Preclinical testing of selective Aurora kinase inhibitors on a medullary thyroid carcinoma-derived cell line.

Deregulated expression of the Aurora kinases (Aurora-A, B, and C) is thought to be involved in cell malignant transformation and genomic instability in several cancer types. Over the last decade, a number of small-molecule inhibitors of Aurora kinases have been developed, which have proved to efficiently restrain malignant cell growth and tumorigenicity. Regarding medullary thyroid carcinoma (MTC), we previously showed the efficacy of a pan-Aurora kinase inhibitor (MK-0457) in impairing growth and survival of the MTC-derived cell line TT. In the present study, we sought to establish if one of the Aurora kinases might represent a preferential target for MTC therapy. The effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on TT cell proliferation, apoptosis, cell cycle, and ploidy. The two inhibitors reduced TT cell proliferation in a time- and dose-dependent manner, with IC50 of 19.0 ± 2.4 nM for MLN8237 and 401.6 ± 44.1 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited phosphorylation of histone H3 (Ser10) by Aurora-B, while it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Cytofluorimetry experiments showed that both inhibitors induced accumulation of cells in G2/M phase and increased the subG0/G1 fraction and polyploidy. Finally, both inhibitors triggered apoptosis. We demonstrated that inhibition of either Aurora-A or Aurora-B has antiproliferative effects on TT cells, and thus it would be worthwhile to further investigate the therapeutical potential of Aurora kinase inhibitors in MTC treatment.

Severe hypoglycemia in patients with known diabetes requiring emergency department care: A report from an Italian multicenter study.

AIMS: To describe the characteristics and associated risk factors of patients with established diabetes who required Emergency Department (ED) care for severe hypoglycemia. METHODS: We performed an observational retrospective study to identify all cases of severe hypoglycemia among attendees at the EDs of three Italian University hospitals from January 2010 to December 2014. RESULTS: Overall, 520 patients with established diabetes were identified. Mean out-of-hospital blood glucose concentrations at the time of the hypoglycemic event were 2.2 ± 1.3 mmol/L. Most of these patients were frail and had multiple comorbidities. They were treated with oral hypoglycemic drugs (43.6%), insulin (42.8%), or both (13.6%). Among the oral hypoglycemic drugs, glibenclamide (54.5%) and repaglinide (25.7%) were the two most frequently used drugs, followed by glimepiride (11.3%) and gliclazide (7.5%). Hospitalization rates and in-hospital deaths occurred in 35.4% and in 2.3% of patients, respectively. Cirrhosis (odds ratio [OR] 6.76, 95% confidence interval [CI] 1.24-36.8, p < 0.05), chronic kidney disease (OR 2.42, 95% CI 1.11-8.69, p < 0.05) and center (Sapienza University OR 3.70, 95% CI 1.57-8.69, p < 0.05) were the strongest predictors of increased rates of hospital admission. CONCLUSIONS: Severe hypoglycemia is a remarkable burden for patients with established diabetes and increases the risk of adverse clinical outcomes (in-hospital death and hospitalization), mainly in elderly and frail patients. This study further reinforces the notion that careful attention should be taken by health care providers when they prescribe drug therapy in elderly patients with serious comorbidities.

Association of thyroid diseases with primary extra-thyroidal malignancies in women: results of a cross-sectional study of 6,386 patients.

