistry in 148 primary breast cancers and 66 breast cancer metastases as well as in 90 benign mammary lesions. The effects of insulin, IGF-I, estradiol, and hypoxia on leptin and ObR mRNA expression were assessed by reverse transcription-PCR in MCF-7 and MDA-MB-231 breast cancer cell lines. RESULTS: Leptin and ObR were significantly overexpressed in primary and metastatic breast cancer relative to noncancer tissues. In primary tumors, leptin positively correlated with ObR, and both biomarkers were most abundant in G3 tumors. The expression of leptin mRNA was enhanced by insulin and hypoxia in MCF-7 and MDA-MB-231 cells, whereas IGF-I and estradiol stimulated leptin mRNA only in MCF-7 cells. ObR mRNA was induced by insulin, IGF-I, and estradiol in MCF-7 cells and by insulin and hypoxia in MDA-MB-231 cells. CONCLUSIONS: Leptin and ObR are overexpressed in breast cancer, possibly due to hypoxia and/or overexposure of cells to insulin, IGF-I, and/or estradiol.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Leptina/metabolismo , Obesidad/complicaciones , Receptores de Superficie Celular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Estudios de Casos y Controles , Hipoxia de la Célula , Progresión de la Enfermedad , Estradiol/farmacología , Femenino , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Leptina/genética , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Obesidad/metabolismo , Receptores de Superficie Celular/genética , Receptores de Leptina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Purpose: Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested.Experimental Design: We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer.Results: Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1-blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression.Conclusions: These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors. Clin Cancer Res; 23(17); 5149-61. ©2017 AACR.
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Neoplasias Óseas/tratamiento farmacológico , Dioxoles/efectos adversos , Neoplasias Pulmonares/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Tetrahidroisoquinolinas/efectos adversos , Neoplasias Óseas/genética , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/inmunología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Dioxoles/administración & dosificación , Humanos , Inmunoterapia/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Osteosarcoma/genética , Osteosarcoma/inmunología , Osteosarcoma/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tetrahidroisoquinolinas/administración & dosificación , Trabectedina , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In breast cancer cells, 17-beta-estradiol (E2) upregulates the expression of insulin receptor substrate 1 (IRS-1), a molecule transmitting insulin-like growth factor-I (IGF-I) signals through the PI-3K/Akt survival pathways. The stimulation of IRS-1 by E2 has been documented on the transcriptional level. Here we studied whether the expression of estrogen receptor (ER)-alpha affects IRS molecules post-transcriptionally. We used ER-alpha-negative MDA-MB-231 breast cancer cells and MDA-MB-231 cells with re-expressed ER-alpha. In MDA-MB-231 cells cultured under serum-free conditions, IRS-1 and IRS-2 were degraded through the 26S proteasome and calpain pathways. Re-expression of ER-alpha in MDA-MB-231 cells correlated with enhanced stability of IRS molecules. This effect coincided with significantly reduced ubiquitination of IRS-1 and IRS-2, but did not involve increased IRS-1 and IRS-2 transcription. The interference of ER-alpha with IRS-1 and IRS-2 turnover could rely on the competition for common degradation pathways, as in MDA-MB-231/ER cells, ER-alpha processing was blocked by proteasome and calpain inhibitors. Notably, a fraction of the cytosolic ER-alpha colocalized and coprecipitated with IRS-1 and IRS-2, indicating a possible common destination for these proteins. The stabilization of IRS-1 in MDA-MB-231/ER cells was paralleled by the upregulation of the IRS-1/Akt/GSK-3 pathway and improved survival in the presence of IGF-I, whereas IRS-2 was not involved in IGF-I signaling.
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Neoplasias de la Mama/metabolismo , Fosfoproteínas/metabolismo , Receptores de Estrógenos/metabolismo , Western Blotting , División Celular , Supervivencia Celular , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico , Receptor alfa de Estrógeno , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Microscopía Confocal , Microscopía Fluorescente , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Células Tumorales Cultivadas , Ubiquitina/metabolismoRESUMEN
Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptor alpha (ERalpha)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-beta-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERalpha; (4) the IRS-1:ERalpha complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERalpha-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERalpha. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERalpha activity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERalpha and that this interaction might influence ERalpha transcriptional activity.
