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OBJECTIVE: Fine needle cytology (FNC) is the first-line diagnostic method to determine the benign or malignant nature of thyroid nodules. The gray zone of cytological classifications remains, however, a crucial and challenging area for cytopathologists. DESIGN, PATIENTS AND MEASUREMENTS: In the present study, 141 thyroid cytological samples, with ultrasonographic suspicious features, have been prospectively analysed. Molecular analyses were performed by an innovative technology using two multiplex PCRs for the amplification of BRAF, N-H-K-RAS and RET exon genes. RNA samples were studied for RET/PTC1 and RET/PTC3 rearrangements by PCR amplification, and the conditions were set-up to study, with a single experiment, both wild-type PAX8 and PAX8/PPARÉ£ rearrangements. In total, 111 samples were examined for BRAF, N-H-KRAS and RET genes. An ultrasonographic, cytological and molecular correlation was also carried out in an attempt to suggest a possible way to manage the patients with thyroid nodules. Cyto-histological correlation was available in 115 cases, and it was used to verify the global diagnostic accuracy of this combined approach. RESULTS: According to the histopathological diagnosis, FNC accuracy was 100% for TIR5 and metastases; 89% for TIR4; 84% for TIR3A and 58% for TIR3B. About 11% of the studied samples showed either RET-PTC1 or RET/PTC3 chromosomal rearrangements, and only one sample simultaneously presented RET/PTC1 and RET/PTC3 rearrangements. PAX8/PPARÉ£ rearrangement was found in 6% of the samples. CONCLUSIONS: A multidisciplinary approach to the thyroid is therefore necessary to develop innovative methods suitable for an improved diagnostic and prognostic definition of thyroid cancer.
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Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/diagnóstico , Nódulo Tiroideo/diagnóstico por imagen , Nódulo Tiroideo/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Análisis Mutacional de ADN , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor de Transcripción PAX8/genética , Estudios Prospectivos , Proteínas Proto-Oncogénicas B-raf/genética , Cáncer Papilar Tiroideo/diagnóstico , Cáncer Papilar Tiroideo/diagnóstico por imagen , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Nódulo Tiroideo/genética , Adulto JovenRESUMEN
PATZ1 is a transcriptional factor downregulated in thyroid cancer whose re-expression in thyroid cancer cells leads to a partial reversion of the malignant phenotype, including the capacity to proliferate, migrate, and undergo epithelial-to-mesenchymal transition. We have recently shown that PATZ1 is specifically downregulated downstream of the Ras oncogenic signaling through miR-29b, and that restoration of PATZ1 in Ha-Ras transformed FRTL5 rat thyroid cells is able to inhibit their capacities to proliferate and migrate in vitro. Here, we analyzed the impact of PATZ1 expression on the in vivo tumorigenesis of these cells. Surprisingly, FRTL5-Ras-PATZ1 cells showed enhanced tumor initiation when engrafted in nude mice, even if their tumor growth rate was reduced compared to that of FRTL5-Ras control cells. To further investigate the cause of the enhanced tumor engraftment of FRTL5-Ras-PATZ1 cells, we analyzed the stem-like potential of these cells through their capacity to grow as thyrospheres. The results showed that restoration of PATZ1 expression in these cells increases stem cell markers' expression and self-renewal ability of the thyrospheres while limiting their growth capacity. Therefore, we suggest that PATZ1 may play a role in enhancing the stem cell potential of thyroid cancer cells, but, at the same time, it impairs the proliferation of non-stem cells.
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Carcinogénesis/genética , Neoplasias de la Tiroides/genética , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Animales , Carcinogénesis/metabolismo , Línea Celular Tumoral , Células Cultivadas , Femenino , Ratones , Ratones Desnudos , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Ratas , Neoplasias de la Tiroides/metabolismo , Factores de Transcripción/genética , Proteínas ras/genéticaRESUMEN
Thyroid cancer is the most common malignancy of the endocrine system and includes well-differentiated forms, namely papillary and follicular carcinomas, and the poorly differentiated and undifferentiated forms that result from the transformation of thyroid follicular cells (anaplastic carcinomas). Notably, 5-10% of all thyroid cancers are medullary thyroid cancers that arise from parafollicular cells also known as C cells. The most common genetic mutations in papillary and follicular thyroid cancers are point mutations of the BRAF or RAS genes, while the most common chromosomal alterations are RET/PTC and PAX8/PPARγ rearrangements. The most frequent initial manifestation of thyroid cancer is the appearance of a nodule most of which are benign; indeed, less than 5% are malignant. However, some cases are misdiagnosed, and many patients undergo unnecessary surgery. Therefore, an accurate pre-surgery evaluation is crucial. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA) cytology, which accurately distinguishes between a benign and malignant lesion in most cases. However, cytological discrimination between malignant and benign follicular cancer is often difficult because of poor quality samples. Here we describe rapid methods to create a positive control and identify the PAX8/PPARγ rearrangement in FNA thyroid samples by molecular biology.
