Massimino D'Armiento

The Urokinase Plasminogen Activator System: A Target for Anti-Cancer Therapy

The urokinase plasminogen activator (uPA) system (uPAS) consists of the uPA, its cognate receptor (uPAR) and two specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and 2 (PAI-2). The uPA converts the proenzyme plasminogen in the serine protease plasmin, involved in a number of physiopathological processes requiring basement membrane (BM) and/or extracellular matrix (ECM) remodelling, including tumor progression and metastasis. Data accumulated over the past years have made increasingly clear that the uPAS has a multifunctional task in the neoplastic evolution, affecting tumor angiogenesis, malignant cell proliferation, adhesion and migration, intravasation and growth at the metastatic site. In agreement with their role in cancer progression and metastasis, an increased expression of uPA, uPAR, and PAI-1 has been documented in several malignant tumors, and a positive correlation between the levels of one or more uPAS members and a poor prognosis has been frequently reported. This is particularly evident in breast cancer, for which uPA has been demonstrated to be the most potent independent prognostic factor described to date. The involvement of the uPAS in cancer progression identifies its components as suitable targets for anti-cancer therapy. Several therapeutical approaches aimed at inhibiting the uPA/uPAR functions have been shown to possess anti-tumor effects in xenograft models, including selective inhibitors of uPA activity, antagonist peptides, monoclonal antibodies able to prevent uPA binding to uPAR and gene therapy techniques silencing uPA/uPAR expression. All these strategies, however, although promising, need definitive confirmation in humans as, up to now, only few uPA inhibitors entered clinical trial.

Expression of Aurora kinases in human thyroid carcinoma cell lines and tissues

The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. In the present study, we demonstrated that human thyrocytes express all 3 Aurora kinases (A, B and C) at both protein and mRNA level and this expression is cell cycle‐regulated. An increase in the protein level of the 3 kinases was found, with respect to normal human thyrocytes (HTU5), in the human cell lines derived from follicular (FTC‐133), papillary (B‐CPAP) and anaplastic (8305C) thyroid carcinomas, but not in cells derived from a follicular adenoma (HTU42). These observations were mirrored in RT‐PCR experiments for Aurora‐A and B. In contrast, Aurora‐C mRNA levels were not significantly different among the different cell types analyzed, suggesting that posttranscriptional mechanism(s) modulate its expression. The expression at the protein level of all 3 Aurora kinases was significantly higher in 3 thyroid papillary carcinomas with respect to normal matched tissues obtained from the same patients. Similar modifications, at the mRNA level, could be observed in 7 papillary carcinoma tissues for Aurora‐A and B, but not for Aurora‐C. In conclusion, we demonstrated that normal human thyrocytes express all 3 members of the Aurora kinase family, and their expression is amplified in malignant thyroid cell lines and tissues. These results suggest that the Aurora kinases may play a relevant role in malignant thyroid cancers, and may represent a putative therapeutic target for thyroid neoplasms.

Thyroid hormone stimulates glucose transport and GLUT1 mRNA in rat Sertoli cells

The transport of 2-deoxyglucose (dGlc) by cultured rat Sertoli cells was stimulated by L-triiodothyronine (T3) in a time and dose-dependent manner. The lag-time was of about 6 h, the half-maximal dose (ED50) was 0.47 nM, which correlates with the Kd of the nuclear T3 receptor of rat Sertoli cells (Kd = 1-2 nM), and the stimulation was maintained up to 24 h. The effect was specific, as judged by the order of potency of T3 analogs. Cycloheximide prevented the stimulatory effect without affecting the basal uptake. T3 stimulated the uptake of the glucose analog 3-O-methylglucose (MeGlc) with the same order of potency as that of dGlc. The ontogenetic profile of the T3 effect coincides with that of T3 nuclear receptors in rat Sertoli cells. Northern blot analysis demonstrated that Sertoli cells express the erythrocyte/brain glucose transporter isoform (GLUT1) but not the adipose/muscle isoform (GLUT4). T3 treatment (10(-7) M for 24 h) induces an increase of GLUT1 mRNA level comparable to that of glucose analog uptake. These results suggest that thyroid hormone stimulates glucose transport by increasing the synthesis of new glucose transporter units and give further evidence for a direct effect of thyroid hormone in the modulation of Sertoli cell functions.

Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines

Anaplastic thyroid cancers (ATC) are aggressive tumors, which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy, and most patients die within a few months of diagnosis. In the present study, we evaluated the in vitro effects on ATC cells of VX-680, an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy were tested on the ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time- and dose-dependent manner, with the IC50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with > or =4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion, our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment.

Iodine deficiency in pregnant women residing in an area with adequate iodine intake

OBJECTIVE To prevent iodine deficiency disorders, the World Health Organization, United Nations Childrens Fund, and International Council for the Control of Iodine Deficiency Disorders established that for a given population median urinary iodine concentrations (UIC) must be 100-199 microg/L in clinically healthy subjects and 150-249 microg/L in clinically healthy pregnant women. We evaluated whether in the urban area of Rome, Italy, where a salt iodination program (30 mg/kg) was introduced since 2005, an increased demand of iodine during pregnancy is guaranteed. METHODS During 2006, 51 pregnant women at first trimester of a physiologic gestation were consecutively enrolled on presentation to evaluate UIC in morning spot urine samples. As controls, 100 age-matched clinically healthy non-pregnant women were evaluated. RESULTS The median UICs were 182 microg/L (range 85-340 microg/L) and 74 microg/L (range 17-243 microg/L), respectively, in the control and pregnant groups. This difference was highly significant (P < 0.001). In particular, the UIC was found to be lower than adequate in 4% of control women compared with 92% of pregnant women. This difference of occurrences was highly significant (P < 0.001). CONCLUSION This observational study demonstrated that, despite the adequate supplementation of iodine intake, most pregnant women appear not to be protected against iodine deficiency. If confirmed in larger case studies, this finding claims the attention of relevant professionals to monitor iodine nutrition during gestation, assuming that ordinary supplementation of iodine intake seems to be sufficient only in non-gestational conditions.

Erk-dependent cytosolic phospholipase A2 activity is induced by CD95 ligand cross-linking in the mouse derived Sertoli cell line TM4 and is required to trigger apoptosis in CD95 bearing cells.

In the present study we demonstrated that CD95L cross-linking generated reverse signalling in the mouse derived Sertoli cell line TM4. Treatment of TM4 cells with mAb anti-CD95L induced activation of the cytosolic phospholipase A2 (cPLA2). Cytosolic PLA2 activation was controlled by the MAPK pathway as indicated by the ability of the specific MEK inhibitor, PD098059, to abolish cPLA2 activation. In addition, Western blot experiments showed a rapid increase in phosphorylated Erk1/2 following CD95L cross-linking, while no effect on the phosphorylation of other MAPK, p38 or JNK, was observed. CD95L cross-linking by mAb increased the levels of soluble CD95L and apoptotic activity of TM4 cell supernatants, which was blocked by co-incubation with the PLA2 inhibitor, AACOCF3 or PD098059. Finally, pre-treatment of TM4 cells with AACOCF3 or PD098059 completely abolished TM4-induced apoptosis of Jurkat T cells, thus indicating that the Erk/cPLA2 pathway is required for CD95L-induced apoptosis. Cell Death and Differentiation (2000) 7, 916–924

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.

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.

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

It has been suggested that patients with papillary thyroid cancer (PTC) harbouring the BRAFV600E 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).

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

BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsWe demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.