Overexpression and Clinicopathological Contribution of DcR3 in Bladder Urothelial Carcinoma Tissues

Bladder urothelial carcinoma (BUC) is the fifth most common cancer worldwide, with an estimated incidence of 73, 510 cases and 14, 880 deaths in the United States in 2012. In China, the morbidity of urothelial carcinoma is 6.61/100,000, ranked the ninth in all malignant tumors (O’Keeffe et al., 2008; Yang et al., 2011; Siegel et al., 2012; Bracha et al., 2014). The decoy receptor 3 (DcR3), locating on chromosome 20q13, is a member of the tumor necrosis factor receptor (TNFR) superfamily. Previously, we have reported the overexpression of DcR3 mRNA and protein in sera or tissues of several human malignancies, including hepatocellular carcinoma, gastric carcinoma and glioma (Chen and Luo, 2008a; 2008b; Chen et al., 2010; Huang et al., 2014). DcR3 has been considered as an oncogene for the aforementioned malignancies. So far, there has been only one report studying the relationship between DcR3 and urothelial carcinoma (UC). Yamana et al. (2005) investigated the amplification and protein expression of DcR3 in the tissues and cell lines of UC by using real-time quantitative PCR and immunohistochemistry in a small number of patients. However, no study has been performed to explore the relationship between DcR3 and progression of UC patients. Furthermore, the role of DcR3 on BUC has not been fully clarified. Thus, the aim of the current study was to explore


Introduction
Bladder urothelial carcinoma (BUC) is the fifth most common cancer worldwide, with an estimated incidence of 73, 510 cases and 14, 880 deaths in the United States in 2012.In China, the morbidity of urothelial carcinoma is 6.61/100,000, ranked the ninth in all malignant tumors (O'Keeffe et al., 2008;Yang et al., 2011;Siegel et al., 2012;Bracha et al., 2014).The decoy receptor 3 (DcR3), locating on chromosome 20q13, is a member of the tumor necrosis factor receptor (TNFR) superfamily.Previously, we have reported the overexpression of DcR3 mRNA and protein in sera or tissues of several human malignancies, including hepatocellular carcinoma, gastric carcinoma and glioma (Chen and Luo, 2008a;2008b;Chen et al., 2010;Huang et al., 2014).DcR3 has been considered as an oncogene for the aforementioned malignancies.So far, there has been only one report studying the relationship between DcR3 and urothelial carcinoma (UC).Yamana et al. (2005) investigated the amplification and protein expression of DcR3 in the tissues and cell lines of UC by using real-time quantitative PCR and immunohistochemistry in a small number of patients.
However, no study has been performed to explore the relationship between DcR3 and progression of UC patients.Furthermore, the role of DcR3 on BUC has not been fully clarified.Thus, the aim of the current study was to explore

Patients population and histologic analysis
We studied 166 consecutive patients with BUC who had undergone partial or complete cystectomy and 56 cases of normal bladder from autopsies.The FFPE tissues were made into 2 tissue microarrays (TMAs).All cases were diagnosed at the First Affiliated Hospital, Guangxi Medical University, China, from January 2003 to October 2006.The age of BUC patients ranged from 27 to 96 years (mean age: 61.62 years) and the male/female ratio was 141/25.The age of normal bladder samples ranged from 44 to 66 years (mean age: 52.43) and the male/female ratio was 46/10.The histological sections were processed from tissues fixed in 10% formalin with standard methods and stained using hematoxylin and eosin (H&E).H&Estained slides were used to evaluate histological grade as low or high grade (Jankovic Velickovic et al., 2009;2011) for non-invasive BUCs, pathologic stage (pT) (Jankovic Velickovic et al., 2009;2011), growth pattern of tumor (papillary/solid) for all BUCs.The invasive status of the tumors was divided into two stages: low-stage non-muscle invasive (pTa-pT1) and high-stage muscle invasive (pT2-pT4) tumors (Jankovic Velickovic et al., 2009;2011).The status of lymph node metastasis, distant metastasis, tumor number and tumor size were also collected (Table 1).None of the BUC patients had received prior chemotherapy, intravesical instillation or radiation therapy.Of all 166 patients, 85 were followed up to 6~40 months.Till the end of follow-up, 19 cases had recurrence and 12 candidates were dead.The study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Guangxi Medical University.Written informed consent was obtained from the patients and clinicians for the usage of the samples for research.

