S100A4 suppresses cancer stem cell proliferation via interaction with the IKK/NF-κB signaling pathway

Background Bladder cancer often recurs due to incomplete elimination of the cancer stem cells (CSCs). Therefore, new strategies targeting bladder CSCs are needed and the aim of this study was to investigate the effect of S100A4 on the proliferation capacity of MB49 bladder cancer stem cells (MCSCs). Methods MCSCs were established and validated. The expression level of S100A4 in MCSCs and MB49 cells was evaluated using Western blotting and quantitative polymerase chain reaction (QPCR). S100A4 was overexpressed or knocked-down by transfection of pCMV6-XL5-S100A4 plasmid or RNA interference (RNAi) respectively. Proliferation capacity of MCSC was evaluated by cell proliferation assay and in vivo tumorigenicity study. Transcriptional activity of nuclear factor kappa B (NF-κB) was analyzed using luciferase reporter assay, and the level of interleukin (IL)-2 as well as tumor necrosis factor (TNF) was quantified by QPCR. Protein-protein interaction of S100A4 and inhibitor of nuclear factor kappa B NF-κB kinase (IKK) was analyzed by immunoprecipitation. Results S100A4 was significantly up-regulated in MCSCs, which positively associated with the proliferation capacity, as well as the level of NF-κB, IKK, IL-2 and TNF in MCSCs. Knock-down of S100A4 could reverse such effects. Using immunoprecipitation assay, an interaction between S100A4 and IKK could be observed. Conclusions S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway, and S100A4 maybe a hopeful therapeutic target for MCSCs.


Background
Bladder cancer is a most common urological cancer in China, and the rest of the world [1]. Radical cystectomy with pelvic lymphadenectomy is the standard treatment for bladder cancer. However, bladder cancer may recur due to the incomplete elimination of cancer stem cells (CSCs). Therefore, new strategies targeting bladder CSCs are urgently needed. S100A4, also known as metastasin/FSP1/pEL98/mts-1, is a gene encoding a small calcium binding protein that interacts with other proteins to enhance apoptosis, cell motility, and tumorigenesis [2]. S100A4 is overexpressed in most cancers, including breast cancer, gastric cancer, and non-small cell lung cancer (NSCLC) [3]. In addition, S100A4 expression is correlated with patients' outcome and cancer metastasis [4]. It has been recently reported that S100A4 is a novel marker and a critical regulator of glioma stem cells, with the enhanced S100A4 expression contributing to the presentation of a metastatic phenotype [5]. These findings indicate that S100A4 may be a promising therapeutic target for bladder CSCs. Through bioinformatics analysis in preliminary experiments, we found that there was a close relationship between S100A4 protein and the nuclear factor kappa B (NF-κB) signaling pathway.
In the present study, we demonstrate that S100A4 is up-regulated in MB49 bladder cancer stem cells (MCSCs). Additionally, overexpression of S100A4 enhances the proliferation capacity of MCSCs in vitro, and also upregulates inhibitor of nuclear factor kappa B NF-κB kinase (IKK) and activates the NF-κBsignaling pathway, whereas knockdown of S100A4 resulted in the opposite effects. The findings of this study suggest that S100A4 may promote the proliferation capacity and upregulate IKK in MCSCs by activating the NF-κB signaling pathway. Therefore, S100A4 may have the potential to be a therapeutic target in MCSCs.
The validation of MCSCs was performed as previously reported [6]. Cancer stem cell markers CD133 and CD44 was detected by flow cytometry analysis, Western blotting, and quantitative polymerase chain reaction (QPCR). The proliferative ability and susceptibility to chemotherapy were examined by Cell Counting Kit-8 reagent assay. Cell migration ability was examined with the transwell assay. The tumorigenic ability was verified using nude mice.

Western blot analysis
The MB49 cells and MCSCs were respectively harvested. Equal amount proteins were extracted from cells, and separated by 10% sodium dodecyl sulfate -polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in PBS, and incubated overnight at 4°C with the primary antibodies including anti-S100A4 (Abcam, Cambridge, MA), anti-IKK (Abcam) and anti-β-actin antibody (Abcam) followed by secondary antibodies (Abcam). Bands were visualized using Fluor Chem FC2 (Alpha Innotech, San Leandro, CA).

Quantitative polymerase chain reaction
Total RNA was extracted using the Arcturus PicoPure RNA isolation kit (Applied Biosciences, Carlsbad, NM). RNA quality was tested using the Bioanalyzer RNA Pico Chip (Agilent Technologies, Santa Clara, CA). Total RNA were transcribed reversely with Superscript III (Invitrogen), followed by synthesizing the first-strand cDNA which was amplified using a SYBR green PCR master mix (Bio-Rad, Hercules, CA) performed on a 7500 real-time PCR system (AB Applied Biosystems, Singapore). The cycling systems were denaturation (95°C for 10 s), annealing and extension (60°C for 60 s). The primers were designed using Primer Express version 2.0 (Applied Biosystems, Foster City, CA), and are shown in Table 1. The relative expression level were analyzed using the ΔΔC t method. GAPDH was used as the internal control.