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POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is an emerging cancer-related gene that is downregulated in different human malignancies, including thyroid cancer, where its levels gradually decrease going from papillary thyroid carcinomas (PTC) to poorly differentiated and undifferentiated highly aggressive anaplastic carcinomas (ATC). The restoration of PATZ1 expression in thyroid cancer cells reverted their malignant phenotype by inducing mesenchymal-to-epithelial transition, thus validating a tumor suppressor role for PATZ1 and suggesting its involvement in thyroid cancer progression. Here, we investigated the consequences of the homozygous and heterozygous loss of PATZ1 in the context of a mouse modeling of PTC, represented by mice carrying the RET/PTC1 oncogene under the thyroid specific control of the thyroglobulin promoter RET/PTC1 (RET/PTC1TG). The phenotypic analysis of RET/PTC1TG mice intercrossed with Patz1-knockout mice revealed that deficiency of both Patz1 alleles enhanced thyroid cancer incidence in RET/PTC1TG mice, but not the heterozygous knockout of the Patz1 gene. However, both RET/PTC1TG;Patz1+/- and RET/PTC1TG;Patz1-/- mice developed a more aggressive thyroid cancer phenotype-characterized by higher Ki-67 expression, presence of ATCs, and increased incidence of solid variants of PTC-than that shown by RET/PTC1TG; Patz1+/+ compound mice. These results confirm that PATZ1 downregulation has a critical role in thyroid carcinogenesis, showing that it cooperates with RET/PTC1 in thyroid cancer progression.
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BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.
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We have previously reported a critical role of HMGA proteins in pituitary tumorigenesis since either the Hmga1 or Hmga2 gene overexpression/activation induces the development of mixed growth hormone/prolactin cell pituitary adenomas by activating the E2F transcription factor 1, and then enhancing the G1/S transition of the cell cycle. Consistently, amplification and overexpression of the HMGA2 gene was found in human pituitary prolactinomas. Since impairment of the cell cycle control represents a feature of experimental and human pituitary adenomas, we have investigated the possible synergism between the alterations of other cell cycle regulators, such as p27 deficiency or Cdk4R24C mutation, with Hmga2 overexpression in pituitary tumorigenesis. Therefore, we crossed the Hmga2/T mice, overexpressing the truncated/active form of the Hmga2 gene, either with the knockout mice for p27kip1, or with the knockin mice for the Cdk4R24C mutation, both developing pituitary adenomas. Increased incidence and decreased latency in the development of pituitary lesions appeared in double mutant Hmga2/T;Cdk4R24C mice, and increased features of invasiveness and atypia were observed in pituitary tumors of both Hmga2/T;p27-ko and Hmga2/T;Cdk4R24C double mutant mice as compared with single mutant compounds. Interestingly, most of these mice develop pituitary adenomas with high Ki67 index, extrasellar expansion and brain tissue infiltration, representing good mouse models for human aggressive pituitary adenomas. Taken together, the results reported here indicate a cooperation between HMGA2 overexpression and either p27kip1 or CDK4 impairment in promoting pituitary tumor development and progression.
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Quinasa 4 Dependiente de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteína HMGA2/genética , Neoplasias Hipofisarias/patología , Animales , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Femenino , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/mortalidadRESUMEN
Breast cancer is a heterogeneous disease that is characterized by a high grade of cell plasticity arising from the contribution of a diverse range of factors. When combined, these factors allow a cancer cell to transition from an epithelial to a mesenchymal state through a process of dedifferentiation that confers stem-like features, including chemoresistance, as well as the capacity to migrate and invade. Understanding the complex events that lead to the acquisition of a mesenchymal phenotype will therefore help to design new therapies against metastatic breast cancer. Here, we recapitulate the main endogenous molecular signals involved in this process, and their cross-talk with paracrine factors. These signals and cross-talk include the extracellular matrix; the secretome of cancer-associated fibroblasts, macrophages, cancer stem cells, and cancer cells; and exosomes with their cargo of miRNAs. Finally, we highlight some of the more promising therapeutic perspectives based on counteracting the epithelial-to-mesenchymal transition in breast cancer cells.
