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In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations.
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BACKGROUND: Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons - ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium. MAIN BODY: In this paper we describe the main features of the EurOPDX Data Portal ( https://dataportal.europdx.eu/ ), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views. (SHORT) CONCLUSION: EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users' interests.
Assuntos
Neoplasias , Animais , Xenoenxertos , Humanos , Disseminação de Informação , Camundongos , Neoplasias/genética , Medicina de Precisão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patient-derived tumor xenograft (PDX) mouse models are a versatile oncology research platform for studying tumor biology and for testing chemotherapeutic approaches tailored to genomic characteristics of individual patients' tumors. PDX models are generated and distributed by a diverse group of academic labs, multi-institution consortia and contract research organizations. The distributed nature of PDX repositories and the use of different metadata standards for describing model characteristics presents a significant challenge to identifying PDX models relevant to specific cancer research questions. The Jackson Laboratory and EMBL-EBI are addressing these challenges by co-developing PDX Finder, a comprehensive open global catalog of PDX models and their associated datasets. Within PDX Finder, model attributes are harmonized and integrated using a previously developed community minimal information standard to support consistent searching across the originating resources. Links to repositories are provided from the PDX Finder search results to facilitate model acquisition and/or collaboration. The PDX Finder resource currently contains information for 1985 PDX models of diverse cancers including those from large resources such as the Patient-Derived Models Repository, PDXNet and EurOPDX. Individuals or organizations that generate and distribute PDXs are invited to increase the 'findability' of their models by participating in the PDX Finder initiative at www.pdxfinder.org.
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Biologia Computacional/métodos , Bases de Dados Factuais , Neoplasias/genética , Neoplasias/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Armazenamento e Recuperação da Informação/métodos , Armazenamento e Recuperação da Informação/estatística & dados numéricos , Internet , Metadados/estatística & dados numéricos , CamundongosRESUMO
Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62-66. ©2017 AACR.
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Neoplasias , Ensaios Antitumorais Modelo de Xenoenxerto/estatística & dados numéricos , Animais , Bases de Dados como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , PacientesRESUMO
NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.
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Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Alelos , Animais , Diferenciação Celular , Autorrenovação Celular , Sobrevivência Celular/genética , Progressão da Doença , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Mielopoese , Proteínas Nucleares/metabolismo , Nucleofosmina , Penetrância , Fenótipo , Fatores de Transcrição/genética , Transcriptoma/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
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Modelos Animais de Doenças , Técnicas de Inativação de Genes , Animais , Feminino , Doenças Genéticas Inatas , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.
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Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Adulto , Células Cultivadas , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1(DK)) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53(+/-) background we observed that Mps1(DK/DK) mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3-4 mo after birth.
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Aneuploidia , Instabilidade Cromossômica/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Serina-Treonina Quinases/genética , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/genética , Animais , Aberrações Cromossômicas , Células Clonais , Dosagem de Genes , Regulação Leucêmica da Expressão Gênica , Heterozigoto , Humanos , Cariotipagem , Pontos de Checagem da Fase M do Ciclo Celular/genética , Camundongos , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.
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Transformação Celular Neoplásica/genética , Instabilidade Genômica/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Linfócitos T/enzimologia , Animais , Animais Recém-Nascidos , Transformação Celular Neoplásica/imunologia , Cromatina/genética , Aberrações Cromossômicas , Dano ao DNA/genética , Dano ao DNA/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Instabilidade Genômica/imunologia , Haploinsuficiência/genética , Haploinsuficiência/imunologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timo/citologia , Transcriptoma/imunologiaRESUMO
Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability.
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Elementos de DNA Transponíveis , Genes Neoplásicos , Testes Genéticos/métodos , Mutagênese Insercional , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/genética , Oncogenes , Regiões Promotoras GenéticasRESUMO
Taxins are a family of centrosomal proteins important for the regulation of mitosis and microtubule dynamics. Cytokinesis, the last step of M phase, is essential for chromosomal integrity and cell division. It is highly regulated and involves a reorganization of microtubules and actin filaments. We show here that TACC1 localizes diffusely to the midzone spindle in anaphase and strongly to the midbody during cytokinesis, indicating a possible involvement of this protein in the exit of M phase. TACC1 also relocalizes to the nucleolus in interphase. We demonstrate that TACC1 and the mitotic kinase Aurora B belong to the same complex during cytokinesis. We further show that Aurora B knocked down by RNA-mediated interference prevents the formation of the midbody - and consequently affects TACC1 localization at this site - and leads to abnormal cell division and multinucleated cells.
