Tenascin-C promotes bladder cancer progression, depends on syndecan-4 and involves NF-κB signaling activation

Bladder cancer (BCa) is an unfortunately critical genitourinary tract disease with an uncertain pathology. Increasing evidence indicates that the tumor microenvironment is decisive with respect to cancer progression, and that this is driven by tumor cell interactions with stromal components. Tenascin-C (TN-C) is an important extracellular matrix (ECM) component and TN-C has been reported to be involved in other cancers, i.e. breast cancer. Expression of TN-C in BCa tissue is reported to positively correlate to BCa pathologic grade, yet the presence of urine TN-C is regarded as an independent risk factor for BCa. Thus, we assessed the value of TN-C in BCa tissues and noted that it also was increased according to tumor grade and was an independent risk factor for BCa. In fact, TN-C contributes to BCa cell migration, invasion and proliferation and this is dependent on syndecan-4 and involves NF-κB signaling activation. How syndecan-4 is linked to activation of NF-κB signaling is unclear. Our data provide a foundation for future investigations into TN-C’s contribution to BCa progression. IHC was performed with a Dako Autostainer Plus system (Dako Corporation, Carpinteria, CA). Tissues were de-paranized, rehydrated and subjected to 5-min pressure-cooking antigen retrieval, 15-min endogenous enzyme block, 60-min primary antibody (1:300) incubation and 30-min DakoCytomation EnVision-HRP reagent incubation with rabbit antibodies (1:200). Signals were measured according to substrate hydrogen peroxide using DAB as a chromogen followed by hematoxylin counterstaining. Negative controls were prepared by omitting primary antibody. Stained (brown) cells were quantied by counting the positive cells X 100/total cells in 10 random microscopic (400X) elds in each slice.


Introduction
Bladder cancer (BCa) accounts for 90-95% of urothelial carcinomas and is the most common urinary tract malignancy 1 . Almost 80% BCas initially are diagnosed as non-muscle invasive BCa (NMIBC) which have better prognosis, but some of these tumors will progress to muscle invasive BCa (MIBC). Even with surgical interventions, 30% of BCas become invasive 2 and worsen patient prognosis 3 . The remaining 20% of BCas are MIBC at initial diagnosis and have less favorable prognosis-5% of patients have metastatic BCa 4 . Complete resection of all tumor tissue by transurethral bladder tumor resection (TURBT) is recommended for NMIBC, followed by chemotherapeutic instillation 5 . However, for special types of BCa, such as T 1 G 3 or Carcinoma in situ (CIS), special treatments are available. Radical cystectomy (RC) with extended lymphadenectomy is considered the standard treatment for MIBC 6 , followed by cisplatin-based adjuvant chemotherapy. Two different pathological pathways 7,8 are thought to contribute to MIBC and this is responsible for different prognoses between initially diagnosed MIBC and MIBC that is derived from NMBIC 9 . Therefore, understanding how BCa progression occurs is needed to establish better BCa therapy.
Cancer cell interplay with stromal components, such as broblasts, macrophages, bronectin, and the initiation of brosis is thought to be needed for tumor recurrence, drug-resistance, and poor prognosis 12,[14][15][16] . A vital component of the extracellular matrix (ECM) in tumor cells, tenascin-C (TN-C) may have multifaceted and complicated roles in tumor progression.
TN-C is large (~300 kDa) as an intact monomer and ~1800 kDa when assembled as a hexamer 17 . After an initial identi cation in gliomas in 1983, TN-C has since been noted to appear in head and neck squamous cell carcinoma, breast cancer 18 , prostate 19 , thyroid 20 , and pancreatic cancers 21 , melanomas, gastric cancer 17 , and osteosarcomas 22 , and in most of these cancers, TN-C is considered to be a tumor promoter that worsens prognosis.
Four syndecan family members are found in mammals, and of these three (syndecan-1, 2 and 3) have a restricted tissue distribution. Syndecan-4 is expressed ubiquitously and is a member of the membraneintercalated proteoglycans 28,35 . Binding to bronectin within two independent sites with syndecan-4 and α5β1 is key to homeostasis of normal tissue 36 , and involves activation of downstream signals related to cytoskeletal organization and cell proliferation. TN-C is reported to compete with the binding site of bronectin with syndecan-4 and this interaction with syndecan-4 partially destroys the effects of this coreceptor, as well as attenuates the interaction of syndecan-4 with bronectin, enhancing tumor cell malignancy. This process also includes FAK and Rho signaling 36 .
Activation of NF-κB signaling, manifested by the nuclear translocation of P65 37,38 was demonstrated with IHC staining in BCa tissues and this is reported to positively correlate to tumor progression. EMT progression is another aspect of this signaling 39,40 , promoting tumor malignancy. Previous work suggests that TN-C is crucial to cancer progression 17 and urinary TN-C may be a useful biomarker of BCa progression 41-43 . To assess this idea, we used IHC to quantify TN-C expression in BCa tissues and noted that it was elevated with worsening tumor grade and was associated with shorter survival. Thus, TN-C expression is an independent risk factor for BCa patients. Additional studies suggest that TN-C promotes BCa cell line migration, invasion and proliferation via NF-κB signal activation that depends on syndecan-4.