We here analyzed the prevalence of extra-thyroidal malignancies (EM) in 6,386 female patients affected by different thyroid disease (TD). At first, an age-matched analysis of EM in all patients was performed. We then evaluated EM prevalence in four TD diagnostic categories: non-nodular TD (n = 2,159); solitary nodule (n = 905); multinodular TD (n = 2,871); differentiated thyroid cancers (n = 451). Finally, patients were grouped based on the absence (n = 3,820) or presence of anti-thyroglobulin (TgAb) and/or anti-thyroperoxidase (TPOAb) (n = 2,369), or anti-Thyroid Stmulating Hormone (TSH) receptor autoantibodies (n = 197). A total of 673 EM were recorded. EM prevalence in TD patients was higher compared to the general population (Odds Ratio, OR 3.21) and the most frequent EM was breast cancer (OR 3.94), followed by colorectal (OR 2.18), melanoma (OR 6.71), hematological (OR 8.57), uterus (OR 2.52), kidney (OR 3.40) and ovary (OR 2.62) neoplasms. Age-matched analysis demonstrated that the risk of EM was maximal at age 0-44 yr (OR 11.28), remaining lower, but significantly higher that in the general population, in the 45-59 and 60-74 year age range. Breast and hematological malignancies showed an increased OR in all TD, while other cancers associated with specific TD. An increased OR for melanoma, breast and hematological malignancies was observed in both TPOAb and/or TgAb autoantibody negative and positive patients, while colorectal, uterus, kidney and ovary cancers showed an increased OR only in thyroid autoantibody negative patients. In conclusions, women affected by both benign and malignant TD, especially at a younger age and in absence of thyroid autoimmunity, have an increased risk of developing primary EM, thus requiring a careful follow-up and surveillance.

Deregulated expression of Aurora kinases is not a prognostic biomarker in papillary thyroid cancer patients.

A number of reports indicated that Aurora-A or Aurora-B overexpression represented a negative prognostic factor in several human malignancies. In thyroid cancer tissues a deregulated expression of Aurora kinases has been also demonstrated, but no information regarding its possible prognostic role in differentiated thyroid cancer is available. Here, we evaluated Aurora-A and Aurora-B mRNA expression and its prognostic relevance in a series of 87 papillary thyroid cancers (PTC), with a median follow-up of 63 months. The analysis of Aurora-A and Aurora-B mRNA levels in PTC tissues, compared to normal matched tissues, revealed that their expression was either up- or down-regulated in the majority of cancer tissues. In particular, Aurora-A and Aurora-B mRNA levels were altered, respectively, in 55 (63.2%) and 79 (90.8%) out of the 87 PTC analyzed.A significant positive correlation between Aurora-A and Aurora-B mRNAs was observed (p=0.001). The expression of both Aurora genes was not affected by the BRAFV600E mutation. Univariate, multivariate and Kaplan-Mayer analyses documented the lack of association between Aurora-A or Aurora-B expression and clinicopathological parameters such as gender, age, tumor size, histology, TNM stage, lymph node metastasis and BRAF status as well as disease recurrences or disease-free interval. Only Aurora-B mRNA was significantly higher in T(3-4) tissues, with respect to T(1-2) PTC tissues. The data reported here demonstrate that the expression of Aurora kinases is deregulated in the majority of PTC tissues, likely contributing to PTC progression. However, differently from other human solid cancers, detection of Aurora-A or Aurora-B mRNAs is not a prognostic biomarker in PTC patients.

A new aurora in anaplastic thyroid cancer therapy.

Anaplastic thyroid cancers (ATC) are among the most aggressive human neoplasms with a dire prognosis and a median survival time of few months from the diagnosis. The complete absence of effective therapies for ATC renders the identification of novel therapeutic approaches sorely needed. Chromosomal instability, a feature of all human cancers, is thought to represent a major driving force in thyroid cancer progression and a number of mitotic kinases showing a deregulated expression in malignant thyroid tissues are now held responsible for thyroid tumor aneuploidy. These include the three members of the Aurora family (Aurora-A, Aurora-B, and Aurora-C), serine/threonine kinases that regulate multiple aspects of chromosome segregation and cytokinesis. Over the last few years, several small molecule inhibitors targeting Aurora kinases were developed, which showed promising antitumor effects against a variety of human cancers, including ATC, in preclinical studies. Several of these molecules are now being evaluated in phase I/II clinical trials against advanced solid and hematological malignancies. In the present review we will describe the structure, expression, and mitotic functions of the Aurora kinases, their implications in human cancer progression, with particular regard to ATC, and the effects of their functional inhibition on malignant cell proliferation.

Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.

Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway.

Emerging molecular markers for the prognosis of differentiated thyroid cancer patients.

