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Even with systemic chemotherapy, cytoreductive surgery (CRS), and hyperthermic intraperitoneal chemotherapy (HIPEC), peritoneal metastases (PM) remain a common site of disease progression for colorectal cancer (CRC) and are frequently associated with a poor prognosis. The mass spectrometry (MS) method known as Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-TOF) is frequently used in medicine to identify structural compounds and biomarkers. It has been demonstrated that lipids are crucial in mediating the aggressive growth of tumors. In order to investigate the lipid profiles, particularly with regard to histological distribution, we used MALDI-TOF MS (MALDI-MS) and MALDI-TOF imaging MS (MALDI-IMS) on patient-derived tumor organoids (PDOs) and PM clinical samples. According to the MALDI-IMS research shown here, the predominant lipid signature of PDOs in PM tissues, glycosphingolipid (GSL) sulfates or sulfatides, or STs, is unique to the areas containing tumor cells and absent from the surrounding stromal compartments. Bioactive lipids are derived from arachidonic acid (AA), and AA-containing phosphatidylinositol (PI), or PI (18:0-20:4), is shown to be highly expressed in the stromal components. On the other hand, the tumor components contained a higher abundance of PI species with shorter and more saturated acyl chains (C34 and C36 carbons). The cellular subversion of PI and ST species may alter in ways that promote the growth, aggressiveness, and metastasis of tumor cells. Together, these findings suggest that the GSL/ST metabolic programming of PM may contain novel therapeutic targets to impede or halt PM progression.
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BACKGROUND: Currently used CA15-3 and CEA have found their clinical application particularly in the follow-up of patients with advanced disease. Novel biomarkers are urgent, especially for improving early diagnosis as well as for discriminating between benign and malignant disease. PATIENTS AND METHODS: In the present study, we used a proteomic approach based on surface-enhanced laser desorption/ionization-time of flight-mass spectrometry screening with the aim of identifying differentially expressed 2-30 kDa proteins in plasma of patients with malignant (65 cases) and benign (88 cases) breast lesions with respect to 121 healthy controls. RESULTS: We found that the most promising SELDI peaks were those corresponding to hepcidin-25 and ferritin light chain. We evaluated the capability of these peaks in predicting malignant and benign breast lesions using the area under the receiver operating characteristic curve (AUC). The results showed a good capacity to predict malignant breast lesions for hepcidin-25 [AUC: 0.82; 95% confidence interval (CI) 0.75-0.90] and ferritin light chain (AUC: 0.86; 95% CI 0.79-0.92). Conversely, a weak and satisfactory capability to predict benign breast lesion was observed for hepcidin-25 (AUC: 0.63; 95% CI 0.41-0.85) and ferritin light chain (AUC: 0.73; 95% CI 0.49-0.97). A significant association between HER2 status and hepcidin-25 was observed and the distribution of transferrin and ferritin were found significantly different in patients with breast cancer when compared with that of controls. CONCLUSIONS: This study provides evidence that hepcidin and ferritin light chain level in plasma may be of clinical usefulness to predict malignant and benign disease with respect to healthy controls.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Ferritinas/sangue , Hepcidinas/sangue , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Curva ROC , Adulto JovemRESUMO
OBJECTIVES: Breast cancer is the most common form of cancer affecting women, and the strongest risk factor remains family history. Although screening in asymptomatic women seems able to reduce breast-cancer related mortality, it is of limited usefulness in young women and patients with familial breast cancer syndrome. New diagnostic tools useful for breast cancer management are urgently needed. The aim of the present paper is to look for new candidate tumor markers useful for diagnosis in these patients. DESIGN AND METHODS: In this prospective study 292 serum samples (100 from healthy people, 100 from sporadic breast cancer patients and 92 from familial breast cancer patients) were analyzed by SELDI-TOF-MS. All samples both from cancer patients and healthy subjects were run in duplicate and randomly spotted on CM10 and IMAC30 protein chip array. Data were analyzed using the expression differential mapping (EDM) tool, decisional tree and multivariate analysis. A further in silico investigation was performed in order to hypothesize the identity of evidenced peptides. RESULTS: