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BACKGROUND INFORMATION: Arpin, an Arp2/3 inhibitory protein, inhibits lamellipodial protrusions and cell migration. Arpin expression is lost in tumor cells of several cancer types. RESULTS: Here we analyzed expression levels of Arpin and various markers using Reverse Phase Protein Array (RPPA) in human mammary carcinomas. We found that Arpin protein levels were correlated with those of several DNA damage response markers. Arpin-null cells display enhanced clustering of double stand breaks (DSBs) when cells are treated with a DNA damaging agent, in line with a previously described role of the Arp2/3 complex in promoting DSB clustering for homologous DNA repair (HDR) in the nucleus. Using a specific HDR assay, we further showed that Arpin depletion increased HDR efficiency two-fold through its ability to inactivate the Arp2/3 complex. CONCLUSIONS: Arpin regulates both cell migration in the cytosol and HDR in the nucleus. SIGNIFICANCE: Loss of Arpin expression coordinates enhanced cell migration with up-regulated DNA repair, which is required when DNA damage is induced by active cell migration.
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BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.
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Neoplasias Hepáticas , Melanoma , Humanos , Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Melanoma/patología , Neoplasias Hepáticas/patología , Linfocitos Infiltrantes de Tumor , Pronóstico , Biomarcadores de Tumor/análisisRESUMEN
The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds' effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.
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Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Estrona/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/genética , Autofagia/genética , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Estrenos/farmacología , Estrona/análogos & derivados , Estrona/química , Femenino , Células HeLa , Humanos , Microtúbulos/química , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.
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Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Medicina de Precisión/métodos , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Toma de Decisiones Clínicas , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Selección de Paciente , Fenotipo , Mapas de Interacción de Proteínas , Transducción de Señal , TranscriptomaRESUMEN
Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.
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Dosificación de Gen , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias de la Mama Triple Negativas/genética , Animales , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Proteínas de la Membrana/genética , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , Trasplante de Neoplasias , Medicina de Precisión , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The analytic platform described in this chapter uses proteins extracted from cultured cells as an infinite source of material to set up, validate, and quality control an RPPA platform. Readout of the arrays uses near-infrared fluorescence labeling and data normalization is performed using the bioinformatics package NormaCurve.In the first part, we will describe the advantages, drawbacks, and different applications of cell line material for RPPA. In the second part, we will describe how the staining protocol, the method of readout, and the normalization method applied afterward are interconnected and should be considered together. Finally, we will describe the NormaCurve package, which is freely available, and its requirements for implementation.Four protocols are provided in this chapter: (1) Protein lysis of cell lines using a homemade Laemmli buffer, (2) RPPA staining for fluorescent readout including a signal amplification step, (3) total protein staining in the visible spectrum for normalization purposes, and (4) total protein staining in the near-infrared spectrum for normalization purposes.
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Análisis por Matrices de Proteínas , Proteínas , Línea Celular , Fluorescencia , Análisis por Matrices de Proteínas/métodos , Análisis por Matrices de Proteínas/normas , Proteínas/química , Control de CalidadRESUMEN
Supplementary Table 1 and the Supplementary Figure legends were not included when this manuscript was first published. The files are now available here.
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BACKGROUND: Metastatic colorectal cancer (mCRC) patients with mutant KRAS or NRAS are ineligible for anti-epidermal growth factor receptor (anti-EGFR) therapy, as RAS mutations activate downstream pathways independently of EGFR and induce primary resistance. However, even among RAS wild-type (WT) patients, only a fraction responds to anti-EGFR therapy, suggesting that other mechanisms of resistance exist. We hypothesise that different (epi)genetic alterations can lead to primary anti-EGFR resistance and that the crucial end point is the activation of protein signalling pathways. METHODS: We analysed the expression and activation of proteins involved in cell signalling, using reverse phase protein arrays, on a multicentre French cohort of RAS WT mCRC treated with anti-EGFR treatment. RESULTS: We identify activated EGFR and HER3 as protein biomarkers predictive for better overall survival. Active EGFR signalling and downstream PI3K, but not MAPK, pathway activation are associated with response to anti-EGFR treatment. Left-sided mCRC displays active ErbB2/3 and Wnt pathways and a better response to anti-EGFR therapy compared to right-sided mCRC. CONCLUSIONS: We identify active EGFR and PI3K signalling as a key factor for response to anti-EGFR treatment in mCRC and highlight the importance of developing these biomarkers in clinical practice for the selection of RAS WT mCRC patients that would benefit from anti-EGFR treatment.
