221 research outputs found

    Identification of signaling pathways related to drug efficacy in hepatocellular carcinoma via integration of phosphoproteomic, genomic and clinical data

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    Hepatocellular Carcinoma (HCC) is one of the leading causes of death worldwide, with only a handful of treatments effective in unresectable HCC. Most of the clinical trials for HCC using new generation interventions (drug-targeted therapies) have poor efficacy whereas just a few of them show some promising clinical outcomes [1]. This is amongst the first studies where the mode of action of some of the compounds extensively used in clinical trials is interrogated on the phosphoproteomic level, in an attempt to build predictive models for clinical efficacy. Signaling data are combined with previously published gene expression and clinical data within a consistent framework that identifies drug effects on the phosphoproteomic level and translates them to the gene expression level. The interrogated drugs are then correlated with genes differentially expressed in normal versus tumor tissue, and genes predictive of patient survival. Although the number of clinical trial results considered is small, our approach shows potential for discerning signaling activities that may help predict drug efficacy for HCC.National Institutes of Health (U.S.) (Grant U54-CA119267)National Institutes of Health (U.S.) (Grant R01-CA96504

    Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC

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    Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells

    Clinical application of genomics- and phosphoproteomics-based selection of targeted therapy in patients with advanced solid tumors

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    Precision oncology has come a long way since the introduction of the first targeted drug (trastuzumab) in 1999. Broad molecular testing of tumor tissue has vastly expanded our knowledge of the biology of cancer, leading to a steep increase in the number of approved targeted drugs and an expansion of the labeled indications of these drugs. Off-label use of these new classes of targeted drugs is nowadays better documented and often performed in clinical trials to maximize the learning potential of these experimental treatments for the medical community. As long as no “cure for cancer” exists, there will be room for improvement of our knowledge and approach to treating patients with cancer. General improvements in the logistics, availability of targeted drugs and access to diagnostics and expertise will likely have the greatest impact on direct benefit for patients. In the future, standardized processing and conservation of tumor tissue/biopsies should be possible in all healthcare facilities, and collaborations and sharing of knowledge and resources with the academic institutes will be viable to delivering precision oncology to all patients. If these conditions are met, more patients may potentially benefit from the knowledge and new treatment options resulting from the precision oncology trials. Also, medical oncologists may learn more about molecular testing and interpreting test results from participation in MTBs. To maximize the impact of precision oncology, international collaborations are of utmost importance and research groups throughout the world are encouraged to share best practices and creative solutions to overcome the hurdles that still hamper new initiatives in the field today. Future clinical research may focus on prospective therapy selection using molecular information from other –omics fields, such as phosphoproteomics, especially in patients where no clear monogenetic driver mutations is identified and a comprehensive pathway analysis may give more direction for potential therapeutic strategies. More knowledge on the best method of prioritizing targets for treatments will be essential, as well as clinical trials investigating new combinations of targeted agents. With an increasing understanding of cancer biology and improved strategies for treatment selection, precision oncology will be accessible for patients with advanced cancer and more patients will benefit from the knowledge that we gain today and tomorrow. In the future, treatments based on histology alone may be considered old-fashioned, and multi-omics diagnostics may result in a comprehensible report that can be easily interpreted, and will directly guide treatment decisions for individual patients

    In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple- negative breast cancer

