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Background: Identification of cancer metastasis-relevant molecular networks is desired to provide the basis for understanding and developing intervention strategies. Here we address the role of GIPC1 in the process of MACC1-driven metastasis. MACC1 is a prognostic indicator for patient metastasis formation and metastasis-free survival. MACC1 controls gene transcription, promotes motility, invasion and proliferation of colon cancer cells in vitro, and causes tumor growth and metastasis in mice. Methods: By using yeast-two-hybrid assay, mass spectrometry, co-immunoprecipitation and peptide array we analyzed GIPC1 protein binding partners, by using the MACC1 gene promoter and chromatin immunoprecipitation and electrophoretic mobility shift assay we probed for GIPC1 as transcription factor. We employed GIPC1/MACC1-manipulated cell lines for in vitro and in vivo analyses, and we probed the GIPC1/MACC1 impact using human primary colorectal cancer (CRC) tissue. Results: We identified MACC1 and its paralogue SH3BP4 as protein binding partners of the protein GIPC1, and we also demonstrated the binding of GIPC1 as transcription factor to the MACC1 promoter (TSS to -60 bp). GIPC1 knockdown reduced endogenous, but not CMV promoter-driven MACC1 expression, and diminished MACC1-induced cell migration and invasion. GIPC1 suppression reduced tumor growth and metastasis in mice intrasplenically transplanted with MACC1-overexpressing CRC cells. In human primary CRC specimens, GIPC1 correlates with MACC1 expression and is of prognostic value for metastasis formation and metastasis-free survival. Combination of MACC1 and GIPC1 expression improved patient survival prognosis, whereas SH3BP4 expression did not show any prognostic value. Conclusions: We identified an important, dual function of GIPC1 - as protein interaction partner and as transcription factor of MACC1 - for tumor progression and cancer metastasis.
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Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.
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Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.
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In colorectal cancer (CRC), the prevalence of NRAS mutations (5-9%) is inferior to that of KRAS mutations (40-50%). NRAS mutations feature lately during tumour progression and drive resistance to anti-EGFR therapy in KRAS wild-type tumours. To elucidate specific functions of NRAS mutations in CRC, we expressed doxycycline-inducible G12D and Q61K mutations in the CRC cell line Caco-2. A focused phospho-proteome analysis based on the Bio-Plex platform, which interrogated the activity of MAPK, PI3K, mTOR, STAT, p38, JNK and ATF2, did not reveal significant differences between Caco-2 cells expressing NRASG12D, NRASQ61K and KRASG12V. However, phenotypic read-outs were different. The NRAS Q61K mutation promoted anchorage-independent proliferation and tumorigenicity, similar to features driven by canonical KRAS mutations. In contrast, expression of NRASG12D resulted in reduced proliferation and apoptosis. At the transcriptome level, we saw upregulation of cytokines and chemokines. IL1A, IL11, CXCL8 (IL-8) and CCL20 exhibited enhanced secretion into the culture medium. In addition, RNA sequencing results indicated activation of the IL1-, JAK/STAT-, NFκB- and TNFα signalling pathways. These results form the basis for an NRASG12D-driven inflammatory phenotype in CRC.
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Neoplasias Colorretais/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Apoptose , Células CACO-2 , Proliferação de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/fisiopatologia , Citocinas/genética , Citocinas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Mutação , Oncogenes , Transdução de SinaisRESUMO
Patient-derived xenograft (PDX) tumor models represent a valuable platform for identifying new biomarkers and novel targets, to evaluate therapy response and resistance mechanisms. This study aimed at establishment, characterization and therapy testing of colorectal carcinoma-derived PDX. We generated 49 PDX and validated identity between patient tumor and corresponding PDX. Sensitivity of PDX toward conventional and targeted drugs revealed that 92% of PDX responded toward irinotecan, 45% toward 5-FU, 65% toward bevacizumab, and 61% toward cetuximab. Expression of epidermal growth factor receptor (EGFR) ligands correlated to the sensitivity toward cetuximab. Proto-oncogene B-RAF, EGFR, Kirsten rat sarcoma virus oncogene homolog gene copy number correlated positively with cetuximab and erlotinib sensitivity. The mutational analyses revealed an individual mutational profile of PDX and mainly identical profiles of PDX from primary tumor vs corresponding metastasis. Mutation in PIK3CA was a determinant of accelerated tumor doubling time. PDX with wildtype Kirsten rat sarcoma virus oncogene homolog, proto-oncogene B-RAF, and phosphatidylinositol-4,5-bisphosphate 3-kinaseM catalytic subunit alfa showed higher sensitivity toward cetuximab and erlotinib. To study the molecular mechanism of cetuximab resistance, cetuximab resistant PDX models were generated, and changes in HER2, HER3, betacellulin, transforming growth factor alfa were observed. Global proteome and phosphoproteome profiling showed a reduction in canonical EGFR-mediated signaling via PTPN11 (SHP2) and AKT1S1 (PRAS40) and an increase in anti-apoptotic signaling as a consequence of acquired cetuximab resistance. This demonstrates that PDX models provide a multitude of possibilities to identify and validate biomarkers, signaling pathways and resistance mechanisms for clinically relevant improvement in cancer therapy.
