RESUMO
We performed post-mortem studies on two patients with advanced Parkinson's disease 8 and10 years following AAV2-neurturin (CERE120) gene therapy, the longest post-mortem trophic factor gene therapy cases reported to date. CERE120 was delivered to the putamen bilaterally in one case (10 years post-surgery), and to the putamen plus the substantia nigra bilaterally in the second (8 years post-surgery). In both patients there was persistent, albeit limited, neurturin expression in the putamen covering â¼3-12% of the putamen. In the putamen, dense staining of tyrosine hydroxylase-positive fibres was observed in areas that contained detectable neurturin expression. In the substantia nigra, neurturin expression was detected in 9.8-18.95% and 22.02-39% of remaining melanin-containing neurons in the patient with putamenal and combined putamenal and nigral gene delivery, respectively. Melanized neurons displayed intense tyrosine hydroxylase and RET proto-oncogene expression in nigral neurons in the patient where CERE120 was directly delivered to the nigra. There was no difference in the degree of Lewy pathology in comparison to untreated control patients with Parkinson's disease, and α-synuclein aggregates were detected in neurons that also stained for neurturin, RET, and tyrosine hydroxylase. These changes were not associated with antiparkinsonian benefits likely due to the limited neurturin expression. This study provides the longest term evidence of persistent transgene expression following gene delivery to the CNS and the first human results when targeting both the terminal fields in the putamen as well as the originating nigral neurons.
Assuntos
Terapia Genética , Neurturina/biossíntese , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Humanos , Corpos de Lewy/metabolismo , Melaninas/imunologia , Pessoa de Meia-Idade , Neurônios/imunologia , Neurturina/administração & dosagem , Doença de Parkinson/imunologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret/biossíntese , Putamen/imunologia , Putamen/metabolismo , Substância Negra/imunologia , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/imunologia , alfa-Sinucleína/metabolismoRESUMO
Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1â¯mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5-11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starchiodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.
Assuntos
Cloridrato de Fingolimode/análogos & derivados , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Atrofia de Múltiplos Sistemas/patologia , Fármacos Neuroprotetores/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Cloridrato de Fingolimode/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/efeitos dos fármacos , alfa-Sinucleína/genéticaRESUMO
POURPOSES: We investigated the expression of RET9 and RET51 isoforms in medullary (MTC), papillary (PTC) thyroid carcinoma, normal thyroid tissues, and pheochromocytoma (PHEO) to verify if these isoforms are present also in follicular thyroid cell-derived tissues, and if there is a differential expression of RET9 and RET51 in MTC. METHODS: Nineteen patients with MTC, 18 patients with PTC, 18 samples of contralateral normal thyroid tissues, and 5 cases of PHEO were included in this study. RET isoform expression was studied by real-time RT-PCR. RESULTS: All MTCs and PHEOs were positive for RET9 and RET51. Fourteen/eighteen (77.7%) PTC cases were positive for RET9 and/or RET51, and four were positive for only one of the genes. In normal thyroid tissues, 3/18 (16.7%) cases were negative for both isoforms, 4/18 (22.2%) were positive for both, and 11/18 (61.1%) were positive for only one. RET isoforms were expressed at different levels in MTC, PHEO, PTC, and normal thyroid tissues: RET9 expression was higher in PHEO than in MTC, PTC, and normal thyroid tissues. RET9 expression was also higher in MTC than in PTC and normal thyroid tissues. No difference was observed between PTC and normal thyroid tissues. A similar pattern of expression was observed for RET51. In addition, RET51 was significantly more expressed than RET9 in MTC, while RET9 was the predominant isoform in PHEO. CONCLUSIONS: Our study documented the expression of the RET9 and RET51 isoforms in normal thyroid and PTC tissues. RET9 and RET51 isoforms were also present in MTC and PHEO. RET51 expression was higher than RET9 expression in MTC, while there was no difference in the expression of these two isoforms in PTC and normal thyroid tissues. RET9 was more highly expressed than RET51 in PHEOs.
Assuntos
Carcinoma Neuroendócrino/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação/genética , Feocromocitoma/genética , Feocromocitoma/metabolismo , Isoformas de Proteínas/genéticaRESUMO
Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.