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Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum-based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro, in xenografts in vivo, and in primary tumor cells derived from platinum-treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients.
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Quinasa 6 Dependiente de la Ciclina/metabolismo , Proteína Forkhead Box O3/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Platino (Metal)/farmacología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Carcinoma Epitelial de Ovario , Muerte Celular , Línea Celular Tumoral , Quinasa 6 Dependiente de la Ciclina/genética , Daño del ADN , Femenino , Proteína Forkhead Box O3/genética , Humanos , Ratones , Ratones Desnudos , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Ováricas/enzimología , Piperazinas/farmacología , Piperazinas/uso terapéutico , Platino (Metal)/uso terapéutico , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The most frequent initial manifestation of thyroid cancer is the appearance of a nodule. More than 20% of the general population has a palpable thyroid nodule and the percentage rises to 70% based on ultrasound identification. In 95% of cases the nodule is simply a hyperplastic or benign lesion. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA), but cytological discrimination between malignant and benign follicular neoplasms remains difficult. Cytological analysis is now, almost routinely, being combined with molecular genetics to enable the pathologist to make a more objective diagnosis. In this study, we performed the molecular analysis using a new simplified procedure that involves a panel of BRAF, RAS, RET and RET/PTC gene mutations in easily obtainable FNA samples, in the attempt to improve the efficacy of the FNA diagnosis of thyroid nodules and thus patient management. In this new procedure, PCR and sequencing analysis are used to detect point mutations, and, in parallel, RT-PCR is used to detect the chimeric RET/PTC1 and RET/PTC3 transcripts in RNA extracted from FNA.
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Reacción en Cadena de la Polimerasa Multiplex , Mutación , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Biomarcadores de Tumor , Biopsia con Aguja Fina , Análisis Mutacional de ADN/métodos , Exones , Reordenamiento Génico , Genes Esenciales , Humanos , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas ras/genéticaRESUMEN
BACKGROUND: No biomarker is available to predict prognosis of patients with advanced ovarian cancer (AOC) and guide the choice of chemotherapy. We performed a prospective-retrospective biomarker study within the MITO2 trial on the treatment of AOC. PATIENTS AND METHODS: MITO2 is a randomised multicentre phase 3 trial conducted with 820 AOC patients assigned carboplatin/paclitaxel (carboplatin: AUC5, paclitaxel: 175 mg/m², every 3 weeks for 6 cycles) or carboplatin/PLD-pegylated liposomal doxorubicin (carboplatin: AUC5, PLD: 30 mg/m², every 3 weeks for 6 cycles) as first line treatment. Sixteen biomarkers (pathways of adhesion/invasion, apoptosis, transcription regulation, metabolism, and DNA repair) were studied in 229 patients, in a tissue microarray. Progression-free and overall survival were analysed with multivariable Cox model. RESULTS: After 72 months median follow-up, 594 progressions and 426 deaths were reported; there was no significant difference between the two arms in the whole trial. No biomarker had significant prognostic value. Statistically significant interactions with treatment were found for DNA-dependent protein kinase (DNA-PK) and phosphorylated acetyl-coenzymeA carboxylase (pACC), both predicting worse outcome for patients receiving carboplatin/paclitaxel. CONCLUSION: These data show that in presence of DNA-PK or pACC overexpression, carboplatin/paclitaxel might be less effective than carboplatin/PLD as first line treatment of ovarian cancer patients. Further validation of these findings is warranted.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteína Quinasa Activada por ADN/genética , Neoplasias Ováricas/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Ováricas/patología , PronósticoRESUMEN
