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1.
Nature ; 553(7686): 96-100, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29258294

RESUMO

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.


Assuntos
Reprogramação Celular , Senescência Celular , Linfoma de Células B/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Feminino , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Via de Sinalização Wnt/efeitos dos fármacos
2.
EMBO J ; 35(22): 2399-2416, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27572462

RESUMO

Unfavorable patient survival coincides with lineage plasticity observed in human acute leukemias. These cases are assumed to arise from hematopoietic stem cells, which have stable multipotent differentiation potential. However, here we report that plasticity in leukemia can result from instable lineage identity states inherited from differentiating progenitor cells. Using mice with enhanced c-Myc expression, we show, at the single-cell level, that T-lymphoid progenitors retain broad malignant lineage potential with a high capacity to differentiate into myeloid leukemia. These T-cell-derived myeloid blasts retain expression of a defined set of T-cell transcription factors, creating a lymphoid epigenetic memory that confers growth and propagates myeloid/T-lymphoid plasticity. Based on these characteristics, we identified a correlating human leukemia cohort and revealed targeting of Jak2/Stat3 signaling as a therapeutic possibility. Collectively, our study suggests the thymus as a source for myeloid leukemia and proposes leukemic plasticity as a driving mechanism. Moreover, our results reveal a pathway-directed therapy option against thymus-derived myeloid leukemogenesis and propose a model in which dynamic progenitor differentiation states shape unique neoplastic identities and therapy responses.


Assuntos
Transdiferenciação Celular , Leucemia Mieloide/patologia , Células Progenitoras Linfoides/fisiologia , Linfócitos T/fisiologia , Animais , Humanos , Camundongos
3.
BMC Cancer ; 20(1): 427, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32408898

RESUMO

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application. METHODS: To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis. RESULTS: Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium. CONCLUSIONS: This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep.


Assuntos
Adenosina/análogos & derivados , Adenosil-Homocisteinase/genética , Apoptose , Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Linfoma de Células B/patologia , Adenosina/farmacologia , Proliferação de Células , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Células Tumorais Cultivadas
4.
Mod Pathol ; 32(6): 855-865, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30723296

RESUMO

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Neoplasias Colorretais/diagnóstico , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/patologia , Metástase Neoplásica/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adenocarcinoma de Pulmão/genética , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Feminino , Humanos , Neoplasias Pulmonares/genética , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade
5.
Brief Bioinform ; 18(5): 837-850, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27473063

RESUMO

Differential network analysis (DiNA) denotes a recent class of network-based Bioinformatics algorithms which focus on the differences in network topologies between two states of a cell, such as healthy and disease, to identify key players in the discriminating biological processes. In contrast to conventional differential analysis, DiNA identifies changes in the interplay between molecules, rather than changes in single molecules. This ability is especially important in cases where effectors are changed, e.g. mutated, but their expression is not. A number of different DiNA approaches have been proposed, yet a comparative assessment of their performance in different settings is still lacking. In this paper, we evaluate 10 different DiNA algorithms regarding their ability to recover genetic key players from transcriptome data. We construct high-quality regulatory networks and enrich them with co-expression data from four different types of cancer. Next, we assess the results of applying DiNA algorithms on these data sets using a gold standard list (GSL). We find that local DiNA algorithms are generally superior to global algorithms, and that all DiNA algorithms outperform conventional differential expression analysis. We also assess the ability of DiNA methods to exploit additional knowledge in the underlying cellular networks. To this end, we enrich the cancer-type specific networks with known regulatory miRNAs and compare the algorithms performance in networks with and without miRNA. We find that including miRNAs consistently and considerably improves the performance of almost all tested algorithms. Our results underline the advantages of comprehensive cell models for the analysis of -omics data.


Assuntos
Redes Reguladoras de Genes , Algoritmos , Biologia Computacional , Perfilação da Expressão Gênica , MicroRNAs
6.
Nature ; 501(7467): 421-5, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-23945590

RESUMO

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.