Assuntos
Carcinoma Neuroendócrino/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Animais , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Técnicas de Introdução de Genes/métodos , Mutação em Linhagem Germinativa , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Hiperplasia do Timo/genética , Hiperplasia do Timo/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismoRESUMO
The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.
Assuntos
Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Nociceptores/metabolismo , Idoso , Animais , Feminino , Humanos , Hibridização In Situ , Masculino , Camundongos , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.8/biossíntese , Canal de Sódio Disparado por Voltagem NAV1.9/biossíntese , Proteínas de Neurofilamentos/biossíntese , Dor/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Receptor trkA/biossíntese , Especificidade da Espécie , Canais de Cátion TRPV/biossínteseRESUMO
HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.
Assuntos
Sequência de Bases , Regulação da Expressão Gênica , Predisposição Genética para Doença , Doença de Hirschsprung/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Deleção de Sequência , Fatores de Transcrição/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Feminino , Doença de Hirschsprung/genética , Doença de Hirschsprung/patologia , Proteínas de Homeodomínio/genética , Humanos , Masculino , Peptídeos/genética , Proteínas Proto-Oncogênicas c-ret/genética , Fatores de Transcrição/genéticaAssuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Bucais/patologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Intervalo Livre de Doença , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Neoplasias Bucais/metabolismo , Neoplasias Bucais/mortalidade , Proteínas Proto-Oncogênicas c-ret/genética , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Germline and somatic mutations play a crucial role in breast cancer (BC), driving the initiation, progression, response to therapy and outcome of the disease. Hormonal therapy is limited to patients with tumors expressing steroid hormone receptors, such as estrogen receptor (ER), nevertheless resistance often limits its success.The RET gene is known to be involved in neurocristopathies such as Hirschsprung disease and Multiple Endocrine Neoplasia type 2, in the presence of loss-of-function and gain-of-function mutations, respectively. More recently, RET over-expression has emerged as a new player in ER-positive (ER+) BC, and as a potential target to enhance sensitivity and avoid resistance to tamoxifen therapy.Therefore, targeting the RET pathway may lead to new therapies in ER+ BC. To this end, we have investigated the molecular mechanisms which underlie RET overexpression and its possible modulation in two BC cell lines, MCF7 and T47D, showing different RET expression levels. Moreover, we have carried out a pilot association study in 93 ER+ BC patients. Consistent with the adverse role of RET over-expression in BC, increased overall survival was observed in carriers of the variant allele of SNP rs2435357, a RET polymorphism already known to be associated with reduced RET expression.
Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Proto-Oncogênicas c-ret/biossíntese , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Projetos Piloto , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
INTRODUCTION: ROS1 and RET gene fusions were recently discovered in non-small-cell lung cancer (NSCLC) as potential therapeutic targets with small-molecule kinase inhibitors. The conventional screening methods of these fusions are time-consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing polymerase chain reaction and the sensitivity of mass spectrometry. METHODS: The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false-negative results. To flag false-positives, the system also comprises two independent assays for each fusion gene junction. RESULTS: The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using complimentary DNA from lung tissue of healthy individuals. The system was further validated using complimentary DNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors. CONCLUSION: The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Proteínas de Fusão Oncogênica/biossíntese , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-ret/biossínteseRESUMO
RET gene is crucial for the development of enteric nervous system, and dys-regulation of RET expression causes Hirschsprung disease. HOXB5 regulates RET transcription, and perturbations in transcriptional regulation by HOXB5 caused reduced RET expression and defective enteric nervous system development in mice. The mechanisms by which HOXB5 regulate RET transcription are unclear. Thus, unraveling the regulatory mechanisms of HOXB5 on RET transcription could lead to a better understanding of the etiology of Hirschsprung disease. In this study, we identified and confirmed HOXB5 binding to the multi-species conserved sequence (MCS+9.7) in the first intron of the RET gene. We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos. The deletion of HOXB5 binding site interfered with HOXB5 trans-activation. Furthermore, transfection of HOXB5 induced endogenous RET transcription, enhanced the co-precipitation of TATA-box binding protein with the transcription start site of RET, and induced histone H3K4 trimethylation in chromatin regions upstream and downstream of RET transcription start site. In conclusion, (i) HOXB5 physically interacted with MCS+9.7 and enhanced RET transcription, (ii) HOXB5 altered chromatin conformation and histone modification of RET locus, which could facilitate the formation of transcription complex, and enhance RET transcription, (iii) expression of RET was mediated by a complex regulatory network of transcription factors functioning in a synergistic, additive and/or independent manners. Hence, dys-regulation of RET expression by HOXB5 could result in insufficient RET expression and Hirschsprung disease.