The urokinase plasminogen activating system (upas)

Transcription

1 ORIGINAL Endocrine ARTICLE Research High Expression of the Urokinase Plasminogen Activator and Its Cognate Receptor Associates with Advanced Stages and Reduced Disease-Free Interval in Papillary Thyroid Carcinoma Salvatore Ulisse,* Enke Baldini,* Salvatore Sorrenti, Susi Barollo, Lucio Gnessi, Antonio Catania, Maria Rosa Pellizzo, Francesco Nardi, Caterina Mian, Enrico De Antoni, Massimino D Armiento, and Luigi Frati Departments of Experimental Medicine (S.U., E.B., L.G., F.N., M.D., L.F.) and Surgical Sciences (S.S., A.C., E.D.A.), Sapienza University of Rome, Rome, Italy; Veneto Institute of Oncology (S.B.), Instituto di Ricovero e Cura a Carattere Scientifico, Padova, Italy; and Department of Medical and Surgical Sciences (M.R.P., C.M.), University of Padova, Padova Italy 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 ,29-, , and 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. (J Clin Endocrinol Metab 96: , 2011) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2011 by The Endocrine Society doi: /jc Received July 22, Accepted October 25, First Published Online November 24, 2010 * S.U. and E.B. contributed equally to this work. The urokinase plasminogen activating system (upas) consists of the urokinase plasminogen activator (upa), its cognate receptor (upar), and two main plasminogen activator inhibitors, PAI-1 and PAI-2. The upas is involved in the extracellular conversion of the inactive plasminogen to the serine protease plasmin, which is implicated in nu- Abbreviations: DFI, Disease-free interval; FNA, fine-needle aspiration; PAI, plasminogen activator inhibitor; PTC, papillary thyroid cancer; Tg, thyroglobulin; TNM, tumor node metastasis; upa, urokinase plasminogen activator; upar, upa cognate receptor; upas, urokinase plasminogen activating system. 504 jcem.endojournals.org J Clin Endocrinol Metab, February 2011, 96(2):

