RESUMO
BACKGROUND: Breast cancer is the most common malignancy in women, and is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers express normal levels of REST (repressor element 1 silencing transcription factor) mRNA but lack functional REST protein. Loss of REST function is seen in ~ 20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. METHODS: TCGA data was analyzed for the expression of REST target genes in breast cancer patient samples. We then utilized gene knockdown in MCF-7 cells in the presence or absence of steroid hormones estrogen and/ progesterone followed by RNA sequencing, as well as chromatin immunoprecipitation and PCR in an attempt to understand the tumor suppressor role of REST in breast cancer. RESULTS: We show that REST directly regulates CEMIP (cell migration-inducing and hyaluronan-binding protein, KIAA1199) and MMP24 (matrix metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cells leads to significant upregulation of CEMIP and MMP24. In addition, we found REST binds to RE-1 sites (repressor element-1) within the genes and influences their transcription. Furthermore, we found that the estrogen receptor (ESR1) signaling pathway is activated in the absence of REST, regardless of hormone treatment. CONCLUSIONS: We demonstrate a critical role for the loss of REST in aggressive breast cancer pathogenesis and provide evidence for REST as an important diagnostic marker for personalized treatment plans.
Assuntos
Neoplasias da Mama/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Hialuronoglucosaminidase/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Biomarcadores Tumorais/genética , Feminino , Humanos , Mutação com Perda de Função/genética , Células MCF-7 , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Processos Neoplásicos , Fenótipo , Prognóstico , RNA Mensageiro/genética , Proteínas Repressoras , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Expression of functional breast cancer susceptibility gene 1 (BRCA1) in human breast and ovarian cancers is associated with resistance to platinum-based chemotherapeutics and poly(ADP ribose) polymerase (PARP) inhibitors. BRCA1 is a nuclear tumor suppressor that is critical for resolving double-strand DNA breaks (DSBs) and interstrand crosslinks (ICLs) by homologous recombination (HR). In vitro, animal and human clinical data have demonstrated that BRCA1-deficient cancers are highly sensitive to ICL-inducing chemotherapeutic agents, are amenable to synthetic lethal approaches that exploit defects in DSB/ICL repair, and may be associated with improved survival. Conversely, high or restored expression of BRCA1 in breast and ovarian cancer is associated with therapeutic resistance and poor prognosis. There has been much interest in identifying agents that interfere with BRCA1-dependent DSB/ICL repair to restore or enhance sensitivity to cancer therapeutics. We demonstrate that the heat-shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin [17-AAG (Tanespimycin)], currently in Phase II/III clinical evaluation for several cancers, induces BRCA1 ubiquitination and proteasomal degradation, resulting in compromised repair of ionizing radiation- and platinum-induced DNA damage. We show that loss of HSP90 function abolishes BRCA1-dependent DSB repair and that BRCA1-deficient cells are hypersensitive to 17-AAG due to impaired Gap 2/Mitosis (G2/M) checkpoint activation and resultant mitotic catastrophe. In summary, we document an upstream HSP90-dependent regulatory point in the Fanconi anemia/BRCA DSB/ICL repair pathway, illuminate the role of BRCA1 in regulating damage-associated checkpoint and repair responses to HSP90 inhibitors, and identify BRCA1 as a clinically relevant target for enhancing sensitivity in refractory and/or resistant malignancies.
Assuntos
Proteína BRCA1/genética , Proteína BRCA1/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Choque Térmico HSP90/metabolismo , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2 , Células HeLa , Recombinação Homóloga , Humanos , Lactamas Macrocíclicas/farmacologiaRESUMO
MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
Assuntos
Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Ribonuclease III/metabolismo , Proteínas Argonautas , Linhagem Celular , Fator de Iniciação 2 em Eucariotos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Coativadores de Receptor Nuclear , Fatores de Iniciação de Peptídeos/genética , Ligação Proteica , Ribonuclease III/genética , Transcrição GênicaRESUMO
BACH1 (also known as FANCJ and BRIP1) is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1. Previous biochemical and functional analyses have suggested a role for the BACH1 homolog in Caenorhabditis elegans during DNA replication. Here, we report the association of BACH1 with a distinct BRCA1/BRCA2-containing complex during the S phase of the cell cycle. Depletion of BACH1 or BRCA1 using small interfering RNAs results in delayed entry into the S phase of the cell cycle. Such timely progression through S phase requires the helicase activity of BACH1. Importantly, cells expressing a dominant negative mutation in BACH1 that results in a defective helicase displayed increased activation of DNA damage checkpoints and genomic instability. BACH1 helicase is silenced during the G(1) phase of the cell cycle and is activated through a dephosphorylation event as cells enter S phase. These results point to a critical role for BACH1 helicase activity not only in the timely progression through the S phase but also in maintaining genomic stability.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fase S/fisiologia , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Dano ao DNA , Ativação Enzimática , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Humanos , Complexos Multiproteicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BRCA1 (breast cancer 1, early onset), a well-known breast cancer susceptibility gene, is a highly alternatively spliced gene. BRCA1 alternative splicing may serve as an alternative regulatory mechanism for the inactivation of the BRCA1 gene in both hereditary and sporadic breast cancers, and other BRCA1-associated cancers. The alternative transcripts of BRCA1 can mimic known functions, possess unique functions compared with the full-length BRCA1 transcript, and in some cases, appear to function in opposition to full-length BRCA1 In this review, we will summarize the functional "naturally occurring" alternative splicing transcripts of BRCA1 and then discuss the latest next-generation sequencing-based detection methods and techniques to detect alternative BRCA1 splicing patterns and their potential use in cancer diagnosis, prognosis, and therapy.