Epithelial thyroid cancers are represented by the differentiated papillary and follicular thyroid carcinomas which, following dedifferentiation, are thought to give rise to the highly aggressive and incurable anaplastic thyroid carcinomas. Although derived from the same cell type, the different thyroid tumors show specific histological features, biological behavior and degree of differentiation as a consequence of different genetic alterations. Over the last few years, our knowledge regarding the molecular alterations underlying thyroid cell malignant transformation and cancer progression has considerably increased; however, the prognosis of differentiated thyroid cancer patients still relies on high-risk clinic-pathological variables. In particular, the actual staging systems provides only a rough prediction for cancer mortality and risk of recurrences, including in each risk group patients with highly different tumor-specific progression, disease-free interval and survival time. In order to improve DTC patient's risk stratification, both the European and the American Thyroid Associations proposed practical guidelines to integrate the actual staging systems with additional clinical features such as the tumor histological variant, the results of post-ablative whole body scan and the serum thyroglobulin levels. Despite that, patients within the same risk group still show a very heterogeneous behavior in terms of disease-free interval. As a consequence, the identification of new prognostic molecular biomarkers able to testify tumor aggressiveness is highly required. Here we'll review recently characterized new molecular markers potentially able to ameliorate the prognosis in DTC patients.

Prevalence of breast cancer in thyroid diseases: results of a cross-sectional study of 3,921 patients.

Results from national cancer registries reveal an association of thyroid cancers with extra-thyroidal malignancies. In this study, we evaluated the prevalence of breast cancer (BC) in women affected by both benign and malignant thyroid diseases (TD) in comparison to the general population. To this end, 3,921 female patients from central and southern regions of Italy were evaluated. Age-matched analysis of the prevalence of BC was carried out after dividing the patients into three diagnostic categories: (1) 1,149 patients with non-nodular TD; (2) 2350 patients with nodular TD; (3) 422 patients affected by differentiated thyroid cancers. Furthermore, the patients were grouped according to the absence (2,344 patients) or presence (1,453 patients) of anti-thyroglobulin (TgAb) and/or anti-thyroperoxidase (TPOAb) or anti-TSH receptor auto-antibodies (124 patients). BC prevalence in TD patients as a whole was significantly higher compared to the general population, with an odds ratio (OR) of 3.33. Age-matched analysis showed that the risk of a BC in TD patients was higher in younger patients (age 0-44 years), with an OR of 15.24, which decreased with increasing age. Patients without thyroid auto-antibodies showed a higher OR for BC (p = 0.0005) than TD patients with TgAb and/or TPOAb. The results demonstrate that women affected by either benign or malignant thyroid disease have a significantly greater risk of BC, which is higher at a younger age. Furthermore, thyroid auto-antibodies appear to be protective against BC. These findings may contribute to the identification of common genetic and environmental factors underlying this disease association.

Effects of ultraviolet radiation on FRTL-5 cell growth and thyroid-specific gene expression.

During space missions, radiation represents a major hazard for human health and involves all body organs and tissues. Regarding thyroid function, it has been shown that ultraviolet radiation (UVC) has dose-dependent apoptotic effects on FRTL-5 cells, a normal strain of rat thyrocytes. We examined the effects of a sublethal dose of UVC on FRTL-5 cell growth and gene expression. Cells exposed to 10 J/m(2) UVC showed no differences in viability compared to control cells after 24 h, but the BrdU incorporation was reduced, indicating a cytostatic effect. Quantitative RT-PCR carried out at 24 and 48 h after irradiation demonstrated that the mRNA levels of thyroglobulin (Tg), thyroperoxidase (Tpo), and sodium/iodide symporter (Nis) were transiently decreased at 24 h in treated cells, while the mRNAs of the thyroid transcription factors TTF1, Foxe1, and Pax8 were not affected. In cells cultured with TSH-free medium, the basal transcription of Tg, Tpo, and Nis genes was equally impaired by radiation and no longer stimulated by TSH. Overall, the results demonstrate that a sub-apoptotic dose of UVC compromises not only thyrocyte proliferation but also the expression of genes involved in thyroid hormone production. These findings might contribute to explaining the histological, biochemical, and clinical features of hypothyroidism observed in both animals and humans during spaceflight, and suggest that free thyroxine levels of astronauts during prolonged space missions should be monitored.

In papillary thyroid carcinoma BRAFV600E is associated with increased expression of the urokinase plasminogen activator and its cognate receptor, but not with disease-free interval.

