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BACKGROUND: The optimal surgical margins assessment is capital in oral squamous cell carcinoma (OSCC) management. We evaluated the clinical benefits of integrating intraoperative macroscopic margin (MM) assessment and narrow band imaging (NBI). METHODS: Sixteen OSCC patients eligible for surgery were prospectively enrolled. For each patient, 2 to 6 bioptic samples of MM and NBI margins were obtained and histologically analyzed for the presence of dysplasia and lymphocytes. Microvessel density was investigated by CD34 immunohistochemistry. RESULTS: Taken together, 104 specimens were analyzed, including 15% tumors, 33% MM, 33% NBI margins, and 19% MM-NBI overlapping margins. The NBI margins were closer to the lesion in 50% cases, while the same number of MM were more conservative than NBI, irrespective of the tumor site. The rate of histologically positive margins was similar among the two methods, akin to the microvessel density. CONCLUSIONS: MM assessment should be integrated but not replaced with the NBI technology to allow for more conservative surgery.
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Carcinoma de Células Escamosas/diagnóstico por imagen , Márgenes de Escisión , Neoplasias de la Boca/diagnóstico por imagen , Imagen de Banda Estrecha , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34 , Carcinoma de Células Escamosas/cirugía , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/cirugía , Estudios ProspectivosRESUMEN
Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.
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Apoptosis , Respuesta al Choque Térmico , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Estrés Oxidativo , Perfusión , Animales , Supervivencia Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.
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Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , Invasividad Neoplásica , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transporte Biológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Metabolismo Energético , Humanos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Sorafenib is the standard of care in advanced hepatocellular carcinoma (HCC), however no criteria have been established to select patients likely to benefit from this therapy. In this study, we evaluated the predictive role of microRNAs (miRNAs) in this setting of patients. METHODS: We profiled 522 miRNA in a series of 26 HCC patients treated with sorafenib (training set) and validated the results in an independent series of 58 patients (validation set). Formalin-fixed paraffin-embedded tumour and cirrhotic liver biopsies were used for RNA extraction and miRNAs profiling with TaqMan Arrays technology. Statistical analyses were used to correlate miRNA levels with clinical outcome, including time to progression (TTP), progression free (PFS), and overall survival. Cell viability and cell motility of HuH-7 or HepG2 HCC cells were tested in vitro after transfection with specific miRNA precursor, inhibitor or controls and sorafenib treatment. RESULTS: Six miRNAs were significantly associated with clinical variables in the training set and only miR-425-3p could be further validated. Higher levels of miR-425-3p were associated with longer TTP and PFS (P = 0.0008; HR = 0.4; 95% CI = 0.2-0.7 and P = 0.007; HR = 0.5; 95% CI = 0.3-0.9 respectively). Multivariate analysis confirmed the predictive significance of miR-425-3p. Furthermore, an association between increased miR-425-3p, cell death and reduced cell motility was defined in vitro in HCC cell lines treated with sorafenib. CONCLUSIONS: Assessment of miR-425-3p levels in liver biopsies could help in stratifying patients with advanced HCC for sorafenib treatment. These promising results need to be confirmed in a large prospective study.
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Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , MicroARNs/metabolismo , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Niacinamida/uso terapéutico , SorafenibRESUMEN
INTRODUCTION: The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown. METHODS: We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types. RESULTS: Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis. CONCLUSIONS: Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.
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Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos/genética , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/genética , Naftoquinonas/farmacología , Empalme Alternativo , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Camptotecina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Daño del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Etopósido/farmacología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Células MCF-7 , Paclitaxel/farmacología , Receptor ErbB-2/metabolismo , Survivin , Proteína p53 Supresora de Tumor/genéticaRESUMEN
miR-296-5p is a central regulator of signalling pathways affecting development, stem cell differentiation and cancer. We hypothesized that miR-296-5p is involved in breast cancer onset and progression possibly through regulation of its target SCRIB (Scribble), a polarity protein recently implicated in the acquisition of cancer stem cell traits and in cell motility. We found that miR-296-5p levels were consistently reduced in human breast cancer tissues compared with non-neoplastic mammary parenchyma, and low expression of this miRNA predicted shorter disease-free survival independently of classic clinicopathological parameters. Further, reduced miR-296-5p levels were significantly correlated with an earlier spread of cancer in the overall series and with distant metastases in the subset. In contrast with its regulator, SCRIB was overexpressed and mislocalized in primary breast cancers or locoregional or distant metastatic lesions compared with normal parenchyma. Notably, SCRIB mislocalization was associated with overall survival, metastatic spread and organ tropism in patients with breast cancer. Finally, direct injection of a precursor miR-296-5p into tumours of a breast cancer xenograft model significantly decreased tumour growth. Our results show that the miR-296-5p/SCRIB axis plays a role in breast carcinogenesis and an miR-296-5p-based therapeutic approach hampers breast cancer tumour growth in vivo. Modulation of miR-296-5p may represent a new therapeutic option for patients with breast cancer.