Results
TN-C expression in BCa tissue increases with tumor grade and is an independent risk factor for BCa TN-C expression in tumor, such as breast cancer, is negatively associated with survival and is an independent risk factor 44 , but this relationship is uncon rmed in other tumors 17 . We noted that urinary TN-C is an independent risk factor for BCa in the presence of other limited conditions (such as exclusion of in ammation) and these data agree with published studies 41 . Whether TN-C expression in BCa tissues in local Chinese patients is critical is uncertain although its expression has been noted in BCa patients of other regions 45 . To understand this signi cance, we measured TN-C expression in BCa tissues from Chinese patients. Figures 1 A, B show that TN-C expression across different grades of BCa tissue differs and increases with severity of tumor grade. In addition, we observed that BCa tumor grade was an independent risk factor for BCa patients (Fig. 1 C). To understand TN-C expression and patient survival, patients were strati ed as high-or low-TN-C expressers. Figure 1 D indicates that TN-C expression exceeding the mean suggests poor survival and these data agree with published information 46 . Finally, a correlation analysis suggested that TN-C expression in BCa tissue is negatively correlated to tumor-free survival (Fig 1 E).

Preparation of stable high-and low-TN-C expressing cell lines
Previous results indicate that TN-C expression may drive BCa progression, so we measured TN-C in BCa cell lines from different tumor grades. We know that TN-C is secreted into the ECM and we measured TN-C in cell media to monitor this secretion. Data indicate diverse expression of TN-C in BCa cell lines (Fig. 2  A, B), and that T24 and J82 had higher TN-C expression and 5637 and 253J had lower TN-C expression.
ELISA data agreed as did Western blot and real time PCR results, suggesting that TN-C may function as a secreted protein (Fig. 2 C). Research suggests a complicated role for TN-C as an ECM component, but our TN-C staining data from BCa tissues indicates that ECM deposition of TN-C occurs beyond the cytoplasm. Thus, tumor cells exposed to exogenous TN-C may be modi ed. We added human TN-C peptide to media of TN-C-negative cell lines (T24 siTN-C , J82 siTN-C , 5637 Vec , and 253J Vec ) as well as added human TN-C neutralizing antibody to TN-C-positive cell lines (T24 Sc , J82 Sc , 5637 TN-C , and 253J TN-C ) and a Boyden chamber assay, a wound healing study and BrdU incorporation was assessed to measure malignancy.
Exogenous TN-C (Ex TN-C, Ex) enhanced migration, invasion, and proliferation of 5637 Vec (Fig. 4 A TN-C contributes to elevated expression of EMT-related markers and expression of MMP 2 /MMP 9 by activating NF-κB signaling Our data show that TN-C promotes BCa cell line migration, invasion, and proliferation, but how this happens is uncertain. TN-C may execute this function as a component of the ECM, at least partially. In transitional cell carcinoma, enhanced migration and invasion of tumor cells is often accompanied with epithelial to mesenchymal transition (EMT) 48 , and this can be used to monitor malignant behavior of BCa cells. Activation of NF-κB has been causally linked to an