Endocrine Surgery Unit

Barcelona, Spain

Endocrine Surgery Unit

Barcelona, Spain
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Cirello V.,Endocrine Unit | Cirello V.,University of Milan | Vaira V.,Fondazione IRCCS Ca Granda | Grassi E.S.,Instituto Auxologico Italiano IRCCS | And 12 more authors.
Oncotarget | Year: 2017

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients' tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and "stem-like" components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and β-catenin turnover. Consistently, SP treatment resulted in a significant decrease in β-catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells. In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.


PubMed | Endocrine Surgery Unit, IRCCS Instituto Clinico Humanitas, Endocrinology and Diabetology Unit, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico and University of Milan
Type: Journal Article | Journal: Histology and histopathology | Year: 2015

The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in both cell growth and cell differentiation control. Mutations of the PRKAR1A gene have been found in patients with Carney complex and in a minority of sporadic anaplastic thyroid carcinomas. The aim of the study was to retrospectively evaluate the expression of different PKA regulatory subunits in benign and non benign human thyroid tumours and to correlate their expression with clinical phenotype. Immunohistochemistry demonstrated a significant increase in PRKAR2B expression in both differentiated and undifferentiated (anaplastic) thyroid tumors in comparison with normal thyroid tissues. Conversely, a significant increase in PRKAR1A expression was only demonstrated in undifferentiated thyroid carcinomas in comparison with normal thyroid tissue and differentiated thyroid tumors. In thyroid cancers without lymph nodal metastases PRKAR1A expression was higher in tumours of more than 2 cm in size (T2 and T3) compared to smaller ones (T1). In conclusion, our data shows that an increased PRKAR1A expression is associated with aggressive and undifferentiated thyroid tumors.


Muzza M.,Endocrine Unit | Muzza M.,University of Milan | Colombo C.,Endocrine Unit | Colombo C.,University of Milan | And 21 more authors.
Molecular and Cellular Endocrinology | Year: 2015

Telomerase-reverse-transcriptase (TERT) promoter mutations have been recently described in tumors. In the present large series, TERT mutations were found in 12% of papillary thyroid cancers (PTCs) and in 14% of follicular thyroid cancers (FTCs), and were found to significantly correlate with older age at diagnosis and poorer outcome. Interestingly, the prognostic value of TERT mutations resulted to be significantly stronger than that of BRAFV600E. Moreover, the outcome was not different among tumors with isolated TERT mutation and those with coexistent mutations (TERT/BRAF in PTCs or TERT/RAS in FTCs). TERT rs2853669 polymorphism was found in 44.4% of tumors. At WB, TERT was significantly more expressed in tumors than in normal samples, being the highest levels of expression recorded in TERT mutated cases. At IHC, in tumors and in metastatic lymph-nodes TERT staining was significantly higher in the cytoplasm than in the nucleus, whereas in normal tissue the degree of staining did not differ in the two cellular compartments.In conclusion, TERT mutations were shown to strongly correlate with a poorer outcome in differentiated thyroid tumors, and neither BRAF nor RAS mutation were found to confer an additional effect in the disease persistence. TERT protein was found to be more expressed in neoplastic than in normal tissues, and to display a different cellular localization, suggesting that it could contribute to thyroid cancer progression by mechanisms taking place in the cytoplasm. © 2014.


PubMed | IRCCS Instituto Ortopedico Galeazzi, Internal Medicine Unit, G Vezzoli, Sidra Research Laboratory and 4 more.
Type: | Journal: Journal of molecular endocrinology | Year: 2016

Parathyroid tumors display reduced sensitivity to extracellular calcium ([Ca2+]o). [Ca2+]o activates calcium-sensing receptor (CASR), which interacts with the scaffold protein filamin A (FLNA). The study aimed to investigate: 1) the FLNA expression in human parathyroid tumors, 2) its effects on the CASR mRNA and protein expression, and 3) on ERK signaling activation, 4) the effect of the carboxy-terminal CASR variants and 5) of the treatment with the CASR agonist R568 on FLNA-mediated ERK phosphorylation in HEK293 cells. Full-length FLNA immunostaining was variably reduced in parathyroid tumors. Immunofluorescence showed that FLNA localized in membrane and cytoplasm and co-localized with CASR in parathyroid adenomas (PAds)-derived cells. Cleaved C-terminus FLNA fragment could also be detected in PAds nuclear protein fractions. In HEK293 cells transfected with 990R-CASR or 990G-CASR variants, silencing of endogenous FLNA reduced CASR mRNA levels and total and membrane-associated CASR proteins. In agreement, FLNA mRNA levels positively correlated with CASR expression in a series of 74 PAds; however, any significant correlation with primary hyperparathyroidism severity could be detected and FLNA transcript levels did not differ between PAds harboring 990R or 990G CASR variants. R568 treatment was efficient in restoring 990R-CASR and 990G-CASR sensitivity to [Ca2+]o in absence of FLNA. In conclusion, FLNA is down-regulated in parathyroid tumors and parallels the CASR expression levels. Loss of FLNA reduces CASR mRNA and protein expression levels and the CASR-induced ERK phosphorylation. FLNA is involved in receptor expression, membrane localization and ERK signaling activation of both 990R and 990G CASR variants.