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Neoplasias de la Mama/genética , Movimiento Celular/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , MicroARNs/genética , Persona de Mediana Edad , Transducción de Señal/fisiologíaRESUMEN
Adams-Oliver syndrome is a rare inherited condition characterized by scalp defects and limb abnormalities. It is caused by variants in different genes such as ARHGAP31. Here, we used an interdisciplinary approach to study a family with lower limb anomalies. We identified a novel variant in the ARHGAP31 gene that is predicted to result in a truncated protein with a constitutively activated catalytic site due to the loss of 688 amino acids involved in the C-terminal domain, essential for protein auto-inhibition. Pathogenic variants in ARHGAP31 exon 12, leading to a premature protein termination, are associated with Adams-Oliver syndrome. Bioinformatic analysis was useful to elucidate the impact of the identified genetic variant on protein structure. To better understand the impact of the identified variant, 3D protein models were predicted for the ARHGAP31 wild type, the newly discovered variant, and other pathogenetic alterations already reported. Our study identified a novel variant probably involved in Adams-Oliver syndrome and increased the evidence on the phenotypic variability in patients affected by this syndrome, underlining the importance of translational research, including experimental and bioinformatics analyses. This strategy represents a successful model to investigate molecular mechanisms involved in syndrome occurrence.
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Displasia Ectodérmica , Proteínas Activadoras de GTPasa , Deformidades Congénitas de las Extremidades , Fosfoproteínas , Dermatosis del Cuero Cabelludo , Femenino , Humanos , Masculino , Displasia Ectodérmica/genética , Displasia Ectodérmica/patología , Proteínas Activadoras de GTPasa/genética , Deformidades Congénitas de las Extremidades/genética , Mutación , Linaje , Fenotipo , Dermatosis del Cuero Cabelludo/genética , Dermatosis del Cuero Cabelludo/congénito , Dermatosis del Cuero Cabelludo/patologíaRESUMEN
Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial-stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.
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Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Biopsia , Forma de la Célula , Supervivencia Celular , Perfilación de la Expresión Génica , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.
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Polaridad Celular , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Supresoras de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Neoplasias/genética , Análisis de Matrices Tisulares , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Despite significant improvement in screening programs for cancers of the respiratory district, especially in at-risk subjects, early disease detection is still a major issue. In this scenario, new molecular and non-invasive biomarkers are needed to improve early disease diagnosis. METHODS: We profiled the miRNome in exhaled breath condensate (EBC) and plasma samples from fourteen patients affected by lung AdCa, nine healthy subjects. miRNA signatures were then analyzed in another neoplasia of the respiratory district, i.e. pleural mesothelioma (n = 23) and subjects previously exposed to asbestos were used as controls for this cohort (n = 19). Selected miRNAs were analyzed in purified pulmonary neoplastic or normal epithelial and stromal cell subpopulation from AdCa patients. Finally, the plasmatic miRNA signature was analyzed in a publicly available cohort of NSCLC patients for data validation and in silico analysis was performed with predicted miRNA targets using the multiMiR tool and STRING database. RESULTS: miR-597-5p and miR-1260a are significantly over-expressed in EBC from lung AdCa and are associated with AdCa. Similarly, miR-1260a is also up-regulated in the plasma of AdCa patients together with miR-518f-3p and correlates with presence of lung cancer, whereas let-7f-5p is under-expressed. Analysis of these circulating miRNAs in pleural mesothelioma cases confirmed that up-regulation of miR-518f-3p, -597-5p and -1260a, is specific for lung AdCa. Lastly, quantification of the miRNAs in laser-assisted microdissected lung tissues revealed that miR-518f-3p, 597-5p and miR-1260a are predominantly expressed in tumor epithelial cells. Validation analysis confirmed miR-518f-3p as a possible circulating biomarker of NSCLC. In silico analysis of the potentially modulated biological processes by these three miRNAs, shows that tumor bioenergetics are the most affected pathways. CONCLUSIONS: Overall, our data suggest a 3-miRNAs signature as a non-invasive and accurate biomarker of lung AdCa. This approach could supplement the current screening approaches for early lung cancer diagnosis.