invasive phenotype and can directly or indirectly induce expression of Snail, Slug, Twist, Zeb1, and Zeb2 49 , all of which are markers of EMT. Thus, we hypothesize that TN-C-induced effects may involve NF-κB signaling. Data from Western blot and real time PCR to monitor expression of EMT-related markers, and immuno uorescent staining to quantify NF-κB signaling con rmed data from the Boyden chamber assay and wound healing studies. Also, knock down data for TN-C with T24 cells con rmed decreased expression of MMP 2 /MMP 9 , vimentin, N-cadherin and Snail, and this was accompanied by elevated expression of E-cadherin ( Fig. 5 A, B, T24 Sc -Con Vs T24 siTN-C -Con), which is a reversal of EMT. Also, TN-C induced activation of NF-κB signaling. P65 is the functional subunit of the NF-κB dimer (P65/P50), and nuclear translocation of this subunit is thought to activate this signaling. Fig. 5 C shows that TN-C induces nuclear translocation in T24 siTN-C Vs T24 Sc cells and 5637 Vec Vs 5637 TN-C cells, just like T24 siTN-C -Con Vs T24 siTN-C -Ex and 5637 Vec -Con Vs 5637 Vec -Ex. However, nuclear translocation is inhibited by TN-C functional inhibition (Fig. 5 C, T24 Sc -Con Vs T24 Sc -Anti, 5637 TN-C -Con Vs 5637 TN-C -Anti).
TN-C induced activation of NF-κB signaling depends on syndecan-4 TN-C chie y functions as a component of ECM, indicating an interaction between TN-C and tumor cells and syndecan-4 is reported to be the receptor involved in those interactions 50 . Brie y, syndecan-4 is regarded as a co-receptor of syndecan-4/α5β1, which is important to cell adhesion. Interference with this co-receptor causes tumor cell proliferation and metastases, so TN-C functions may depend on this membrane receptor in BCa cell line. To assess this, we measured expression of syndecan-4 in all BCa cell lines and Fig. 6 A shows that signi cant syndecan-4 expression was observed in all lines. There was no apparent difference among cell lines. Thus, T24 and 5637 cells were selected to represent TN-C-positive and negative-cell lines, respectively and we studied the role of syndecan-4 by knocking down its expression with siRNA.
Two TN-C related stable cell clones, T24 Sc/siTN-C and 5637 Vec/TN-C cells were also treated to knock down syndecan-4 expression and study the effect of TN-C on that expression. Data show that syndecan-4 expression was knocked down in both cell lines but knocking down of TN-C did not change syndecan-4 expression (Fig. 6 B).
To determine whether syndecan-4 knock down in both cell lines could modify migration and invasion, a Boyden chamber assay was employed and syndecan-4 knock down was noted to decrease migration and invasion in both cell lines. Effects of overexpression of TN-C (in 5637 cells) or the exogenous TN-C addition (in T24 siTN-C and 5637 Vec cells) were attenuated (Fig. 6 C).
Also, syndecan-4 interference inhibits P65 nuclear translocation, blocking signal activation as shown by immuno uorescent staining (Fig. 6 D). Effects of overexpression of TN-C (in 5637 cells) or addition of Ex TN-C (in T24 siTN-C and 5637 Vec cells) were blocked as well. Thus, TN-C enhances cancer cell line migration, invasion and proliferation, as well as activation of NF-κB signaling and this depends on syndecan-4.

