Salvatore Sorrenti

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.

Q-elastography in the presurgical diagnosis of thyroid nodules with indeterminate cytology.

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.

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.

Cervical lymph node metastases from thyroid cancer: does thyroglobulin and calcitonin measurement in fine needle aspirates improve the diagnostic value of cytology?

BackgroundMeasurement of thyroglobulin (Tg) protein in the washout of the needle used for fine needle aspiration biopsy cytology (FNAB-C) has been shown to increase the sensitivity of FNAB-C in identifying cervical lymph node (CLN) metastasis from well-differentiated thyroid cancer (TC). In this study, we evaluated whether routine measurement of Tg protein (FNAB-Tgp), Tg mRNA (FNAB-Tgm) and calcitonin (CT) mRNA (FNAB-CTm) in the FNAB washout of CLN increases the accuracy of FNAB-C in the diagnosis of suspicious metastatic CLN.MethodsIn this prospective study 35 CLN from 28 patients were examined. Histology showed metastatic papillary TC (PTC) in 26 CLN, metastatic medullary TC (MTC) in 3 CLN, metastatic anaplastic TC (ATC) in 3 CLN and 3 metastatic CLN from extra-thyroidal cancers.ResultsThe overall accuracy of FNAB-C was 84.4%, reaching 95.7% when the analysis was restricted to PTC. Both FNAB-Tgp and FNAB-Tgm compared favorably with FNAB-C and shown diagnostic performances not statistically different from that of FNAB-C. However, FNAB-Tgp and FNAB-Tgm/FNAB-CTm were found useful in cases in which cytology results were inadequate or provided diagnosis inconsistent with patients clinical parameters.ConclusionsWe demonstrated that FNAB-C, Tg/CT mRNA and Tg protein determination in the fine-needle washout showed similar accuracy in the diagnosis of metastatic CLN from TC. The results of this study suggest that samples for Tg protein and Tg/CT mRNA measurements from CLN suspicious for metastatic TC should be collected, but their measurements should be restricted to cases in which FNAB-C provides uninformative or inconsistent diagnosis with respect to patients clinical parameters.

Analysis of clinical, ultrasound and colour flow‐Doppler characteristics in predicting malignancy in follicular thyroid neoplasms

Fine-needle aspiration cytology (FNAC) represents the main diagnostic tool in the evaluation of thyroid nodules because of its high sensitivity, specificity and accuracy. However, while FNAC is very reliable in detecting papillary thyroid carcinomas (PTC) and anaplastic thyroid carcinomas, follicular neoplasms (FN) represent the grey zone in which cytology cannot discriminate malignant from benign tumours. 1 Consequently, in presence of nodules cytologically classified as FN, thyroidectomy is usually required and the histological evaluation reveals that about 80% are benign lesions. Thus, identification of parameters associated with malignancy is needed. In this context, several studies have focused on clinical, ultrasonographic and cytological aspects, with controversial results. 2,3

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.

Galectin-3 expression in thyroid fine needle cytology (t-FNAC) uncertain cases: validation of molecular markers and technology innovation.

Thyroid cancer is not very common, accounting for 1–2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, galectin‐3 (Gal‐3) has been proposed as a diagnostic marker with varied success. In this paper, we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM‐IGL) to investigate the expression of Gal‐3 on Thin‐Prep fine needle aspiration cytology (FNAC). We optimized the ESEM‐IGL method on thyroid cell lines (RO‐82 and FTC‐133) comparing our membrane Gal‐3 labeling data with Western blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre‐surgical diagnosis. ESEM‐IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Gal‐3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin‐Prep ESEM‐IGL shows the levels of Gal‐3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, Gal‐3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre‐surgery diagnosis. J. Cell. Physiol.

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.