Therapeutic targeting Netrin-1-positive invasive bladder cancer cells with oridonin

Background Small compound oridonin acts as an effective anti-tumor agent used for a wide variety of human malignancies, while the antitumor efficacy and molecular mechanism of oridonin against bladder cancer remains unclear. Methods Four independent cohorts of bladder cancer were employed to assess the correlation between netrin-1 expression and progression and prognosis of bladder cancer. Clinical potential of netrin-1 as a biomarker and oridonin-mediated netrin-1-targeting anti-tumor mechanism were investigated by QRT-PCR, Western blot and ELISA. Regulatory mechanisms of Netrin-1 were investigated by luciferase reporter assay and Western blot. The EJ-inoculated nude mice xenograft model was used for the in vivo study. High expression of netrin-1 was significantly correlated with a poor overall survival in three independent bladder cancer cohorts. Urinary netrin-1 level was significantly elevated in bladder cancer patients correlating with tumor invasion and grade. Netrin-1 promoted cell proliferation, tumorsphere-forming, migration and invasion in bladder cancer cells. Oridonin treatment strikingly suppressed these malignant phenotypes and induced cell death in TCC cell lines and murine xenografts. Oridonin markedly decreased netrin-1 through inhibiting NF-κB transcriptional activity and shortening the half-life of NTN1 mRNA via inducing IRE1α , resulting in repression of its downstream signaling. Together, these results strongly suggest that netrin-1 promotes the progression of bladder cancer, and acts as a potential urinary biomarker for the diagnosis as well as the prediction of the tumor progression. Oridonin exerts its strong anti-tumor activity against the invasive bladder cancer by suppressing netrin-1 mRNA production and stability. Cell anti-p65 Cell anti-phospho-Src anti-Src Cell Signaling), anti-phospho-Akt Cell Signaling), anti-Akt Cell Signaling) and anti-actin (1:4000, Sigma) antibodies.


Introduction
Transitional cell carcinoma (TCC) is the most predominant pathologic subtype of bladder cancer, which accounts for ~ 90% of the tumors. The patients with the invasive diseases display a high risk of the progression and commonly the poor prognosis. To date, TP53 mutation [1], p27Kip1 [1,2], surviving [3], EFGRs [4], Integrin-linked kinase [5] and several microRNAs [6] have been identified to be related to the progression and the unfavorable outcome of the muscle-invasive tumors. Owing to the heterogeneity of the property of bladder cancer, the treatment of the invasive and the advanced TCCs remains a clinical challenge.
NTN1 (Netrin-1) is a secreted extracellular matrix protein that plays a fundamental role in axon guidance of the developing brain, through binding to deleted in colorectal cancer (DCC) and UNC5 (UNC5A, UNC5B, UNC5C and UNC5D) receptors [7]. DCC and UNC5 have been considered to be the dependence receptors that transduce the distinct signals, dependent on their availability of netrin-1 [8,9]. Recently, the aberrant expressions of netrin-1 and its receptors DCC and UNC5 have been documented to promote tumor progression and metastasis. Overexpression of netrin-1 have been described in the metastatic breast cancer, non-small-cell lung cancer, aggressive neuroblastoma, pancreatic adenocarcinoma, glioblastoma and colorectal cancers [10][11][12][13][14][15], indicating that netrin-1 might be a potential target for cancer therapy. Nevertheless, the precise role of netrin-1 in the progression of bladder cancer remains to be elucidated.
Oridonin, a cell-permeable diterpenoid compound purified from the herb Rabdosia rubescens, has been shown to possess the wide pharmacological activities against inflammation, bacteria and tumors. Recent studies have demonstrated that oridonin functions as an effective anti-tumor agent in a wide variety of human malignancies [16]. Oridonin-mediated anti-tumor activity was implicated in cell cycle arrest, inhibition of cell growth, induction of senescence, apoptosis and autophagy [16].
However, it has not yet investigated whether oridonin could effectively inhibit the malignant behaviors of bladder cancer cells and also its potential target(s) in bladder cancer is unknown.
In this study, we have addressed the clinical and the functional significance of netrin-1 in TCC of bladder and elucidated the molecular mechanisms behind the anti-tumor activity of oridonin targeting netrin-1against the invasive bladder cancer.

