RESUMO
Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Mutação , Neoplasias/genética , Animais , Animais Recém-Nascidos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Mutação com Ganho de Função , Hemangioma Cavernoso do Sistema Nervoso Central/irrigação sanguínea , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação com Perda de Função , MAP Quinase Quinase Quinase 3/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Patients with schizophrenia are at increased risk of diabetes, cardiovascular disease (CVD) and associated mortality versus the general population. Increased intra-abdominal and pericardial adipose tissue are associated with elevated CVD and mortality in the general population, but little is known about these in patients with schizophrenia. This study examined pericardial and intra-abdominal adipose tissue in schizophrenia and compared this to healthy controls. Thirty-one patients with schizophrenia (mean age 41.2 years, 76% males) and 30 healthy volunteers (CTRL) were examined in this study. The primary outcomes were the volumes of pericardial adipose tissue and intra-abdominal adipose tissue, measured using magnetic resonance imaging. Secondary outcomes included diabetes and cardiac event risk assessed by established instruments. Volumes of pericardial adipose tissue were increased in male and female patients with schizophrenia compared to healthy controls after the adjustment of age, sex and body mass index (P < 0.005). The 10-year risk of a cardiac event was significantly higher in patients with schizophrenia. Furthermore, the risk for developing type-2 diabetes mellitus was slightly increased in schizophrenia. Volumes of intra-abdominal adipose tissue were slightly increased in male and female patients with schizophrenia, albeit not statistically significant. This study demonstrates that patients with schizophrenia have increased pericardial adipose tissue versus controls. This increased fat deposit around the heart is highly relevant for understanding the comorbidity between heart disease and schizophrenia. Interventions aiming to reduce pericardial and intra-abdominal adipose tissue, such as exercise, may be essential to reduce the burden of heart disease in schizophrenia.
Assuntos
Tecido Adiposo Branco/diagnóstico por imagem , Doenças Cardiovasculares/sangue , Diabetes Mellitus/sangue , Pericárdio/diagnóstico por imagem , Esquizofrenia/fisiopatologia , Adulto , Doenças Cardiovasculares/epidemiologia , Comorbidade , Diabetes Mellitus/epidemiologia , Feminino , Humanos , Gordura Intra-Abdominal/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Risco , Esquizofrenia/epidemiologiaRESUMO
The high mobility group box protein SOX9 and the GLI1 transcription factor play protumorigenic roles in pancreatic ductal adenocarcinoma (PDA). In Kras transgenic mice, each of these factors are crucial for the development of PDA precursor lesions. SOX9 transcription is directly regulated by GLI1, but how SOX9 functions downstream of GLI1 is unclear. We observed positive feedback, such that SOX9-deficient PDA cells have severely repressed levels of endogenous GLI1, attributed to loss of GLI1 protein stability. SOX9 associated with the F-box domain of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase component, ß-TrCP (also known as F-box/WD repeat-containing protein 1A), and suppressed its association with SKP1 and GLI1, a substrate of SCF-ß-TrCP. SOX9 also tethered ß-TrCP within the nucleus and promoted its degradation. SOX9 bound to ß-TrCP through the SOX9 C-terminal PQA/S domain that mediates transcriptional activation. Suppression of ß-TrCP in SOX9-deficient PDA cells restored GLI1 levels and promoted SOX9-dependent cancer stem cell properties. These studies identify SOX9-GLI1 positive feedback as a major determinant of GLI1 protein stability and implicate ß-TrCP as a latent SOX9-bound tumor suppressor with the potential to degrade oncogenic proteins in tumor cells.