Sitges-Serra A.,Endocrine Surgery Unit | Garcia L.,Institute Municipal dInvestigacio Medica | Prieto R.,Endocrine Surgery Unit | Pena M.J.,Institute Municipal dInvestigacio Medica | And 2 more authors.
British Journal of Surgery | Year: 2010

Background: The bone mineral density (BMD) response to parathyroidectomy is heterogeneous and difficult to predict. Available data come from mixed populations of men and women, of different age and degrees of disease severity, and preoperative BMD loss. Methods: This was a longitudinal, prospective cohort study of 103 postmenopausal women, with. osteopenia or osteoporosis at the femoral neck site, successfully operated on for primary hyperparathyroidism. BMD and metabolic variables were recorded before and 1 year after parathyroidectomy. Results: After surgery, there was a 1-3 percent increase in the median BMD at the femoral neck site (0-615 versus 0-623 g/cm2; P = 0-001). Overall, positive responses were also observed at total hip (0-4 per cent) and lumbar spine (2-3 per cent) sites. Analysing the individual responses, however, only 45 (46 per cent) of 97 patients showed a significant (at least 3-7 per cent) increase in BMD at the femoral neck site compared with the preoperative value and 52 had a decreased (15) or unchanged (37) femoral neck BMD. Patients who gained BMI) were younger, had more severe hyperparathyroidism, and better renal function. Conclusion: Almost half of the postmenopausal women with hyperparathyroidism and low BMD have a significant remineralization response 1 year after parathyroidectomy. Differential mineralization responses of BMD after surgery appear to be related to severity of primary hyperparathyroidism, age and renal function. Copyright © 2010 British Journal of Surgery Society Ltd.


Nordenstrom E.,Lund University | Nordenstrom E.,Skåne University Hospital | Sitges-Serra A.,Endocrine Surgery Unit | Sancho J.J.,Endocrine Surgery Unit | And 2 more authors.
International Journal of Endocrinology | Year: 2013

Aim. The interaction between vitamin D deficiency and primary hyperparathyroidism (PHPT) is not fully understood. The aim of this study was to investigate whether patients with PHPT from Spain and Sweden differed in vitamin D status and PHPT disease activity before and after surgery. Methods. We compared two cohorts of postmenopausal women from Spain (n=126) and Sweden (n=128) that had first-time surgery for sporadic, uniglandular PHPT. Biochemical variables reflecting bone metabolism and disease activity, including levels of 25-hydroxy vitamin D3 (25(OH)D) and bone mineral density, BMD, were measured pre- and one year postoperatively. Results. Median preoperative 25(OH)D levels were lower, and adenoma weight, PTH, and urinary calcium levels were higher in the Spanish cohort. The Spanish patients had higher preoperative levels of PTH (13.5 versus 11.0 pmol/L, P<0.001), urinary calcium (7.3 versus 4.1 mmol/L, P<0.001), and heavier adenomas (620 versus 500 g, P<0.001). The mean increase in BMD was higher in patients from Spain and in patients with vitamin D deficiency one year after surgery. Conclusion. Postmenopasual women with PHPT from Spain had a more advanced disease and lower vitamin 25(OH)D levels. Improvement in bone density one year after surgery was higher in patients with preoperative vitamin D deficiency. © 2013 Erik Nordenström et al.


PubMed | Endocrine Surgery Unit, University of Milan, Instituto Auxologico Italiano IRCCS, Fondazione IRCCS Ca Granda and Endocrine Unit
Type: | Journal: Oncotarget | Year: 2016

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and stem-like components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and -catenin turnover. Consistently, SP treatment resulted in a significant decrease in -catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells.In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.


