Urotensin II receptor determines prognosis of bladder cancer regulating cell motility/invasion

A progressive Tissue Micro-Array (TMA), has been constructed using 130 tissue samples (113 tumour and 17 normal tissues), after pathologic re-evaluation according WHO/ISUP 2007[17].

Tumor tissues included 36 NMIBC and 77 MIBC. All tumours and controls have been reviewed by two experienced pathologists (RF, GB). Tissue cylinders were brought into one recipient paraffin block using a semiautomated tissue arrayer (Galileo TMA).

The bladder cancer samples were collected from the National Institute of Tumours of Naples after Internal Ethical Institutional approval in compliance with the Helsinki Declaration

159 patients, undergone to bladder biopsy from 2000 to 2008 at the National Cancer Institute “Fondazione Giovanni Pascale” of Naples have been included in this study. 84 (53%) of 159 patients showed relapses. All cases were reviewed according to WHO classification criteria [17].

Medical records have been reviewed for clinical information, including histologic parameters assessed on standard H&E-stained slides.

Immunohistochemical staining was done on slides from formalin-fixed, paraffin embedded tissues, to evaluate UTR expression. After protein block, slides were incubated with primary anti-UTR antibody followed by secondary antibody (Novocastra Streptavidin-HRP) and then visualized using a 3,3’-diaminobenzidine. Sections were counterstained with hematoxylin and mounted.

Total RNA was isolated from FFPE biopsies of prognostic series whole section, collected in National Cancer Institute “Fondazione G. Pascale” Institutional Bio-Bank, using High pure FFPE RNA Micro Kit (Roche). RNA was subjected to cDNA synthesis using the Ready To Go You-Primer First-Strand Beads kit (Amersham Biosciences) in a reaction mixture containing random hexamers (Applied Biosystems).

Quantitative RT–PCR was performed in a LightCycler system (Roche) using TaqMan® analysis. All reactions were performed in triplicate. The thermal cycling conditions included a step of 20 sec at 95°C followed by 40 cycles of 95°C for 1 sec and 60°C for 20 sec. Comparative C_t method was used to determine human UTR gene variation, using as reference gene TaqMan Endogenous Controls Human ACTB (β-actin) Endogenous Control (RealTime Designer Assay, Roche). We identified a calibrator cell line (LNCaP) that represents the unitary amount of the target, consequently the samples express n-fold mRNA relative to the calibrator.

UTR immunohistochemistry expression was evaluated in bladder cancer TMA including normal, NMIBC and invasive samples. The mean and median tissue UTR expression, expressed as a percentage of immunoreactive cells, was calculated. Kruskal-Wallis test identified differences in median expression values. Selection of the median value as cut-off score was based on evaluation of the distribution of UTR scores. Differences in the number of negative and positive cases were analyzed using a test of equal proportions.

UTR expression was then evaluated on a prognostic series of NMIBC with complete clinical-pathological information.

Association between UTR expression and other molecular and clinical-pathological parameters was calculated using contingency table methods and tested for significance using the Pearson chi-squared test. Univariate and multivariate relative risks have been calculated using the COX proportional hazards regression. All calculations have been performed using the SPSS (Statistical Package for the Social Science rel.13) software (Chicago, IL) and the results have been considered statistically significant when P-value has been ≤ 0.05.

hUII and urantide, the agonist–antagonistic compounds of UII, UPG83, UPG84 and UPG85 were all provided by Prof. P. Grieco[18].

HT1376, MCR, T24 and RT112, cell lines of human bladder cancer, were provided by ATCC. HT1376 and T24 are a grade 3 whereas RT112 is a grade 2 urinary bladder cell line. Cell lines were plated in 96-well plates and one day later were treated with different compounds at concentrations ranging from 10 to 1,000 nM (urantide, UII, UPG83 and UPG85) or with concentrations ranging from 10 to 2,000 nM (UPG84). Cell proliferation was evaluated by MTT assay[19].

Total proteins were prepared as described[19]. Membranes were incubated with the following primary antibodies: (a) anti-UTR; (b) anti- α-tubulin. Bound antibodies were detected by horseradish peroxidase-conjugated secondary antibodies, followed by enhanced chemiluminescence[19].

For determination of cell surface UTR expression, analysis was performed using indirect UTR staining at FACS. We have seeded and treated or not cells with 10 nM urantide or UPG84 for 72 h. After treatment, cells were centrifuged and 4% paraformaldehyde was added for 15 min at 4°C in the dark. Cells were incubated in PBS/BSA for 10 min at 4°C and subsequently with a primary rabbit polyclonal antibody raised against human UTR (GPR14) or with an irrelevant immunoglobulin (IgG1) or in PBS and processed as previously described[19].

For invasion assays, 8 μm inserts (Falcon) were employed and Matrigel TM (Sigma) was diluted in serum-free medium. Subsequently, assays were performed as previously reported[20].