Assuntos
Autofagia , Senescência Celular , Glucose/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 12/metabolismo , Caspase 3/metabolismo , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Feminino , Linfoma de Células B/genética , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteólise , Estresse Fisiológico , Taxa de Sobrevida
7.
Genes Dev ; 25(20): 2137-46, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21979374

RESUMO

In malignancies, enhanced nuclear factor-κB (NF-κB) activity is largely viewed as an oncogenic property that also confers resistance to chemotherapy. Recently, NF-κB has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-κB. Using a mouse lymphoma model and analyzing transcriptome and clinical data from lymphoma patients, we show here that therapy-induced senescence presents with and depends on active NF-κB signaling, whereas NF-κB simultaneously promotes resistance to apoptosis. Further characterization and genetic engineering of primary mouse lymphomas according to distinct NF-κB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes guided us to identify Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL as a clinically relevant subgroup with significantly superior outcome when NF-κB is hyperactive. Our data illustrate the power of cross-species investigations to functionally test genetic mechanisms in transgenic mouse tumors that recapitulate distinct features of the corresponding human entity, and to ultimately use the mouse model-derived genetic information to redefine novel, clinically relevant patient subcohorts.


Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , NF-kappa B/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma não Hodgkin/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
8.
J Dtsch Dermatol Ges ; 17(8): 800-808, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31437373

RESUMO

BACKGROUND AND OBJECTIVES: Overall survival (OS) in patients with early-stage malignant melanoma differs. To date, there are no established prognostic markers. We aimed to contribute to a better understanding of potential prognostic immunohistochemical markers for risk stratification. PATIENTS AND METHODS: 161 surgically resected early-stage malignant melanomas (stage pT1 and pT2) were analyzed for expression of 20 different proteins using immunohistochemistry. The results were correlated with OS. The cohort was randomly split into a discovery and a validation cohort. RESULTS: High Bcl-2 expression, high nuclear S100A4 expression as well as a Ki67 proliferation index of ≥ 20 % were associated with shorter OS. Strong MITF immunoreactivity was a predictor for favorable prognosis. A combination of these four markers resulted in a multi-marker score with significant prognostic value in multivariate survival analysis (HR: 3.704; 95 % CI 1.484 to 9.246; p = 0.005). Furthermore, the score was able to differentiate a low-risk group with excellent OS rates (five-year survival rate: 100 %), an intermediate-risk group (five-year survival rate: 81.8 %) and a high-risk group (five-year survival rate: 52.6 %). The prognostic value was confirmed within the validation cohort. CONCLUSIONS: Combined immunohistochemical analysis of Bcl-2, nuclear S100A4, Ki67 and MITF could contribute to better risk stratification of early-stage malignant melanoma patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Imuno-Histoquímica/métodos , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Melanoma/mortalidade , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Pessoa de Meia-Idade , Índice Mitótico , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Medição de Risco , Fatores de Risco , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia
9.
Blood ; 128(23): 2666-2670, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27670424

RESUMO

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.


Assuntos
Biomarcadores Tumorais/genética , Deleção de Genes , Proteínas I-kappa B/genética , Linfoma de Células B , Neoplasias do Mediastino , Proteínas Proto-Oncogênicas/genética , Adolescente , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Linfoma de Células B/genética , Linfoma de Células B/mortalidade , Masculino , Neoplasias do Mediastino/genética , Neoplasias do Mediastino/mortalidade , Pessoa de Meia-Idade , Taxa de Sobrevida
10.
BMC Cancer ; 18(1): 1158, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30466405

RESUMO

BACKGROUND: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma. METHODS: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series. RESULTS: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript. CONCLUSIONS: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Fusão Oncogênica/metabolismo , Sensibilidade e Especificidade , Transcriptoma
11.
Exp Mol Pathol ; 104(1): 76-81, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29337243