BACKGROUND: Risk of relapse or progression remains high in the treatment of most patients with epithelial ovarian cancer, and development of a molecular predictor could be a valuable tool for stratification of patients by risk. We aimed to develop a microRNA (miRNA)-based molecular classifier that can predict risk of progression or relapse in patients with epithelial ovarian cancer. METHODS: We analysed miRNA expression profiles in three cohorts of samples collected at diagnosis. We used 179 samples from a Multicenter Italian Trial in Ovarian cancer trial (cohort OC179) to develop the model and 263 samples from two cancer centres (cohort OC263) and 452 samples from The Cancer Genome Atlas epithelial ovarian cancer series (cohort OC452) to validate the model. The primary clinical endpoint was progression-free survival, and we adapted a semi-supervised prediction method to the miRNA expression profile of OC179 to identify miRNAs that predict risk of progression. We assessed the independent prognostic role of the model using multivariable analysis with a Cox regression model. FINDINGS: We identified 35 miRNAs that predicted risk of progression or relapse and used them to create a prognostic model, the 35-miRNA-based predictor of Risk of Ovarian Cancer Relapse or progression (MiROvaR). MiROvaR was able to classify patients in OC179 into a high-risk group (89 patients; median progression-free survival 18 months [95% CI 15-22]) and a low-risk group (90 patients; median progression-free survival 38 months [24-not estimable]; hazard ratio [HR] 1·85 [1·29-2·64], p=0·00082). MiROvaR was a significant predictor of progression in the two validation sets (OC263 HR 3·16, 95% CI 2·33-4·29, p<0·0001; OC452 HR 1·39, 95% CI 1·11-1·74, p=0·0047) and maintained its independent prognostic effect when adjusted for relevant clinical covariates using multivariable analyses (OC179: adjusted HR 1·48, 95% CI 1·03-2·13, p=0·036; OC263: adjusted HR 3·09 [2·24-4·28], p<0·0001; and OC452: HR 1·41 [1·11-1·79], p=0·0047). INTERPRETATION: MiROvaR is a potential predictor of epithelial ovarian cancer progression and has prognostic value independent of relevant clinical covariates. MiROvaR warrants further investigation for the development of a clinical-grade prognostic assay. FUNDING: AIRC and CARIPLO Foundation.
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Adenocarcinoma de Células Claras/patología , Adenocarcinoma Mucinoso/patología , Cistadenocarcinoma Seroso/patología , Neoplasias Endometriales/patología , MicroARNs/genética , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/patología , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/cirugía , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Estudios de Cohortes , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/cirugía , Progresión de la Enfermedad , Neoplasias Endometriales/genética , Neoplasias Endometriales/cirugía , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/cirugía , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/cirugía , Pronóstico , Tasa de SupervivenciaRESUMEN
Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC.
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Proteína HMGA2/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , ARN Mensajero/sangre , ARN Mensajero/genética , Adulto , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Femenino , Proteína HMGA2/metabolismo , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Ováricas/sangreRESUMEN
Fine-needle cytology (FNC) is frequently used to diagnose thyroid nodules discovered by palpation or imaging studies. Molecular tests on FNC material may increase its diagnostic accuracy. We report a case of a classic papillary thyroid carcinoma combined with a mucoepidermoid carcinoma correctly identified on FNC. The papillary component had a classic immunophenotype (CK19+, TTF1+), while the mucoepidermoid one was only focally CK19+. Point mutations (BRAF and RAS) and rearrangements (RET/PTC) of the papillary component have been also investigated on FNC samples, with resulting concurrent rearrangements of RET/PTC1 and RET/PTC3, but no point mutations. The histogenesis of combined papillary and mucoepidermoid carcinoma of the thyroid still remains partly unsettled, and further genomic studies are needed to shed some more light on this peculiar neoplasm.