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Regulação da Expressão Gênica , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Humanos , Íntrons , Mutagênese Sítio-Dirigida , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-ret/biossíntese , Transcrição Gênica , TransfecçãoRESUMO
RET is a gene playing a key role during embryogenesis and in particular during the enteric nervous system development. High levels of RET gene expression are maintained in different human tissues also in adulthood, although their physiological role remains unclear. In particular, collected evidences of a RET contribution in the development and maintenance of the immune system prompted us to investigate its levels of surface expression on peripheral blood mononuclear cells (PBMCs) from adult healthy donors. Despite variability among samples, RET expression was conserved at similar levels in the different immune cell subsets, with higher correlations in similar lymphocyte populations (i.e. CD4(+) and CD8(+) T cells). Conversely, no correlation was found between the amount of RET receptor, the expression of its putative ligands and co-receptors and the genotypes at the RET locus. Moreover, we investigated the RET-associated inflammatory pathways in PBMCs from healthy donors both in resting conditions and upon glial cell derived neurotrophic factor (GDNF) and GPI-linked co-receptors alpha 1 (GFRα1) mediated RET activation. RET mRNA levels positively correlated with the transcript amount of interleukin-8 (IL-8), a cytokine produced by monocytes and macrophages, though we could not demonstrate its direct effect on RET expression by in vitro experiments on THP1 human monocytic cells. These results imply that RET expression might be influenced by either cis- and/or trans-factors, which together would account for its high variability within the general population, and suggest a putative functional role of the RET gene in modulating immune cell responses during inflammation and carcinogenesis.
Assuntos
Carcinogênese/genética , Interleucina-8/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Adulto , Regulação da Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Interleucina-8/genética , Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/biossíntese , Linfócitos T/metabolismoRESUMO
RET (rearranged during transfection) is a receptor tyrosine kinase overexpressed in a subset of oestrogen receptor (ER)-positive breast cancers whose expression is regulated by ER signalling. The article from the Hynes group has reported for the first time that RET expression can also be regulated by the inflammatory cytokine IL-6. Importantly, RET and IL-6 interact at a functional level to control migration and the metastatic potential of ER-positive breast cancer cells, in a process that is mediated by FAK activation. Further, targeting RET with receptor tyrosine kinase inhibitors was reported to be more effective than endocrine therapies in impairing metastatic dissemination in vivo, thereby indicating a level of RET regulation that is independent of ER.
Assuntos
Neoplasias da Mama/patologia , Interleucina-6/metabolismo , Metástase Neoplásica/patologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Movimento Celular/genética , Ativação Enzimática , Feminino , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/biossíntese , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Estrogênio/metabolismoRESUMO
We show that elevated levels of Ret receptor are found in different sub-types of human breast cancers and that high Ret correlates with decreased metastasis-free survival. The role of Ret in ER+ breast cancer models was explored combining in vitro and in vivo approaches. Our analyses revealed that ligand-induced Ret activation: (i) stimulates migration of breast cancer cells; (ii) rescues cells from anti-proliferative effects of endocrine treatment and (iii) stimulates expression of cytokines in the presence of endocrine agents. Indeed, we uncovered a positive feed-forward loop between the inflammatory cytokine IL6 and Ret that links them at the expression and the functional level. In vivo inhibition of Ret in a metastatic breast cancer model inhibits tumour outgrowth and metastatic potential. Ret inhibition blocks the feed-forward loop by down-regulating Ret levels, as well as decreasing activity of Fak, an integrator of IL6-Ret signalling. Our results suggest that Ret kinase should be considered as a novel therapeutic target in subsets of breast cancer.
