RESUMO
Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.
Assuntos
Anticorpos Antivirais/análise , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Estudos Soroepidemiológicos , Antígenos Virais/imunologia , Austrália , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunoglobulina G/análise , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Over the last decade, our appreciation of the importance of the nucleolus for cellular function has progressed from the ordinary to the extraordinary. We no longer think of the nucleolus as simply the site of ribosome production, or a dynamic subnuclear body noted by pathologists for its changes in size and shape with malignancy. Instead, the nucleolus has emerged as a key controller of many cellular processes that are fundamental to normal cell homeostasis and the target for dysregulation in many human diseases; in some cases, independent of its functions in ribosome biogenesis. These extra-nucleolar or new functions, which we term "non-canonical" to distinguish them from the more traditional role of the nucleolus in ribosome synthesis, are the focus of this review. In particular, we explore how these non-canonical functions may provide novel insights into human disease and in some cases new targets for therapeutic development.
Assuntos
Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Neoplasias/metabolismo , Doenças do Sistema Nervoso/metabolismo , Biogênese de OrganelasRESUMO
Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.
Assuntos
Benzotiazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Naftiridinas/farmacologia , Células-Tronco Neoplásicas/enzimologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Fase G2/efeitos dos fármacos , Fase G2/genética , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Células-Tronco Neoplásicas/patologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Assuntos
Anemia de Diamond-Blackfan/tratamento farmacológico , Eritropoese/efeitos dos fármacos , Prednisolona/uso terapêutico , Proteínas Ribossômicas/deficiência , Proteína Supressora de Tumor p53/fisiologia , Adolescente , Anemia de Diamond-Blackfan/sangue , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dexametasona/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prednisolona/farmacologia , Quimera por Radiação , Proteínas Ribossômicas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.
Assuntos
Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Glândulas Mamárias Humanas/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Ativação Transcricional , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Linhagem Celular Transformada , Colina Quinase/antagonistas & inibidores , Colina Quinase/genética , Colina Quinase/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais , Trombina/metabolismo , Trombina/farmacologiaRESUMO
ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.
Assuntos
DNA Helicases/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Telômero/metabolismo , Adenosina Trifosfatases/química , Animais , Cromatina/metabolismo , Cromatina/fisiologia , Montagem e Desmontagem da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Genes , Histonas/genética , Humanos , Deficiência Intelectual/genética , Interfase/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Mutação , Proteínas Nucleares/genética , Proteína Nuclear Ligada ao X , Talassemia alfa/genéticaRESUMO
Povidone-iodine (PVP-I) inactivates a broad range of pathogens. Despite its widespread use over decades, the safety of PVP-I remains controversial. Its extended use in the current SARS-CoV-2 virus pandemic urges the need to clarify safety features of PVP-I on a cellular level. Our investigation in epithelial, mesothelial, endothelial, and innate immune cells revealed that the toxicity of PVP-I is caused by diatomic iodine (I2), which is rapidly released from PVP-I to fuel organic halogenation with fast first-order kinetics. Eukaryotic toxicity manifests at below clinically used concentrations with a threshold of 0.1% PVP-I (wt/vol), equalling 1 mM of total available I2 Above this threshold, membrane disruption, loss of mitochondrial membrane potential, and abolition of oxidative phosphorylation induce a rapid form of cell death we propose to term iodoptosis. Furthermore, PVP-I attacks lipid rafts, leading to the failure of tight junctions and thereby compromising the barrier functions of surface-lining cells. Thus, the therapeutic window of PVP-I is considerably narrower than commonly believed. Our findings urge the reappraisal of PVP-I in clinical practice to avert unwarranted toxicity whilst safeguarding its benefits.
Assuntos
Anti-Infecciosos Locais , COVID-19 , Iodo , Humanos , Povidona-Iodo/farmacologia , Povidona-Iodo/uso terapêutico , Anti-Infecciosos Locais/farmacologia , Anti-Infecciosos Locais/uso terapêutico , Iodo/farmacologia , SARS-CoV-2 , Morte CelularAssuntos
Anemia de Diamond-Blackfan/genética , Mutação , Criança , Humanos , Masculino , Linhagem , Fenótipo , Proteínas Ribossômicas/genéticaRESUMO
SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8-3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.