2 J Clin Endocrinol Metab, February 2011, 96(2): jcem.endojournals.org 505 merous pathophysiological processes including tumor progression (1). Aberrant expression of upas components has a pivotal role in extracellular matrix degradation and affects multiple aspects of the neoplastic evolution including tumor cell proliferation, adhesion, migration, growth at the metastatic sites, and neoangiogenesis (1). Tumor overexpression of upas components is associated with poor clinical outcome in several human malignancies. In particular, their proven ability to guide therapeutic decision making for breast cancer patients prompted the American Society of Clinical Oncology to include them among the recommended breast tumor markers for clinical use (2 5). Thyroid carcinoma is the most common endocrine malignancy accounting for 1% of all human cancers, and it represents the fifth most common cancer in women in the United States (6, 7). The majority of thyroid tumors are of the differentiated papillary thyroid cancer (PTC) histotype comprising about 90% of all thyroid cancers (7 9). The prognosis is favorable with a 10-yr survival rate of 90% (6, 10). However, 20% of patients face the morbidity of disease recurrences and PTC-related deaths (6, 11). As a consequence, the identification of molecular marker(s) to refine the stratification risk of PTC patients would be of particular interest (12 14). In this context recent reports from our own and other laboratories (1, 15 18) have demonstrated the increased expression of the upas components in PTC, which prompted us to analyze the potential prognostic values of the expression of upas components in patients affected by PTC. Patients and Methods Tissue samples, histology, and patients staging Normal and matched tumor thyroid tissue was obtained from surgical specimens of 99 patients (21 males and 78 females, age range yr, median 45 yr) who underwent total thyroidectomy for PTC at the Department of Surgical Sciences, Sapienza University of Rome (Rome, Italy) (n 42), or at the Department of Medical and Surgical Sciences, University of Padova, (Padua, Italy) (n 57). All the patients gave their informed consent. Tissue samples were collected, frozen in liquid nitrogen, and stored at 80 C. Patients older than 45 yr of age underwent total thyroidectomy with dissection of the lymph nodes of the central compartment (level VI). Patients younger than 45 yr of age had total thyroidectomy with dissection of the lymph nodes of the central compartment limited to patients with nodal disease. Surgical resection of the lymph nodes from the lateral neck compartments (level II-V) was performed in patients with nodal disease diagnosed by preoperative ultrasound-guided fine-needle aspiration (FNA) cytology and/or thyroglobulin (Tg) measurement in the FNA washout. Of the 99 PTC patients, 77 exhibited classical, 15 follicular, four tall cell, and three oncocytic variants. The histological diagnoses were made independently by two different histopathologists according to the World Health Organization classification (19). At the time of surgery, lymph node metastases were found in 41 patients. After tumor node metastasis (TNM) staging, 65 patients were at stage I, three at stage II, 24 at stage III, and seven at stage IV (15). Approximately d later all the patients underwent radioiodine therapy followed by thyroid hormone replacement therapy. To ascertain their disease-free condition, 4 5 months later all the patients underwent neck ultrasound and serum Tg measurement. At this time three patients presented persistent disease. Recurrences were diagnosed by measurement of serum Tg levels either in basal conditions or after recombinant human TSH stimulation; FNA cytology and/or Tg determination in the FNA washout from lymph nodes; 131 I whole-body scan; histological analysis after surgical resection of the lesion. During the follow-up, 16 recurrences were recorded, 12 being cervical lymph nodes and four being lung metastases. The follow-up included 76 patients (mean months, range 6 64 months), 50 of which were at TNM stage I. The lower limit of time to recurrence started at 6 months. Two patients died because of the persistence of the disease within 4 months of the diagnosis. Extraction and analysis of mrna Total RNA was extracted from thyroid tissue as previously described and reverse transcribed (15). The cdnas were used for the quantitative PCR amplifications of the upas components, and seven different housekeeping genes using the following set of primers: upa forward, 5 -GCTCAAGGCTTAACTCCAACAC-3, reverse, 5 -TCCTTGGAACGGATCTTCAG-3 ; upar forward, 5 - GAAGATCACCAGCCTTACCG-3, reverse, 5 -AACGGC- TTCGGGAATAGG-3 ; PAI-1 forward, 5 -ATACTGAGTTCAC- CACGCCC-3, reverse, 5 -GTGGAGAGGCTCTTGGTCTG-3 ; -actin forward, 5 -CAAGAGATGGCCACGGCTGCT-3, reverse, 5 -TCCTTCTGCATCCTGTCGGCA-3 ; glyceraldehyde- 3-phosphate dehydrogenase forward, 5 -ATCATCAGCAATGC- CTCCTG-3, reverse, 5 -GGCCATCCACAGTCTTCTG-3 ; hypoxanthine phosphoribosyltransferase 1 forward, 5 -ATGCT- GAGGATTTGGAAAGG-3, reverse, 5 -AATCCAGCAGGT- CAGCAAAG-3 ; 2 -microglobulin forward, 5 -CAGCAGA- GAATGGAAAGTC-3, reverse, 5 -CATGCTGCTTACATG- TCTCG-3 ; ribosomal protein L13a forward, 5 -ACCGTGC- GAGGTATGCTG-3, reverse, 5 -TAGGCTTCAGACGCAC- GAC-3 ; succinate dehydrogenase complex, subunit A forward, 5 -GCATAAGAACATCGGAACTGC-3, reverse, 5 -GGTC- GAACGTCTTCAGGTG-3 ; TATA box binding protein forward, 5 -GCAAGGGTTTCTGGTTTGC-3, reverse, 5 - GGTCAGTCCAGTGCCATAAG-3. Real-time PCR was performed with a LightCycler instrument using the FastStart DNA master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). Amplicon specificities were checked by automated DNA sequencing (Primm; San Raffaele Biomedical Science Park, Milano, Italy). Determination of the most stable reference genes and data analysis Seven candidate reference genes above reported were amplified by quantitative RT-PCR in a sample subgroup composed of 15 normal and 15 matched tumor tissues and ranked according to their stability with the genorm visual basic application for Microsoft Excel (Richmond, CA) (20). The three