Assuntos
Neoplasias da Mama/fisiopatologia , Movimento Celular , Proliferação de Células , Metástase Neoplásica/fisiopatologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/mortalidade , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-6/metabolismo , Camundongos , Análise em Microsséries , Análise de SobrevidaRESUMO
RET has recently been identified as a potential new oncogenic driver in a subset of patients with non-small cell lung cancer (NSCLC). In this issue of Cancer Discovery, Drilon and colleagues report preliminary trial data with a RET inhibitor in RET fusion-positive NSCLC, validating RET as a therapeutic target in lung cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anilidas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ret/biossíntese , Piridinas/uso terapêutico , Adenocarcinoma de Pulmão , HumanosRESUMO
The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.
Assuntos
Proteínas de Homeodomínio/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Neurotransmissores/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Contagem de Células , Feminino , Feto , Deleção de Genes , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação/fisiologia , Gravidez , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/genética , Somatostatina/genética , Somatostatina/fisiologia , Gânglio Estrelado/citologia , Gânglio Estrelado/crescimento & desenvolvimento , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/embriologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/fisiologia , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
The discovery of RET fusions in lung cancers has uncovered a new therapeutic target for patients whose tumors harbor these changes. In an unselected population of non-small cell lung carcinomas (NSCLCs), RET fusions are present in 1% to 2% of cases. This incidence increases substantially, however, in never-smokers with lung adenocarcinomas that lack other known driver oncogenes. Although preclinical data provide experimental support for the use of RET inhibitors in the treatment of RET fusion-positive tumors, clinical data on response are lacking. We report preliminary data for the first three patients treated with the RET inhibitor cabozantinib on a prospective phase II trial for patients with RET fusion-positive NSCLCs (NCT01639508). Confirmed partial responses were observed in 2 patients, including one harboring a novel TRIM33-RET fusion. A third patient with a KIF5B-RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remain progression-free on treatment.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anilidas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ret/biossíntese , Piridinas/uso terapêutico , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Anilidas/efeitos adversos , Intervalo Livre de Doença , Fusão Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-ret/genética , Piridinas/efeitos adversosRESUMO
Today there is a better understanding of the events involved in the initiation and progression of thyroid cancer. It is indeed now known that BRAF and RAS mutations and RET/PTC and PAX8/PPARγ rearrangements account for the majority of molecular alterations detected in differentiated thyroid cancers. Abnormal regulation of microRNAs (miRNAs) is also a promising way of research. The diagnostic utility and prognostic value of detecting these molecular events has been analyzed in several recent studies. BRAF mutation analysis improves the performance of fine-needle aspiration diagnosis by increasing specificity in "indeterminate" cytologies and sensitivity in false negatives. Testing for a "panel of mutations" (BRAF, RAS, RET/PTC and PAX8/PPARγ) improves the performance, detecting papillary carcinomas with non-classic histology. The specificity of these analyzes is excellent but their sensitivity is still insufficient. In the future, specific miRNAs expression profiles in thyroid carcinoma and identification of new mutations might provide interesting information. Several studies have found that BRAF mutations are associated with a more aggressive tumor behavior, a higher risk of recurrence and treatment failure. With regard to the other mutations and rearrangements, current data are conflicting and it seems premature to draw practical conclusions applicable in routine practice. Lastly, targeted therapy with tyrosine kinase inhibitors, based on our understanding of the molecular mechanisms of thyroid oncogenesis, has shown promise in metastatic, progressive, and radioactive iodine-refractory differentiated thyroid carcinomas.