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Antineoplásicos/farmacologia , Biomarcadores Tumorais , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Xenoenxertos , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos , Imunofluorescência , Perfilação da Expressão Gênica , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Camundongos , Terapia de Alvo Molecular , Mutação , Medicina de Precisão/métodos , Proteoma , Proteômica , Proto-Oncogene Mas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide and is primarily treated with radiation, surgery, and platinum-based drugs like cisplatin and carboplatin. The major challenge in the treatment of NSCLC patients is intrinsic or acquired resistance to chemotherapy. Molecular markers predicting the outcome of the patients are urgently needed. METHODS: Here, we employed patient-derived xenografts (PDXs) to detect predictive methylation biomarkers for platin-based therapies. We used MeDIP-Seq to generate genome-wide DNA methylation profiles of 22 PDXs, their parental primary NSCLC, and their corresponding normal tissues and complemented the data with gene expression analyses of the same tissues. Candidate biomarkers were validated with quantitative methylation-specific PCRs (qMSP) in an independent cohort. RESULTS: Comprehensive analyses revealed that differential methylation patterns are highly similar, enriched in PDXs and lung tumor-specific when comparing differences in methylation between PDXs versus primary NSCLC. We identified a set of 40 candidate regions with methylation correlated to carboplatin response and corresponding inverse gene expression pattern even before therapy. This analysis led to the identification of a promoter CpG island methylation of LDL receptor-related protein 12 (LRP12) associated with increased resistance to carboplatin. Validation in an independent patient cohort (n = 35) confirmed that LRP12 methylation status is predictive for therapeutic response of NSCLC patients to platin therapy with a sensitivity of 80% and a specificity of 84% (p < 0.01). Similarly, we find a shorter survival time for patients with LRP12 hypermethylation in the TCGA data set for NSCLC (lung adenocarcinoma). CONCLUSIONS: Using an epigenome-wide sequencing approach, we find differential methylation patterns from primary lung cancer and PDX-derived cancers to be very similar, albeit with a lower degree of differential methylation in primary tumors. We identify LRP12 DNA methylation as a powerful predictive marker for carboplatin resistance. These findings outline a platform for the identification of epigenetic therapy resistance biomarkers based on PDX NSCLC models.
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Biomarcadores Tumorais/genética , Carboplatina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA/genética , Epigenômica , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores Tumorais/metabolismo , Carboplatina/farmacologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Genes Supressores de Tumor , Genoma Humano , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neoplasias Pulmonares/genética , Camundongos Nus , Regiões Promotoras Genéticas , Resultado do TratamentoRESUMO
Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. Translational activation of proto-oncogenes through loss-of-uORF mutations has been demonstrated, yet a systematic search for cancer-associated genetic alterations in uORFs is lacking. Here, we applied a PCR-based, multiplex identifier-tagged deep sequencing approach to screen 404 uORF translation initiation sites of 83 human tyrosine kinases and 49 other proto-oncogenes in 308 human malignancies. We identified loss-of-function uORF mutations in EPHB1 in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma. Both mutations were associated with enhanced translation, suggesting that loss-of-uORF-mediated translational induction of the downstream main protein coding sequence may have contributed to carcinogenesis. Computational analysis of whole exome sequencing datasets of 464 colon adenocarcinomas subsequently revealed another 53 non-recurrent somatic mutations functionally deleting 22 uORF initiation and 31 uORF termination codons, respectively. These data provide evidence for somatic mutations affecting uORF initiation and termination codons in human cancer. The insufficient coverage of uORF regions in current whole exome sequencing datasets demands for future genome-wide analyses to ultimately define the contribution of uORF-mediated translational deregulation in oncogenesis.