EDM highlighted thirteen and sixteen significant differentially expressed peaks by CM10 and IMAC30 protein chip respectively. Subsequent analysis showed that two peaks at m/z 11730 and 5066 were differentially expressed in sporadic and familial breast cancer patients respectively, while a peak at m/z 8127 was overexpressed only in familial breast cancer patients. The diagnostic power of protein peaks was tested by decisional tree; sensitivity and specificity ranged from 17% to 91.67%. CONCLUSIONS: We show that the serum profile of familial breast cancer patients was different when compared with that of sporadic breast cancer patients. We hypothesized the identity of the most significant peaks, and further studies are now planned in order to definitively establish the identity.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Predisposição Genética para Doença , Proteômica/métodos , Proteínas Sanguíneas/análise , Neoplasias da Mama/sangue , Estudos de Casos e Controles , Detecção Precoce de Câncer , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Estudos Prospectivos , Análise Serial de Proteínas , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não ParamétricasRESUMO
OBJECTIVES: The purpose of this study was to evaluate the efficacy of a protein-based pattern in serum previously determined by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization-Time of Flight) and considered potentially useful for prediction of clinical outcome of EGFR (epidermal growth factor receptor) TKIs (tyrosine kinase inhibitors) treated patients. DESIGN AND METHODS: We generated SELDI-TOF (Surface Enhanced Laser Desorption Ionization-Time of Flight) spectra in sera of 11 advanced NSCLC treated with Gefitinib. We detected the clusters with m/z 5843, 11445, 11529, 11685, 11759 and 11903 which were previously reported to be potential predictors of response to Gefitinib treatment. RESULTS: Four cluster peaks with m/z 5843, 11445, 11529, 11685 corresponded to SAA (serum amyloid A) protein on the basis on their calculated molecular weight, peptide fingerprinting and antibodies recognition. CONCLUSIONS: We confirm that several proteins already reported were isoforms of SAA but further studies are in development in order to evaluate the predictive value of such algorithm.
Assuntos
Proteínas Sanguíneas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Idoso , Algoritmos , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , Progressão da Doença , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Incidental/therapeutic thyroid irradiation causes hypothyroidism and nodular disease. As increasing numbers of children, adolescents and young adults are being cured of cancer after being treated with radiation therapy that has included the thyroid bed, it is important to understand whether early diagnosis and treatment of any radiation-related thyroid changes has an impact on their evolution and outcome. In this review the authors discuss main epidemiologic data, morbidity events, radiation cancerogenic effects and also original data about patients surveillance and evolution control of iatrogenic effects, together with some experiences of prospectively reducing the occurrence of post-radiation hypothyroidism.
Assuntos
Doença Iatrogênica , Radioterapia/efeitos adversos , Doenças da Glândula Tireoide/etiologia , Criança , Humanos , Hipotireoidismo/epidemiologia , Hipotireoidismo/etiologia , Doenças da Glândula Tireoide/epidemiologia , Glândula Tireoide/patologia , Glândula Tireoide/efeitos da radiação , Neoplasias da Glândula Tireoide/epidemiologia , Neoplasias da Glândula Tireoide/etiologiaRESUMO
Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.
Assuntos
Mutação em Linhagem Germinativa/genética , Proteínas Oncogênicas/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Medular/tratamento farmacológico , Carcinoma Medular/genética , Carcinoma Medular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Humanos , Camundongos , Camundongos Nus , Neoplasia Endócrina Múltipla Tipo 2a/tratamento farmacológico , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Neoplasia Endócrina Múltipla Tipo 2b/tratamento farmacológico , Neoplasia Endócrina Múltipla Tipo 2b/genética , Neoplasia Endócrina Múltipla Tipo 2b/metabolismo , Fosforilação/efeitos dos fármacos , Quinazolinas/farmacologia , Transdução de Sinais , Neoplasias da Glândula Tireoide/tratamento farmacológico , Tirosina/metabolismoRESUMO
The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células Cultivadas , Humanos , Camundongos , Fosforilação , Isoformas de Proteínas/fisiologia , Ratos , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Domínios de Homologia de src/fisiologiaRESUMO
We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.