Assuntos
Genes Neoplásicos , Técnicas de Diagnóstico Molecular/métodos , Proteínas de Neoplasias/genética , Neoplasias da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/diagnóstico , Adenocarcinoma Folicular/diagnóstico , Adenocarcinoma Folicular/tratamento farmacológico , Adenocarcinoma Folicular/epidemiologia , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Adenoma/diagnóstico , Adenoma/epidemiologia , Adenoma/genética , Adenoma/patologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma Medular/diagnóstico , Carcinoma Medular/tratamento farmacológico , Carcinoma Medular/epidemiologia , Carcinoma Medular/genética , Carcinoma Medular/patologia , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/epidemiologia , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Ensaios Clínicos como Assunto , Análise Mutacional de DNA/métodos , Perfilação da Expressão Gênica , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX8 , PPAR gama/biossíntese , PPAR gama/genética , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Receptores Patched , Valor Preditivo dos Testes , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/biossíntese , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Sensibilidade e EspecificidadeRESUMO
The glial cell line-derived neurotrophic factor (GDNF) has multiple functions that promote cell survival, proliferation and migration in different cell types. The experimental over-expression of GDNF in mouse testis leads to infertility and promotes seminomatous germ cell tumours in older animals, which suggests that deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis. GDNF activates downstream pathways upon binding to its specific co-receptor GDNF family receptor-a 1 (GFRA1). This complex then interacts with Ret and other co-receptors to activate several intracellular signalling cascades. To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell tumours, we analysed GFRA1 and Ret expression patterns in seminoma samples. We demonstrated, via immunohistochemistry, that GFRA1, but not Ret, is over-expressed in in situ carcinoma (CIS) and in intratubular and invasive seminoma cells compared with normal human germ cells. Functional analysis of the GDNF biological activity was performed on TCam-2 seminoma cell line. Reverse transcription-PCR (RT-PCR) and immunohistochemical analyses demonstrate that TCam-2 cells express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level. In TCam-2 cells, although GDNF is not mitogenic, it is able to induce migration, as demonstrated by a Boyden chamber assay, possibly through the Src and MEK pathways. Moreover, GDNF promotes invasive behaviour, an effect dependent on pericellular protease activity, possibly through the activity of matrix metalloproteinases. GFRA1 over-expression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer.
Assuntos
Seminoma/patologia , Adulto , Carcinoma in Situ/metabolismo , Linhagem Celular Tumoral , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/fisiopatologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , RNA Mensageiro/metabolismo , Seminoma/metabolismo , Neoplasias Testiculares/patologiaRESUMO
OBJECTIVE: To examine the role of RET in renal malignancy, in particular papillary renal cell carcinoma (RCC). MATERIALS AND METHODS: A cohort of 111 archival renal samples was used consisting of 94 renal cancers (66 papillary RCC, 18 conventional clear cell carcinoma, 10 chromophobe RCC), 4 benign oncocytomas, and 13 normal kidney tissues. RET protein expression was examined by immunohistochemistry and expression levels were correlated with clinicopathologic and patient survival data. RESULTS: Positive RET staining was seen in 34/66 (52%) papillary RCCs, 4/10 (40%) chromophobe carcinomas, 4/4 (100%) oncocytomas, and 11/13 (85%) normal kidney samples. All 18 cases of conventional clear cell carcinoma had negative RET staining. RET expression was associated with low Fuhrman nuclear grade. CONCLUSIONS: RET protein may be contributing in part to an adaptation of a papillary growth pattern in certain renal malignancies. Given the possible therapeutic benefit of small molecule inhibitors of RET activation, further work needs to be done to highlight the functional relevance of RET protein expression in papillary RCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-ret/biossíntese , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-ret/análiseRESUMO
The RET tyrosine kinase receptor plays an important role in the development and maintenance of the nervous system. Although the ligand-induced RET signaling pathway has been well described, little is known about the regulation of RET surface expression, which is integral to the cell ability to control the response to ligand stimuli. We found that in dorsal root ganglion (DRG) neurons, which co-express RET and TrkB, the receptor surface levels of RET are significantly higher than that of TrkB. Using a sequence substitution strategy, we identified a key motif (Box1), which is necessary and sufficient for the differential RET and TrkB surface levels. Furthermore, pharmacological and mutagenesis assays revealed that protein kinase C (PKC) and high K(+) depolarization increase RET surface levels through phosphorylation of the Thr(675) residue in the Box1 motif. Finally, we found that the phosphorylation status of the Thr(675) residue influences RET mediated response to GDNF stimulation. In all, these findings provide a novel mechanism for the modulation of RET surface expression.