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Carcinoma Mucoepidermoide/diagnóstico , Carcinoma/diagnóstico , Tumor Mixto Maligno/diagnóstico , Neoplasias de la Tiroides/diagnóstico , Adulto , Biopsia con Aguja Fina , Carcinoma/metabolismo , Carcinoma/patología , Carcinoma Mucoepidermoide/metabolismo , Carcinoma Mucoepidermoide/patología , Carcinoma Papilar , Diagnóstico Diferencial , Femenino , Humanos , Inmunohistoquímica , Tumor Mixto Maligno/metabolismo , Tumor Mixto Maligno/patología , Técnicas de Diagnóstico Molecular , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
PATZ1, a POZ-Zinc finger protein, is emerging as an important regulator of development and cancer, but its cancer-related function as oncogene or tumor-suppressor is still debated. Here, we investigated its possible role in thyroid carcinogenesis. We demonstrated PATZ1 is down-regulated in thyroid carcinomas compared to normal thyroid tissues, with an inverse correlation to the degree of cell differentiation. In fact, PATZ1 expression was significantly further down-regulated in poorly differentiated and anaplastic thyroid cancers compared to the papillary histotype, and it resulted increasingly delocalized from the nucleus to the cytoplasm proceeding from differentiated to undifferentiated thyroid carcinomas. Restoration of PATZ1 expression in three thyroid cancer-derived cell lines, all characterized by fully dedifferentiated cells, significantly inhibited their malignant behaviors, including in vitro proliferation, anchorage-independent growth, migration and invasion, as well as in vivo tumor growth. Consistent with recent studies showing a role for PATZ1 in the p53 pathway, we showed that ectopic expression of PATZ1 in thyroid cancer cells activates p53-dependent pathways opposing epithelial-mesenchymal transition and cell migration to prevent invasiveness. These results provide insights into a potential tumor-suppressor role of PATZ1 in thyroid cancer progression, and thus may have potential clinical relevance for the prognosis and therapy of thyroid cancer.
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Carcinoma Papilar/patología , Movimiento Celular , Transición Epitelial-Mesenquimal , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Proliferación Celular , Inmunoprecipitación de Cromatina , Femenino , Genes Supresores de Tumor , Humanos , Técnicas para Inmunoenzimas , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Desnudos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serpinas/genética , Serpinas/metabolismo , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have previously studied the function of microRNAs (miRNAs) in thyroid cells using the differentiated rat thyroid PC Cl 3 cells that need thyrotropin (TSH) for their growth. The miRNA expression profile examination allowed the detection of a set of miRNAs downregulated and upregulated by TSH. Here, we first demonstrated that upregulation of miR-130b-3p occurs through a protein kinase A-cAMP-responsive element binding protein (CREB)-dependent mechanism. Then, we analyzed its expression in human thyroid follicular adenomas, where a constitutive CREB activation is frequently present. miR-130b-3p results in upregulation with a high fold-change in most thyroid follicular adenomas. Then, we identified CCDC6, coding for a protein that interacts with CREB1 leading to the transcriptional repression of CREB1 target genes, as a target of this miRNA. The targeting of CCDC6 by miR-130b-3p likely accounts for the mechanism by which its upregulation contributes to the development of thyroid adenomas increasing CREB1 activity.
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CONTEXT: A previous micro-RNA expression profile of thyroid follicular adenomas identified miR-142 precursor among the miRNAs downregulated in the neoplastic tissues compared to normal thyroid gland. OBJECTIVE: The aim of this work has been to assess the expression of miR-142-3p in a large panel of follicular thyroid adenomas and carcinomas and evaluate its effect on thyroid cell proliferation and target expression. DESIGN: The expression of miR-142-3p was analyzed by qRT-PCR in thyroid follicular adenomas and carcinomas, compared to normal thyroids. MiR-142-3p expression was restored in WRO cells and the effects on cell proliferation and target expression were evaluated. RESULTS: Here we show that miR-142-3p is downregulated in FTAs, FTCs, and FVPTCs. MiR-142-3p was demonstrated to reduce the proliferation rate of WRO and FTC133 cells, supporting its tumor suppressor role in thyroid cancerogenesis. Moreover, this microRNA was able to downregulate the expression of ASH1L and MLL1, by direct and indirect mechanisms, respectively. Consistently, an inverse correlation between miR-142-3p expression and ASH1L and MLL1 proteins was found in thyroid follicular adenomas and carcinomas. ASH1L and MLL1, which belong to the Trithorax group (TrxG) proteins and are major regulators of Homeobox gene expression, maintain active target gene transcription by histone 3 lysine 4 methylation. Interestingly, we found that FTCs and FTC cell lines express tumor specific, shorter forms of the two proteins. The capability of miR-142-3p to modulate the levels of these tumor-associated forms and to reactivate thyroid-specific Hox gene expression, likely contributes to its tumor suppressive function. CONCLUSIONS: These data demonstrate that miR-142-3p downregulation has a role in thyroid tumorigenesis, by regulating ASH1L and MLL1.
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Adenocarcinoma Folicular/genética , Carcinogénesis/genética , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , MicroARNs/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Neoplasias de la Tiroides/genética , Factores de Transcripción/genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patología , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , MicroARNs/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Factores de Transcripción/metabolismoRESUMEN
BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.