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BACKGROUND: Breast cancers that harbor mismatch-repair (MMR) deficiency and/or microsatellite instability (MSI) might be sensitive to immune checkpoint blockade, but there are currently no specific guidelines for assessing MMR status in breast cancer. Here, we sought to define the clinical value of MMR immunohistochemistry (IHC) and MSI analysis in breast cancers. METHODS: We subjected 444 breast cancers to MMR IHC and MSI analysis. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and MMR-heterogeneous (hMMR) based on the loss of immunoreactivity; MSI was defined by instability in the five indicators recommended by the National Cancer Institute for endometrial and colorectal cancers. Correlation of MMR status with patients' survival was assessed using the Kaplan-Meier estimator. Statistical tests were two-sided. RESULTS: Loss of MMR proteins was homogeneous (dMMR) in 75 patients (17%) and heterogeneous (hMMR) in 55 (12%). Among luminal breast cancers, there were similar frequencies of dMMR and hMMR tumors. Overall, the rate of discrepancy between IHC and MSI analysis was high (91%). Women with Luminal B-like dMMR carcinomas (n = 44) showed shorter overall survival (median = 77 months, range = 0-115 months) than those with pMMR (n = 205) or hMMR (n = 35) tumors (median = 84 months, range = 0-127 months) (P = .008). On the contrary, patients with estrogen receptor-negative breast cancers treated with chemotherapy lived longer in cases of dMMR (n = 9) than pMMR (n = 33) or hMMR (n = 7) tumors, with 87 months of median survival (range = 73-123 months) for the former compared with 79 months (range = 8-113 months) for the latter two categories (P < .001). CONCLUSIONS: Immunohistochemistry and MSI are not interchangeable tests in breast carcinomas. MMR protein loss is a more common event than MSI and shows intra-tumor heterogeneity. MMR IHC allows the identification of clinically relevant subclasses of breast cancer patients, provided that multiple areas of the tumor are analyzed.
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Lung cancer is the leading cause of tumor-related death worldwide and more efforts are needed to elucidate lung carcinogenesis. Here we investigated the expression of 641 miRNAs in lung tumorigenesis in a K-Ras(+/LSLG12Vgeo);RERTn(ert/ert) mouse model and 113 human tumors. The conserved miRNA cluster on chromosome 12qF1 was significantly and progressively upregulated during murine lung carcinogenesis. In particular, miR-494-3p expression was correlated with lung cancer progression in mice and with worse survival in lung cancer patients. Mechanistically, ectopic expression of miR-494-3p in A549 lung cancer cells boosted the tumor-initiating population, enhanced cancer cell motility, and increased the expression of stem cell-related genes. Importantly, miR-494-3p improved the ability of A549 cells to grow and metastasize in vivo, modulating NOTCH1 and PTEN/PI3K/AKT signaling.Overall, these data identify miR-494-3p as a key factor in lung cancer onset and progression and possible therapeutic target.
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Transformación Celular Neoplásica/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Células A549 , Animales , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Genes ras , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Ratones Desnudos , Ratones Transgénicos , MicroARNs/metabolismo , Mutación , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Células de Población Lateral/metabolismo , Células de Población Lateral/patología , Transducción de Señal , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
BACKGROUND: Tumor epigenetic defects are of increasing relevance to clinical practice, because they are 'druggable' targets for cancer therapy using chromatin-remodeling agents (CRAs). New evidences highlight the importance of the microenvironment on the epigenome regulation and the need to use culture models able to preserve tissue morphology, to better understand the action of CRAs. Methods & methods: We studied the epigenetic response induced by culturing and CRAs in a preclinical model, preserving ex vivo the original tissue microenvironment and morphology, assessing different epigenetic signatures. Our overall findings suggest that culturing and CRAs cause heterogeneous effects on the genes methylation; CRAs affect the global DNA methylation and can trigger an active DNA demethylation; the culture induces alterations in the histone deacetylase expression. CONCLUSION: Despite the limited number of cases, these findings can be considered a proof of concept of the possibility to test CRAs epigenetic effects on ex vivo tissues maintained in their native tissue architecture.