Cell counting Kit-8 (CCK-8) assay
Cells were plated at a density of 1 × 10 3 cells per well in a 96-well plate, followed by incubating for 72 h. After incubation, cell counting kit-8 reagent (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) was added to each well with 10 μl at a time periods of 24, 48, and 72 h. Incubated for 4 h, the absorbance value was read at 450 nm performing in EnSpire 2300 multilabel reader (PerkinElmer, Singapore).

In vivo tumorigenicity study
All animal experiments were obeyed the Chinese animal protection laws and guidelines, and approved by the Ethics Committee of Southern Medical University (Contract 1,116,904).
Four-week-old immune deficient nude mice were purchased from Experimental Animals Center (Southern Medical University, Guangzhou, China), and fed under specific, pathogen-free conditions. Cells (1 × 10 4 ) were injected into mice subcutaneously. Tumor xenograft formation was recorded at 10, 20, 30, and 45 days, calculated the tumor volume according to the formula d 2 × D/2, where D and d were the longest and the shortest diameters, respectively. Then mice were sacrificed after CO 2 anesthesia.
Luciferase reporter assay NF-κB transcriptional activity was examined using the pNF-κB-luciferase reporter and control plasmids (Clontech, Mountain View, CA). The cells were plated at a sub confluent density, followed by co-transfecting with 0.5 μg of NF-κB luciferase reporter plasmid or negative plasmid, and 0.02 μg of Renilla luciferase pRL-TK plasmid (Promega, Madison, WI) performing in Lipofectamine 2000 reagent (Invitrogen). Cell lysates were prepared 24 h after transfection, and the reporter activity was measured using the Dual-luciferase reporter assay system (Promega).

Statistical analysis
SPSS19.0 software was used for all statistical analyses. Numeric data were described as the mean value ± standard deviation. Comparisons were performed by Students t-test. A value of P < 0.05 was considered to indicate statistical significance.

Results
Expression of S100A4 in MCSC MB49 cells S100A4 level in MCSCs was increased as detected by Western blotting (Fig. 1a), and S100A4 mRNA expression level in MCSCs was also significantly increased as detected by QPCR (Fig. 1b). Fig. 1 S100A4 was upregulated in MCSCs. a Western blotting analysis of S100A4 protein expression in MCSCs and MB49 cells; β-actin was used as a loading control. b Quantitative PCR of S100A4 mRNA expression in MCSCs and MB49 cells. Transcript levels were normalized to GAPDH, and expressed relative to MB49 cells. Data is mean ± SD of three independent experiments. *P < 0.05

Effects of transfection on MCSCs
MCSCs were transfected with S100A4-vector or S100A4-siRNA, and the transfection efficiency was examined in vitro. As shown in Fig. 2a, protein level of S100A4 was increased in S100A4-vector group, while which was inhibited in S100A4-siRNA group. S100A4 mRNA expression was increased by transfecting S100A4-vector (Fig. 2b, P < 0.05), while suppressed by S100A4-siRNA transfection (Fig. 2c, P < 0.05).

Effects of S100A4 on MCSC proliferation
The proliferation capacity of transfected MCSCs was evaluated by Cell Counting Kit-8 and tumorigenicity assay. The cell proliferation curve was increased by S100A4 overexpression (Fig. 3a), while was decreased by S100A4 suppression (Fig. 3b)., Tumor volume was increased by S100A4 overexpression, while was decreased by S100A4 suppression (Fig. 3c and d, respectively).

S100A4 promotes the activity of NF-κB and the transcription of its target genes
Overexpression of S100A4 enhanced the transcriptional activity of a NF-κB reporter gene (Fig. 4a), which was suppressed when knockdown of S100A4 (Fig. 4b).
Western blotting indicated that overexpression of S100A4 increased the level of IKK (Fig. 4c). In addition, several NF-κB target genes, including IL-2 and TNF, were up-regulated in S100A4-overexpressing cells (Fig. 4d), and downregulated in S100A4-silenced MCSCs (Fig. 4e). Taken together, these results indicated that the NF-κB pathway may participate in the proliferation effect of S100A4 in MCSCs.