Materials And Methods
Tumor tissue samples Thirty-five freshly-frozen primary bladder tumors were obtained from First Hospital of China Medical University (CMU) with an informed consent. The present study was approved by the Institutional Research Ethics Committee. The clinical parameters of these 35 TCCs were described in Supplemental Table 1. Human TCC tumor tissue array containing 65 primary TCCs and 22 paired normal tissues was provided by Shanghai Biochip (Shanghai, China). The clinical parameters of these samples were summarized in Supplemental Table 2. Two independent open-access datasets of bladder cancer were used: The Cancer Genome Atlas (TCGA) dataset (n = 406) (http://www.oncolnc.org/; https://portal.gdc.cancer.gov) and the TumorBladder-Hoglund-308 dataset (n = 308) in the R2 platform (http://hgserver1.amc.nl/cgi-bin/r2/main.cg).

Cell culture and reagents
Human TCC-derived T24, EJ and J82 cells were cultured in RPMI 1640 medium supplemented with heat-inactivated 10% FBS. The reagents, expression plasmids and siRNAs used in this study were described in detail in Supplementary Materials.

Enzyme-linked immunosorbent assay (ELISA)
Urine samples were preoperatively obtained from 30 primary TCC patients who did not receive any neoadjuvant therapies. Among them, the urine samples were collected again from 10 patients with muscle-invasive tumors 4 weeks after the radical cystectomy. Ten age-and the gender-matched nontumor patients (ureteral calculus: 8; benign prostatic hyperplasia: 2) and 10 normal individuals were recruited as control. ELISA was performed as described in Supplementary Material.

Western blot analysis
For western blot analysis, whole cell lysates were prepared by lysing cells with 1 × SDS sample buffer.

In vivo tumor xenograft study
The 6-week-old female BALB/cA Jcl-nu/nu mice (CLEA Japan) were used in our current studies, which were fed and housed according to the institutional guidelines for laboratory animal research. EJ cells (1.0 × 10 6 cells) in the exponential growth phase were subcutaneously inoculated together with a Matrigel Basement Membrane Matrix (1:1 in dilution, BD Biosciences) into the right flank of the mice.
After tumor volume reached 50 mm 3 , the mice were randomly separated into two groups (6 mice/group) and treated with vehicle or with oridonin (15 mg/kg/day) by the intraperitoneal injection daily for 20 days. Tumor volume was estimated based on the following formula: tumor volume (mm 3 ) = (length × width 2 ) / 2. On day 21, the mice were sacrificed and xenograft tumors were excised and weighted.

Other assays
Quantitative RT-PCR, immunohistochemcial staining, WST-8 cell proliferation assay, flow cytometry analysis, migration/invasion assay, sphere formation assay, luciferase reporter assay, colony formation assay and monodansylcadaverine (MDC) vital staining were conducted as described in Supplementary Material.