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Carcinogênese/genética , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , Fases de Leitura Aberta , Proto-Oncogenes , Regiões 5' não Traduzidas , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Códon de Terminação , Genes Reporter , Estudo de Associação Genômica Ampla , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Luciferases/genética , Luciferases/metabolismo , MAP Quinase Quinase 6 , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Iniciação Traducional da Cadeia Peptídica , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptor EphB1/genética , Receptor EphB1/metabolismoRESUMO
PURPOSE: Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults. The epigenetically active ribonucleoside analog 5-azacitidine is a new therapy option that changes tumor cell chromatin, which is frequently modified by methylation and deacetylation in malignant gliomas. METHODS: In vitro, we analyzed cell viability, cell apoptosis, and migration of human GBM cells. In vivo, we established subcutaneous and intracerebral GBM mouse models originating from U87MG, U373MG, and primary GBM cells as well as one patient-derived xenograft. Xenografts were treated with 5-azacitidine as well as valproic acid, bevacizumab, temozolomide, and phosphate buffered saline. The tumor sizes and Ki67 proliferation indices were determined. Glioma angiogenesis was examined immunohistochemically by expression analysis of endothelial cells (CD31) and pericytes (PDGFRß). RESULTS: In vitro, 5-azacitidine treatment significantly reduced human glioblastoma cell viability, increased cellular apoptosis, and reduced cellular migration. In vivo, 5-azacitidine significantly reduced growth in two intracerebral GBM models. Notably, this was also shown for a xenograft established from a patient surgery sample; whereas, epigenetically acting valproic acid did not show any growth reduction. Highly vascularized tumors responded to treatment, whereas low-vascularized xenografts showed no response. Furthermore, intracerebral glioblastomas treated with 5-azacitidine showed a clearly visible reduction of tumor angiogenesis and lower numbers of endothelial cells and tumor vessel pericytes. CONCLUSIONS: Our data show significant growth inhibition as well as antiangiogenic effects in intracerebral as well as patient-derived GBM xenografts. This encourages to investigate in detail the multifactorial effects of 5-azacitidine on glioblastomas.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Apoptose/efeitos dos fármacos , Azacitidina/administração & dosagem , Azacitidina/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glioblastoma/patologia , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Carga Tumoral/efeitos dos fármacosRESUMO
Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer.Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors.Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP.Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR.
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Neoplasias da Mama/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Resistencia a Medicamentos Antineoplásicos , Processamento de Proteína Pós-Traducional , Receptores de Estrogênio/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The European NCI compounds programme, a joint initiative of the EORTC Research Branch, Cancer Research Campaign and the US National Cancer Institute, was initiated in 1993. The objective was to help the NCI in reducing the backlog of in vivo testing of potential anticancer compounds, synthesised in Europe that emerged from the NCI in vitro 60-cell screen. METHODS: Over a period of more than twenty years the EORTC-Cancer Research Campaign panel reviewed â¼2000 compounds of which 95 were selected for further evaluation. Selected compounds were stepwise developed with clear go/no go decision points using a pharmacologically directed programme. RESULTS: This approach eliminated quickly compounds with unsuitable pharmacological properties. A few compounds went into Phase I clinical evaluation. The lessons learned and many of the principles outlined in the paper can easily be applied to current and future drug discovery and development programmes. CONCLUSIONS: Changes in the review panel, restrictions regarding numbers and types of compounds tested in the NCI in vitro screen and the appearance of targeted agents led to the discontinuation of the European NCI programme in 2017 and its transformation into an academic platform of excellence for anticancer drug discovery and development within the EORTC-PAMM group. This group remains open for advice and collaboration with interested parties in the field of cancer pharmacology.
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Antineoplásicos/uso terapêutico , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Pesquisa , União Europeia , Humanos , National Cancer Institute (U.S.) , Estados UnidosRESUMO
Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts.
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Carcinoma Basocelular/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Carboplatina/farmacologia , Carcinoma Basocelular/enzimologia , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Análise Mutacional de DNA , Docetaxel , Receptores ErbB/genética , Everolimo/farmacologia , Fluoruracila/farmacologia , Expressão Gênica , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos NOD , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Taxoides/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) as well as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea).
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Metilação de DNA , Genômica/métodos , Análise de Sequência de DNA/métodos , Animais , Teorema de Bayes , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Camundongos , Regiões Promotoras Genéticas , Sulfitos , Fluxo de TrabalhoRESUMO
Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment-refractory metastatic colorectal cancer. In clinical studies, steady-state exposure to the M-2 and M-5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M-2, and M-5 in vitro and in murine xenograft models. M-2 and M-5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell-based assays. In murine xenograft models, oral regorafenib, M-2, and M-5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M-2 and M-5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M-2, and M-5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M-2, but not M-5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M-2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.
