RESUMO
The mechanism by which the 8q24 MYC enhancer region, including cancer-associated variant rs6983267, increases cancer risk is unknown due to the lack of protein-coding genes at 8q24.21. Here we report the identification of long noncoding RNAs named cancer-associated region long noncoding RNAs (CARLos) in the 8q24 region. The expression of one of the long noncoding RNAs, CARLo-5, is significantly correlated with the rs6983267 allele associated with increased cancer susceptibility. We also found the MYC enhancer region physically interacts with the active regulatory region of the CARLo-5 promoter, suggesting long-range interaction of MYC enhancer with the CARLo-5 promoter regulates CARLo-5 expression. Finally, we demonstrate that CARLo-5 has a function in cell-cycle regulation and tumor development. Overall, our data provide a key of the mystery of the 8q24 gene desert.
Assuntos
Cromossomos Humanos Par 8/genética , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Neoplasias/genética , RNA Longo não Codificante/genética , Sequência de Bases , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Malignant gliomas have a distinctive ability to infiltrate the brain parenchyma and disrupt the neural extracellular matrix that inhibits motility of axons and normal neural cells. Chondroitin sulfate proteoglycans (CSPGs) are among the major inhibitory components in the neural matrix, but surprisingly, some are up-regulated in gliomas and act as pro-invasive signals. In the normal brain, CSPGs are thought to associate with hyaluronic acid and glycoproteins such as the tenascins and link proteins to form the matrix scaffold. Here, we examined for the first time the expression of link proteins in human brain and malignant gliomas. Our results indicate that HAPLN4 and HAPLN2 are the predominant members of this family in the adult human brain but are strongly reduced in the tumor parenchyma. To test if their absence was related to a pro-invasive gain of function of CSPGs, we expressed HAPLN4 in glioma cells in combination with the CSPG brevican. Surprisingly, HAPLN4 increased glioma cell adhesion and migration and even potentiated the motogenic effect of brevican. Further characterization revealed that HAPLN4 expressed in glioma cells was largely soluble and did not reproduce the strong, hyaluronan-independent association of the native protein to brain subcellular membranes. Taken together, our results suggest that the tumor parenchyma is rich in CSPGs that are not associated to HAPLNs and could instead interact with other extracellular matrix proteins produced by glioma cells. This dissociation may contribute to changes in the matrix scaffold caused by invasive glioma cells.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteoglicanas/metabolismo , Adulto , Western Blotting , Encéfalo/metabolismo , Brevicam , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas da Matriz Extracelular/genética , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas do Tecido Nervoso/genética , Proteoglicanas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Non-small cell lung carcinoma (NSCLC) is leading cause of cancer-related deaths in the world. The Tumor Suppressor Candidate 3 (TUSC3) at chromosome 8p22 known to be frequently deleted in cancer is often found to be deleted in advanced stage of solid tumors. However, the role of TUSC3 still remains controversial in lung cancer and context-dependent in several cancers. Here we propose that miR-224/-520c-dependent TUSC3 deficiency enhances the metastatic potential of NSCLC through the alteration of three unfolded protein response pathways and HRD1-dependent ERAD. ATF6α-dependent UPR is enhanced whereas the affinity of HRD1 to its substrates, PERK, IRE1α and p53 is weakened. Consequently, the alteration of UPRs and the suppressed p53-NM23H1/2 pathway by TUSC3 deficiency is ultimately responsible for enhancing metastatic potential of lung cancer. These findings provide mechanistic insight of unrecognized roles of TUSC3 in cancer progression and the oncogenic role of HRD1-dependent ERAD in cancer metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Degradação Associada com o Retículo Endoplasmático/fisiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Degradação Associada com o Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Hibridização In Situ , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools. Here, we describe the generation, validation and characterization of a new mouse model that expresses Cre recombinase driven by the Prokr2 promoter, using CRISPR-Cas9 technology. Cre expression was visualized using reporter genes, tdTomato and GFP, in males and females. Expression of Cre-induced reporter genes was found in brain sites previously described to express Prokr2, e.g., the paraventricular and the suprachiasmatic nuclei, and the area postrema. The Prokr2-Cre mouse model was further validated by colocalization of Cre-induced GFP and Prokr2 mRNA. No disruption of Prokr2 expression, GnRH neuronal migration or fertility was observed. Comparative analysis of Prokr2-Cre expression in male and female brains revealed a sexually dimorphic distribution confirmed by in situ hybridization. In females, higher Cre activity was found in the medial preoptic area, ventromedial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral parabrachial nucleus. In males, Cre was higher in the amygdalo-hippocampal area. The sexually dimorphic pattern of Prokr2 expression indicates differential roles in reproductive function and, potentially, in other physiological systems.
Assuntos
Encéfalo/patologia , Síndrome de Kallmann/patologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Caracteres Sexuais , Animais , Modelos Animais de Doenças , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrases , Síndrome de Kallmann/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismoRESUMO
Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Targeting of the soluble factors that trigger invasion and resistance, therefore, could have a significant impact against the infiltrative glioma cells that are a major source of recurrence. Fibulin-3 is a matrix protein that is absent in normal brain but upregulated in gliomas and promotes tumor invasion by unknown mechanisms. Here, we show that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor or Notch, and promotes tumor cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we found that controlled downregulation of fibulin-3 reduced Notch signaling and led to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. In addition, fibulin-3 expression correlated with expression levels of Notch-dependent genes and was a marker of Notch activation in patient-derived glioma samples. These findings underscore a major role for the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this work describes a noncanonical, soluble activator of Notch in a cancer model and shows how Notch signaling can be reduced by targeting tumor-specific accessible molecules in the tumor microenvironment.
Assuntos
Neoplasias Encefálicas/patologia , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/fisiologia , Glioma/patologia , Receptor Notch1/fisiologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Comunicação Parácrina/genética , Comunicação Parácrina/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant gliomas are highly invasive tumors with an almost invariably rapid and lethal outcome. Surgery and chemoradiotherapy fail to remove resistant tumor cells that disperse within normal tissue, which are a major cause for disease progression and therapy failure. Infiltration of the neural parenchyma is a distinctive property of malignant gliomas compared with other solid tumors. Thus, glioma cells are thought to produce unique molecular changes that remodel the neural extracellular matrix and form a microenvironment permissive for their motility. Here, we describe the unique expression and proinvasive role of fibulin-3, a mesenchymal matrix protein specifically upregulated in gliomas. Fibulin-3 is downregulated in peripheral tumors and is thought to inhibit tumor growth. However, we found fibulin-3 highly upregulated in gliomas and cultured glioma cells, although the protein was undetectable in normal brain or cultured astrocytes. Overexpression and knockdown experiments revealed that fibulin-3 did not seem to affect glioma cell morphology or proliferation, but enhanced substrate-specific cell adhesion and promoted cell motility and dispersion in organotypic cultures. Moreover, orthotopic implantation of fibulin-3-overexpressing glioma cells resulted in diffuse tumors with increased volume and rostrocaudal extension compared with controls. Tumors and cultured cells overexpressing fibulin-3 also showed elevated expression and activity of matrix metalloproteases, such as MMP-2/MMP-9 and ADAMTS-5. Taken together, our results suggest that fibulin-3 has a unique expression and protumoral role in gliomas, and could be a potential target against tumor progression. Strategies against this glioma-specific matrix component could disrupt invasive mechanisms and restrict the dissemination of these tumors.