Discussion
High recurrence of BCa is likely attributed to interactions of tumor cells with the surrounding microenvironment to drive progression, metastasis and drug resistance. Macrophages from prostate cancer tissue can induce cancer phenotypes of normal prostate epithelial cells when co-cultured 51 . Also, broblasts, in ammatory cells, and the ECM have vital roles in cancer, among which, TN-C is the least understood.
TN-C is reported to be important to embryogenesis, in ammation, and wound healing, and behaves in a similar manner in tumorigenesis. TN-C expression in cancerous tissue has been documented in various tumors and is regarded to be an independent risk factor for cancer patients. Consistent with the literature, TN-C expression data for BCa tissue in our study suggests a positive role in BCa progression, but TN-C expression across different BCa cell lines is diverse and does not correlate with tumor grade. For example, in 5637 cells (ATCC ® HTB-9™, www.atcc.org) TN-C expression is not observed in grade II BCa, this may be explained by different sources of TN-C. In BCa cell lines cancer is the sole TN-C source, but tumor cell secretions or mesenchymal cells can also produce TN-C.
Previous reports suggest a vital role of TN-C in tumor progression and TN-C content in BCa cell lines is consistent with tumor cell TN-C expression. (Fig. 2 A, C). Modifying TN-C expression in BCa cell lines causes the same effect to TN-C concentration in the corresponding BCa cell lines (Fig. 2 D, F). Thus, secreted TN-C may be a primary source of TN-C tumor activity. TN-C neutralizing antibody reduces TN-C overexpression, as does human TN-C peptide (Fig. 4). Thus, in BCa cell lines, TN-C executes its role mainly as a component of ECM, perhaps by binding with membrane receptors. Investigations have con rmed that the co-receptor of syndecan-4/α5β1 is important to tumor cell adhesion to bronectin of ECM, and that interfering with this by TN-C decreases tumor cell adhesion and enhances metastasis and proliferation. Syndecan-4 is the sole syndecan family member that is ubiquitously expressed in the cell membrane. Many downstream signals of syndecan-4 are known, including PKCα, PKCδ, PI3K/Akt, and synectin 29,52 . Previous reports suggest that interference with syndecan-4/α5β1 co-receptor in the cell membrane inhibits normal cell proliferation and enhances tumor cell proliferation, but why this happens is not certain 26,27 .
We found that knocking down syndecan-4 expression attenuates TN-C-induced tumor migration, invasion and proliferation, suggesting that TN-C contributes to metastasis and proliferation in a manner that depends on syndecan-4. However, how TN-C binds to membrane syndecan-4 and how this is connected to metastasis and proliferation is not known. Forced expression of TN-C or in the presence of Ex TN-C upregulates mesenchymal markers (elevated expression of vimentin, Snail, N-cadherin, MMP 2 /MMP 9 ) and decreases expression of E-cadherin (Fig. 5 A, B) in BCa cell lines, indicating that alternative expression of these genes may be related to the activation of NF-κB signaling.
We offer evidence that in BCa cell lines, activation of the NF-κB signal leads to EMT, manifested as previously depicted 53 . Immuno uorescent staining con rmed binding of TN-C, either by forced expression or exogenous TN-C, with syndecan-4 induces nuclear translocation of P65, a process that can be inhibited by syndecan-4 knock down (Fig. 6 D). Thus, binding of TN-C with syndecan-4 induces NF-κB signal activation and promotes tumor cell metastasis and proliferation.
Our present work is summarized in Figure 7. NF-κB is the downstream of the PI3K/Akt pathways, suggesting that TN-C binding with syndecan-4 may induced activation of the NF-κB signal is involved in PI3K/Akt pathway activation via the cytoplasmic domain of syndecan-4. Whether binding of TN-C with syndecan-4 involves the co-receptor α5β1 is unknown, but we suggest that TN-C promotes tumor cell metastasis and proliferation and this depends on syndecan-4. These data offer a solid foundation for future studies into the role of TN-C in BCa progression and may be a potential therapeutic target for treating BCa.