Cell culture
Three human BUC cell lines (T24, HT1376 and RT4) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).All cell lines were cultured in Dulbecco's modified essential medium (DMEM, Invitrogen Corp., Grand Island, NY, USA) and supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Corp., Grand Island, NY, USA), 2mM glutamine, and gentamicin at 37℃ in a humidified incubator with 5% CO 2 .Experiments were performed in triplicate.

Preparation of primary BUC cell cultures
Preparation of primary BUC cell cultures was performed as previously reported (Huang et al., 2014).Briefly, human BUC cells were achieved from 8 patients who underwent partial or complete cystectomy.
After surgery, the tissues were placed immediately in Petri dishes, minced mechanically, and digested enzymatically using collagenase (1h, 37℃).Next, the dissociated cells were filtered through 100mm cell strainers to remove cell debris.The BUC cells were centrifuged and washed with hypotonic water for the lysis of erythrocytes.Subsequently, the BUC cells were washed and re-suspended in full medium of DMEM.Conditioned medium was harvested after less than 5 passages.The diagnosis of BUC was confirmed by pathology.

Western blotting
BUC cells (5×10 6 ) were cultured in serum-free DMEM for 24h.The supernatants were collected and then concentrated by centrifugal filter devices.
Cell lysis sample from known DcR3 positive hepatocellular carcinoma cell line HepG2 was used as a positive control for western blot as previously reported (Huang et al., 2014).

Statistical analysis
SPSS20.0 (Munich, Germany) was used for statistical analysis.Mann-Whitney U test was employed to assess the difference of DcR3 expression between bladder tumor and non-cancer tissues, as well as in corresponding groups of each clinicopathological parameter.Values were presented as the mean ± standard deviation (SD) for Ki-67, PCNA LIs and P53 signals.
The Spearman Test was used to reveal the relationship between the expression of DcR3 and other markers (caspase-3, Bcl-2, VEGF, Ki-67, PCNA and P53).Kaplan-Meier and log-rank test were performed for the survival analysis.Statistical significance was determined at a p<0.05 level.

Relationship between DcR3 expression and survival
In the 85 patients with full follow-up information, the survival of the DcR3 negative group (n=41) was 35.57±1.72 months, longer than that of the DcR3 positive group (n=44, 24.52±1.163months, Figure 3A).However, the difference showed no significance (p=0.07).In the subgroup of invasive BUC (n=59), the survival presented similar trend as above.The survival of the DcR3 negative group (n=25) was 34.43±2.05months, longer than that of the DcR3 positive group (n=34, 23.76±1.369months, Figure 3B).However, the difference showed either no significance (p=0.152).

Expression of DcR3 in the supernatants of BUC cells
DcR3 protein was expressed in the supernatants from all 3 cell lines (T24, HT1376 and RT4) tested, without significantly difference between each other (Figure 4).BUC cells were also freshly prepared from 8 BUC specimens and the supernatants were collected before the 5th passage.Soluble DcR3 could be detected in all samples tested by using western blot (Figure 4).

