Cigarette smoke induced urocystic epithelial mesenchymal transition via MAPK pathways

Cigarette smoke has been shown to be a major risk factor for bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial process in cancer development. The role of MAPK pathways in regulating cigarette smoke-triggered urocystic EMT remains to be elucidated. Human normal urothelial cells and BALB/c mice were used as in vitro and in vivo cigarette smoke exposure models. Exposure of human normal urothelial cells to cigarette smoke induced morphological change, enhanced migratory and invasive capacities, reduced epithelial marker expression and increased mesenchymal marker expression, along with the activation of MAPK pathways. Moreover, we revealed that ERK1/2 and p38 inhibitors, but rather JNK inhibitor, effectively attenuated cigarette smoke-induced urocystic EMT. Importantly, the regulatory function of ERK1/2 and p38 pathways in cigarette smoke-triggered urocystic EMT was further confirmed in mice exposed to CS for 12 weeks. These findings could provide new insight into the molecular mechanisms of cigarette smoke-associated bladder cancer development as well as its potential intervention.


INTRODUCTION
Bladder cancer is the fifth most common cancer in Western countries and the most common cancer in urinary tract [1]. In 2013 there were estimated 382,700 new cases of bladder cancer worldwide, with 143,000 resultant deaths all over the world [2]. Cigarette smoking (CS) has been proven as the major risk factors for bladder cancer [3,4]. It is estimated that CS causes-almost half of male patients and a quarter of female patients of bladder cancer in the United State [5]. Although enormous progresses have been made in understanding the molecular mechanisms leading to bladder cancer development, the tumorigenic process is still poorly understood.
Epithelial-to-mesenchymal transition (EMT), a process characterized by loss of homotypic cadhesion and cell polarity and increased invasion and migration, plays essential roles in development and wound healing [6]. Numerous in vitro and in vivo studies suggest that EMT is associated with cancer cell invasion and metastasis in various malignancies, including bladder cancer [6,7]. EMT is also a critical player in the early stage of tumorigenesis [8][9][10][11]. CS-induced EMT has been found to regulate early events in carcinogenesis [12]. Yet the mechanisms regarding how CS induces EMT remain to be elucidated.
The present study aimed to examine the role of ERK1/2, p38 and JNK pathways in CS-elicited EMT in both normal urothelial cells and bladder tissues. Findings from this study could provide important information for the molecular mechanisms of CS-related bladder tumorigenesis.

CSE elicited EMT in normal urothelial cells
Following the treatment of human SV-HUC-1 cells with various concentrations of CSE for 5 days, the cell viability was determined by MTT assay. The results showed that 2% or higher concentrations of CSE were cytotoxic to SV-HUC-1 cells since the cell viability was significantly reduced when compared with the control group ( Figure 1A). Consequently, we chose 1% CSE as the highest CSE concentration for the subsequentexperiments.
The EMT process is characterized by alterations of cell morphology, migrative and invasive capacity, as well as epithelial and mesenchymal markers' expression. CSE treatment for 5 days led to significant morphological change of SV-HUC-1 cells, i.e., from a urothelial oblateshape to a spindle-like mesenchymal form ( Figure 1B). To examine the alterations of EMT markers, Western blot and qRT-PCR were carried out. We found that the protein levels of epithelial markers E-cadherin and ZO-1 were significantly decreased by CSE treatment. On the contrary, CSE treatment significantly increased the expression levels of mesenchymal proteins Vimentin and N-cadherin ( Figure 1D). Similar changes were observed for the mRNA expression of epithelial and mesenchymal markers in CSE-treated SV-HUC-1 cells ( Figure 1E). Likewise, immunofluorescence staining confirmed that CSE reduced E-cadherin expression and elevated Vimentin expression ( Figure 1F). Futhermore, transwell assays revealed that CSE enhanced the invasion of SV-HUC-1 cells through reconstituted matrigel matrices( Figure 1C). Together, these results demonstrated that CSE elicited EMT in normal urothelial cells.

CSE-triggered urocystic EMT was associated with activation of MAPK pathways
The activation status of MAPK pathways was determined in SV-HUC-1 cells following CSE treatment for 5 days. It was shown that CSE remarkably activated the level of phosphorylated ERK1/2, p38 and JNK in the cells in a dose-dependent manner ( Figure 2A). Moreover, CSE provoked the activation of AP-1 proteins in SV-HUC-1 cells, as evidenced by increased levels of AP-1 components (p-c-Jun, p-c-Fos, FosB and JunB) ( Figure 2B).

