RESUMO
The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.
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Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.
Assuntos
Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/sangue , Metilação de DNA , Genes Supressores de Tumor , Proteínas de Membrana/sangue , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Regulação para CimaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.
Assuntos
Hemangioblastos/metabolismo , Proteínas de Membrana/genética , Peixe-Zebra/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Desenvolvimento Embrionário , Hematopoese , Morfolinos/farmacologia , Família Multigênica , Peixe-Zebra/sangue , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The reprimo (RPRM) gene family is a group of single exon genes present exclusively within the vertebrate lineage. Two out of three members of this family are present in humans: RPRM and RPRM-Like (RPRML). RPRM induces cell cycle arrest at G2/M in response to p53 expression. Loss-of-expression of RPRM is related to increased cell proliferation and growth in gastric cancer. This evidence suggests that RPRM has tumor suppressive properties. However, the molecular mechanisms and signaling partners by which RPRM exerts its functions remain unknown. Moreover, scarce studies have attempted to characterize RPRML, and its functionality is unclear. Herein, we highlight the role of the RPRM gene family in gastric carcinogenesis, as well as its potential applications in clinical settings. In addition, we summarize the current knowledge on the phylogeny and expression patterns of this family of genes in embryonic zebrafish and adult humans. Strikingly, in both species, RPRM is expressed primarily in the digestive tract, blood vessels and central nervous system, supporting the use of zebrafish for further functional characterization of RPRM. Finally, drawing on embryonic and adult expression patterns, we address the potential relevance of RPRM and RPRML in cancer. Active investigation or analytical research in the coming years should contribute to novel translational applications of this poorly understood gene family as potential biomarkers and development of novel cancer therapies.
Assuntos
Proteínas de Ciclo Celular/genética , Metilação de DNA/genética , Glicoproteínas/genética , Proteínas de Membrana/genética , Neoplasias Gástricas/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Regiões Promotoras Genéticas , Neoplasias Gástricas/patologiaRESUMO
The Reprimo (RPRM) family is composed of highly conserved single-exon genes. The expression pattern of this gene family has been recently described during zebrafish (Danio rerio) embryogenesis, and primarily locates in the nervous system. Its most characterized member, RPRM, which duplicated to give rise rprma and rprmb in the fish lineage, is known to act as a tumor-suppressor gene in mammalian models. Here, we describe in detail the spatiotemporal expression of three rprm genes (rprma, rprmb, and rprml) within distinct anatomical structures in the developing peripheral and central nervous system. In the zebrafish, rprma mRNA is expressed in the olfactory placodes (OP) and olfactory epithelium (OE), rprmb is observed in the tectum opticum (TeO) and trigeminal ganglion (Tg), whereas rprml is found primarily in the telencephalon (Tel). At protein level, RPRM is present in a subset of cells in the OP, and neurons in the OE, TeO, hindbrain and sensory peripheral structures. Most importantly, the expression of RPRM has been conserved between teleosts and mammals. Thus, we provide a reference dataset describing the expression patterns of RPRM gene products during zebrafish and mouse development as a first step to approach the physiological role of the RPRM gene family.
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Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Hibridização in Situ Fluorescente , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologiaRESUMO
Genes related to human diseases should be natural targets for evolutionary studies, since they could provide clues regarding the genetic bases of pathologies and potential treatments. Here we studied the evolution of the reprimo gene family, a group of tumor-suppressor genes that are implicated in p53-mediated cell cycle arrest. These genes, especially the reprimo duplicate located on human chromosome 2, have been associated with epigenetic modifications correlated with transcriptional silencing and cancer progression. We demonstrate the presence of a third reprimo lineage that, together with the reprimo and reprimo-like genes, appears to have been differentially retained during the evolutionary history of vertebrates. We present evidence that these reprimo lineages originated early in vertebrate evolution and expanded as a result of the two rounds of whole genome duplications that occurred in the last common ancestor of vertebrates. The reprimo gene has been lost in birds, and the third reprimo gene lineage has been retained in only a few distantly related species, such as coelacanth and gar. Expression analyses revealed that the reprimo paralogs are mainly expressed in the nervous system. Different vertebrate lineages have retained different reprimo paralogs, and even in species that have retained multiple copies, only one of them is heavily expressed.
Assuntos
Proteínas de Ciclo Celular/genética , Evolução Molecular , Genes Supressores de Tumor , Família Multigênica , Filogenia , Vertebrados/genética , Sequência de Aminoácidos , Animais , Duplicação Gênica , Humanos , Funções Verossimilhança , Alinhamento de Sequência , Sintenia/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Niemann-Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal-lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c-Abl/p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c-Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c-Abl-specific inhibitor imatinib. The proapoptotic c-Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c-Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c-Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c-Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.
Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebelar/efeitos dos fármacos , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Benzamidas , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebelar/metabolismo , Córtex Cerebelar/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Mesilato de Imatinib , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/patologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismoRESUMO
Se realiza una revisión de diversos procederes diagnósticos utilizados en el estudio de la hipertensión arterial debido a la elevaba morbimortalidad que se observa en la actualidad por esta afección. Un mejor conocimiento de su utilidad relativa permitirá la búsqueda de casos secundarios de hipertensión potencialmente curables. Se señalan las principales alteraciones informadas en el pielograma minutado, el renograma isotópico y la arteriografía renal. Se hacen algunas consideraciones sobre otros métodos de estudio más novedosos, como la digital substraction angiography, (DSA) y las técnicas angiográficas de angulación combinada