RESUMO
The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/genética , Nucléolo Celular/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top 'hits' was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of ß-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.
Assuntos
Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Mutações Sintéticas Letais/genética , Via de Sinalização Wnt/fisiologia , Animais , Divisão Celular , Linhagem Celular , Humanos , Peixe-Zebra , CoesinasRESUMO
Exquisite regulation of PI3K/AKT/mTORC1 signaling is essential for homeostatic control of cell growth, proliferation, and survival. Aberrant activation of this signaling network is an early driver of many sporadic human cancers. Paradoxically, sustained hyperactivation of the PI3K/AKT/mTORC1 pathway in nontransformed cells results in cellular senescence, which is a tumor-suppressive mechanism that must be overcome to promote malignant transformation. While oncogene-induced senescence (OIS) driven by excessive RAS/ERK signaling has been well studied, little is known about the mechanisms underpinning the AKT-induced senescence (AIS) response. Here, we utilize a combination of transcriptome and metabolic profiling to identify key signatures required to maintain AIS. We also employ a whole protein-coding genome RNAi screen for AIS escape, validating a subset of novel mediators and demonstrating their preferential specificity for AIS as compared with OIS. As proof of concept of the potential to exploit the AIS network, we show that neurofibromin 1 (NF1) is upregulated during AIS and its ability to suppress RAS/ERK signaling facilitates AIS maintenance. Furthermore, depletion of NF1 enhances transformation of p53-mutant epithelial cells expressing activated AKT, while its overexpression blocks transformation by inducing a senescent-like phenotype. Together, our findings reveal novel mechanistic insights into the control of AIS and identify putative senescence regulators that can potentially be targeted, with implications for new therapeutic options to treat PI3K/AKT/mTORC1-driven cancers.
Assuntos
Senescência Celular/genética , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/genéticaRESUMO
Neuritic abnormalities are a major hallmark of Alzheimer's disease (AD) pathology. Accumulation of beta-amyloid protein (Abeta) in the brain causes changes in neuritic processes in individuals with this disease. In this study, we show that Abeta decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Abeta alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Abeta increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Y cells. This increase in GTP-RhoA correlated with an increase in an alternatively spliced form of CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Abeta stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Abeta but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice. We found that there was an increase in threonine phosphorylation of CRMP-2 in Tg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin. The results suggest that Abeta-induced neurite outgrowth inhibition may be initiated through a mechanism in which Abeta causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/fisiologia , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Tamanho Celular/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Neuritos/ultraestrutura , Fosforilação , Transdução de Sinais , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The nucleolus is a dynamic subnuclear compartment that has a number of different functions, but its primary role is to coordinate the production and assembly of ribosomes. For well over 100 years, pathologists have used changes in nucleolar number and size to stage diseases such as cancer. New information about the nucleolus' broader role within the cell is leading to the development of drugs which directly target its structure as therapies for disease. Traditionally, it has been difficult to develop high-throughput image analysis pipelines to measure nucleolar changes due to the broad range of morphologies observed. In this study, we describe a simple high-content image analysis algorithm using Harmony software (PerkinElmer), with a PhenoLOGIC™ machine-learning component, that can measure and classify three different nucleolar morphologies based on nucleolin and fibrillarin staining ("normal," "peri-nucleolar rings" and "dispersed"). We have utilized this algorithm to determine the changes in these classes of nucleolar morphologies over time with drugs known to alter nucleolar structure. This approach could be further adapted to include other parameters required for the identification of new therapies that directly target the nucleolus.