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Proteínas Fetais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase , Aurora Quinase B , Aurora Quinases , Divisão Celular/fisiologia , Núcleo Celular/genética , Proteínas Fetais/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/genética , Mitose/fisiologia , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Interferência de RNA , Fuso Acromático/metabolismoRESUMO
The three human TACC (transforming acidic coiled-coil) genes encode a family of proteins with poorly defined functions that are suspected to play a role in oncogenesis. A Xenopus TACC homolog called Maskin is involved in translational control, while Drosophila D-TACC interacts with the microtubule-associated protein MSPS (Mini SPindleS) to ensure proper dynamics of spindle pole microtubules during cell division. We have delineated here the interactions of TACC1 with four proteins, namely the microtubule-associated chTOG (colonic and hepatic tumor-overexpressed gene) protein (ortholog of Drosophila MSPS), the adaptor protein TRAP (tudor repeat associator with PCTAIRE2), the mitotic serine/threonine kinase Aurora A and the mRNA regulator LSM7 (Like-Sm protein 7). To measure the relevance of the TACC1-associated complex in human cancer we have examined the expression of the three TACC, chTOG and Aurora A in breast cancer using immunohistochemistry on tissue microarrays. We show that expressions of TACC1, TACC2, TACC3 and Aurora A are significantly correlated and downregulated in a subset of breast tumors. Using siRNAs, we further show that depletion of chTOG and, to a lesser extent of TACC1, perturbates cell division. We propose that TACC proteins, which we also named 'Taxins', control mRNA translation and cell division in conjunction with microtubule organization and in association with chTOG and Aurora A, and that these complexes and cell processes may be affected during mammary gland oncogenesis.
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Neoplasias da Mama/enzimologia , Proteínas Fetais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Aurora Quinase A , Aurora Quinases , Neoplasias da Mama/metabolismo , Células CACO-2 , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Feminino , Células HeLa , Humanos , Filogenia , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases , Proteínas de XenopusRESUMO
Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining two-thirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing approximately 600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.
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Biomarcadores Tumorais/análise , Neoplasias da Mama/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , Sequência de Bases , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Citoplasma/patologia , Primers do DNA , DNA Complementar/genética , Feminino , Humanos , Proteínas de Neoplasias/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/patologia , Transcrição GênicaRESUMO
Alterations of chromosomal region 8p11-21 are very frequent in human cancers, and especially in breast cancer; yet, most of the genes involved have not been identified. We performed laser capture microdissection in a series of 52 consecutive breast tumor samples to obtain pure tumor cells without surrounding normal breast. To determine genomic subregions in which some of the cancer genes may be located, we conducted a search for loss of heterozygosity (LOH) at 13 microsatellite markers from this region. Two-thirds of the tumors showed LOH at least at one marker. Microdissection of pure tumor samples was helpful to precisely define four LOH subregions. No LOH was observed in the corresponding peritumoral tissues. We studied by immunohistochemistry (IHC) on tissue-microarrays the expression in the same tumors, of the protein product of three potential tumor genes lying close to or within the subregions of LOH. In most samples, the TACC1 gene product was downregulated in tumor cells as compared to normal cells. Our results show that the centromeric portion of chromosome arm 8p is frequently altered in breast tumor cells.
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Neoplasias da Mama/genética , Cromossomos Humanos Par 8 , Perda de Heterozigosidade , Repetições de Microssatélites , Neoplasias da Mama/química , Feminino , Proteínas Fetais/análise , Marcadores Genéticos , Humanos , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/análise , Pessoa de Meia-Idade , Neuregulina-1/análise , Proteínas Nucleares/análiseRESUMO
All molecular alterations that lead to breast cancer are not precisely known. We are evaluating the frequency and consequences of reciprocal translocations in breast cancer. We surveyed 15 mammary cell lines by multicolor fluorescence in situ hybridization (M-FISH). We identified nine apparently reciprocal translocations. Using mBanding FISH and FISH with selected YAC clones, we identified the breakpoints for four of them, and cloned the t(3;20)(p14;p11) found in the BrCa-MZ-02 cell line. We found that the breakpoint targets the potential tumor-suppressor gene FHIT (fragile histidine triad) in the FRA3B region; it is accompanied by homozygous deletion of exon 5 of the gene and absence of functional FHIT and fusion transcripts, which leads to the loss of FHIT protein expression. Additional experiments using comparative genomic hybridization provided further information on the genomic context in which the t(3;20)(p14;p11) reciprocal translocation was found.
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Hidrolases Anidrido Ácido , Neoplasias da Mama/genética , Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 3/genética , Clonagem Molecular , Proteínas de Neoplasias/genética , Translocação Genética/genética , Sequência de Bases , Bandeamento Cromossômico , Quebra Cromossômica/genética , Deleção Cromossômica , Fragilidade Cromossômica/genética , Mapeamento Cromossômico , Coloração Cromossômica , Cromossomos Artificiais de Levedura/genética , Clonagem Molecular/métodos , Éxons/genética , Genes Supressores de Tumor , Marcadores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Células Tumorais CultivadasRESUMO
The three human TACC genes encode a family of proteins that are suspected to play a role in carcinogenesis. Their function is not precisely known; a Xenopus TACC protein called Maskin is involved in translational control, while the Drosophila D-TACC associates with microtubules and centrosomes. We have characterized the human TACC1 gene and its products. The TACC1 gene is located in region p12 of chromosome 8; its mRNA is ubiquitously expressed and encodes a protein with an apparent molecular mass of 125 kDa, which is cytoplasmic and mainly perinuclear. We show that TACC1 mRNA gene expression is down-regulated in various types of tumors. Using immunohistochemistry of tumor tissue-microarrays and sections, we confirm that the level of TACC1 protein is down-regulated in breast cancer. Finally, using the two-hybrid screen in yeast, GST pull-downs and co-immunoprecipitations, we identified two potential binding partners for TACC1, LSM7 and SmG. They constitute a conserved subfamily of Sm-like small proteins that associate with U6 snRNPs and play a role in several aspects of mRNA processing. We speculate that down-regulation of TACC1 may alter the control of mRNA homeostasis in polarized cells and participates in the oncogenic processes.