Materials And Methods
Tissue preparation and patient follow up BCa tissue samples (N = 57) were obtained from the Department of Urology, at the First A liated Hospital of Xi'an Jiaotong University (32 males; age range 39-78 years-of-age; mean 63.7 ± 7.5 years). Pathological grading was monitored by three independent hospital pathologists and 15, 18, and 24 samples of grade I-III were noted, and all were transitional cell carcinomas. Samples were xed in 4% formalin and para n-embedded.
To assess any TN-C expression and tumor grade correlation, TN-C and survival time was assessed and patients who gave the tumor samples were contacted by telephone. Survival was a normal distribution as demonstrated by Shapiro-Wilk test. This study was approved by the Ethics Committee of Xi'an Jiaotong University.

Immunohistochemical (IHC) staining of TN-C in BCa tissues
IHC was performed with a Dako Autostainer Plus system (Dako Corporation, Carpinteria, CA). Tissues were de-para nized, rehydrated and subjected to 5-min pressure-cooking antigen retrieval, 15-min endogenous enzyme block, 60-min primary antibody (1:300) incubation and 30-min DakoCytomation EnVision-HRP reagent incubation with rabbit antibodies (1:200). Signals were measured according to substrate hydrogen peroxide using DAB as a chromogen followed by hematoxylin counterstaining. Negative controls were prepared by omitting primary antibody. Stained (brown) cells were quanti ed by counting the positive cells X 100/total cells in 10 random microscopic (400X) elds in each slice.

Real-time PCR
Total RNA was isolated from frozen tissues and cell lines using Trizol reagent (Invitrogen) and quanti ed by reading the absorbance at 260 nm. RNA (2 μg) was reverse transcribed using Revert Aid TM First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer's protocol.
For real-time PCR, we used the SYBR R Premix Ex Taq TM II system (TaKaRa Biotechnology Co., Ltd, Dalian, China) and a Bio-Rad CFX96 TM Real-time system (Bio-Rad, city, CA). Then, 12.5 μl SYBR R Premix Ex Taq TM II, 1 μl primer (10 μM, primers; Table 1), 200 ng cDNA and 9.5 μl double de-ionized water were mixed. Then, pre-degeneration was conducted at 95 °C, 30 sec, for one repeat, and PCR was carried out at 95 °C for 5 sec followed by a 60 °C incubation for 30 sec, and 35 repeats. Next, dissociation was carried out at 95 °C for 15 sec followed by a 60 °C incubation for 30 sec, and another 95 °C incubation for 15 sec and data collection. GAPDH was a loading control.

Western blot
Cells are harvested when 80% con uent and washed with cold PBS three times. Total cellular protein lysates were prepared with RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate] containing proteinase inhibitors (1% Cocktail and 1 mM PMSF, Sigma, St Louis, MO). Protein (30 μg) was separated with 6% (for TN-C) or 10% (for others) SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h with 5% skim milk in Tris-buffered saline without Tween 20 (pH 7.6, TBS). Polyclonal antibody TN-C was applied (1:300 dilution; Table 2) with 5% skim milk in TBS at 4 °C overnight, followed by washing with TBST (with Tween 20, pH 7.6), and membranes were incubated with secondary antibodies (Licor, Rockford, IL) coupled to the rst antibody at room temperature in the dark for 1 h. Then, membranes were washed in the dark room, dried with neutral absorbent paper, and scanned using an Odyssey detection system (Licor).
GAPDH was a loading control.
Boyden chamber assay and wound healing assay Migration and invasion were tested with a Boyden chamber assay (Millipore, city, Switzerland). For the migration assay, 0. Wound healing was assessed by scratching 6-well dishes with a 10 μl pipette tip when cells were 80% con uent. Scratch widths were compared at 0, 12, and 36 h.