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Ensamble y Desensamble de Cromatina/efectos de los fármacos , Epigénesis Genética , Neoplasias Pulmonares/genética , Pulmón/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/secundario , Epigénesis Genética/efectos de los fármacos , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Pulmón/citología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Regiones Promotoras Genéticas , Microambiente TumoralRESUMEN
INTRODUCTION: Lung cancer remains the leading cause of tumor-related deaths, despite advances in the understanding of the disease pathogenesis and in its clinical treatment. It is crucial to develop novel technologies to discover disease biomarkers and predict individual therapy response. MATERIALS AND METHODS: We established 48 patients-derived tumor xenografts (PDTXs) implanted in the subrenal capsule of immunodeficient mice using thin, precision-cut tumor tissue slices, derived from five patients affected by non-small cell lung cancer. Twenty-six tissue slices were immediately processed and implanted at sample recovery [patients-derived tumor xenografts derived from fresh tissue (dPDTX)], whereas the remaining sections were cultured on specific organotypic supports at 37°C and 5% CO2 for 24 h before grafting [patients-derived tumor xenografts derived from cultured tissue (cPDTX)]. At sacrifice, xenografts tissue morphology, proliferation (Ki67), and histotype markers were analyzed. Oncogenic miRNAs profiles were assessed in PDTXs, human tumors, and serum from one patient. RESULTS: Xenografts retained the original cancer features and there were no differences between dPDTXs and cPDTXs. Squamous cell carcinoma (SCC) xenografts showed a higher engraftment rate than adenocarcinoma (AC)-derived tumors. At basal time, Ki67 levels were higher in SCCs than in ACs, and the expression levels of genes associated to a stem cell-like phenotype were also more expressed in SCC samples. The analysis of oncogenic miRNAs showed that circulating miR-19b, -21, and -210 levels were correlated with higher Ki67 expression in xenografts. CONCLUSION: Our study implemented the PDTX model with thin, precision-cut tumor slices from small tumors, which could be useful for clinical applications and predictive purposes. The different engraftment success is likely determined by tumor histotype, high proliferation index, and the expression of genes essential for cancer stem cells maintenance. Our PDTXs model could be a valid tool to expand primary tumors for the discovery of new biomarkers and explore therapeutic options.
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BACKGROUND: Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. METHODS: We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4-5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. RESULTS: PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). CONCLUSIONS: Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial adaptation can dramatically improve on the efficacy of PI3K therapy in the clinic.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Reprogramación Celular , Resistencia a Antineoplásicos , Elafina/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Guanidinas/farmacología , Mitocondrias/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Ciclofilinas/efectos de los fármacos , Ciclofilinas/metabolismo , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Guanidinas/uso terapéutico , Humanos , Huésped Inmunocomprometido , Ratones , Fosforilación/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. RESULTS: In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras(v12) transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II). CONCLUSIONS: Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.
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Metastatic traits seem to be acquired by transformed cells with progenitor-like cancer-initiating properties, but there remains little mechanistic insight into this linkage. In this report, we show that the polarity protein Numbl, which is expressed normally in neuronal progenitors, becomes overexpressed and mislocalized in cancer cells from a variety of human tumors. Numbl overexpression relies on loss of the tumor suppressor miRNA-296-5p (miR-296), which actively represses translation of Numbl in normal cells. In turn, deregulated expression of Numbl mediates random tumor cell migration and invasion, blocking anoikis and promoting metastatic dissemination. In clinical specimens of non-small cell lung cancer, we found that Numbl overexpression correlated with a reduction in overall patient survival. Mechanistically, Numbl-mediated tumorigenesis involved suppression of a "stemness" transcriptional program driven by the stem cell programming transcription factor Klf4, thereby preserving a pool of progenitor-like cells in lung cancer. Our results reveal that Numbl-Klf4 signaling is critical to maintain multiple nodes of metastatic progression, including persistence of cancer-initiating cells, rationalizing its therapeutic exploitation to improve the treatment of advanced lung cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Transducción de Señal , Animales , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/genética , Análisis por Conglomerados , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , MicroARNs/química , MicroARNs/genética , Metástasis de la Neoplasia , Células Madre Neoplásicas/metabolismo , Interferencia de ARN , Transcripción GenéticaRESUMEN
Survivin is an oncogene that functions in cancer cell cytoprotection and mitosis. Here we report that differential expression in cancer cells of a C-terminal splice variant of survivin, termed survivin-ΔEx3, is tightly associated with aggressive disease and markers of unfavorable prognosis. In contrast to other survivin variants, survivin-ΔEx3 localized exclusively to nuclei in tumor cells and was phosphorylated at multiple residues by the checkpoint kinase Chk2 during DNA damage. Mutagenesis of the Chk2 phosphorylation sites enhanced the stability of survivin-ΔEx3 in tumor cells, inhibited the expression of phosphorylated H2AX (γH2AX) in response to double-strand DNA breaks, and impaired growth after DNA damage. DNA damage induced Chk2 phosphorylation, stabilization of p53, induction of the cyclin-dependent kinase inhibitor p21, and homologous recombination-induced repair were not affected. In vivo, active Chk2 was detected at the earliest stages of the colorectal adenoma-to-carcinoma transition, persisted in advanced tumors, and correlated with increased survivin expression. Together, our findings suggest that Chk2-mediated phosphorylation of survivin-ΔEx3 contributes to a DNA damage-sensing checkpoint that may affect cancer cell sensitivity to genotoxic therapies.