S100A4 regulates NF-κB activation through IKK
We hypothesized that S100A4 may regulate NF-κB signaling pathway through a direct interaction with IKK. To verify this hypothesis, interaction between S100A4 and IKK were analyzed using immunoprecipitation assays. As shown in Fig. 5, S100A4 physically interacted with IKK. Discussion S100A4 has been reported to be an important regulator for modulating the cell cycle, proliferation, apoptosis, and migration in different kinds of cells through various mechanisms [7]. In adult animals, S100A4 expression is restricted to very few kinds of normal tissue or cells, whereas it is usually overexpressed in cancerous tissues [2]. However, little is known about the function of S100A4 on bladder CSCs. In this research, we demonstrate firstly that S100A4 is able to enhance the proliferation capacity of mouse bladder MCSCs. Another study Fig. 2 Effects of transfection on MCSCs. a S100A4 protein level of was detected by Western blotting. b The mRNA expression of S100A4 in MCSCs transfected with S100A4 vector was determined by quantitative PCR. c The mRNA expression of S100A4 in MCSCs transfected with S100A4-siRNA was determined by quantitative PCR. Data is mean ± SD of three independent experiments. *P < 0.05 has indicated that S100A4 could be a novel marker and regulator of glioma stem cells in human and murine malignant gliomas [5]. Different expression levels of S100A4 lead to different stem cell characteristics: it promotes self-renewal at a lower level, while promotes quiescence through asymmetric stem progenitor divisions at a higher level [5]. Several works have also demonstrated that S100A4 may have oncogenic effects in a multitude of tumor types [8]. However, further researches are needed to manifest the exact function of S100A4 in various cancer stem cells.
In the present research, we found that cell proliferation was significantly prompted by S100A4 overexpression, while was inhibited by S100A4 suppression. Luciferase reporter assays manifested that the transcriptional activity of NF-κB was enhanced significantly by overexpression of S100A4, implying NF-κB may play an crucial part in the S100A4-induced proliferation capacity of MCSCs.
NF-κB is the collective name of a family of transcription factors consisting of seven proteins, encoded by five genes: c-Rel, RelA, RelB, p100/p52 and p105/p50 [9]. NF-κB has been widely known for its regulatory effects on immunological and inflammatory processes, like a serious of other pathological and physiological responses containing of the development and progression of cancer. NF-κB activation is mediated frequently by plenty of chemotherapeutic agents, which generally means inducing a strong anti-apoptotic response which limits the efficacy of treatments [10]. As shown in this research, activation of NF-κB signaling is regulated by IKK in a negative way. The IκB family of inhibitory proteins generally holds the NF-κB pathway in an inactive status by sequestering NF-κB in the cytoplasm. Many extracellular stimuli could result in the activation of IKK [11]. Following stimulation, IKK is recruited to the combined signaling complex of late around membrane receptors, which affording a platform in phosphorylation and activation subsequently [12]. As many works have reported, the accurate regulation of IKK activity is an important procedure in activating NF-κB pathway [13]. Accordingly, the regulation of IKK recruitment is crucial in activating NF-κB induced by an extracellular stimulation.
Consistent with those previous works, our research also demonstrated that overexpression of S100A4 upregulated the level of IKK, followed by raising the activation Fig. 3 S100A4 enhanced the proliferation ability of MCSCs. a The cell proliferation growth curve using the Cell Counting Kit (CCK)-8 assay showed that MCSCs transfected with S100A4-vector exhibit a higher absorbance value. b The CCK-8 assay showed that MCSCs transfected with S100A4-siRNA exhibited a lower absorbance value. c MCSCs transfected with S100A4-vector caused an increased tumor volume. d MCSCs transfected with S100A4-siRNA caused a decreased tumor volume. Data is mean ± SD of three independent experiments. *P < 0.05 of NF-κB ultimately. In addition, overexpression of S100A4 upregulated some genes, IL-2 and TNF, which downstream of the NF-κB signaling pathway. IL-2 has been demonstrate to play an crucial role in tumor proliferation [14]. TNF-α takes a paramount role in proliferation during the development and progression in different kinds of cancer [15]. Accordingly, it would be attractive to detect whether IL-2 or TNF act a part in proliferation and disease progression in MCSCs.
There are some limitations to this study that needed to be taken into account. The IKK complex is formed by three subunits: IKKα, IKKβ, and IKKγ [16], and it would be better to test the individual subunits separately rather than IKK alone. In addition, our study did not test whether S100A4 enhanced the stemness of MB49 cells or not, and it would be better to overexpressed or knocked-down S100A4 not only in MCSCs but also in MB49 cells. Fig. 4 S100A4 promoted NF-κB transcriptional activity. a Luciferase reporter assay of NF-κB transcriptional activity showed that MCSCs transfected with S100A4-vector exhibited enhanced transcriptional activity of a NF-κB reporter gene. b Luciferase reporter assay showed that MCSCs transfected with S100A4-siRNA exhibited decreased NF-κB transcriptional activity. c Western blotting analysis of the expression of IKK; β-actin was used as a negative control. d Quantitative PCR analysis showed that the expression of IL-2 and TNF were up-regulated in MCSCs transfected with S100A4-vector. e Quantitative PCR analysis showed that the expression of IL-2 and TNF were down-regulated in MCSCs transfected with S100A4-siRNA. Data is mean ± SD of three independent experiments. * P < 0.05 Fig. 5 S100A4 interacted with IKK. MCSCs extracts were immunoprecipitated with mouse IgM or anti-S100A4 antibody, and then subjected to immunoblot analysis of the IKK protein

Conclusion
In summary, the present work showed that S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway. These results may offer a mechanisms for regulation of proliferation in MCSCs, and S100A4 maybe a hopeful therapeutic target for MCSCs.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
Author's contributions YTZ, CYW and LMC conceived and designed the study; YZ, XZ, YCG, DXH and JLZ performed experimental work; YTZ and CYW performed data analysis; YTZ and YZ prepared the manuscript; YTZ and LMC provided administrative support and funded experiments; all authors read and approved the final manuscript.

Ethics approval
All animal related experiments were authorized by the Ethics Committee of Southern Medical University (Contract 1,116,904).