HMGN5 promotes IL-6-induced epithelial-mesenchymal transition of bladder cancer by interacting with Hsp27

Bladder cancer (BC) is one of the most common cancers worldwide, with a high rate of recurrence and poor outcomes. High-mobility group nucleosome-binding domain 5 (HMGN5) is overexpressed in many cancers and could cause carcinogenesis in BC. By protein-protein-interaction (PPI) analysis, we found that heat shock protein 27 (Hsp27), also a crucial functional factor in BC carcinogenesis, is significantly related to HMGN5. Hsp27 is required for IL-6-mediated EMT via STAT3/Twist signaling in prostate cancer. Here, we hypothesize that HMGN5 may interact with Hsp27 to affect IL-6-induced EMT and invasion in BC via STAT3 signaling. In the present study, we found that HMGN5 and Hsp27 are highly expressed in BC tissues and positively correlated with each other. HMGN5 interacts with Hsp27 in vitro, to modulate the cell invasion and EMT in BC. Moreover, HMGN5 could modulate IL-6-Hsp27-induced EMT and invasion in BC cells by regulating STAT3 phosphorylation and STAT3 targeting of the Twist promoter. HMGN5 interacts with Hsp27 to promote tumor growth in a human BC xenograft model in nude mice. In summary, HMGN5 interacts with Hsp27 to promote IL-6-induced EMT, therefore promoting invasion in BC and contributing to the progression of BC.


INTRODUCTION
Bladder cancer is one of the most common malignant tumors globally with a high rate of recurrence and poor outcomes, as a result of relapse. Of all primary bladder cancers, approximately 90% are transitional cell carcinomas, 5% are squamous cell carcinomas, and 1-2% are adenocarcinomas [1,2]. Developing an in-depth knowledge of the molecular and cellular mechanisms of tumorigenesis may provide new directions for bladder cancer therapy.
High-mobility group nucleosome-binding domain 5 (HMGN5) is one of the HMGNs found recently [3,4]. Recent emerging research has reported that HMGN5 is overexpressed in many human cancers and that HMGN5 is related to carcinogenesis in a variety of can-cer models [5], including bladder cancer [6]. In bladder cancer, HMGN5 silencing could block the PI3K/Akt signaling pathway, therefore improving the chemical sensitivity of human bladder cancer cells to cisplatin, whereby the viability and invasion of these cells could be inhibited in vitro and in vivo [7]. To study how HMGN5 exerts its effects on bladder cancer cell invasion and epithelial-mesenchymal transition (EMT), a crucial issue during cancer metastasis, we performed protein-protein-interaction (PPI) analysis to search for factors related to HMGN5. Of the selected candidates, heat shock protein 27 (Hsp27), another crucial functional factor in bladder cancer carcinogenesis [8,9], drew our attention.
As molecular chaperones, Hsps exert their biological effects by holding protein and serving as the folding of AGING the newly synthesized protein to maintain cellular homeostasis [10]. A small Hsp, Hsp27 responds to heat shock and other cellular stressors such as cancers [11,12]. Additionally, Hsp27 expression has been reported to be significantly associated with bladder cancer invasiveness and prognosis in bladder cancer patients [13,14]. Based on its properties, Hsp27 has been considered a therapeutic target for many malignant tumors. For example, OGX-427 is a second-generation antisense oligonucleotide sequence designed to bind to Hsp27 mRNA. It can induce apoptosis, improve the sensitivity to chemotherapy drugs and suppress cellular proliferation, indicating its clinical potential in bladder cancer therapy [14][15][16]. In prostate cancer, silencing Hsp27 can decrease IL-6-dependent STAT3 phosphorylation, nuclear translocation, and STAT3 targeting of the Twist promoter, indicating that Hsp27 is required for IL-6-mediated EMT via regulation of STAT3/Twist signaling [17]. Thus, we hypothesize that HMGN5 may interact with Hsp27 to affect IL-6-induced EMT and invasion in bladder cancer by modulating STAT3 phosphorylation and STAT3 targeting of the Twist promoter.
Here, the expression of HMGN5 and Hsp27 and their correlation in tissue samples were evaluated. The predicted interaction between the HMGN5 and Hsp27 proteins was examined. Next, the dynamic effects of HMGN5 and Hsp27 on IL-6-independent-and IL-6dependent EMT and invasion in bladder cancer cells were evaluated. Furthermore, we examined whether HMGN5 could modulate IL-6-Hsp27-induced STAT3 phosphorylation and STAT3 targeting of Twist promoter. Finally, the dynamic effects of HMGN5 and Hsp27 on tumor growth were investigated in nude mice. In summary, we provide a novel mechanism and experimental evidence showing how the interaction between HMGN5 and Hsp27 affects bladder cancer cell invasion and EMT with or without IL-6.