CS altered EMT markers' expression and activated MAPKs in mouse bladder tissue
Following the illustration of the function of ERK1/2 and p38 in CS-induced urocystic EMT in vitro, we further investigated whether CS induced EMT-like changes and modulated MAPK activation in a mouse exposure model. We exposed mice to CS for 12 conseutive weeks and the expression levels of epithelial and mesenchymal markers were determined in mouse bladder tissues. We found that CS significantly reduced the protein levels of E-cadherin and ZO-1, and increased Vimentin and N-cadherin proteins in the bladder tissues of mouse ( Figure 4A). Silimar changes in mRNA expression of EMT markers was showed by qRT-PCR assays ( Figure 4B). In addition, we revealed that exposure of mice to CS resulted in significant increased ERK1/2, p38 and JNK activity and increased c-Jun and c-Fos activation in bladder tissues ( Figure 4C). These data were in line with our in vitro findings.

Inhibitors of MAPK pathways diminished CSinduced EMT change in mouse bladder tissue
To explore the role of MAPK pathway in CSmediated EMT-like change in the bladder, mice were received MAPK inhibitors and exposed to CS for 12 conseutive weeks. Safety and toxicity examination, including body weight, diet consumption, hematology www.impactjournals.com/oncotarget MTT assay showed cell viability decreased below 80% when cells were exposed to 2% or higher CSE concentrations in SV-HUC-1 cells. B. CSE induced morphological change from epithelial to spindle-like mesenchymal shape. SV-HUC-1 cells became longer and thinner, some of which generated slender tails. C. Transwell invasion assay revealed CS made a strong stimulative effect on the invasion capacity of SV-HUC-1 cells. The subsequent absorbance assay confirmed this change. D. CSE decreased the expression of epithelial markers E-cadherin and ZO-1, and increased expression of mesenchymal markers Vimentin and N-cadherin in SV-HUC-1 cells by Western blotting. E. CSE decreased the expression of E-cadherin and ZO-1 mRNAs, and enhanced the expression of Vimentin and N-cadherin mRNAs, detected by qRT-PCR. Data are expressed as mean ± SD. *p < 0.05, ** p < 0.01, compared with control group. F. Immunofluorescent staining also showed that CSE decreased E-cadherin protein expression and increased Vimentin expression in SV-HUC-1 cells. and blood biochemistry, showed that no obvious adverse effects were observed in mice treated with MAPK inhibitors when compared with the control group mice (data not shown). We showed that EKR1/2 and p38 inhibitors significantly attenuated p-c-Jun and p-c-Fos expression levels in the bladder after exposure to CS for 12 weeks (Figure 5A-5B). Moreover, our results revealed that CS-induced decrease of E-cadherin protein and increase of Vimentin protein were effectively supressed by inhibitor of ERK1/2 and p38. Similar results were observed in mRNA level ( Figure 5D-5E). These data indicated that inhibitors of ERK1/2 and p38 reversed CSinduced urocystic EMT in vivo. However, we found that JNK inhibitor SP600125 failed to attenuate CS-eliciated EMT change in the bladder of mouse ( Figure 5C-5E). These results were in accordance with our in vitro findings.