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Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Receptores de Estrógenos/metabolismo , Animales , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Receptor alfa de Estrógeno , Humanos , Proteínas Sustrato del Receptor de Insulina , Pruebas de Precipitina , Unión Proteica , Transporte de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
PURPOSE: Obesity is a risk factor for breast cancer development in postmenopausal women and correlates with shorter disease-free and overall survival in breast cancer patients, regardless of menopausal status. Adipose tissue is a major source of leptin, a cytokine regulating energy balance and controlling different processes in peripheral tissues, including breast cancer cell growth. Here, we investigated whether leptin can counteract antitumorigenic activities of the antiestrogen ICI 182,780 in breast cancer cells. EXPERIMENTAL DESIGN: Mitogenic response to leptin and the effects of leptin on ICI 182,780-dependent growth inhibition were studied in MCF-7 estrogen receptor alpha-positive breast cancer cells. The expression of leptin receptor and the activation of signaling pathways were studied by Western immunoblotting. The interference of leptin with ICI 182,780-induced estrogen receptor alpha degradation was probed by Western immunoblotting, fluorescence microscopy, and pulse-chase experiments. Leptin effects on estrogen receptor alpha-dependent transcription in the presence and absence of ICI 182,780 were studied by luciferase reporter assays and chromatin immunoprecipitation. RESULTS: MCF-7 cells were found to express the leptin receptor and respond to leptin with cell growth and activation the signal transducers and activators of transcription 3, extracellular signal-regulated kinase-1/2, and Akt/GSK3/pRb pathways. The exposure of cells to 10 nmol/L ICI 182,780 blocked cell proliferation, induced rapid estrogen receptor alpha degradation, inhibited nuclear estrogen receptor alpha expression, and reduced estrogen receptor alpha-dependent transcription from estrogen response element-containing promoters. All of these effects of ICI 182,780 were significantly attenuated by simultaneous treatment of cells with 100 ng/mL leptin. CONCLUSIONS: Leptin interferes with the effects of ICI 182,780 on estrogen receptor alpha in breast cancer cells. Thus, high leptin levels in obese breast cancer patients might contribute to the development of antiestrogen resistance.
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Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Leptina/farmacología , Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/farmacología , Femenino , Fulvestrant , Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Ubiquitina/metabolismoRESUMEN
Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction.
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Proteínas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Xanthomonas/metabolismo , Secuencia de Aminoácidos , Arginina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Virulencia/genética , Xanthomonas/genética , Xanthomonas/patogenicidadRESUMEN
Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Several studies have confirmed the involvement of the insulin-like growth factor (IGF) system in the regulation of OS cell proliferation and differentiation as well as in the protection of cells from chemotherapy. Insulin receptor substrate (IRS)-1 is a critical mediator of IGF-1R signaling, and we recently reported that its overexpression in OS cells increases proliferation, migration, and metastasis both in vitro and in vivo. In this study, we evaluated the efficacy of NT157, a selective inhibitor of IRS-1/2, in a panel of OS cells. A strong dose-dependent inhibition of growth was observed in the MG-63, OS-19, and U-2OS OS cell lines, displaying IC50 values at sub-micromolar doses after 72 h of treatment. Exposure to NT157 elicited dose- and time-dependent decreases in IRS-1 levels. Moreover, a protein analysis showed that the degradation of IRS-1 inhibited the activation of principal downstream mediators of the IGF pathway. NT157 significantly affected the cells' migratory ability, as confirmed by a wound-healing assay. The inhibitor induced cytostatic effects, as evidenced by G2/M cell cycle arrest, and did not affect apoptosis. Consequently, NT157 was combined with drugs used to treat OS in order to capitalize on its therapeutic potential. Simultaneous treatments were made in association with chemotherapeutic agents in a fixed ratio for 72 h and cell proliferation was determined by MTT assay. Synergistic or addictive effects with respect to single agents are expressed as the combination index. Significant synergistic effects were obtained with several targeted drugs, such as Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, and NVP-BEZ235, a dual inhibitor of PI-3K/mTOR. Overall, these findings provide evidence for the effectiveness of a selected inhibitor of IRS-1/2 NT157 in OS cells, displaying a promising approach based on the targeting of IRS-1 combined with other therapies for the treatment of this pediatric solid tumor.
RESUMEN
Identifying patients who may benefit from targeted therapy is an urgent clinical issue in prostate cancer (PCa). We investigated the molecular relationship between TMPRSS2-ERG (T2E) fusion gene and insulin-like growth factor receptor (IGF-1R) to optimize the use of IGF-1R inhibitors.IGF-1R was analyzed in cell lines and in radical prostatectomy specimens in relation to T2E status. ERG binding to IGF-1R promoter was evaluated by chromatin immunoprecipitation (ChIP). Sensitivity to anti-IGF-1R agents was evaluated alone or in combination with anti-androgen abiraterone acetate in vitro at basal levels or upon ERG modulation.IGF-1R analysis performed in PCa cells or clinical samples showed that T2E expression correlated with higher IGF-1R expression at mRNA and protein levels. Genetic modulation of ERG directly affected IGF-1R protein levels in vitro. ChIP analysis showed that ERG binds IGF-1R promoter and that promoter occupancy is higher in T2E-positive cells. IGF-1R inhibition was more effective in cell lines expressing the fusion gene and combination of IGF-1R inhibitors with abiraterone acetate produced synergistic effects in T2E-expressing cells.Here, we provide the rationale for use of T2E fusion gene to select PCa patients for anti-IGF-1R treatments. The combination of anti-IGF-1R-HAbs with an anti-androgen therapy is strongly advocated for patients expressing T2E.