Discussion
In the present study, we established 2 TMAs of BUC and normal bladder epithelial tissues.Then we analyzed the expression of DcR3 and studied its correlation with clinicopathological parameters and various markers, which play vital roles in cell proliferation, apoptosis and angiogenesis.
DcR3 is a member of the tumor necrosis factor receptor superfamily, which locates on the chromosome 20q13.3.The expression of DcR3 protein was detected in various types of malignant tumors by our and other groups, especially in glioma, gastric, breast, liver and pancreatic cancer (Chen and Luo, 2008a;2008b;Chen et al., 2010;Huang et al., 2014;Wu et al., 2014;Zhou et al., 2014).DcR3 is also closely related to the occurrence, development and prognosis of various malignant tumors (Yang et al., 2010;Toda et al., 2013;Zhou et al., 2013;Wu et al., 2014;Zhou et al., 2014).Moreover, it is absent or lowly expressed in normal tissues except cancerization.However, the role of DcR3 in bladder cancer has not yet been totally clarified.By far, only one research group has reported the relationship between DcR3 expression and urothelial cancers, including bladder, ureteral and renal pelvic cancer (Yamana et al., 2005).By using real-time quantitative PCR and immunohistochemistry, Yamana, et al. (2005) detected the amplification and expression of DcR3 in the tissues and cell lines of urothelial carcinoma (UC) and found a visible gene amplification in one UC cell line (T24) and five cases UC tissues (25%, 5/20).DcR3 expression was detected in UC (90.0%) and normal urothelium (80.7%) with immunohistochemical detection.This was inconsistent with the result from the current study.We found that the positive ratio of DcR3 expression was 36.7%, much lower than what was detected by Yamana et al. (2005).The difference of sample sources, sample sizes and detecting methods may partially lead to the discrepancy.However, even more noticeably low expression of DcR3 was found in the normal bladder epithelial tissues, indicating that the DcR3 protein expression increased in BUC compared to the normal tissues, which is similar to other malignancies.Furthermore, DcR3 protein could be detected in 3 BUC cell lines by using western blot (data not shown).Therefore, DcR3 might act as a proto-oncogene being involved in the process of BUC.DcR3 is a secretory protein, since the amino acid sequence of DcR3 protein lacks of membrane structure.The overexpression of serum DcR3 has also been detected in various types of body fluid of cancers and showed great potential to become a biomarker for the early non-invasive diagnosis for malignant tumors.In the current study, we found the expression of DcR3 in the supernatants of different BUC cell lines, as well as the freshly cultured BUC cells, which indicates the soluble characteristic of DcR3.However, the expression and clinical significance of serum DcR3 in BUC have never been investigated.Further work is warranted.
To explore the relationship between DcR3 expression and clinicopathological parameters in UC, Yamana et al. (2005) found no significant correlation between the expression of DcR3 protein and differentiation, stage or prognosis of UC.Distinct results were found in the current study.DcR3 expression was closely related to muscular invasion, lymph node metastasis and recurrence, which implies that BUC with higher DcR3 expression would progress faster and cause a poorer prognosis.Moreover, we, for the first time, have investigated the role of DcR3   doi.org/10.7314/APJCP.2014.15.21.9137Overexpression and Clinicopathological Contribution of DcR3 in Bladder Urothelial Carcinoma Tissues on patient survival.The average survival time in the DcR3 positive group was shorter than in the DcR3 negative group, even it had no significant value, it still suggests that the DcR3 expression could possess the potential to become an indicator of prognosis for BUC.However, a larger cohort with more patients is required to support the current finding.
DcR3 can block LIGHT, FasL and TL1A mediated cell apoptosis and proliferation (Aiba et al., 2013;Fukuda et al., 2013;Toda et al., 2013;Zhou et al., 2013).To further investigate the potential mechanism of DcR3 to influence the biological function of BUC, we compared the DcR3 expression and other common biomarkers, including caspase-3, Bcl-2, VEGF, Ki-67 and PCNA labeling indexes (LIs) and P53.Among these biomarkers, sequential activation of caspase-3 plays a central role in the execution-phase of cell apoptosis (Cheng et al., 2013).The Bcl-2 gene is a proto-oncogene, and it can inhibit the tumor cells apoptosis (Jeon and Yoon, 2012;Kouri et al., 2012;Song et al., 2014).VEGF is a signal protein that stimulates vasculogenesis and angiogenesis (Chuangsuwanich et al., 2014;Nakamura et al., 2014).Ki-67 and PCNA are considered as the classical markers of cell proliferation and are in routine used by pathologists in diagnosis of some malignancies.P53 is the most prominent tumor suppressor, and many types of cancers result from functionally impaired P53 that loses its capability in DNA repairing and cell apoptosis induction (Shin and Kim, 2014;Song et al., 2014).In the current study, we found an inverse correlation between the expressions of DcR3 and caspase-3, confirming that higher DcR3 can block the pathway of apoptosis.This is also supported by the positive relationships between DcR3 and Bcl-2, P53.Additionally, DcR3 also leads to faster cell growth due to the positive correlation between DcR3 expression and Ki-67, PCNA LIs.More interestingly, we also found that DcR3 was related to the expression of VEGF, suggesting a role of DcR3 in prompting the angiogenesis of BUC.The relationship between DcR3 and these aforementioned biomarkers helps to explain its overexpression in the clinical BUC samples.DcR3 could influence the outcome of BUC patients via the related pathways of inhibiting apoptosis, promoting proliferation and inducing angiogenesis.
In conclusions, The current findings suggest the role of DcR3 as an oncogene in the process of carcinogenesis and deterioration of human BUC.The detection of DcR3 protein in the FFPE samples could assist to predict the progression of BUC patients.However, a larger cohort is needed to verify the evidence.DcR3 might influence the related pathways of apoptosis, proliferation and angiogenesis, thus to impact patients outcome.Further in vitro and in vivo functional experiments are warranted to investigate the mechanism of DcR3 in the malignant phenotype of BUC.