CONTEXT: It has been suggested that patients with papillary thyroid cancer (PTC) harbouring the BRAF(V600E) mutation have a worse prognosis. We showed in PTC that high levels of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) inversely correlate with disease-free interval (DFI). OBJECTIVES: To investigate the effects of BRAF(V600E) on the expression of uPA and uPAR and to evaluate the prognostic relevance of BRAF(V600E) alone or in combination with uPA and uPAR. DESIGN/SETTING/PATIENTS/INTERVENTION: The case study included 91 patients with PTC. All patients underwent thyroidectomy and radioiodine therapy. Follow-up was available for 75 patients. MAIN OUTCOME MEASURES: The BRAF(V600E) mutation was analysed by sequencing and mutant allele-specific PCR amplification; uPA and uPAR expression by quantitative RT-PCR. RESULTS: BRAF(V600E) was found in 44 of the 91 patients and associated with older age, but not with high-risk clinicopathological features. Urokinase PA and uPAR mRNA levels were higher in tumour tissues by 9·51 ± 1·30 and 4·64 ± 0·44 fold, respectively, compared to normal matched tissues, being significantly higher in BRAF(V600E) -positive patients. In vitro induction of BRAF(V600E) in PCCL3 cells caused a significant increase in both uPA and uPAR mRNAs. Higher levels of uPA and uPAR correlated with lymph node metastases, TNM stage and disease recurrences. Kaplan-Meier and multivariate analyses demonstrated that uPA and uPAR were associated with shorter DFI, while the BRAF(V600E) was not. CONCLUSION: In PTC, BRAF(V600E) induces uPA and uPAR expression. The latter, but not BRAF(V600E) , associates with advanced stages and shorter DFI. If confirmed in larger case studies, they may represent reliable prognostic markers for more accurate risk stratification and postoperative decision-making in patients with PTC.

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT.

BACKGROUND: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets. METHODS: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy. RESULTS: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar. CONCLUSIONS: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.

Aurora-C interacts with and phosphorylates the transforming acidic coiled-coil 1 protein.

Aurora-C, a member of the Aurora kinase family, is implicated in the regulation of mitosis. In contrast to Aurora-A and Aurora-B its cellular localization and functions are poorly characterized. TACC1 protein belongs to the transforming acidic coiled-coil family shown to interact with the Aurora kinases. In the present study we analyzed the interaction between Aurora-C and TACC1 by means of immunofluorescence (IF), co-immunoprecipitation (IP) and in vitro phosphorylation experiments. We demonstrated that Aurora-C and TACC1 proteins co-localize to the midbody of HeLa cells during cytokinesis. Immunoprecipitated TACC1 from HeLa cell extracts was associated with Aurora-C. In addition, the interaction of the two proteins was tested by analyzing the phosphorylation of TACC1 in vitro. The results demonstrated that TACC1 is phosphorylated by Aurora-C on a serine at position 228. In conclusion, the study demonstrated that TACC1 localizes at the midbody during cytokinesis and interacts with and is a substrate of Aurora-C, which warrant further investigation in order to elucidate the functional significance of this interaction.

High expression of the urokinase plasminogen activator and its cognate receptor associates with advanced stages and reduced disease-free interval in papillary thyroid carcinoma.

CONTEXT: The urokinase plasminogen activating system is implicated in neoplastic progression, and high tissue levels of urokinase plasminogen activating system components correlate with poor prognosis in various human cancers. OBJECTIVE: The objective of the study was to investigate the prognostic relevance of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR), and the plasminogen activator inhibitor 1 (PAI-1) in human papillary thyroid cancer (PTC). DESIGN: The expression of uPA, uPAR, and PAI-1 genes was analyzed in PTC and normal matched tissues by quantitative RT-PCR. The case study consisted of 99 patients (21 males and 78 females) affected by PTC including 77 classical, 15 follicular, four tall cell, and three oncocytic variants. Forty-one patients had lymph node metastases at the time of diagnosis. All the patients underwent thyroidectomy and radioiodine therapy followed by thyroid hormone replacement therapy. Follow-up data were available for 76 patients up to 64 months. RESULTS: The uPA, uPAR, and PAI-1 mRNA levels were significantly higher in PTC compared with normal matched tissues by 9.63 ± 1,29-, 4.82 ± 0.45-, and 5.64 ± 0.71-fold, respectively. The increased expression of uPA and uPAR correlated statistically with advanced pT and N status. The uPA was also significantly associated with advanced tumor node metastasis stages. The Kaplan-Meier analysis showed a significant association of uPA and uPAR levels with reduced patient disease-free interval (DFI), and this association was stronger in stage I patients. CONCLUSION: The study demonstrated that in PTC the increased gene expression of uPA and uPAR is associated with tumor invasiveness, advanced stages, and shorter DFI, suggesting their prognostic relevance. These observations warrant further investigation in larger patient populations with longer follow-up.