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Carcinoma/genética , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Genes ras , Receptor ErbB-3/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Biomarcadores de Tumor/metabolismo , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Carcinoma/tratamiento farmacológico , Carcinoma/secundario , Cetuximab/administración & dosificación , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Epigénesis Genética , Receptores ErbB/antagonistas & inhibidores , Femenino , Fluorouracilo/administración & dosificación , Humanos , Leucovorina/administración & dosificación , Sistema de Señalización de MAP Quinasas , Masculino , Persona de Mediana Edad , Mutación , Compuestos Organoplatinos/administración & dosificación , Panitumumab , Fosforilación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Receptor ErbB-2/metabolismo , Criterios de Evaluación de Respuesta en Tumores Sólidos , Estudios Retrospectivos , Vía de Señalización WntRESUMEN
BACKGROUND: The insulin growth factor (IGF) pathway has been proposed as a potential therapeutic target in bladder cancer. We characterized the expression of components of the IGF pathway - insulin growth factor receptors (INSR, IGF1R, IGF2R), ligands (INS, IGF1, IGF2), and binding proteins (IGFBP1-7, IGF2BP1-3) - in bladder cancer and its correlation with IGF1R activation, and the anti-proliferative efficacy of an IGF1R kinase inhibitor in this setting. METHODS: We analyzed transcriptomic data from two independent bladder cancer datasets, corresponding to 200 tumoral and five normal urothelium samples. We evaluated the activation status of the IGF pathway in bladder tumors, by assessing IGF1R phosphorylation and evaluating its correlation with mRNA levels for IGF pathway components. We finally evaluated the correlation between inhibition of proliferation by a selective inhibitor of the IGF1R kinase (AEW541), reported in 13 bladder cancer derived cell lines by the Cancer Cell Line Encyclopedia Consortium and mRNA levels for IGF pathway components. RESULTS: IGF1R expression and activation were stronger in non-muscle-invasive than in muscle-invasive bladder tumors. There was a significant inverse correlation between IGF1R phosphorylation and IGFBP5 expression in tumors. Consistent with this finding, the inhibition of bladder cell line viability by IGF1R inhibitor was also inversely correlated with IGFBP5 expression. CONCLUSION: The IGF pathway is activated and therefore a potential therapeutic target for non muscle-invasive bladder tumors and IGFBP5 could be used as a surrogate marker for predicting tumor sensitivity to anti-IGF therapy.
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Regulación Neoplásica de la Expresión Génica , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Receptor IGF Tipo 1/agonistas , Receptor IGF Tipo 1/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Anciano , Anciano de 80 o más Años , Proteínas Portadoras , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ligandos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Unión Proteica , ARN Mensajero/genética , Transducción de Señal , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide. CC pathogenesis is triggered when human papillomavirus (HPV) inserts into the genome, resulting in tumour suppressor gene inactivation and oncogene activation. Collecting tumour and blood samples is critical for identifying these genetic alterations. METHODS: BIO-RAIDs is the first prospective molecular profiling clinical study to include a substantial biobanking effort that used uniform high-quality standards and control of samples. In this European Union (EU)-funded study, we identified the challenges that were impeding the effective implementation of such a systematic and comprehensive biobanking effort. RESULTS: The challenges included a lack of uniform international legal and ethical standards, complexities in clinical and molecular data management, and difficulties in determining the best technical platforms and data analysis techniques. Some difficulties were encountered by all investigators, while others affected only certain institutions, regions, or countries. CONCLUSIONS: The results of the BIO-RAIDs programme highlight the need to facilitate and standardise regulatory procedures, and we feel that there is also a need for international working groups that make recommendations to regulatory bodies, governmental funding agencies, and academic institutions to achieve a proficient biobanking programme throughout EU countries. This represents the first step in precision medicine.
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Bancos de Muestras Biológicas , Neoplasias del Cuello Uterino/patología , Femenino , HumanosRESUMEN
BACKGROUND: Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known. METHODS: We used RT-PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome. RESULTS: MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway. CONCLUSIONS: Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target.
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Neoplasias de la Mama/genética , ARN Largo no Codificante/genética , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/genética , Epigenómica , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The Arp2/3 complex is required for cell migration and invasion. The Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. Here we investigate the expression of Arpin, the Arp2/3 inhibitory protein that antagonises the WAVE complex. METHODS: We used qRT-PCR and reverse phase protein arrays in a patient cohort with known clinical parameters and outcome, immunofluorescence in breast biopsy cryosections and breast cancer cell lines. RESULTS: Arpin was downregulated at the mRNA and protein levels in mammary carcinoma cells. Arpin mRNA downregulation was associated with poor metastasis-free survival (MFS) on univariate analysis (P=0.022). High expression of the NCKAP1 gene that encodes a WAVE complex subunit was also associated with poor MFS on univariate analysis (P=0.0037) and was mutually exclusive with Arpin low. Arpin low or NCKAP1 high was an independent prognosis factor on multivariate analysis (P=0.0012) and was strongly associated with poor MFS (P=0.000064). CONCLUSIONS: Loss of the Arp2/3 inhibitory protein Arpin produces a similar poor outcome in breast cancer as high expression of the NCKAP1 subunit of the Arp2/3 activatory WAVE complex.