Preparing stable clone cell lines
PsiCHECK-2 TNC plasmids (Addgene plasmid 26995, http://www.addgene.org) and a vector were transfected into 5637 and 253J (TN-C negative) BCa cell lines, respectively. Lipofectamine TM 2000 (Life Technologies, city, state) was used for transfection according to kit instructions and stable cell clones highly expressing TN-C were selected quanti ed with Western blot and real time PCR.
Short hairpin RNA (shRNA/Sc) to knock down TN-C expression in T24 and J82 cells (TN-C positive) was measured. pGPU-6-GFP TN-C/Sc was transfected into cell lines as mentioned above, low-TN-C expressing lines were chosen using G418 (600 μg/ml), and Western blot and real time PCR was used to assess shRNA effects. siRNA to knock down syndecan-4 expression was used (Table 1). A protocol for transfection using Lipofectamine TM 2000 is the same as mentioned above.

BrdU incorporation assay
BrdU was added to cell media (3 μg/ml) after cells reached 60-70% con uence on coverslips and incubated for 4 h. Then, coverslips were rinsed three times with PBS for 10 min to remove free BrdU and samples were xed in 4% paraformaldehyde for 45 min, followed by rinsing ve times with PBS for 20 min. Then, 0.1% Triton X-100 was added to destroy the cell membrane (15 min) and 2N HCl (25 min) was added to separate DNA into single strands for primary antibody access to incorporated BrdU. Before blocking nonspeci c epitopes with 10% BSA for 20 min, cells were rinsed three times with PBS for 10 min to remove HCl and Triton. Then 10% BSA with anti-BrdU antibody (1:200) was added and incubated overnight at 4 °C.
The next day, cells were rinsed ve times with PBS to remove free antibody, and cells were incubating with TRTIC-labeled second antibody for 1 h at room temperature and rinsed another three times with PBS to remove free antibody. Fluorescent intensity of TRITC was monitored with a Super Micro Ori ce Plate Spectrophotometer (BioTek, city, state) at 547 nm.
ELISA TN-C in cell media was measured with ELISA. Brie y, cell line (different groups) media were collected and tested with ELISA analysis according kits instructions (Human TN-C ELISA Kit, Shanghai Westang Biological Technology Co., Ltd. Shanghai, PRC), and optical density was measured at 450 nm. Data are expressed in μg/ml. Immuno uorescent staining for nuclear translocation of NF-κB Prepared cells were washed three times in cold PBS (pH = 7.4) and xed with 4% paraformaldehyde for 15 min, followed by permeabilization in 0.5% Triton X-100 for 10 min and blocking with 1% BSA for 1 h. Rabbit anti-human-P65 in 1% BSA was added to media and incubated overnight at 4 °C. Mouse antirabbit TRITC (Red) IgG antibody (Santa Cruz, city, CA) diluted 1:100 in blocking buffer was added to media and incubated 1 h. Then cells were washed with cold PBS three times and cell nuclei were stained with DAPI (10 μg/ml, Sigma) for 3 min. Cells were observed under an Image Pro Plus System mounted on a uorescent microscope (Olympus, Japan).
Other reagent and experiments TN-C peptide (Millipore, location) was an exogenous TN-C added to media (1 μg/ml), and TN-C neutralizing antibody (1 μg/ml) (MAB2138, Novus, location) was used to neutralize TN-C in the media.

Statistical analysis
Statistical analysis was performed with a SPSS 15.0 statistic package (Chicago, IL). For comparisons among different grades, one-way ANOVA was used, and for comparisons between two different grades, The Student's t-test was used. A Shapiro-Wilk test was used to con rm distribution of survival time of BCa patients. P-values <0.05 were considered to be statistically signi cant.

Declarations
Author contributions statement   Correlation analysis suggests that tumor-free survival is negatively associated with TN-C expression in BCa patients, P < 0.05.