Effect of supra-physiological dose administration of rhGH on pituitary-thyroid axis in healthy male athletes.

The effect of recombinant human growth hormone (rhGH) administration on hypothalamic-pituitary-thyroid (HPT) system in healthy trained humans still needs to be fully clarified. Furthermore, whether rhGH abuse could exert undesirable or noxious effect on health is still unclear. In order to evaluate changes in HPT axis variables in time after rhGH administration, 14 well-trained healthy male athletes were treated with rhGH (0.03 mg/kg body weight/day, sc) administration, 6 days/week for 3 weeks. Morning blood samples were collected immediately before and 3, 4, 8, 15, and 21 days after rhGH administration. A further set of blood samples was taken 3, 6 and 9 days after drug withdrawal. Samples were analyzed for GH-IGF and HPT axis. Significant TSH serum decrease and IGF-I increase occurred early after rhGH administration, without FT(3) content modification and with FT(4) reduction delayed in time. Serum TSH concentrations negatively correlated with IGF-I, IGFBP-3 and IGF-I/IGFBP-3 ratios. rhGH short-term administration in healthy trained subjects induced an early TSH suppression--likely acting at central level through IGF-I--without thyroid function alteration. Further investigations in athletes are necessary to verify whether prolonged TSH suppression, i.e. rhGH intake for longer time, could induce pathologic condition, such as hypothyroidism.

Is minimally invasive surgery appropriate for small differentiated thyroid carcinomas?

PURPOSE: To retrospectively evaluate the surgical completeness of minimally invasive total thyroidectomy for small (<20 mm) differentiated thyroid carcinoma (DTC). METHODS: The subjects of this study were 30 patients who underwent minimally invasive total thyroidectomy as a single procedure. We registered the following postoperative measurements in the LT4 withdrawal period: serum thyroglobulin level, 6-h radioiodine uptake diagnostic test results, and neck ultrasound (US) findings. RESULTS: The mean serum thyroglobulin level was 4.99 +/- 4.67 ng/ml, the mean radioiodine uptake diagnostic test after 6 h was 3.11% +/- 2.90%, and US showed no thyroid remnant. CONCLUSIONS: The short-term outcome measures showed adequate resection of thyroid tissue, comparable with that reported after conventional surgery. Our findings suggest that small nodules with suspicious or malignant cytology are one of the best indications for minimally invasive surgery.

Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas.

BACKGROUND: The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas. METHODS: The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry. RESULTS: Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 +/- 0.74 (p < 0.01) and 6.25 +/- 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 +/- 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 +/- 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 +/- 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size. CONCLUSIONS: We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression.

Inhibition of the aurora kinases suppresses in vitro NT2-D1 cell growth and tumorigenicity.

The aurora kinase family members, Aurora-A, -B, and -C (listed as AURKA, AURKB and AURKC respectively in the HUGO Database), are serine/threonine kinases involved in the regulation of chromosome segregation and cytokinesis, and alterations in their expression are associated with malignant cell transformation and genomic instability. Deregulation of the expression of the aurora kinases has been shown to occur also in testicular germ cell tumors (TGCTs) identifying them as putative anticancer therapeutic targets. We here evaluated the in vitro effects of MK-0457, an aurora kinases inhibitor, on cell proliferation, cell cycle, ploidy, apoptosis, and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment with MK-0457 inhibited cell proliferation in a time- and dose-dependent manner, with IC(50)=17.2+/-3.3 nM. MK-0457 did not affect the expression of the three aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it, presenting after short time the typical features of apoptotic cells. Cytofluorimetric analysis confirmed that the treatment with MK-0457 for 6 h induced NT2-D1 cells accumulation in the G(2)/M phase of the cell cycle and the subsequent appearance of sub-G(0) nuclei. The latter result was further supported by the detection of caspase-3 activation following 24-h treatment with the inhibitor. Finally, MK-0457 prevented the capability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of aurora kinase activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment.