Assuntos
Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , RNA/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento Alternativo , Animais , Transporte Biológico , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Camundongos , Modelos Biológicos , Neoplasia Endócrina Múltipla Tipo 2b/genética , Neoplasia Endócrina Múltipla Tipo 2b/metabolismo , Células NIH 3T3 , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , TransfecçãoRESUMO
Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.
Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Sobrevivência Celular/fisiologia , Primers do DNA , Regulação Neoplásica da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/patologiaRESUMO
The RET proto-oncogene encodes a receptor tyrosine kinase for transforming growth factor-beta-related neurotrophic factors, which include GDNF and neurturin. The expression of RET proto-oncogene was detected in several tissues, such as spleen, thymus, lymph nodes, salivary gland, and spinal cord, and in several neural crest-derived cell lines. RET expression in the thyroid gland was reported to be restricted to neural crest-derived C cells. The presence of RET mRNA or protein has not yet been reported in thyroid follicular cells. We previously demonstrated the expression of oncogenic rearranged versions of RET in papillary thyroid carcinomas: tumors derived from thyroid follicular cells. To assess the expression of the normal RET proto-oncogene in follicular cells, we analyzed its expression in a panel of neoplasias originating from thyroid follicular epithelial cells: papillary carcinomas and both follicular adenomas and carcinomas. We also demonstrated the presence of RET normal transcripts in two follicular thyroid carcinoma lymph node metastases. Moreover, we found the presence of the RET/ELE1 transcript, the reciprocal complementary form of the oncogenic fusion transcript ELE1/RET, in a papillary thyroid carcinoma specimen expressing the RET/PTC3 oncogene, thus demonstrating that the RET promoter is active in those cells after rearrangement. Finally, we show that in a papillary carcinoma-derived cell line expressing the proto-RET receptor and the related GFRalpha2 co-receptor, GDNF treatment induced RET tyrosine phosphorylation and subsequent signal transduction pathway, indicating that RET could be active in thyroid follicular cells.
Assuntos
Adenocarcinoma Folicular/metabolismo , Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Papilar/metabolismo , Sequência de Bases , Western Blotting , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Testes de Precipitina , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Experimental evidence has shown, both in vitro and in animal models, that neoplastic growth and subsequent metastasis formation depend on the tumor's ability to induce an angiogenic switch. This requires a change in the balance of angiogenic stimulators and inhibitors. To assess the potential role of angiogenesis factors in human thyroid tumor growth and spread, we analyzed their expression by semiquantitative RT-PCR and immunohistochemistry in normal thyroid tissues, benign lesions, and different thyroid carcinomas. Compared to normal tissues, in thyroid neoplasias we observed a consistent increase in vascular endothelial growth factor (VEGF), VEGF-C, and angiopoietin-2 and in their tyrosine kinase receptors KDR, Flt-4, and Tek. In particular, we report the overexpression of angiopoietin-2 and VEGF in thyroid tumor progression from a prevascular to a vascular phase. In fact, we found a strong association between tumor size and high levels of VEGF and angiopoietin-2. Furthermore, our results show an increased expression of VEGF-C in lymph node invasive thyroid tumors and, on the other hand, a decrease of thrombospondin-1, an angioinhibitory factor, in thyroid malignancies capable of hematic spread. These results suggest that, in human thyroid tumors, angiogenesis factors seem involved in neoplastic growth and aggressiveness. Moreover, our findings are in keeping with a recent hypothesis that in the presence of VEGF, angiopoietin-2 may collaborate at the front of invading vascular sprouts, serving as an initial angiogenic signal that accompanies tumor growth.
Assuntos
Indutores da Angiogênese/metabolismo , Inibidores da Angiogênese/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Angiopoietina-1 , Angiopoietina-2 , Northern Blotting , Regulação para Baixo , Fatores de Crescimento Endotelial/metabolismo , Humanos , Imuno-Histoquímica , Metástase Linfática , Linfocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Invasividade Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Neovascularização Fisiológica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/análise , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondinas/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Cisteína/metabolismo , Proteínas de Drosophila , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Deleção de Sequência , Células 3T3/efeitos dos fármacos , Animais , Membrana Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Dimerização , Retículo Endoplasmático/metabolismo , Ácido Glutâmico , Leucina , Mercaptoetanol/farmacologia , Camundongos , Fosforilação , Polissacarídeos/metabolismo , Testes de Precipitina , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).