Statistical analysis
Statistical analysis was performed using SPSS Statistics ver.19 (IBM). Survival distributions were compared using the log-rank test. Mann-Whitney U Test was used to explore the differences in mRNA expressions of netrin-1 and UNC5 family between the non-invasive and the invasive samples. Cox regression analysis was used for the impact of the several risk factors on survival. Two-tailed Student's t-test was used to explore the differences between the other experiment groups. Statistical significance was declared if P-value was < 0.05. containing 65 primary TCCs and paired normal tissues. Compared to normal adjacent tissues, netrin-1 expression level was significantly higher in TCC tumors ( Fig. 1A-B), and significantly associated with the high tumor stage (Supplementary Table 1), consistent with our previous observations on a cohort of 160 primary TCCs [17]. At mRNA level, netrin-1 expression levels were remarkably higher in the high-grade (Mann-Whitney U Test, low-grade vs. high-grade, P = 0.027, Fig. 1C) and the muscleinvasive subsets (Mann-Whitney U Test, non-invasive vs. invasive, P = 0.035, Fig. 1C) in an independent cohort of 35 primary TCCs, however no big difference in the expression levels of UNC5 family members was observed between 2 subsets in those samples ( Supplementary Fig. 1). Kaplan-Meier survival analysis showed that the high expression level of netrin-1 is significantly correlated to a poor overall survival (OS) (P = 0.009, Fig. 1C).
The correlation of netrin-1 expression to TCC was further validated in 2 independent open access datasets: the TCGA bladder cancer RNA sequencing dataset (n = 406) and the R2 bladder cancer microarray expression dataset (Hoglund-308-custom-ilmnht12v3, n = 308). In accordance with our observations, netrin-1 expression levels were obviously higher in the high-grade as well as the invasive TCCs and predicted a poor OS in both datasets (the TCGA dataset, P = 0.006, Fig. 1D; Hoglund-308-custom-ilmnht12v3, P = 0.012, Fig. 1E). Moreover, the univariate and the multivariate cox regression analyses demonstrated that netrin-1 expression level is independently associated with a poor outcome in our and the TCGA cohorts (Table 1). Further clinical microarray data analysis showed that there exists a clear correlation between netrin-1 and the oncogenic signals such as Wnt/ β-catenin and apoptosis inhibition in human bladder cancer (Fig. 1F,G and Supplementary Fig. 2), indicating that netrin-1 might be a universal biomarker of bladder cancer with the malignant behaviors.
Consistent with the previous observations, netrin-1 expression level was markedly higher in the invasive TCC-derived T24 cells and their highly malignant subline EJ cells [18,19], whereas J82 cells expressed netrin-1 at extremely low level, which were in line with their growth capabilities (Supplementary Fig. 3A-D). ELISA assays further revealed that these TCC cells secret netrin-1 in an autocrine manner, which is in proportion to their growth capabilities ( Supplementary Fig. 3E). In contrast, UNC5A and UNC5D were consistently expressed at various levels, while DCC, UNC5B and Urinary Netrin-1 is a potential diagnostic biomarker of TCC To ask its clinical utility, urinary netrin-1 was measured by ELISA. Compared to normal individuals and the patients with non-tumor urologic diseases, TCC patients showed a significantly higher urinary netrin-1 level ( Fig. 2A), which were further elevated in the patients with the invasive and the highgrade tumors ( Fig. 2B-C). Moreover, urinary netrin-1 concentrations of 10 patients with the invasive TCC remarkably reduced 4 weeks after the operation relative to their preoperative ones (Fig. 2D).
These results indicate the potential of urinary netrin-1 as a diagnostic biomarker of TCC. Notably, netrin-1 overexpression induced the activating phosphorylation of Src and Akt in T24 cells ( Fig. 3K). Similarly, the phosphorylation level of IκBα, a key inhibitor of NF-κB, was also up-regulated ( Fig. 3H), which might trigger its proteolytic degradation and thereby activating NF-κB dimer [20].

Netrin-1 promotes the malignant behaviors of TCC cells
These observations were supported by the results obtained from netrin-1-knockdown cells (Fig. 3L).
Oridonin has a strong antitumor activity against TCC cells expressing Netrin-1 We tested the anti-tumor effectiveness of the nature small compound oridonin on the invasive TCC.
As shown in Fig These results were supported by oridonin-mediated induction of cleavage of caspase-3 as well as poly(ADP-ribose) polymerase (PARP) and the increase in the amount of LC3-II, which act as the molecular markers of apoptosis and autophagy, respectively (Fig. 4D) [21]. In netrin-1 highly expressing T24 and EJ cells, netrin-1 was strongly down-regulated in response to oridonin in a time- Oridonin suppresses Netrin-1 at mRNA level It has been reported that netrin-1 is a direct transcriptional target of NF-κB, whose up-regulation in response to NF-κB activation contributes to the development and the progression of colorectal cancer [12,22]. Indeed, netrin-1 was up-regulated in T24 and J82 cells treated with TNFα, a classic NF-κB inducer, in a time-dependent manner (Fig. 5A). While, TNFα-mediated stimulation of netrin-1 was largely attenuated in the presence of PDTC, a potent NF-κB inhibitor (Fig. 5B). Therefore, it is likely that netrin-1 might be transcriptionally up-regulated through the activation of NF-κB in TCC cells.
Intriguingly, the reporter assay demonstrated that oridonin significantly inhibits TNFα-mediated transactivation activity of NF-κB (Fig. 5C). In addition, p65 subunit-mediated netrin-1 induction was suppressed by oridonin (Fig. 5D) and netrin-1 promoter activity was down-regulated by oridonin (Fig. 5E). These results were consistent with the previous observations showing that the sequencespecific binding of NF-κB to netrin-1 promoter is prohibited in the presence of oridonin [23]. We have also found that the phosphorylation of Src, Akt and IκBα was robustly inhibited in oridonin-treated T24 cells with the high netrin-1 expression but not in oridonin-exposed J82 cells with the extremely low netrin-1 level ( Fig. 5F and Supplementary Fig. 6). These observations indicate that oridonin represses NF-κB-mediated transcriptional up-regulation of netrin-1.
Meanwhile, oridonin treatment sharply shortened the half-life of netrin-1 mRNA from approximately 12.6 hours to 1.4 hours in T24 cells (Fig. 6A), indicating that oridonin promotes a rapid degradation of netrin-1 mRNA. Previous study has shown that, the endoribonuclease,IRE1α,specifically recognizes and efficiently degrades netrin-1 mRNA [24]. To explore the molecular mechanisms behind oridoninmediated degradation of netrin-1 mRNA in TCC cells, we sought to examine the possible involvement of IRE1α in this phenomenon. As expected, knockdown of the endogenous IRE1α increased the amount of netrin-1 protein (Supplementary Fig. 7). Additionally, we have found that oridonin, as an effective inducer of ER-stress [25] induces the expression of IRE1α and thereby reducing the amount of netrin-1 (Fig. 6B). On the other hand, knockdown of IRE1α partially attenuated the inhibitory effect of oridonin on netrin-1 mRNA expression (Fig. 6C). These results suggest that oridonin might reduce the stability of netrin-1 mRNA at least in part by activating RNA hydrolase IRE1α in TCC cells (Fig. 6D).