DISCUSSION
By using in vitro and in vivo models, we demonstrated in the present study that CS induced urocystic EMT. We further showed that CS-triggered EMT was associated with activation of MAPK pathways.
Moreover, our data indicated that inhibitors of ERK1/2 and p38, but not JNK inhibitor, effectively diminished CSinduced urocystic EMT in vitro and in vivo. Our findings suggested the critical role of ERK1/2 and p38 activity in CS-associated urocystic EMT and the underlying mechanism of CS-associated bladder cancer development.
EMT is a crucial process in cancer development [12]. In line with previous reports, we revealed that CS exposure induced EMT in human urocystic SV-HUC-1 cells, as evidenced by cellular morphological change, increased invasive ability, and altered EMT markers expression, including reduced epithelial markers E-cadherin and ZO-1, and elevated mesenchymal markers Vimentin and N-cadherin. Moreover, similar alterations were found in the bladder of mice, suggestive of EMT induction. Together, these results illustrated that CS triggered urocystic EMT in both in vitro and in vivo settings.
The regulation of EMT involves several critical cellular signalings. However, the potential mechanisms of CS-induced EMT remain unclear. Evidences have shown that ERK1/2, JNK and p38 pathways faciliate EMT [17-20, 25, 26]. Nevertheless, little information    is available regarding MAPK regulation of urocystic EMT induced by CS. We demonstrated in our study that CS triggerd urocystic EMT mainly through ERK1/2 and p38 pathways, while JNK pathway exhibited little role in regulating CS-induced urocystic EMT. Since ERK1/2 is a major determinant in controlling cell growth, survival and invasion, and inhibitors of ERK1/2 have been applicated for anticancer drugs [27][28][29]. It has been reported that butein can inhibit the migrative and invasive capacities of human bladder cancer cells through ERK1/2 suppression [30]. Sunitinib is effective in bladder cancer patients, which suppresses the activation of ERK1/2 [31]. Moreover, evidence revealed that ERK1/2 and p38 play a role in CCDC34mediated bladder cancer cell proliferation and migration [32]. Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. Inhibition of p38 significantly increased Gemcitabine sensitivity in bladder cancer cells [33]. Fang, Y et al reported that JNK inhibitor SP600125 suppressed anchorage-independent growth of T24T bladder cancer cells [34]. In the present study we illustrated that ERK1/2 and p38 inhibitors significantly attenuated CS-induced urocystic EMT in vitro and in vivo, whereas JNK inhibitor failed to exhibit similar effects. Thus, our data suggested the differential riole of MAPK pathways in regulating CS-triggered urocystic EMT.
In summary, our study demonstrated that ERK1/2 and p38 promote CS-induced urocystic EMT in both human urothelial cells and mouse bladder tissue. These novel findings indicate the critical function of ERK1/2 and p38 activity in CS-related bladder cancer development and provide a plausible strategy for the search of potential interventional target of CS-associated bladder oncogenesis.

MATERIALS AND METHODS
See the supplemental material for further details concerning methods.

Cell culture and treatment
SV-HUC-1 cells were cultured in F12K medium containing antibiotics (100 U/ml penicillin and 100 μg/ ml streptomycin). CS extract (CSE) was freshly prepared for each experiment by combusting one filterless 3R4F reference cigarette according to reported method [35,36]. SV-HUC-1 cells were treated with various concentrations of CSE for 5 days.

Cell toxicity assay
MTT assay was used to detect SV-HUC-1 cells viability and appropriate CSE concentrations were chosen for further use.

Mice and CS exposure
Eight-week-old male BALB/c mice weighing 18-22 g were purchased from the Animal Research Center of Nanjing Medical University. Mice were exposed to tobacco smoke in a smoking apparatus. The smoke was delivered to whole-body exposure chambers with target concentration of total particulate matter (TPM) of 85 mg/m 3 . Animals were exposed for 6 hours daily for 12 consecutive weeks. After the last CS exposure, mice were sacrificed and the bladder tissues were isolated, frozen and stored at -80°C until analysis.

Treatment of mice by inhibitors of MAPK pathways
In a separate set of animal study, mice were treated daily with different MAPK inhibitors as previously reported [37][38][39][40]. All the inhibitors (0.5 mg/kg for U0126, 1 mg/kg for SB203580 and 1 mg/kg for SP600125) were dissolved in DMSO and intraperitoneally injected to the mice. Mice were randomly assigned into six groups (n=10 per group): filtered air group; CS-exposed group; CS+DMSO group; CS+U0126 group; CS+SB203580 group; CS+SP600125 group; Following the completion of exposure, mice were sacrificed and bladder tissues were collected for analysis.

Western blot analysis
Proteins were extracted from SV-HUC-1 cells and mouse bladder tissues. Western blot analyses were performed for the determination of protein expression levels. www.impactjournals.com/oncotarget Quantitative real-time PCR Total RNA was isolated by RNAiso Plus according to the manufacturer's instructions (TaKaRa, Japan). qRT-PCR was performed using Power SYBR Green Master Mix (TaKaRa, Japan) and an ABI 7300 realtime PCR detection system (Applied Biosystems). Fold changes in expression of each gene were calculated by a comparative threshold cycle (Ct) method using the formula 2 − (ΔΔCt) .

Transwell assay
Invasive capacities of CSE-treated SV-HUC-1 cells were evaluated using Transwell chambers with matrigel.

Immunofluorescence
Immunofluorescent staining was performed to analyze the expression of E-cadherin and Vimentin in SV-HUC-1 cells treated with CSE.

Statistical analysis
Statistical analyses were performed with SPSS 16.0. All data were expressed as mean ± standard deviation. One-way ANOVA was used for comparison of statistical differences among multiple groups, followed by the LSD significant difference test. Unpaired Student t test was also used for the comparison between two groups. A value of p< 0.05 was considered significantly different.