We have evaluated UTR expression on a progressive bladder TMA. We have found a mean expression of UTR of about 16.67% and 13.57% for NMIBC and MIBC, respectively. Percentage of negative cases was significantly higher in MIBC than NMIBC. Pearson chi-squared test showed significant higher UTR expression in NMIBC (p = 0.0001) (Figure 1). These results suggest a higher expression of UTR in NMIBC.

Our series included 125 males and 34 females, 118 (74%) older than 60 years of age (mean age 68, range from 40 to 88 years). The anatomic sites of the tumour were lateral wall in 87 cases (55%), cupola in 13 cases (8%), trigon in 51 cases (32%). Relapses have been recorded in 75 patients (47%), from 1 (57) to 6 (1) total episodes (Table 1).

High-grade primitive tumours have been observed in 41 patients (26%); the relapse tumor grade was the same of primitive tumors in 71 cases (94%), while 4 cases (6%) of the relapsed tumours showed higher grade than the primary. In 53 cases (33%) lamina propria infiltration was observed (Table 1).High expression was recorded when positive cells were > 30%. Patients with low expression of UTR were 94 (59%), in particular 38 (40%) without recurrences and 56 (60%) with histologically documented recurrences. Moreover, 65 patients (41%) showed high expression of UTR, 46 (71%) without recurrences and 19 (29%) with recurrences. High UTR expression was significantly associated to low grade carcinoma (p = 0.044) while low expression was associated to cases that have developed at least one relapse (p = 0.001). Moreover, UTR expression in relapses was significantly lost respect to primary tumors (p = 0.025) (Figures 2 and3).

The decrease of the correlation between high UTR expression and absence of relapse was likely due to the short time of observation that could explain the presence of some patients who will develop the relapse in the following observation time (likely expressing low UTR levels).

Considering only first relapse, we recorded a significant association between low UTR expression and shorter time to relapse (p = 0.004). As expected tumor grade was associated to recurrence (p = 0.02) (Figure 4). Also considering low grade carcinoma group, shorter DFS was associated to UTR low expression (p = 0.01) (Figure 4).

In a multivariate analysis including UTR expression and grade, only UTR appeared to be an independent prognostic factor (p = 0.025).

UTR gene expression was evaluated on LNCaP cell line and 14 NMIBC and 3 invasive MIBC samples by Real-Time PCR quantification.In LNCaP, UTR expression was moderate. In 4 NMIBC samples the expression was low while in 3 samples the increase of expression was moderate. The most part of NMIBC showed a significant increase in UTR mRNA expression (Figure 5). UTR gene expression was very low in all invasive bTCC selected (Figure 5). Moreover, UTR mRNA levels correlated with the expression of the protein in all examined cancer samples.

In order to evaluate the involvement of UTR-dependent signaling pathway on the growth of bladder cancer cells, biological effects of human agonists (UII and UPG84), and antagonists (urantide, UPG83 and UPG85), were evaluated on proliferation of human bladder cancer cell lines MCR, RT112, T24 and HT1376 (Figure 6) after 72 h of treatment. UII had no significant effects on the proliferation of all cell lines (Figure 7A-D). The 50% growth inhibitory concentration (IC:50) of urantide was 350 nM in T24 and 375 nM in RT112 while it was not achieved in HT1376 and MCR cells (Table 2). UPG84 induced 45-50% growth inhibition at a concentration close to the supposed Kd of UTR (about 10 nM) in all cell lines with the exception of RT112 cells that were almost insensitive (Figure 7E). Interestingly, UPG84 is a superagonist of UTR (Additional file1: Table S1) and the addition to cell culture for 72 h could affect UTR expression on cell surface for internalization process triggering. On this light, we have found that treatment of these cells with UPG84 induced a time-dependent down regulation of UTR expression that reached an about 50% and 60% decrease at 72 h from the beginning of the treatment on HT-1376 and T24 cells, respectively (Figure 7F). On the other hand, urantide induced 30 and 20% decrease of UTR expression in HT-1376 and T24 cells, respectively (Figure 7F).

In order to explore the specific contribution of UTR in regulation of motility and invasion of bladder cancer cells, T24 and RT112 cells were treated with urantide (100 nM) for 48 h and/or were transiently transfected with shRNA for UTR to down-regulate UTR protein expression. Cells were seeded in transwell chambers and allowed to migrate and invade in absence or presence of urantide. After 48 h, urantide induced an about 35% and 50% reduction of cell motility and invasion, respectively, in T24 cells if compared to untreated cells. When T24 cells were transfected with shUTR displayed 45% and 56% inhibition of their ability to migrate and invade, respectively (Figure 8A and B, respectively). Downregulation of UTR in T24 treated with urantide did not increase the effect induced by urantide alone (Figure 8A and B, respectively). Similar results were also obtained in RT112 (Figure 9A and B). These data demonstrated that UTR is involved in both motility and invasion of human bladder cancer cells.