TSH receptor and thyroid-specific gene expression in human skin.

Experimental evidence suggests that in autoimmune thyroid diseases (AITDs) the skin is a target of autoantibodies against thyroid-specific antigens; however, the role of these autoantibodies in skin alterations remains unclear. To gain insight into the function of nominally thyroid-specific genes in skin, we analyzed the expression of thyroid-stimulating hormone-receptor (TSH-R), thyroglobulin (Tg), sodium iodide symporter (NIS), and thyroperoxidase (TPO) genes in normal human skin biopsies and cultured primary keratinocytes and dermal fibroblasts. The results revealed the presence of all the transcripts in skin biopsies. However, in keratinocytes and fibroblasts, only TSH-R messenger RNA was always detected. Western blot and immunohistochemical analyses of skin specimens confirmed the presence of TSH-R protein in keratinocytes and fibroblasts. Moreover, TSH treatment induced the proliferation of cultured keratinocytes and fibroblasts and increased keratinocyte intracellular cAMP. Finally, affinity-purified IgGs from serum of patients affected by Graves' disease, but not by chronic lymphocytic thyroiditis, stimulated cAMP accumulation in cultured keratinocytes, as well as their proliferation. In conclusion, the expression of thyroid-specific genes in cultured keratinocytes and fibroblasts and the mitogenic effects of TSH and IgGs on these cells support the concept that autoantibodies against thyroid-specific antigens may contribute to cutaneous symptoms in AITDs.

Comparison of malignancy rate in thyroid nodules with cytology of indeterminate follicular or indeterminate Hürthle cell neoplasm.

BACKGROUND: Thyroid nodules that are read on cytology as follicular or Hürthle cell neoplasms (FN and HN, respectively) and indeterminate for malignancy require surgery to differentiate benign from malignant nodules. We analyzed FN and HN with indeterminate cytology to determine if there were differences in the rate and types of thyroid malignancy and if the rate of thyroid malignancy was influenced by age or sex. METHODS: We analyzed 463 nodules with an indeterminate cytological diagnosis of FN and 140 nodules with an indeterminate cytological diagnosis of HN. The histopathological diagnosis after thyroidectomy was the method for establishing the diagnosis and type of malignancy. RESULTS: For the entire series of 603 patients there were 106 (17.6%) with thyroid cancer; 80 of these had a cytology reading of FN and 26 had HN. Extrathyroidal invasion in the grouped HN and FN patients who had papillary thyroid carcinoma (PTC) was more common in females than in males (62% vs. 25 %, p < 0.05). The rate of thyroid cancer was similar in FN (17.3%) and HN (18.6%). The rate of Hürthle cell thyroid cancer was significantly higher in HN than in FN (5.0% vs. 0.7%, p < 0.01) and the rate of the oncocytic variant of PTC was also significantly greater in HN compared to FN nodules (23.1% vs. 1.7%, p < 0.05). The rate of follicular thyroid carcinoma was almost identical in patients with HN and FN (19.2% vs. 18.8 %). CONCLUSIONS: There is little difference in the rate of malignancy between thyroid nodules with a cytological reading of FN indeterminate for malignancy and HN indeterminate for malignancy but there is a difference in the types of thyroid cancers in these groups. Hürthle cell thyroid cancer and the oncocytic variant of PTC is more common in nodules with an HN indeterminate for malignancy cytology reading than in nodules with a FN indeterminate for malignancy cytology reading. Since Hürthle cell thyroid cancer and the oncocytic variant of PTC are more aggressive than other thyroid cancers, it is likely that patients with an HN indeterminate for malignancy cytology will, as a group, have more aggressive thyroid cancers than those with an FN indeterminate for malignancy cytology.