Expression and correlation of HMGN5 and Hsp27 in tissue samples
The obtained tissue samples were examined for pathological characteristics by HE staining ( Figure 1A). Proteins related to HMGN5 were analyzed by BioGRID (Biological General Repository for Interaction Datasets), and a total of 9 proteins were identified ( Figure 1B). Among them, Hsp27 mRNA expression was the most upregulated in 10 bladder cancer tissues when compared with noncancerous tissue (Supplementary Figure 1). Moreover, Hsp27 was selected for further experiments because it is one of the Hsps overexpressed in bladder cancer and protects tumor cells from therapeutic stressors [9]. Similar to previous investigations, Hsp27 and HMGN5 mRNA expression was remarkably upregulated in 56 cases bladder cancer tissues ( Figure 1C, 1D), and the expression levels were positively correlated with each other ( Figure 1E). As demonstrated by immunoblotting and IHC staining, the protein levels of HMGN5 and Hsp27 were higher in bladder cancer tissues ( Figure 1F, 1G).
Next, we analyzed the correlation of the overall survival rate with HMGN5 and Hsp27 levels. Bladder cancer patients were divided into two groups based on the expression of Hsp27 or HMGN5 using the med ian expression value as the cutoff. As shown in Figure 1H and I, lower Hsp27 or lower HMGN5 expression was related to a higher rate of overall survival in bladder cancer patients. Moreover, the group with high coexpression of HMGN5 and Hsp27 group showed the lowest overall survival rate ( Figure 1J). These data suggest that HMGN5 may interact with Hsp27 to play a role in bladder cancer progression.

HMGN5 interacts with Hsp27
To validate the predicted interaction between HMGN5 and Hsp27, we first examined their protein levels in four bladder cancer cell lines, J82, HT1376, RT4, and T24 by immunoblotting. Figure 2A shows that the protein levels of both HMGN5 and Hsp27 in the four BC cell lines are all higher than those in the normal cell line, SV-HUC-1. IF staining images showed the fluorescence intensity representing HMGN5 (red) and Hsp27 (green) expression ( Figure 2B). The merged image also revealed that HMGN5 and Hsp27 expression might be colocalized in bladder cancer cells ( Figure 2B). Moreover, we conducted an in vitro GST pull-down assay to validate the predicted interaction between HMGN5 and Hsp27. We overexpressed GST-tagged Hsp27 in bacteria and incubated them with His-tagged HMGN5 at 4 °C for 4 h. HMGN5 was captured by Ni-NTA resin, and Hsp27 was detected in the precipitated product by immunoblotting, suggesting that HMGN5 could interact with Hsp27 ( Figure 2C).

Overexpression of HMGN5 and Hsp27 promotes bladder cancer cell invasion and EMT
We confirmed the interaction between HMGN5 and Hsp27; then, we examined the effect of HMGN5 and Hsp27 overexpression on bladder cancer cell invasion and EMT. HMGN5 and Hsp27 overexpression was achieved in J82 cells, as confirmed by immunoblotting ( Supplementary Figure 2A, 2B). Transwell assays revealed that HMGN5 or Hsp27 overexpression alone could significantly promote bladder cancer cell invasion AGING AGING ( Figure 3A) and decrease E-cadherin expression and increase Vimentin expression ( Figure 3B, 3C). More importantly, Hsp27 overexpression further enhanced the promoting effect of HMGN5 overexpression ( Figure  3A-3C), suggesting that HMGN5 interacts with Hsp27 to modulate bladder cancer cell invasion and EMT.