RESUMO

Anti-EGFR-targeted therapy is used to treat metastatic colorectal cancers with RAS wild-type. However, resistance to targeted therapy is often observed and can be primary or acquired. One reason for primary resistance is the presence of mutations that are undetected due to genetic heterogeneity, which can be expressed by differences present in primary tumor and distant metastasis or recurrence or by an intratumoral heterogeneity (presence of different subclones in the investigated tumor sample). The aim of our study was to investigate if morphological heterogeneity can be an indicator of intratumoral heterogeneity. We analysed 13 samples with homogeneous and six samples with heterogeneous morphology with NGS. We were able to demonstrate that intratumoral genetic heterogeneity is present in all studied tumor samples, independent of homogeneous or heterogeneous morphology. Moreover, one sample of our cohort with morphological and genetic heterogeneity had a genetic wild-type profile in one tumor component. Therefore, we recommend to include each morphologically identifiable tumor component in the mutational analysis to not overlook resistance-inducing or potentially targetable mutations.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Receptores ErbB/genética , Genes erbB-1 , Genes ras , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estudos Retrospectivos
12.
Proc Natl Acad Sci U S A ; 112(38): E5261-70, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26351698

RESUMO

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.


Assuntos
Linfoma de Burkitt/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/metabolismo , Sequência de Bases , Sítios de Ligação , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Perfilação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Neoplasias/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Homologia de Sequência do Ácido Nucleico
13.
Nucleic Acids Res ; 43(20): e135, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26152304

RESUMO

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.


Assuntos
Contaminação por DNA , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Linhagem Celular , Biologia Computacional , Biblioteca Gênica , Humanos
14.
Haematologica ; 101(11): 1380-1389, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27390358

RESUMO

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project "Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing", we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.


Assuntos
Linfoma de Células B/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Adolescente , Linfoma de Burkitt/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Centro Germinativo , Humanos , Lactente , Recém-Nascido , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Masculino , MicroRNAs/genética , Mutação , Edição de RNA
15.
Proc Natl Acad Sci U S A ; 110(30): 12420-5, 2013 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-23840064

RESUMO

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.


Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Estudos de Coortes , Humanos , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/patologia , Transdução de Sinais
16.
Recent Results Cancer Res ; 199: 95-105, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25636433

RESUMO

In this pilot study we changed several pre-analytical variables of bone marrow trephine biopsy processing with the task to achieve not only a preservation of morphology and antigens but also of nucleic acids. The changes involved employment of a newly established decalcification solution in conjunction with a short fixation time (2 h after receiving the specimens) and performance of decalcification at 37 °C. The comparison of the obtained results from three specimens with those of our routinely established protocol unequivocally revealed that the novel decalcification solution results in a superior preservation of nucleic acids, with only slight differences in preservation of morphology and cellular antigens. These encouraging results imply that this novel decalcification solution will allow a widely accepted standardization of bone marrow trephine biopsy processing.


Assuntos
Medula Óssea/patologia , Manejo de Espécimes/métodos , Biópsia/métodos , Medula Óssea/química , Medula Óssea/metabolismo , Evolução Clonal , DNA/isolamento & purificação , DNA/normas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Projetos Piloto , Reação em Cadeia da Polimerase/normas , Controle de Qualidade , Manejo de Espécimes/normas
17.
Exp Mol Pathol ; 99(2): 344-53, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26260902

RESUMO

In order to study molecular similarities and differences of intrahepatic (IH-CCA) and extrahepatic (EH-CCA) cholangiocarcinoma, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay. Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20. Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor- (DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability. Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14. Moreover, due to the fact that mutations in the Isocitrate dehydrogenase (IDH-1 and IDH-2) genes are more frequent in our IH-CCA than in our EH-CCA samples, we suggest that the tumor subtypes have a different molecular profile. In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.