Assuntos
Nucléolo Celular/patologia , Ensaios de Triagem em Larga Escala , Células A549 , Algoritmos , Nucléolo Celular/metabolismo , Humanos , Aprendizado de Máquina , Estresse Oxidativo , Software , Células Tumorais CultivadasRESUMO
Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/tratamento farmacológico , Progressão da Doença , Losartan/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptor Tipo 1 de Angiotensina/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Biópsia , Carcinogênese/metabolismo , Carcinoma Intraductal não Infiltrante/induzido quimicamente , Carcinoma Intraductal não Infiltrante/imunologia , Carcinoma Intraductal não Infiltrante/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Acetato de Medroxiprogesterona/toxicidade , Camundongos , Invasividade Neoplásica , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Sistema Renina-Angiotensina/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Phosphatidylethanolamine binding protein (PEBP) is a multifunctional protein, with proposed roles as the precursor protein of hippocampal cholinergic neurostimulating peptide (HCNP), and as the Raf kinase inhibitor protein (RKIP). Previous studies have demonstrated a decrease in PEBP mRNA in CA1 region of AD hippocampus. The current study demonstrates that PEBP is decreased in the hippocampus of 11 month Tg2576 mice, in the absence of change in mRNA levels compared to non-transgenic littermates. The level of PEBP in transgenic mouse hippocampus significantly decreases at 11 months (a time point when Abeta begins accumulating) and 15 months (when Abeta plaques have formed). There was a significant correlation between decreased PEBP expression and accumulation of Abeta. Immunohistochemical studies on Tg2576 and AD brain sections demonstrate that PEBP immunoreactivities are present at the periphery of dense multicore Abeta plaques, and in selective astrocytes, primarily surrounding plaques. These findings suggest that PEBP expression may be influenced by accumulation of Abeta. Down-regulation of PEBP may result in lower levels of HCNP or altered coordination of signal transduction pathways that may contribute to neuronal dysfunction and pathogenesis in AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Feminino , Expressão Gênica/fisiologia , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica/métodos , Camundongos , Camundongos Transgênicos , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/genética , Placa Amiloide/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets. High-throughput screening provides a powerful platform for target discovery. Here, we describe an approach to use RNAi transfection of a pre-established AKT-induced senescent cell population and subsequent high-content imaging to screen for senescence regulators. We have incorporated multiparametric readouts, including cell number, proliferation, and senescence-associated beta-galactosidase (SA-ßGal) staining. Using machine learning and automated image analysis, we also describe methods to classify distinct phenotypes of cells with SA-ßGal staining. These methods can be readily adaptable to high-throughput functional screens interrogating the mechanisms that maintain and prevent senescence in various contexts.
Assuntos
Senescência Celular/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Oncogenes/fisiologia , Fenótipo , Coloração e Rotulagem/métodos , beta-Galactosidase/análise , Linhagem Celular Transformada , HumanosRESUMO
UNLABELLED: Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eµ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. SIGNIFICANCE: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eµ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer.
Assuntos
Benzotiazóis/administração & dosagem , DNA Ribossômico/antagonistas & inibidores , Everolimo/administração & dosagem , Linfoma de Células B/terapia , Naftiridinas/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzotiazóis/farmacologia , Sinergismo Farmacológico , Everolimo/farmacologia , Humanos , Linfoma de Células B/genética , Camundongos , Naftiridinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proto-Oncogene Mas , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Transcrição Gênica/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Benzotiazóis/farmacologia , Naftiridinas/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Polimerase I/antagonistas & inibidores , Transdução de Sinais , Animais , Apoptose , Crescimento Celular , Nucléolo Celular/metabolismo , Proliferação de Células , Cromatina/metabolismo , Ensaio Cometa , Dano ao DNA , DNA Ribossômico/genética , Fibroblastos/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Polimerase I/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
To influence physiology and pathophysiology, G protein-coupled receptors (GPCRs) have evolved to appropriate additional signalling modalities, such as activation of adjacent membrane receptors. Epidermal growth factor receptors (EGFRs) mediate growth and remodelling actions of GPCRs, although the precise network of gene products and molecular cascades linking GPCRs to EGFRs (termed EGFR transactivation) remains incomplete. In this review, we describe the current view of GPCR-EGFR transactivation, identifying the established models of receptor cross-talk. We consider the limitations in our current knowledge, and propose that recent advances in molecular and cell biology technology, including functional genomics approaches, will allow a renewed focus of efforts to understand the mechanism underlying EGFR transactivation. Using an unbiased approach for identification of the molecules required for GPCR-mediated EGFR transactivation will provide a contemporary and more complete representation from which to extrapolate therapeutic control in diseases from cardiovascular remodelling to cancer.