Silencing of HMGN5 and Hsp27 inhibits bladder cancer cell invasion and EMT
To further validate the dynamic effects of HMGN5 and Hsp27 on bladder cancer cells, we examined how HMGN5 and Hsp27 silencing affected bladder cancer cell invasion and EMT. T24 cells were infected with Lv-sh-HMGN5 or Lv-sh-Hsp27 alone to silence HMGN5 or Hsp27, as confirmed by immunoblotting ( Supplementary Figure 2A, 2B). Next, T24 cells were coinfected with Lv-sh-HMGN5 and Lv-sh-Hsp27 and examined for cell invasion and EMT. In contrast to HMGN5 or Hsp27 overexpression, HMGN5 or Hsp27 silencing significantly inhibited bladder cancer cell invasion ( Figure 4A) and increased the E-cadherin expression and decreased Vimentin expression ( Figure  4B, 4C). Similarly, the effect of HMGN5 silencing could be enhanced by Hsp27 silence ( Figure 4A-4C), further suggesting that HMGN5 interacts with Hsp27 to modulate bladder cancer cell invasion and EMT.

HMGN5 is involved in IL-6-Hsp27-induced cell invasion and EMT in bladder cancer cells
In prostate cancer, Hsp27 is involved in IL-6-mediated EMT [17]. Here, we investigated whether HMGN5 participates in IL-6-Hsp27-induced EMT in bladder cancer. After 24 h of serum starvation, the protein levels of HMGN5 and Hsp27 were induced by IL-6 stimulation in both J82 and T24 cells in a timedependent manner ( Figure 5A). In vitro GST pull-down assays were used to examine the interaction between HMGN5 and Hsp27. AGING Next, J82 and T24 cells were coinfected with Lv-Hsp27 and Lv-sh-HMGN5 and examined for cell invasion and EMT markers with or without 50 ng/mL IL-6 stimulation. As shown in Figure 5B and 5C, cell invasion and EMT in both J82 and T24 cell lines could be induced by IL-6 stimulation but inhibited by HMGN5 knockdown. However, the HMGN5 knockdown-mediated suppression of cell invasion and EMT could be partially rescued by Hsp27 overexpression. These findings suggest that HMGN5 is involved in IL-6-Hsp27-induced cell invasion and EMT in bladder cancer cells. It has been reported that STAT3-mediated Twist transcription is involved in IL-6/Hsp27-mediated EMT via STAT3 targeting of the Twist promoter in prostate cancer [17]. Next, we validated whether HMGN5/Hsp27 could modulate IL-6-induced STAT3 phosphorylation and Twist promoter activity. HMGN5 silencing significantly decreased the phosphorylation of Hsp27 and STAT3 induced by IL-6 stimulation in J82 and T24 cells ( Figure 6A).

AGING
We performed ChIP assays to examine the dynamic effects of HMGN5 silencing and Hsp27 overexpression on STAT3 targeting of the Twist promoter [18], with or without IL-6 stimulation. The inducing effects of IL-6 on STAT3 targeting of Twist promoter DNA could be significantly attenuated by HMGN5 silencing but partially rescued by Hsp27 overexpression ( Figure 6B). Additionally, we performed luciferase reporter analysis to investigate the dynamic effect of HMGN5 silencing and Hsp27 overexpression on IL-6-induced Twist transcriptional activity. As described by Cheng et al., we used serial truncations of the human Twist promoter to analyze Twist transcriptional activity [18] with or without IL-6. Consistent with the previous study, in each assessment of Twist truncation, promoter activation was significantly induced by IL-6, which could be remarkably suppressed by HMGN5 knockdown but significantly rescued by Hsp27 overexpression ( Figure 6C). These findings suggest that HMGN5 interacts with Hsp27 to modulate IL-6-induced STAT3 targeting of Twist promoter and the activation of the Twist transcription. To further validate the above in vitro findings, we established a xenograft model of human bladder cancer in nude mice using HMGN5-and/or Hsp27-overexpressing or silenced J82 cells. Figure 7A shows the appearance of tumors derived from J82 cells with HMGN5-and/or Hsp27-overexpression. As shown in Figure 7B, 7C, HMGN5 or Hsp27 overexpression alone was sufficient to increase the tumor volume and tumor weight, which was further increased by the combination of HMGN5 and Hsp27 overexpression. In contrast, knockdown of Hsp27 or HMGN5 alone clearly decreased restrained the tumor volume and tumor weight, and the inhibitory effect on tumor volume and tumor weight was amplified in the group with combined HMGN5 and Hsp27 silencing ( Figure 7D-7F). The protein levels of EMT markers could be increased by either HMGN5 or Hsp27 overexpression, and further AGING increased by the combination of HMGN5 and Hsp27 overexpression ( Figure 7G). Conversely, the EMT marker protein levels were inhibited by either HMGN5 or Hsp27 silencing, and further inhibited by the combination of HMGN5 and Hsp27 silencing ( Figure  7H). These findings suggest that HMGN5 interacts with Hsp27 to promote tumor growth in a nude mouse model.