Assuntos
Carcinoma Ductal Pancreático/patologia , Núcleo Celular/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Oncogênicas/metabolismo , Neoplasias Pancreáticas/patologia , Fatores de Transcrição SOX9/metabolismo , Transativadores/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Anoikis , Apoptose , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Núcleo Celular/genética , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteínas Oncogênicas/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteólise , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Esferoides Celulares/metabolismo , Transativadores/genética , Proteína GLI1 em Dedos de Zinco , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
Several master transcription factors (TF) can activate the epithelial-to-mesenchymal transition (EMT). However, their individual and combinatorial contributions to EMT in breast cancer are not defined. We show that overexpression of EMT-TFs individually in epithelial cells upregulated endogenous SNAI2, ZEB1/2, TCF4, and TWIST1/2 as a result of positive feedback mediated in part by suppression of their negative regulator miRNAs miR200s/203/205. We identified TCF4 as a potential new target of miR200s. Expression of ZEB1/2 strongly correlated with the mesenchymal phenotype in breast cancer cells, with the CD24-/CD44+ stemness profile, and with lower expression of core epithelial genes in human breast tumors. Knockdown of EMT-TFs identified the key role of ZEB1 and its functional cooperation with other EMT-TFs in the maintenance of the mesenchymal state. Inducible ZEB1+2 knockdown in xenograft models inhibited pulmonary metastasis, emphasizing their critical role in dissemination from primary site and in extravasation. However, ZEB1+2 depletion one-week after intravenous injection did not inhibit lung colonization, suggesting that ZEB1/2 and EMT are not essential for macrometastatic outgrowth. These results provide strong evidence that EMT is orchestrated by coordinated expression of several EMT-TFs and establish ZEB1 as a key master regulator of EMT and metastasis in breast cancer. IMPLICATIONS: The EMT program is orchestrated by coordinated expression of multiple EMT transcription factors, whereas ZEB1 integrates the EMT master regulatory network and plays the major role in promoting EMT and metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição/metabolismo , Animais , Neoplasias da Mama/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Fatores de Transcrição/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.
Assuntos
Carcinoma de Células Pequenas/genética , Amplificação de Genes , Neoplasias Pulmonares/genética , Oncogenes , Carcinoma de Células Pequenas/patologia , Humanos , Neoplasias Pulmonares/patologia , Metástase NeoplásicaRESUMO
Allelic deletions involving chromosome 18q occur in more than 70 percent of colorectal cancers. Such deletions are thought to signal the existence of a tumor suppressor gene in the affected region, but until now a candidate suppressor gene on this chromosomal arm had not been identified. A contiguous stretch of DNA comprising 370 kilobase pairs (kb) has now been cloned from a region of chromosome 18q suspected to reside near this gene. Potential exons in the 370-kb region were defined by human-rodent sequence identities, and the expression of potential exons was assessed by an "exon-connection" strategy based on the polymerase chain reaction. Expressed exons were used as probes for cDNA screening to obtain clones that encoded a portion of a gene termed DCC; this cDNA was encoded by at least eight exons within the 370-kb genomic region. The predicted amino acid sequence of the cDNA specified a protein with sequence similarity to neural cell adhesion molecules and other related cell surface glycoproteins. While the DCC gene was expressed in most normal tissues, including colonic mucosa, its expression was greatly reduced or absent in most colorectal carcinomas tested. Somatic mutations within the DCC gene observed in colorectal cancers included a homozygous deletion of the 5' end of the gene, a point mutation within one of the introns, and ten examples of DNA insertions within a 0.17-kb fragment immediately downstream of one of the exons. The DCC gene may play a role in the pathogenesis of human colorectal neoplasia, perhaps through alteration of the normal cell-cell interactions controlling growth.