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Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Carcinoma/genética , Proteínas Portadoras/genética , Regulación hacia Abajo , ARN Mensajero/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Supervivencia sin Enfermedad , Receptor alfa de Estrógeno/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pronóstico , Análisis por Matrices de Proteínas , Receptor ErbB-2/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga TumoralRESUMEN
Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology.
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Inmunoensayo/métodos , Análisis por Matrices de Proteínas/métodos , Investigación Biomédica Traslacional/métodos , Educación , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Mammalian cells possess two isoforms of the histone H3-H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis.
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Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Femenino , Perfilación de la Expresión Génica , Humanos , Microscopía Fluorescente , Valor Predictivo de las Pruebas , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hyperhomocysteinemia, characterized by high plasma homocysteine levels, is recognized as an independent risk factor for cardiovascular diseases. The increased synthesis of homocysteine, a product of methionine metabolism involving B vitamins, and its slower intracellular utilization cause increased flux into the blood. Plasma homocysteine level is an important reflection of hepatic methionine metabolism and the rate of processes modified by B vitamins as well as different enzyme activity. Lowering homocysteine might offer therapeutic benefits. However, approximately 50% of hyperhomocysteinemic patients due to cystathionine-beta-synthase deficiency are biochemically responsive to pharmacological doses of B vitamins. Therefore, effective treatments to reduce homocysteine levels are needed, and gene therapy could provide a novel approach. We recently showed that hepatic expression of DYRK1A, a serine/threonine kinase, is negatively correlated with plasma homocysteine levels in cystathionine-beta-synthase deficient mice, a mouse model of hyperhomocysteinemia. Therefore, Dyrk1a is a good candidate for gene therapy to normalize homocysteine levels. We then used an adenoviral construct designed to restrict expression of DYRK1A to hepatocytes, and found decreased plasma homocysteine levels after hepatocyte-specific Dyrk1a gene transfer in hyperhomocysteinemic mice. The elevation of pyridoxal phosphate was consistent with the increase in cystathionine-beta-synthase activity. Commensurate with the decreased plasma homocysteine levels, targeted hepatic expression of DYRK1A resulted in elevated plasma paraoxonase-1 activity and apolipoprotein A-I levels, and rescued the Akt/GSK3 signaling pathways in aorta of mice, which can prevent homocysteine-induced endothelial dysfunction. These results demonstrate that hepatocyte-restricted Dyrk1a gene transfer can offer a useful therapeutic targets for the development of new selective homocysteine lowering therapy.
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Aorta/metabolismo , Apolipoproteína A-I/sangre , Terapia Genética , Hepatocitos/metabolismo , Homocisteína , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Aorta/patología , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Homocisteína/sangre , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/genética , Hiperhomocisteinemia/terapia , Ratones , Ratones Mutantes , Especificidad de Órganos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transducción Genética , Quinasas DyrKRESUMEN
The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n = 25, T1 n = 34, T2-T3-T4 n = 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient RS = 0.26; p = 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p = 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (RS = -0.50; p = 0.0088) and miR-222 (RS = -0.48; p = 0.0132), but not miR-21 (RS = -0.27; p = 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (RS = 0.54; p = 0.0056) and T1 (RS = 0.56; p = 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.