DISCUSSION
Here, we demonstrate that HMGN5 and Hsp27 are highly-expressed in bladder cancer tissues and positively correlated with each other. HMGN5 interacts with Hsp27, in vitro and in vivo, to modulate cell invasion and EMT in bladder cancer cells. Moreover, HMGN5 could modulate IL-6-Hsp27-induced EMT and invasion in bladder cancer cells by regulating STAT3 phosphorylation and STAT3mediated Twist transcription. HMGN5 interacts with Hsp27 to promote tumor growth in a bladder cancer xenograft model in nude mice.
HMGN5 and Hsp27 have both been regarded as oncogenic factors in bladder cancer. HMGN5 is highly expressed in human bladder cancer, thus promoting bladder cancer cell proliferation and AGING invasion [6,19]. Similarly, the expression of Hsp27 is upregulated in bladder cancer [13,15,[20][21][22]. In the present study, we observed consistent results that, HMGN5 and Hsp27 mRNA and protein levels were increased in bladder cancer tissues. Moreover, as predicted by BioGRID, HMGN5 could interact with Hsp27 in vivo and in vitro, as revealed by GST pulldown and IF staining.
Regarding the molecular and cellular functions of the interaction between HMGN5 and Hsp27, the dynamic effects of these two factors on bladder cancer cell EMT and invasion were examined. EMT is a complicated process, encompassing changes in the cytoskeleton and a decrease in E-cadherin expression [23]. Both the loss of epithelial markers and gain of mesenchymal markers have been revealed in many cancers, including in bladder cancer [24][25][26][27][28]. During EMT in bladder cancer, the loss of E-cadherin occurs frequently [29]. Furthermore, mesenchymal markers, Twist and Vimentin, are related to the stage and grade of bladder cancer and could strongly affect the progression and metastasis of bladder cancer [27,30,31]. Herein, HMGN5 or Hsp27 overexpression significantly promoted EMT by increasing E-cadherin and decreasing Vimentin, and promoted the invasion of bladder cancer cells. When combined, the effect of HMGN5 or Hsp27 overexpression was enhanced. Conversely, HMGN5 and Hsp27 silencing synergistically inhibited EMT and invasion in bladder cancer cells. Thus, HMGN5 could interact with Hsp27 to promote bladder cancer EMT, therefore enhancing bladder cancer cell invasion.
Chronic inflammation and increased inflammatory mediators could result in tumor progression, invasion, and angiogenesis [32,33]. Proinflammatory cytokines, such as IL-6, IL-8, and TNF-α, play critical roles in the pathogenesis of bladder cancer [34]. Furthermore, as confirmed by the previous study, persistent STAT3 activation could maintain constitutive NF-κB activity, thus proving the relationship between oncogenic signaling pathways in the inflammatory microenvironment [35]. IL-6 is considered as not only a primary activator of STAT3 signaling pathways, but also the central cytokine that could affect the human inflammatory response [36,37]. Moreover, as confirmed by the present study, IL-6 induced HMGN5 and Hsp27 protein levels in a time-dependent manner. HMGN5 silencing attenuated IL-6-induced bladder cancer EMT and bladder cancer cell invasion. Regarding the cellular mechanism, IL-6 could induce the phosphorylation of STAT3 [17], which could be partially attenuated by HMGN5 silencing. Moreover, IL-6-induced STAT3 targeting of the Twist promoter could also be suppressed by HMGN5 silencing. More importantly, the abovedescribed effects of HMGN5 silencing could all be significantly reversed by Hsp27 overexpression, suggesting that HMGN5 is involved in IL-6-Hsp27induced STAT3 phosphorylation and STAT3 targeting of the Twist promoter, therefore modulating IL-6induced bladder cancer EMT and bladder cancer cell invasion.