3 506 Ulisse et al. Plasminogen Activation and Papillary Thyroid Cancer J Clin Endocrinol Metab, February 2011, 96(2): Statistical analysis The nonparametric Wilcoxon rank sum test was used to calculate the statistical significance of differences in the expression levels of upas components in tumor tissues vs. normal matched tissues and in metastatic (N1) vs. nonmetastatic (N0) tumor tissues. The statistical significance of differences in the expression levels of upas components in the PTC variants, pt, and TNM stages was assessed by the ANOVA (one way ANOVA) followed by the Tukey post-anova test. The correlation analysis between each upas component mrna and the others, or tumor size, represented by the major diameter of the lesion, or the patient s age was evaluated by the Spearman correlation. The impact of the expression of upas components on the disease-free interval was assessed by the Kaplan-Meier analysis combined with Mantel- Cox log rank. For the Kaplan-Meier analysis, tumor samples were dichotomized using the following cutoff values: 3.8 for upa; 3.0 for upar; 3.5 for PAI-1. All statistical analyses were carried out using the GraphPad Prism software (La Jolla, CA). The results were considered significantly different if P values were lower than FIG. 1. Expression of upa and upar mrna in the different pt (A and D) and N status (B and E) and TNM stages (C and F) in normal and matched PTC tissues from 99 patients. The statistical significance of differences in the expression levels of upas components in pt and TNM stages was assessed by the ANOVA followed by the Tukey post-anova test, whereas the statistical analysis of differences in the expression levels of upas components in metastatic (N1) vs. nonmetastatic (N0) tumor tissues was performed using the nonparametric Wilcoxon test. The bars in the graph indicate the median values. resulting most stable genes were glyceraldehyde-3-phosphate dehydrogenase, ribosomal protein L13a, and succinate dehydrogenase complex, subunit A, assumed as reference genes for all samples. The relative quantification of tumor samples with respect to their normal counterparts was accomplished by means of the Relative Expression Software Tool (2009) (20), using a normalization factor computed as the geometric media of the three reference genes. The results are reported as mean SEM and median values. Results Measurements of upas component mrnas in PTC and correlations with clinical parameters The results showed a significant (P ) increase in upa, upar, and PAI-1 mrnas by, respectively, (median 4.46), (median 3.47), and (median 3.56)-fold in 99 PTC tissues, compared with normal matched tissues. The upa, upar, and PAI-1 mrnas increases correlated significantly with each other, with correlation coefficient of (P ) for upa vs. upar, (P ) for upa vs. PAI-1, and (P ) for upar vs. PAI-1. No correlation was found between upa, upar, or PAI-1 expression and patients age, tumor size, or PTC variants (data not shown). The mrna levels of both upa (P ) and upar (P ) were significantly higher in pt3 compared with pt1 tumors (Fig. 1, A and D). Significantly higher levels of upa (P 0.013) and upar (P 0.049) mrna were found in metastatic (N1) vs. nonmetastatic (N0) tumors (Fig. 1, B and E), and upa (P ) in stage III patients, compared with stage I patients (Fig. 1, C and F). Regarding PAI-1, no change was observed among the different pt-, N-status, and TNM stages (data not shown). Prognostic relevance of upas mrna components on disease-free interval (DFI) of PTC patients As reported in Fig. 2 (A and B), the results demonstrated that high upa or upar mrna levels are significantly associated with reduced DFI. Because 50 of the 76 patients were at stage I, we used the Kaplan-Meier analysis also for this subgroup and found that overexpression of upa or upar mrna is associated with shorter DFI (Fig. 2, D and E). In addition, the combined overexpression of upa and upar was associated with a reduced DFI both when considering either all patients or only stage I patients (Fig. 2,

4 J Clin Endocrinol Metab, February 2011, 96(2): jcem.endojournals.org 507 FIG. 2. Expression of upas components and DFI in PTC patients. The Kaplan-Meier analysis combined with Mantel-Cox log-rank statistical text was performed either on all patients (n 76; A C) or restricted to stage I patients (n 50; D F). C and F). Finally, no association between PAI-1 mrna level and DFI was observed (data not shown). Discussion Despite the known role of upas components in cancer progression, little information is available on their role in thyroid cancer growth or their prognostic value (1, 15 18). We previously showed that malignant transformation of human thyrocyte is associated with increased mrna and protein expression of upa, upar, and PAI-1 and that upa and upar overexpression was higher in metastatic lymph node PTC than in nonmetastatic disease (15). This prompted us to investigate the prognostic value of upas in a large group of patients affected by PTC. Confirming our own and the previous findings of others (15 18), this study has demonstrated that upa, upar, and PAI-1 gene expression is significantly higher in PTC tissue compared with normal matched tissue and that such increases positively correlate with each other, suggesting the presence of a common molecular mechanism(s) up-regulating their expression. It is worth noting that the study showed that upa mrna increases are associated with advanced TNM stages and that increased tissue levels of both upa and upar are associated with advanced pt and N status and with shorter DFI. Moreover, this association was statistically stronger in stage I patients. These findings may help to establish a more accurate prognosis, make more informed therapeutic decisions, and develop tailored prevention programs, especially for stage I PTC patients, who are considered at low risk of suffering recurrences (10, 11). It is also worth drawing attention to the fact that cdna samples can be used at the same time to analyze the expression of the upas components and to investigate the presence of the V600E BRAF mutation, which is considered a putative prognostic molecular marker in PTC (12 14). Regarding the latter, however, a debate is ongoing about its clinical relevance. In fact, the frequency of BRAF mutations in PTC is high ( 50%), compared with the poor outcomes ( 20%), and as a consequence a large percentage of patients would face the risk of over- or undertreatment based only on the analysis of the BRAF mutation (13, 14). Thus, the combined analysis of the expression of upas components and BRAF status may lead to a more accurate prognosis in these patients. In conclusion, we demonstrated in PTC that the increased gene expression of upa and upar is associated with tumor invasiveness, advanced stages, and shorter DFI, underlining the clinical relevance of their use as prognostic molecular markers, even in TMN stage I patients. Acknowledgments Address all correspondence and requests for reprints to: Professor Salvatore Ulisse, Department of Experimental Medicine, Sapienza University of Rome, Viale del Policlinico, 155, Roma, Italy. salvatore.ulisse@uniroma1.it. This work was supported by Grant PRIN 2007 from the Ministero Italiano dell Università e della Ricerca. Disclosure Summary: The authors have nothing to disclose.

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