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 18 , Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Supressão Genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Moléculas de Adesão Celular Neuronais/genética , Clonagem Molecular , Reações Cruzadas , Sondas de DNA , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Overexpression of the human epidermal growth factor receptor 2 (HER2) is the cause of HER2-positive breast cancer (BC). Although HER2-inactivating therapies have benefited BC patients, development of resistance and disease recurrence have been the major clinical problems, pointing to a need for alternative therapeutic strategies. For that to happen, proteins that play critical roles in the biology of HER2-induced tumorigenesis have to be identified and characterized. Here, we show that the Src homology phosphotyrosyl phosphatase 2 (Shp2) encoded by the Ptpn11 gene is a requisite for ErbB2-induced tumorigenesis. We report that conditional knockout of Shp2 alleles in the ErbB2 BC model mice abrogates mammary tumorigenesis by blocking the expression of the ErbB2 transgene. We also show that inhibition of SHP2 encoded by the PTPN11 gene in the HER2-amplified BC cells induces a normal-like cellular phenotype and suppresses tumorigenesis and metastasis by blocking HER2 overexpression. These findings demonstrate that ErbB2-induced tumors in mice or xenograft tumors induced by transplantation of HER2-amplified BC cells are vulnerable to SHP2 inhibition since it abrogates the expression of the very oncogene that causes of the disease. This report paves the way for developing SHP2-targeting therapies for BC treatment in the future.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama , Carcinogênese , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Oncogenes/efeitos dos fármacos , Oncogenes/genéticaRESUMO
The Hedgehog pathway transcription factor Gli1 induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin (E-cad). E-cad is normally complexed with beta-catenin at the cell membrane. Loss of E-cad during developmental epithelial-mesenchymal transitions can switch beta-catenin from its role at adherens junctions to its role in nuclear transcription. During tumorigenesis it is unclear which pathways trigger this switch. In the current study, gain- and loss-of-function approaches identified E-cad as a selective inhibitor of transformation by Gli1, and Snail knockdown was rescued by downregulation of E-cad. Gli1 induced relocalization of beta-catenin from the cell membrane to the nucleus. The ability of wild-type or mutant alleles of E-cad to modulate transformation by Gli1 correlated with their ability to regulate localization of beta-catenin. Inhibition of Wnt-beta-catenin signaling by dominant negative Tcf4 selectively blocked in vitro transformation by Gli1. In Gli1-transgenic mice, infiltrating skin tumor cells expressed active, unphosphorylated beta-catenin. Our studies identify E-cad as a selective suppressor of transformation by Gli1 and point to the Sonic Hedgehog-Gli1 pathway as a key regulator of the beta-catenin switch in epithelial cells and cancers.
Assuntos
Caderinas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/farmacologia , Células Epiteliais , Rim , Camundongos , Camundongos Transgênicos , Ratos , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fator de Transcrição 4 , Fatores de Transcrição/farmacologia , Proteínas Wnt/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
Gli family members mediate constitutive Hedgehog signaling in the common skin cancer, basal cell carcinoma (BCC). Snail/Snai1 is rapidly induced by Gli1 in vitro, and is coexpressed with Gli1 in human hair follicles and skin tumors. In the current study, we generated a dominant-negative allele of Snail, SnaZFD, composed of the zinc-finger domain and flanking sequence. In promoter-reporter assays, SnaZFD blocked the activity of wild-type Snail on the E-cadherin promoter. Snail loss-of-function mediated by SnaZFD or by one of several short hairpin RNAs inhibited transformation of RK3E epithelial cells by Gli1. Conversely, enforced expression of Snail promoted transformation in vitro by Gli1, but not by other genes that were tested, including Notch1, ErbB2, and N-Ras. As observed for Gli1, wild-type Snail repressed E-cadherin in RK3E cells and induced blebbing of the cytoplasmic membrane. Induction of a conditional Gli1 transgene in the basal keratinocytes of mouse skin led to rapid upregulation of Snail transcripts and to cell proliferation in the interfollicular epidermis. Established Gli1-induced skin lesions exhibited molecular similarities to BCC, including loss of E-cadherin. The results identify Snail as a Gli1-inducible effector of transformation in vitro, and an early Gli1-responsive gene in the skin.