Vannucchi G.,University of Milan | Perrino M.,University of Milan | Rossi S.,University of Milan | Colombo C.,University of Milan | And 4 more authors.
European Journal of Endocrinology | Year: 2010

Objective: Pregnancy represents a favorable condition for the development of thyroid nodules, likely due to the secretion of hormones with stimulatory activity. In particular, differentiated thyroid cancer (DTC) represents the second most frequent tumor among those diagnosed during pregnancy. However, few and discordant data are available about the impact of pregnancy on tumor outcome. Methods: A total of 123 women with DTC were divided into three groups according to the timing of tumor diagnosis (group 1, at least 1 year after the delivery; group 2, during pregnancy or in the first year after delivery; and group 3, before pregnancy or nulliparity) and evaluated according to the international guidelines. Furthermore, immunohistochemical studies of estrogen receptor α (ERα) were performed in 38 papillary thyroid cancer tissues from the three groups. Results: Thyroid cancer diagnosed during pregnancy was associated with a poorer prognosis compared to tumors developed in nongravidic periods (P<0.0001). Accordingly, at the stepwise logistic regression analysis, the diagnosis of DTC during pregnancy or in the first year post partum was the most significant indicator of persistent disease (P=0.001). Interestingly, ERa expression significantly differed among tumors of the three groups, being detected in 31% of group 1, in 87.5% of group 2, and in 0% of group 3 (P=0.01). Conclusions: Present data indicate that pregnancy has a negative impact on the outcome of thyroid cancer. The presence of ERα in the majority of tumors diagnosed during pregnancy indicates that the poorer outcome of these cases could be related to the estrogen-mediated growth stimulus. © 2010 European Society of Endocrinology.


Cirello V.,University of Milan | Perrino M.,University of Milan | Colombo C.,University of Milan | Muzza M.,University of Milan | And 4 more authors.
International Journal of Cancer | Year: 2010

Fetal cell microchimerism (FCM) is defined as the persistence, for decades after pregnancy, of fetal cells in maternal organs and circulation without any apparent rejection. We recently reported evidence, in papillary thyroid cancer (PTC) tissues, supporting a possible role of FCM in tumor damage and repair. To extend those data at the peripheral level, 106 women with a previous male pregnancy, comprising 57 with PTC and 49 healthy controls were enrolled. The presence of circulating male DNA was assessed by the amplification of the Y chromosome-specific gene SRY, with a sensitivity of 1 male cell per 1 million female cells. Moreover, to compare the microchimeric status in blood and in tumors, the neoplastic tissues of 19 women were studied. At the blood level, a significantly lower frequency of FCM was found in parous women with PTC with respect to controls (49.1% vs. 77.6%; p = 0.002). By PCR, male DNA was identified in the tumor tissues of 6 patients, and FISH analyses confirmed the presence of microchimeric cells (range 2.1-6.9 cells/section). In some patients, FCM was negative in the blood, whereas microchimeric cells were identified in the tumor. In conclusion, the prevalence of FCM in peripheral blood was found to be significantly lower in patients than in healthy controls. The presence of microchimeric cells in the tumors, but not at the peripheral level, supports the hypothesis that fetal cells could reside in maternal niches and could be recruited to diseased areas, where they could differentiate to regenerate damaged tissues. © 2009 UICC.


PubMed | Endocrine Surgery Unit and University of Milan
Type: | Journal: Molecular and cellular endocrinology | Year: 2016

During hormonogenesis, thyrocytes are physiologically exposed to high levels of oxidative stress (OS) which could either be involved in the pathogenesis of thyroid cancer or exert a cytotoxic effect. We analyzed the oxidative status of papillary thyroid cancer (PTC) both directly, by measuring H2O2 generation by NADPH oxidases (NOXs), and indirectly, by evaluating the antioxidant activity of glutathione peroxidase (GPX), which neutralizes H2O2 excess, and the lipid peroxidation (LP). Moreover, we investigated the subcellular localization of telomerase reverse transcriptase (TERT), and the H2O2 levels in the mitochondria of tumor and normal tissues. The calcium-dependent and independent H2O2 generation activity was significantly higher in tumors than in normal tissues. The GPX activity was higher in PTCs than in normal tissues, and, consistently, no differences were found in LP levels. Moreover, while TERT nuclear expression was similar in tumor and normal tissues, the mitochondrial localization was significantly higher in tumors. At the mitochondrial level, no differences were found in H2O2 generation between tumor and normal tissues. In conclusion, present data demonstrate that the intracellular H2O2 generation by NOXs is significantly higher in PTCs than in normal thyroid tissues. The increased GPX activity found in tumors counteracts the potential cytotoxic effects of high OS exposure. The significantly higher mitochondrial localization of TERT in tumors is consistent with its shuttling from the nucleus upon exposure to high OS. Finally, mitochondrial OS was not significantly different in tumors and normal tissues, supporting the postulated role of mitochondrial TERT in the control of local H2O2 production.

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