CONCLUSIONS
HMGN5 interacts with Hsp27 to modulate IL-6independent-or IL-6-dependent EMT and cell invasion in bladder cancer cells. Upon IL-6 stimulation, HMGN5/Hsp27 modulates bladder cancer EMT and bladder cancer cell invasion via the STAT3/Twist signaling pathway.

Lentivirus production, titration, and infection
To generate HMGN5 or Hsp27 silenced or overexpressed or control lentivirus, we cotransfected the plasmids encoding HMGN5/Hsp27 or HMGN5 shRNA/Hsp27 AGING shRNA or the control scrambled sequence into 293T cells together with the plasmids pHelper1.0 and pHelper2.0 (GeneChem) for virus packaging using Lipo2000 (Invitrogen) following the manufacturer's protocols. Then, lentivirus was harvested from the supernatants by ultracentrifugation and examined for the viral titer determination.
For lentiviral infection, cells at 40-50% confluence were incubated overnight. Then, replace the culture medium was replaced with 5ml/well viral supernatant at the appropriate titer, and the cells were incubated at 37°C for 10 h. After that, the viral supernatant was replaced with fresh media. Forty-eight hours later, the infected cells were selected with 2mg/ml puromycin. Five days later, the HMGN5 inhibition efficiency was examined by immunoblotting.

Hematoxylin and eosin (HE) staining
Collected bladder urothelial carcinoma tissue specimens were fixed in 10% formalin overnight and then processed for paraffin embedding and sectioning. Sections of 4 μm were deparaffinized, rehydrated, and then stained using HE staining kit (Beyotime, Shanghai, China) according to the protocols.

GST pull-down assay
The HMGN5-Hsp27 interaction was validated by GST pull-down analysis. GST-tagged protein and His-tagged protein were generated and purified following the method described previously [38]. The GST-fused protein was incubated with prepared glutathione Sepharose beads (GE Healthcare, Glutathione Sepharose 4B, 17-0756-01). Thirty minutes later, the beads were collected, washed, and incubated with the input proteins (0.1 mg/mL) or His-fused protein dissolved in the reaction buffer (20 mM Tris, 100 mM NaCl, 1 mM DTT and 1 mM EDTA) for 30 min. The supernatant was discarded and the beads together with the target proteins were washed with the reaction buffer four times. After that, the target proteins were washed with 10% SDS and the eluate was analyzed and detected by SDS-PAGE and immunoblotting.

Immunofluorescence (IF) staining
Cells were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for 15 min. After blocking with 1% BSA in PBS for 2 h, the cells were then incubated with specific antibodies (dilution 1:200) overnight at 4°C followed by incubation with TRITC or FITC-conjugated secondary antibody (dilution 1: 500, Beyotime, China) for 1 h in the dark. Nuclear staining was performed using DAPI (Beyotime, China). A fluorescence microscope (Olympus, Japan) was used for image capture.

In vitro invasion assays
Cells (5 × 10 5 ) were plated on the top side of apolycarbonate Transwell filter coated with Matrigel in the top chamber of the QCM 24-well cell invasion assay system (Cell Biolabs, Inc. Santiago, USA). The medium in the upper chamber was serum-free, and the serumcontaining medium in the bottom chamber was used as a chemoattractant. After incubating the cells at 37 °C for 48 h, noninvasive cells in the upper chambers were discarded and the invaded cells on the bottom membrane surface were fixed in 100 % methanol for 10 min, air-dried, stained with crystal violet (Beyotime), and counted under a microscope. Three independent experiments were conducted.

Statistical analysis
Data are expressed as the means ± SD of at least three independent experiments and statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests or independent sample t-test using SPSS Statistics 17.0 software. The level of significance was based on the probability of P < 0.05 and P < 0.01.

Ethics approval
The study was performed in accordance with the Declaration of Helsinki, and the praotocol was approved by the Ethics Committee of The Third Xiangya Hospital of the Central South University. All of the enrolled bladder cancer patients signed informed consent forms.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.  Tables   Please browse Full Text version to see the data of Supplementary Table 1