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    Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-03-2017Triple-negative breast cancer (TNBC) is am immunohistochemically-defined breast cancer subtype negative for ER, PR and HER2 expression, with dismal prognosis that lacks prognostic and predictive markers. Since all the aberrations that exist and ultimately contribute to the tumor phenotype converge, from a functional point of view, in the final status of the phosphorylation of the proteome in a given moment, we sought to interrogate the phosphoproteome with two aims: 1) establish a taxonomy of TNBC based on measurable markers that predict clinical course; 2) reduce the phosphoproteome that characterizes the bad- from good-prognosis cases to its targetable, driving kinases, in order to define rational therapeutic approaches in TNBC. Therefore we performed mass-spectrometry based quantitative phosphoproteomics from a training set of 34 frozen patient tumor samples paired by tumor size (T), nodal status (N), and grade (G). 13 patients relapsed in less than 3 years and 21 patients were relapse-free for more than 10 years follow up. Moreover in our study we included ten triple-negative cell lines, 3 cell lines that killed recipient mice in less than 100 days and 7 indolent cell lines (no death in more than 400 days). In our study we were able to identify and quantify more than 17000 unique phosphopeptides (pPs) in the patient samples and cell lines. Differential analysis showed that 161 pPs were up-regulated in the relapsed vs non-relapsed group. Furthermore the hyperactivity of 11 kinases accounted for the pPs upregulated in the relapsed group. A mass-spectrometry-toimmunohistochemistry translation step in 113 consecutive TNBC cases, spotted in TMAs, with 14 years follow up revealed that 6/11 kinases (PRKCE, KIT, PNKP, ERK, CDK6 and P70S6K) preserved independent prognostic value. The kinases split the validation set into two patterns: one (0/6 hyperactivated kinases; 29% of the patients) associated to 93.5 % cure rate. The other (≥1 hyperactivated kinase; 37 subpatterns) was associated to a 9.5-fold higher relapse risk. The 15 possible 2-drug combinations targeting these kinases were synergistic in vitro and in vivo. Our results elucidate novel phosposites/kinases implicated in TNBC and provide a ready to use, actionable targetsbased clinical classification system for TNBC.El cáncer de mama triple negativo (TNBC) es un subtipo de cáncer de mama definido inmunohistoquimicamente como negativo para la expresión de ER, PR y HER2, además carece de marcadores pronósticos y predictivos lo que hace que presente un mal pronóstico en general. Dado que todas las aberraciones genómicas presentes en el tumor contribuyen en el fenotipo tumoral y convergen, desde un punto de vista funcional, en el estado final de la fosforilación del proteoma en un momento dado, se intentó interrogar al fosfoproteoma con dos objetivos: 1) establecer una taxonomía de TNBC basado en marcadores medibles capaces de predecir el curso clínico; 2) simplificar el fosfoproteoma que distingue a pacientes con peor y mejor pronóstico llevado a cabo por las quinasas, con el fin de definir distintos enfoques terapéuticos en el TNBC. Por lo tanto hemos realizado un ánalisis fosfoproteómico cuantitativo, por espectrómetría de masas en un grupo de 34 muestras de pacientes, agrupadas por grado (G), tamaño del tumor (T) y estado de los ganglios (N). 13 de los pacientes recayeron en menos de 3 años y los otros 21 no presentaron recaida en más de 10 años de seguimiento. Además en nuestro estudio se incluyeron diez líneas celurares triple negativas. La inyeccion de 3 de estas líneas celurares en ratones receptores fueron capaces de matarlos en menos de 100 días considerándose líneas agresivas, mientras que las 7 líneas celulares restantes no fueron capaces de matar a los ratones receptores en mas de 400 días y por ello se consideraron no agresivas. En nuestro estudio hemos podido identificar y cuantificar más de 17000 fosfopeptidos únicos, (pPs) presentes en las muestras de pacientes y líneas celulares. El análisis diferencial demostró que 161 de estos pPs idetificados estaban hiperregulados en el grupo de pacientes que recaían comparados con el grupo de pacientes sin recaída. Además, se identificaron 11 kinasas hiperactivas responsables de la hiperregulación de estos pPs, en el grupo de los pacientes que recaian. La validación de los resultados obtenidos mediante espectrometría de masas se llevo a cabo en una neuva seríe de 113 muestras de casos de TNBC (con 14 años de seguimiento) que fueron incluidos en TMAs. Mediante técnicas inmunohistoquímicas observamos que 6 de las 11 kinasas identificadas (PRKCE, KIT, PNKP, ERK, CDK6 y P70S6K) conservaban un valor pronóstico independiente. Estas 6 kinasas son capaces de dividir el grupo de validación en dos patrones: uno asociado a la tasa de curación del 93,5% (0/6 kinasas hiperactivadas; 29% de los pacientes) y el otro asociado a un mayor riesgo de recaida 9.5-veces (≥1 kinasas hiperactivadas; 37 sub-patrones). El tratamiento con las 15 posibles combinaciones de 2 fármacos dirigidos contra estas kinasas, fueron sinergisticas en tratamientos vitro y en vivo. Nuestros resultados identifican nuevos sitios de fosforilación y kinasas implicadas en el cáncer de mama triple negativo, proporcionando un nuevo sistema de clasificación basado en estos marcadores y que podría ser utilizado en la práctica clínica

    MTOR cross-talk in cancer and potential for combination therapy

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    The mammalian Target of Rapamycin (mTOR) pathway plays an essential role in sensing and integrating a variety of exogenous cues to regulate cellular growth and metabolism, in both physiological and pathological conditions. mTOR functions through two functionally and structurally distinct multi-component complexes, mTORC1 and mTORC2, which interact with each other and with several elements of other signaling pathways. In the past few years, many new insights into mTOR function and regulation have been gained and extensive genetic and pharmacological studies in mice have enhanced our understanding of how mTOR dysfunction contributes to several diseases, including cancer. Single-agent mTOR targeting, mostly using rapalogs, has so far met limited clinical success; however, due to the extensive cross-talk between mTOR and other pathways, combined approaches are the most promising avenues to improve clinical efficacy of available therapeutics and overcome drug resistance. This review provides a brief and up-to-date narrative on the regulation of mTOR function, the relative contributions of mTORC1 and mTORC2 complexes to cancer development and progression, and prospects for mTOR inhibition as a therapeutic strategy

    Pharmacological approaches to understanding protein kinase signaling networks

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    Protein kinases play vital roles in controlling cell behavior, and an array of kinase inhibitors are used successfully for treatment of disease. Typical drug development pipelines involve biological studies to validate a protein kinase target, followed by the identification of small molecules that effectively inhibit this target in cells, animal models, and patients. However, it is clear that protein kinases operate within complex signaling networks. These networks increase the resilience of signaling pathways, which can render cells relatively insensitive to inhibition of a single kinase, and provide the potential for pathway rewiring, which can result in resistance to therapy. It is therefore vital to understand the properties of kinase signaling networks in health and disease so that we can design effective multi-targeted drugs or combinations of drugs. Here, we outline how pharmacological and chemo-genetic approaches can contribute to such knowledge, despite the known low selectivity of many kinase inhibitors. We discuss how detailed profiling of target engagement by kinase inhibitors can underpin these studies; how chemical probes can be used to uncover kinase-substrate relationships, and how these tools can be used to gain insight into the configuration and function of kinase signaling networks
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