Assuntos
Proteínas de Drosophila , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2b/genética , Mutação , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Células 3T3 , Animais , Dimerização , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Ligantes , Camundongos , Fatores de Crescimento Neural/farmacologia , Proteínas Proto-Oncogênicas c-ret , Proteínas Recombinantes , TransfecçãoRESUMO
The papillary carcinoma family (PCF) of thyroid tumors includes a wide variety of neoplastic entities regarded as well-differentiated, poorly differentiated, and undifferentiated papillary thyroid carcinomas. Recent studies have established the presence of alternative oncogenic rearrangements of the RET and NTRK1 genes in a consistent fraction (< or = 50%) of papillary thyroid tumors. RET oncogenic rearrangements are also very frequent (approximately 60%) in Chernobyl radiation-associated papillary thyroid neoplasias, which show an increased aggressiveness in terms of pathological stage at disease onset. These observations prompted us to study the relationship between the presence or absence of RET and NTRK1 oncogenes and the clinicopathological features (age, sex, histopathology, and pTNMC2 staging) of 76 consecutive, non-radiation-related tumors of the PCF. As previously reported, statistical univariate analysis revealed a correlation between the combination of RET and NTRK1 (RET/NTRK1) positivity and young age of patients at diagnosis. In addition, a significant association was found between RET/NTRK1 positivity and locally advanced stage of disease at presentation (pT4: P < 0.015). The multivariate analysis confirmed that RET/NTRK1 activation parallels an unfavorable disease presentation, which may correlate with a less favorable disease outcome. Furthermore, within the PCF, the frequency of RET/NTRK1 positivity was not influenced by the different neoplastic subtypes or the tumor versus degree of differentiation.
Assuntos
Carcinoma Papilar/genética , Proteínas de Drosophila , Rearranjo Gênico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Papilar/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-ret , Receptor trkA , Neoplasias da Glândula Tireoide/patologiaRESUMO
A combined cytogenetic and molecular analysis of thyroid tumours has indicated that these neoplasms might represent a significant model for analysing human epithelial cell multi-step cancerogenesis. Thyroid tumours comprise a broad spectrum of lesions with different phenotypes and variable biological and clinical behaviour. Molecular analysis has detected specific genetic alterations in these different tumour types. In particular, the well-differentiated carcinomas of the papillary type are characterised by the activation of the tyrosine kinase receptors (TKRs) RET and NTRK1 proto-oncogenes. Cytogenetic analysis of these tumours has contributed to defining the chromosomal mechanisms leading to the TKRs' oncogenic activation. The results have shown that, in the majority of the cases, intra-chromosomal inversions of chromosome 10 and of chromosome 1 lead to the formation of RET-derived and NTRK1-derived oncogenes, respectively. Exposure to ionizing radiation is associated with papillary carcinomas, and RET activation has been suggested to be related to this event. All these findings are contributing to the definition of genetic and environmental factors relevant to the pathogenesis of thyroid tumours. Moreover, the molecular characterisation of specific genetic lesions could provide significant information about the association between ionising radiation and RET oncogene activation.
Assuntos
Carcinoma Papilar/genética , Proteínas de Drosophila , Rearranjo Gênico , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Fatores Etários , Carcinoma Papilar/patologia , Criança , Pré-Escolar , Humanos , Lactente , Proteínas Proto-Oncogênicas c-ret , Receptor trkA , Neoplasias da Glândula Tireoide/patologiaRESUMO
We reclassified 720 nonmedullary invasive thyroid carcinomas diagnosed and treated between 1975 and 1993. Twenty-seven cases met the criteria of insular carcinoma and 29 cases those of widely invasive follicular carcinoma. Comparison of these histotypes with respect to pathologic stage and overall, relative, and visceral metastasis-free survival showed a significant association between histotype and pT and pN categories. In particular, pT4 (p < 0.001) and pN1 (p < 0.001) categories were more frequent in the insular carcinoma histotype. By contrast, no significant differences in overall, relative, or visceral metastasis-free survival were observed between insular carcinoma and widely invasive follicular carcinoma. Molecular analysis by polymerase chain reaction-single-strand conformation polymorphism demonstrated RAS gene family point mutations in five of eight cases analyzed in each of the two histotypes, with a high proportion of CAA-->AAA transversion at codon 61 of the N-RAS gene in insular carcinoma. These findings suggest that insular carcinoma represents a de novo entity distinct from widely invasive follicular carcinoma, that widely invasive follicular carcinoma has biologic characteristics more consistent with poorly differentiated than well-differentiated carcinomas, and that both insular carcinoma and widely invasive follicular carcinoma share similar molecular alterations.