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Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Inibidores da Angiogênese/farmacocinética , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Metaboloma , Metabolômica/métodos , Camundongos , Compostos de Fenilureia/farmacocinética , Ligação Proteica , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/metabolismo , Piridinas/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The anti-cancer drug oxaliplatin (OxP) has rarely been used to treat breast carcinoma, as it cannot cross the BBB to treat the frequently subsequent brain metastases. Here, we encapsulated OxP in liposomes prepared to reduce side effects and to simultaneously treat primary tumor and brain metastasis. The angiopep LRP-receptor ligand was bound to the vesicular surface for targeting. Targeted and non-targeted OxP liposomes were tested in vitro (binding, uptake, and transcytosis) and in vivo. Liposomes contained 0.65 mg OxP/mL, their mean diameter was 165 nm, and they released 50% of OxP within 8 days at 4 degrees C and within 22 h at 36 degrees C. MDCK cells were used for uptake and transcytosis quantification. Compared to non-targeted liposomes, targeted liposomes showed 12-fold greater uptake, and 2.25-fold higher transcytosis. In vivo efficacy was tested using human MT-3 breast cancer cells transplanted subcutaneously and intracerebrally into female nude mice, and tumor growth inhibition was measured. OxP was injected (6 mg OxP/kg) four times. The best results were obtained with targeted liposomes (T/C: 21% for subcutaneous and 50% for intracerebral). OxP liposomes with a fluid membrane all inhibited MT-3 tumors significantly better than free OxP, with no significant difference between targeted and non-targeted liposomes. The therapeutic effect was accompanied with strong leukopenia and mild thrombocytopenia with all formulations. The newly developed OxP liposomes significantly improved the treatment of subcutaneously and intracerebrally growing breast cancer, but the targeted angiopep-equipped liposomes showed no superior effect in vivo.
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Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias da Mama/tratamento farmacológico , Lipossomos/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Compostos Organoplatínicos/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Difusão , Feminino , Humanos , Metalotioneína 3 , Camundongos , Camundongos Nus , Terapia de Alvo Molecular/métodos , Compostos Organoplatínicos/química , Oxaliplatina , Resultado do TratamentoRESUMO
OBJECTIVES: We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis. DESIGN: We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/ß-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems. RESULTS: Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of ß-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways. CONCLUSIONS: We identified new direct Wnt/ß-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Movimento Celular/genética , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas , Proteínas Nucleares/genética , Via de Sinalização Wnt/genética , Animais , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas Experimentais , Camundongos , Metástase Neoplásica , Proteínas de Ligação a RNA , Células Tumorais CultivadasRESUMO
Three strategies to sample volatile organic compounds (VOC) from lung cancer cell lines cultured in vitro were compared. Headspace solid phase microextraction was applied in situ to culture flasks and alternatively to subsamples of headspace gas or to nutrient solution subsamples followed by gas chromatography-mass spectrometry. The direct quantification of 55 VOC in the headspace of cell cultures was validated and is discussed with respect to reproducibility and system-related interferences. The role of the VOC background from culture media and usually employed polystyrene culture vessels is examined and was seen to invoke potentially misleading conclusions. The commercial A549 and two further adenocarcinoma cell lines displayed largely similar VOC profiles with distinct differences regarding certain individual substances. There is evidence for the inappropriateness of the standard cell culturing methods in the search for volatile cancer markers.
Assuntos
Adenocarcinoma/metabolismo , Microextração em Fase Sólida/métodos , Compostos Orgânicos Voláteis/análise , Análise de Variância , Linhagem Celular Tumoral , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Anaplastic large cell lymphoma (ALCL) is a distinct entity of T-cell lymphoma that can be divided into 2 subtypes based on the presence of translocations involving the ALK gene (ALK(+) and ALK(-) ALCL). The interferon regulatory factor 4 (IRF4) is known to be highly expressed in both ALK(+) and ALK(-) ALCLs. However, the role of IRF4 in the pathogenesis of these lymphomas remains unclear. Here we show that ALCLs of both subtypes are addicted to IRF4 signaling, as knockdown of IRF4 by RNA interference was toxic to ALCL cell lines in vitro and in ALCL xenograft mouse models in vivo. Gene expression profiling after IRF4 knockdown demonstrated a significant downregulation of a variety of known MYC target genes. Furthermore, our analyses revealed that MYC is a primary target of IRF4, identifying a novel regulatory mechanism of MYC expression and its target gene network in ALCL. MYC, itself, is essential for ALCL survival, as both knockdown of MYC and pharmacologic inhibition of MYC signaling were toxic to ALCL cell lines. Collectively, our results demonstrate that ALCLs are dependent on IRF4 and MYC signaling and that MYC may represent a promising target for future therapies.