Discussion
More and more attention has been paid to the role of netrin-1 and its receptors in tumorigenesis and as a possible new target for tumor therapy.In this study, we have provided an evidence that netrin-1 expression levels are tightly linked to the aggressive properties and poor outcome of TCC of the bladder. Analyses of 3 independent TCC cohorts, in which netrin-1 expression level was examined by 3 different approaches, demonstrated that netrin-1 is an independent indicator for the prediction of the tumor progression and poor prognosis of TCC patients. In support of these observations, netrin-1 was highly expressed in TCC-derived cells with the high invasive potential. More importantly, urinary netrin-1 levels were significantly elevated in patients with TCC and positively associated with the tumor invasiveness and grade, indicating that netrin-1 might be a promising non-invasive biomarker for the diagnosis and also for the prediction of tumor progression as well as prognosis of TCC. Indeed, several recent studies have described that the evaluated urinary and serum netrin-1 levels might act as a biomarker for monitoring the tumor progression in medulloblastoma and advanced non-small cell lung cancer [26,27]. Although the diagnostic utility of the urinary netrin-1 in bladder cancer should be further confirmed by much more larger sample size, our present study strongly suggests that netrin-1 might be a potential biomarker and an attractive therapeutic target for the highly malignant bladder cancer. Another important finding of this study is that the natural small compound oridonin has a strong antitumor activity against TCC through down-regulation of netrin-1 mRNA. These results indicate that oridonin markedly reduces netrin-1 by suppressing NF-κB-mediated netrin-1 transactivation and promoting the endoribonuclease IRE1α-dependent netrin-1 mRNA degradation, and thereby prohibiting netrin-1 downstream signaling to trigger apoptotic as well as autophagic cell death (Fig. 6D). Notably, the current main approach to target netrin-1 signaling is to block the interaction between netrin-1 and its receptors by taking advantage of a soluble recombinant DCC ectodomain fragment [10,12,15] or netrin-1-interfering antibody [30, 31], whose effectiveness has been experimentally evaluated in several tumor cells and animal models. It is worth pointing out that our present findings uncover the novel inhibitory pharmacological effect of oridonin on mRNA production and stability of netrin-1.

Conclusions
In summary, our current results provided an evidence that netrin-1 might be used as a biomarker and a potential therapeutic target for the invasive bladder cancer. Additionally, oridonin might be a novel promising therapeutic candidate for the invasive TCC as well as the other tumors with the increased netrin-1 expression.

Ethics approval and consent to participate
This study was approved by ethical committees of the China Medical University. Written informed consent was obtained from all patients and data was analyzed anonymously. All Animal experiments complied with the national guidelines for the care and use of laboratory animals.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.