Assuntos
Adenocarcinoma Folicular/classificação , Adenocarcinoma Folicular/patologia , Genes ras , Mutação Puntual , Neoplasias da Glândula Tireoide/classificação , Neoplasias da Glândula Tireoide/patologia , Neoplasias Abdominais/classificação , Neoplasias Abdominais/patologia , Neoplasias Abdominais/secundário , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/mortalidade , Adolescente , Adulto , Idoso , Códon , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Recidiva , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/mortalidade , Fatores de TempoRESUMO
The NTRK1 gene in the q arm of chromosome 1 encodes one of the receptors for the nerve growth factor and is frequently activated as an oncogene in papillary thyroid carcinomas. The activation is due to chromosomal rearrangements juxtaposing the NTRK1 tyrosine kinase domain to 5'-end sequences from different genes. The thyroid TRK oncogenes are activated by recombination with at least three different genes: the gene coding for tropomyosin and TPR, both on chromosome 1,and TFG on chromosome 3. In a previous study, we showed that two tumors carrying the TPR/NTRK1 rearrangement contained structurally different oncogenes named TRK-T1 and TRK-T2. In this paper, we report (1) the cDNA structure of TRK-T2, (2) evidence that TRK-T2 is generated by different rearrangements in two thyroid tumors, and (3) a detailed analysis of the three different TPR/NTRK1 rearrangements. With molecular studies based on Southern blot hybridization, cloning, and sequencing, we show that all the rearrangements are nearly balanced, involving deletion, insertion, or duplication of only few nucleotides. In one case, an additional rearrangement involving sequences derived from chromosome 17 was detected.
Assuntos
Cromossomos Humanos Par 1 , Rearranjo Gênico , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares , Oncogenes , Receptor trkA , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
The RET/PTC3 oncogene is an activated form of the RET protooncogene, which is frequently rearranged in papillary thyroid carcinoma. RET/PTC3 results from a structural rearrangement between the ELE1 and the RET genes, and it has been observed in both sporadic and radiation-associated post-Chernobyl tumors. To understand the molecular basis that predisposes RET and ELE1 genes to be recurrent targets of "illegitimate" recombination, we examined the genomic regions containing the ELE1/RET breakpoints of six sporadic and three post-Chernobyl tumors in two papillary carcinomas of different origins. Our data indicated, in both genes, a clustering of the breakpoints in regions designated ELE1-bcr (1.8 kb) and RET-bcr (1.9 kb). Notably, in all sporadic tumors and in one post-Chernobyl tumor the ELE1/RET recombination corresponded with short sequences of homology (3-7 nt) between the two rearranging genes. In addition, we observed an interesting distribution of the post-Chernobyl breakpoints in ELE1-bcr located within an Alu element, or in between two close Alu elements, and always in A+T-rich regions.
Assuntos
Carcinoma Papilar/genética , Neoplasias Induzidas por Radiação/genética , Oncogenes , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA de Neoplasias/genética , Éxons , Rearranjo Gênico/efeitos da radiação , Humanos , Íntrons , Dados de Sequência Molecular , Oncogenes/efeitos da radiação , Reação em Cadeia da Polimerase , Liberação Nociva de Radioativos , Recombinação Genética/efeitos da radiação , UcrâniaRESUMO
Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.
Assuntos
Cisteína , Proteínas de Drosophila , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Deleção de Sequência , Células 3T3 , Adulto , Animais , Carcinoma Medular/genética , Transformação Celular Neoplásica , Feminino , Humanos , Masculino , Camundongos , Polimorfismo Conformacional de Fita Simples , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Neoplasias da Glândula Tireoide/genética , TransfecçãoRESUMO
The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.