Assuntos
Processamento Alternativo , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
LoxP-Cre technology was used to remove the selenocysteine tRNA gene, trsp, in either endothelial cells or myocytes of skeletal and heart muscle to elucidate the role of selenoproteins in cardiovascular disease. Loss of selenoprotein expression in endothelial cells was embryonic lethal. A 14.5-day-old embryo had numerous abnormalities including necrosis of the central nervous system, subcutaneous hemorrhage and erythrocyte immaturity. Loss of selenoprotein expression in myocytes manifested no apparent phenotype until about day 12 after birth. Affected mice had decreased mobility and an increased respiratory rate, which proceeded rapidly to death. Pathological analysis revealed that mice lacking trsp had moderate to severe myocarditis with inflammation extending into the mediastinitis. Thus, ablation of selenoprotein expression demonstrated an essential role of selenoproteins in endothelial cell development and in proper cardiac muscle function. The data suggest a direct connection between the loss of selenoprotein expression in these cell types and cardiovascular disease.
Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Coração/crescimento & desenvolvimento , Coração/fisiologia , Miocárdio/metabolismo , Selenoproteínas/biossíntese , Animais , Animais Recém-Nascidos/fisiologia , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Gravidez , RNA de Transferência de Cisteína/genética , RNA de Transferência de Cisteína/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Selenocisteína/metabolismo , Comportamento Sexual Animal/fisiologiaRESUMO
Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.
Assuntos
Mama/metabolismo , Epitélio/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Alelos , Animais , Northern Blotting , Western Blotting , Cromatografia , Cruzamentos Genéticos , Deleção de Genes , Genes BRCA1 , Genes p53/genética , Vetores Genéticos , Glutationa Peroxidase/metabolismo , Heterozigoto , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Regiões Promotoras Genéticas , Proteínas/metabolismo , Recombinação Genética , Selenoproteínas , Distribuição Tecidual , TransgenesRESUMO
The familial aggregation of prostate cancer and breast cancer has been observed for almost half a century and about 85% of the inherited breast cancer can be linked to germ-line mutations of BRCA1 (breast cancer 1, early onset) and BRCA2. In this review, we are mainly focusing on the contribution of BRCA1/2 sequence variations to prostate cancer risk and disease progression. We will discuss the biological functions of BRCA1/2 and BRCA1/2-related signaling pathways in prostate cancer biology. The majority of studies supporting the link between BRCA1/2 mutations and prostate cancer are from populations with a high frequency of mutations, such as Ashkenazi Jewish, Icelandic, and U.K. population. BRCA1 can directly interact with the androgen receptor (AR) and Janus kinase (JAK), and can differentially regulate insulin-like growth factor 1 receptor (IGF-IR) expression in an AR-dependent manner. BRCA2 homeostasis in prostate cancer cells has been found to be critical in determining cell fates during prostate cancer progression. This review may be helpful for medical professionals and prostate cancer patients when discussing prostate cancer risks, treatment and prognosis.
Assuntos
Genes BRCA1 , Genes BRCA2 , Neoplasias da Próstata/genética , Humanos , Masculino , Neoplasias da Próstata/patologiaAssuntos
Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Complementar/genética , Expressão Gênica , Humanos , Fígado/química , Masculino , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Polimorfismo Genético , Proteínas/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Selenocisteína/genética , Selenoproteínas , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Reparo do DNA , Subunidades Proteicas/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase , Radiação Ionizante , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificaçãoRESUMO
Two model systems for examining the role of selenoproteins in health are discussed. One utilizes transgenic mice that carry mutant selenocysteine (Sec) tRNA transgenes that result in the reduction of selenoprotein expression in a protein- and tissue-specific manner. The other utilizes loxP-Cre technology to selectively remove the Sec tRNA gene in mammary epithelium that results in the reduction of only certain selenoproteins in this tissue. Both approaches provide important tools for examining the role of selenoproteins in health.
Assuntos
Modelos Biológicos , Proteínas/fisiologia , Aminoacil-RNA de Transferência/genética , Selenocisteína/genética , Animais , Camundongos , Camundongos Knockout , Camundongos Transgênicos , SelenoproteínasRESUMO
Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.