Assuntos
Neoplasias dos Ductos Biliares/genética , Ductos Biliares Extra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Colangiocarcinoma/genética , Variações do Número de Cópias de DNA , Sondas Moleculares/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Extra-Hepáticos/patologia , Ductos Biliares Intra-Hepáticos/patologia , Biomarcadores Tumorais/genética , Colangiocarcinoma/patologia , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Feminino , Genoma Humano , Humanos , Técnicas Imunoenzimáticas , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Exp Mol Pathol ; 98(1): 113-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25576649

RESUMO

In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Interpretação Estatística de Dados , Rearranjo Gênico , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/genética , Receptores Proteína Tirosina Quinases/genética , Algoritmos , Quinase do Linfoma Anaplásico , Automação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Crizotinibe , Estudos de Viabilidade , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Prognóstico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Software
19.
Clin Neuropathol ; 34(1): 19-25, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25250652

RESUMO

Cerebellar liponeurocytoma, first recognized as a distinct entity in the revised WHO classification of Tumors of the Central Nervous System in 2000, is a rare tumor with characteristic histological features and predominant location in the cerebellum. The proliferative index is usually low, and previous reports supported a favorable prognosis. We report a case of a second recurrence of a cerebellar liponeurocytoma with increased proliferative and mitotic activity in which extensive immunohistochemical characterization and genetic profiling were performed. The tumor specimen was characterized in terms of genetic changes frequently associated with gliomas and medulloblastomas. Considering the low number of reported cases, the prognosis of cerebellar liponeurocytoma seems difficult to assess. Our case suggests the existence of different histological grades of cerebellar liponeurocytoma and its possible progression towards a dedifferentiated, malignant phenotype, which has not yet been adequately taken into consideration in the current WHO classification.


Assuntos
Neoplasias Cerebelares/patologia , Lipoma/patologia , Recidiva Local de Neoplasia/patologia , Neurocitoma/patologia , Biomarcadores Tumorais/análise , Neoplasias Cerebelares/genética , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Lipoma/genética , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Neurocitoma/genética
20.
Haematologica ; 99(4): 726-35, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24179151

RESUMO

Chromosomal translocations affecting the MYC oncogene are the biological hallmark of Burkitt lymphomas but also occur in a subset of other mature B-cell lymphomas. If accompanied by a chromosomal break targeting the BCL2 and/or BCL6 oncogene these MYC translocation-positive (MYC(+)) lymphomas are called double-hit lymphomas, otherwise the term single-hit lymphomas is applied. In order to characterize the biological features of these MYC(+) lymphomas other than Burkitt lymphoma we explored, after exclusion of molecular Burkitt lymphoma as defined by gene expression profiling, the molecular, pathological and clinical aspects of 80 MYC-translocation-positive lymphomas (31 single-hit, 46 double-hit and 3 MYC(+)-lymphomas with unknown BCL6 status). Comparison of single-hit and double-hit lymphomas revealed no difference in MYC partner (IG/non-IG), genomic complexity, MYC expression or gene expression profile. Double-hit lymphomas more frequently showed a germinal center B-cell-like gene expression profile and had higher IGH and MYC mutation frequencies. Gene expression profiling revealed 130 differentially expressed genes between BCL6(+)/MYC(+) and BCL2(+)/MYC(+) double-hit lymphomas. BCL2(+)/MYC(+) double-hit lymphomas more frequently showed a germinal center B-like gene expression profile. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In this series of lymphomas, in which immunochemotherapy was administered in only a minority of cases, single-hit and double-hit lymphomas had a similar poor outcome in contrast to the outcome of molecular Burkitt lymphoma and lymphomas without the MYC break. Our data suggest that, after excluding molecular Burkitt lymphoma and pediatric cases, MYC(+) lymphomas are biologically quite homogeneous with single-hit and double-hit lymphomas as well as IG-MYC and non-IG-MYC(+) lymphomas sharing various molecular characteristics.


Assuntos
Linfoma de Burkitt/genética , Genes myc , Linfoma de Células B/genética , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/mortalidade , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/diagnóstico , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Transcriptoma
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