Assuntos
Transformação Celular Neoplásica , Fatores de Transcrição Kruppel-Like/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Carcinoma Basocelular/etiologia , Ciclo Celular , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Interferente Pequeno/farmacologia , Pele/patologia , Neoplasias Cutâneas/etiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
The tumor suppressor protein p53 was first isolated as a simian virus 40 large T antigen-associated protein and subsequently was found to function in cell proliferation control. Tumor-derived mutations in p53 occur predominantly in four evolutionarily conserved regions spanning approximately 50% of the polypeptide. Previously, three of these regions were identified as essential for T-antigen binding. We have examined the interaction between p53 and T antigen by using Escherichia coli-expressed human p53. By a combination of deletion analysis and antibody inhibition studies, a region of p53 that is both necessary and sufficient for binding to T antigen has been localized. This function is contained within residues 94 to 293, which include the four conserved regions affected by mutation in tumors. Residues 94 to 293 of p53 were expressed in both wild-type and mutant forms. T-antigen binding was unaffected by tumor-derived mutations which have been associated with the wild-type conformation of p53 but was greatly reduced by mutations which were previously shown to alter p53 conformation. Our results show that, like T-antigen binding to the Rb tumor suppressor protein, T antigen appears to interact with the domain of p53 that is commonly mutated in human tumors.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Escherichia coli/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Vírus 40 dos Símios/genética , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
The GLI gene was previously isolated by virtue of its amplification in human glioblastomas. We have now found that GLI expression can result in the in vitro transformation of both primary and secondary rodent cells. When coexpressed with adenovirus E1A, the GLI protein functions analogously to RAS, resulting in the formation of dense foci of cells which are tumorigenic in nude mice.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Oncogênicas Virais/fisiologia , Fatores de Transcrição/fisiologia , Dedos de Zinco , Proteínas Precoces de Adenovirus , Animais , Linhagem Celular , Glioma , Técnicas In Vitro , Família Multigênica , RatosRESUMO
The GLI oncogene, discovered by virtue of its amplification in human tumors, encodes a sequence-specific DNA-binding protein containing five zinc fingers. We have now characterized one member of a family of GLI-related zinc finger genes. A previously identified fragment of GLI3 genomic DNA was used to localize GLI3 to chromosome 7p13 and to isolate cDNA clones. Sequence analysis of these clones and identification of the GLI3 protein by using polyclonal antisera demonstrated that GLI3 encodes a protein of 1,596 amino acids and an apparent molecular mass of 190 kilodaltons. Amino acid sequence comparison with GLI demonstrated seven regions of similarity (53 to 88% identity), with the zinc fingers representing the most similar region. Furthermore, when produced in vitro, the GLI3 protein bound specifically to genomic DNA fragments containing GLI-binding sites. Amino acid sequence comparison with the product of another member of the GLI family, the Drosophila segment polarity gene cubitus interruptus Dominant, revealed additional similarity that was not shared with GLI. These studies suggest that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities. In addition, sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.
Assuntos
Proteínas de Ligação a DNA/genética , Metaloproteínas/genética , Proteínas do Tecido Nervoso , Proteínas Repressoras , Fatores de Transcrição/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cromossomos Humanos Par 7 , Clonagem Molecular , DNA/genética , Humanos , Técnicas Imunológicas , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Família Multigênica , RNA Mensageiro/genética , Proteína Gli3 com Dedos de ZincoRESUMO
The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIbeta binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Replicação do DNA , Doxorrubicina/farmacologia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Fator de Transcrição E2F3 , Fator de Transcrição E2F4 , Humanos , Proteínas Nucleares , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S/fisiologia , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas Supressoras de TumorRESUMO
Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.
Assuntos
Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Genes , Glioblastoma/genética , Metaloproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Amplificação de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Plasmídeos , Transplante HeterólogoRESUMO
KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.
Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Células Epiteliais/patologia , Queratinócitos/citologia , Pele/patologia , Fatores de Transcrição/biossíntese , Animais , Apoptose/efeitos dos fármacos , Cruzamentos Genéticos , Primers do DNA , Doxorrubicina/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
We performed a phase I clinical trial in grade IV astrocytoma to assess the safety of a whole-cell vaccine comprising autologous tumor cells genetically modified by a transforming growth factor-beta2 (TGF-beta2) antisense vector. Blocking secretion of the immunosuppressive molecule TGF-beta in this manner should inhibit one of the major mechanisms by which tumor cells evade immune surveillance and should lead to clinically effective antitumor immunity. Six patients with progressive WHO grade IV astrocytoma were enrolled in the trial. Patients received 2-7 subcutaneous injections of 5 x 10(6)-2 x 10(7) autologous tumor cells per injection. TGF-beta2 secretion by the tumor cells used to vaccinate patients was inhibited by 53-98%. Treatment was well tolerated with only low-grade, transient treatment-related toxicities reported. Two patients had partial regressions and two had stable disease following therapy. The overall median survival was 68 weeks. Median survival of the responding patients was 78 weeks, compared to a historic value of 47 weeks for glioma patients treated conventionally. There were indications of humoral and cellular immunity induced by the vaccine. These findings support further clinical evaluation of vaccines comprised of TGF-beta antisense-modified tumor cells.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Glioma/tratamento farmacológico , Oligonucleotídeos Antissenso/genética , Fator de Crescimento Transformador beta2/genética , Adulto , Formação de Anticorpos , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/genética , Neoplasias do Sistema Nervoso Central/imunologia , Neoplasias do Sistema Nervoso Central/patologia , Feminino , Glioma/imunologia , Glioma/patologia , Humanos , Injeções Intradérmicas , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta2/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to noncancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.
Assuntos
Neoplasias da Mama/fisiopatologia , Mama/fisiologia , Proteínas Hedgehog/fisiologia , Alcaloides de Veratrum/farmacologia , Sequência de Bases , Mama/efeitos dos fármacos , Linhagem Celular Tumoral , Primers do DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologiaRESUMO
Most carcinomas of the bladder show loss of heterozygosity for markers on human chromosome 9, which suggests that one or more tumor suppressor genes are located on this chromosome. Several observations suggest that such alterations are an important early step in tumorigenesis. We analyzed the pattern of allelic loss in 46 primary carcinomas of the bladder using 19 polymorphic markers from chromosome 9. While most tumors with allelic loss showed loss of heterozygosity for all informative markers that were tested, six tumors demonstrated only partial loss of chromosome 9. Two tumors with partial loss contained deletions that predominantly involved the q arm, as shown by previous studies. The other four tumors contained deletions that predominantly or exclusively involved the p arm, with a common region of loss between D9S161 (9p21) and the telomere. The results show that there is no single common region of loss on chromosome 9 and identify two distinct regions of loss that may contain bladder tumor suppressor loci.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 9 , Genes Supressores de Tumor , Neoplasias da Bexiga Urinária/genética , Mapeamento Cromossômico , DNA de Neoplasias/análise , Marcadores Genéticos , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
To gain a better understanding of the molecular changes in head and neck squamous cell carcinoma, we tested every autosomal arm of 29 primary head and neck tumors for allelic loss. Fifty-eight microsatellite markers were used with at least two-thirds of patients informative for each chromosomal arm tested. A high frequency of allelic loss was found on chromosome 9p where 21 of 29 (72%) tumors had loss of heterozygosity for at least one polymorphic marker on this arm. Chromosomes 3, 11q, 13q, and 17p exhibited loss in over 50% of all informative cases, while chromosomes 4, 6p, 8, 14q, and 19q displayed loss in greater than 35% of all cases tested. Additionally, several other chromosomal arms exhibited loss of heterozygosity in 20 to 30% of tumors tested. This high frequency of allelic loss in these advanced stage neoplasms suggests multiple genetic steps in the progression of head and neck cancer and identifies several putative tumor suppressor loci on affected chromosomes.
Assuntos
Alelos , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 9 , Deleção de Genes , HumanosRESUMO
Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.