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Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Vejiga Urinaria/enzimología , Anciano , Transformación Celular Neoplásica/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Activación Enzimática , Femenino , GTP Fosfohidrolasas/genética , Humanos , Masculino , Proteínas de la Membrana/genética , MicroARNs/biosíntesis , Persona de Mediana Edad , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Proteína S6 Ribosómica/biosíntesis , Proteína S6 Ribosómica/metabolismo , Eliminación de Secuencia/genética , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Vejiga Urinaria/enzimología , Vejiga Urinaria/patología , Urotelio/enzimología , Urotelio/patología , Proteínas ras/genéticaRESUMEN
The mechanisms driving late relapse in uveal melanoma (UM) patients remains a medical mystery and major challenge. Clinically it is inferred that UM disseminated cancer cells (DCCs) persist asymptomatic for years-to-decades mainly in the liver before they manifest as symptomatic metastasis. Here we reveal using Gαq/11 mut /BAP wt human uveal melanoma models and human UM metastatic samples, that the neural crest lineage commitment nuclear receptor NR2F1 is a key regulator of spontaneous UM DCC dormancy in the liver. Using a quiescence reporter, RNA-seq and multiplex imaging we revealed that rare dormant UM DCCs upregulate NR2F1 expression and genes related to neural crest programs while repressing gene related to cell cycle progression. Gain and loss of function assays showed that NR2F1 silences YAP1/TEAD1 transcription downstream of Gαq/11 signaling and that NR2F1 expression can also be repressed by YAP1. YAP1 expression is repressed by NR2F1 binding to its promoter and changing the histone H3 tail activation marks to repress YAP1 transcription. In vivo CRISPR KO of NR2F1 led dormant UM DCCs to awaken and initiate relentless liver metastatic growth. Cut&Run and bulk RNA sequencing further confirmed that NR2F1 epigenetically stimulates neuron axon guidance and neural lineage programs, and it globally represses gene expression linked to G-protein signaling to drive dormancy. Pharmacological inhibition of Gαq/11 mut signaling resulted in NR2F1 upregulation and robust UM growth arrest, which was also achieved using a novel NR2F1 agonist. Our work sheds light on the molecular underpinnings of UM dormancy revealing that transcriptional programs driven by NR2F1 epigenetically short-circuit Gαq/11 signaling to its downstream target YAP1. Highlights: Quiescent solitary uveal melanoma (UM) DCCs in the liver up- and down-regulate neural crest and cell cycle progression programs, respectively.NR2F1 drives solitary UM DCC dormancy by antagonizing the Gαq/11-YAP1 pathway; small molecule Gαq/11 inhibition restores NR2F1 expression and quiescence. NR2F1 short-circuits oncogenic YAP1 and G-protein signaling via a chromatin remodeling program. Loss of function of NR2F1 in dormant UM DCCs leads to aggressive liver metastasis.
RESUMEN
DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.
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Daño del ADN , Reparación del ADN , Proteínas HSP90 de Choque Térmico/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Humanos , Ratones , Ratones Desnudos , Fosforilación , RatasRESUMEN
Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease, usually in the liver. When metastatic, the prognosis is poor, and few treatment options exist. Here, we investigated the feasibility of establishing patient-derived xenografts (PDXs) from a patient's tumor in order to screen for therapies that the patient could benefit from. Samples obtained from 29 primary tumors and liver metastases of uveal melanoma were grafted into SCID mice. PDX models were successfully established for 35% of primary patient tumors and 67% of liver metastases. The tumor take rate was proportional to the risk of metastases. PDXs showed the same morphology, the same GNAQ/11, BAP1, and SF3B1 mutations, and the same chromosome 3 and 8q status as the corresponding patient samples. Six PDX models were challenged with two compounds for 4 weeks. We show that, for 31% of patients with high or intermediate risk of metastasis, the timing to obtain efficacy results on PDX models derived from their primary tumors was compatible with the selection of the therapy to treat the patient after relapse. PDXs could thus be a valid tool ("avatar") to select the best personalized therapy for one third of patients that are most at risk of relapse.
Asunto(s)
Neoplasias Hepáticas , Recurrencia Local de Neoplasia , Adulto , Animales , Ratones , Humanos , Estudios de Factibilidad , Xenoinjertos , Ratones SCID , Neoplasias Hepáticas/genética , RecurrenciaRESUMEN
There is no strong and reliable predictive biomarker in head and neck squamous cell carcinoma (HNSCC) for EGFR inhibitors. We aimed to identify predictive and pharmacodynamic biomarkers of efficacy of afatinib, a pan-HER tyrosine kinase inhibitor, in a window-of-opportunity trial (NCT01415674). Multi-omics analyses were carried out on pre-treatment biopsy and surgical specimen for biological assessment of afatinib activity. Sixty-one treatment-naïve and operable HNSCC patients were randomised to afatinib 40 mg/day for 21-28 days versus no treatment. Afatinib produced a high rate of metabolic response. Responders had a higher expression of pERK1/2 (P = 0.02) and lower expressions of pHER4 (P = 0.03) and pRB1 (P = 0.002) in pre-treatment biopsy compared to non-responders. At the cellular level, responders displayed an enrichment of tumor-infiltrating B cells under afatinib (P = 0.02). At the molecular level, NF-kappa B signaling was over-represented among upregulated genes in non-responders (P < 0.001; FDR = 0.01). Although exploratory, phosphoproteomics-based biomarkers deserve further investigations as predictors of afatinib efficacy.