Background
Human psoriasin (S100A7) is a small calcium binding protein, which has been shown to be predominantly expressed in high-grade ductal carcinoma
in situ[
1‐
5]. In addition, its expression is significantly associated with estrogen receptor (ER) α-negative and nodal metastasis in invasive ductal tumors [
1,
3‐
5]. Furthermore, S100A7 expression is associated with increased angiogenesis [
6]. S100A7 has been shown to modulate tumor growth by activating several signaling pathways [
7‐
9]. Recently, it has reported that S100A7 enhances breast tumor growth and metastasis in MDA-MB-468 ERα (−) cells by activating proinflammatory and metastatic pathways [
10].
Nuclear factor-kappa B (NF-κB) is an essential transcription factor that not only modulates cellular responses to stress but also plays a pivotal role in inflammation, immunity, cell cycle growth and survival. NF-κB regulated genes have been documented to be involved in cellular proliferation and invasion along with tumor related angiogenesis and lymph angiogenesis [
11‐
13]. Dysregulation of NF-κB associated pathways are seen in multiple malignancies. Its constitutive activation in the clinically aggressive and prognostically poor ER-negative, Her2-neu positive and inflammatory breast cancer could form the basis for its evolution as a potential prognostic and therapeutic target [
14].
In MDA-MB-468 cells, inhibition of NF-κB by adenovirus-mediated expression of a dominant negative NF-κB or by a proteasome inhibitor, MG132, decreased the vascular endothelial growth factor (VEGF) mRNA and prevented angiogenesis [
15].
In addition, targeting NF-κB signaling may also inhibit breast cancer cell invasion through decreasing matrix metalloproteinase 9 (MMP-9) expression [
16]. Emberley
et al. [
17] has recently reported that overexpression of S100A7 in MDA-MB-468 cell lines promotes survival under conditions of anchorage-independent growth. This effect is paralleled in part by increased activity of NF-κB, which is known to mediate prosurvival pathways. The data suggested that there is significant correlation between S100A7 and NF-κB activity.
S100A7 is among the most highly expressed genes in preinvasive breast cancer, is a marker of poor survival when expressed in invasive disease, and promotes breast tumor progression in experimental models [
1‐
3,
5,
10]. However, it has not been established whether S100A7 may be a target for breast cancer therapy. In the present study, we explored the effect and mechanism of S100A7 silencing on growth and invasion in MDA-MB-468 cell lines.
Methods
Cell culture and reagents
MDA-MB-468 cells were obtained from American Type Culture Collection (ATCC, Shanghai Bioleaf Biotech Co., Ltd. Shanghai, China). The MDA-MB-468 cells were grown in (D)MEM with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were grown in a humidified atmosphere of 5% CO2 at 37°C. Cells were seeded in 75 cm2 flasks with 15 ml of growth medium, unless otherwise mentioned. Antibodies were obtained as follows: anti-S100A7, MMP-9, VEGF, NF-κB p65 and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All secondary antibodies were obtained from Pierce (Rockford, IL, USA). S100A7 small interfering RNA (siRNA),VEGF siRNA, MMP-9 siRNA and siRNA control were obtained from Santa Cruz Biotechnology. LipofectAMINE 2000 was purchased from Invitrogen (Burlington, ON, Canada).
Plasmid construction and transfections
cDNA of S100A7 (OriGene Technologies, Shanghai, China) was subcloned into pcDNA3.1. MDA-MB-468 cells were transfected with pcDNA3.1-S100A7 cDNA or pcDNA3.1 using LipofectAMINE 2000 reagent according to the manufacturer’s instructions and stable clones were generated using G418 (400 mg/mL)(CAMBREX (Walkersville, MD). Stably transfected MDA-MB-468 cells (pcDNA3.1-S100A7 cDNA/MDA-MB-468 and pcDNA3.1/MDA-MB-468) were transfected with MMP-9 siRNA (2 μM) or VEGF siRNA (2 μM) and control siRNA for 48 hours using LipofectAMINE 2000.
Electrophoretic mobility shift assay
Nuclear extracts were prepared from cells using the NE-PER nuclear and cytoplasmic extraction reagents kits according to the manufacturer’s directions (Pierce Biotech). For NF-κB DNA binding, the 10,000 cpm of the 22-bp oligonucleotide 5′-AGTTGAGGGGACTTTCCCAGGC-3′ containing the NF-κB consensus sequence that had been labeled with [−32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase was added to 15 μg nuclear extract. The reaction was allowed to proceed for 30 minutes at room temperature. For cold competition experiments, unlabeled NF-κB oligonucleotide as nonspecific competitor gels were dried and directly exposed to a B-1 phosphorimaging screen and visualized with a GS-250 Molecular Imaging System [Bio-Rad (Hercules, CA, USA)]. TATA binding protein (TBP) was used as a nuclear loading control (Abcam Inc. Beijing, China).
MMP-9 activity assay
MMP-9 activities in whole cell lysates were assessed using the colorimetric Biotrak MMP-9 activity assay (Amersham Biosciences, Shanghai, China) in accordance with the manufacturer’s instructions. Optical densities were quantified using a Vmax microplate spectrophotometer at a wavelength of 405 nm, referenced to 650 nm. Three samples were used for each experimental condition and experiments were performed in triplicate and mean values calculated.
VEGF assay
The culture medium of the cells in different groups grown in six-well plates was collected. After collection, the medium was spun at 800 × g for 3 minutes at 4°C to remove cell debris. The supernatant was either frozen at −20°C for VEGF assay later or assayed immediately using commercially available ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA).
Western blot analysis
Cell lysates were prepared from the MDA-MB-468 cells in different conditions and subjected to Western blot analysis using an anti-MMP-9, VEGF, S100A7, NF-κB p65 and an anti-β-actin monoclonal antibody for normalization of protein loading. Immunoreactivity was detected using the ECL chemiluminescence system (Amersham, Piscataway, NJ, USA) and quantified using an imaging densitometer (Model GS-670; Bio-Rad, Hercules, CA, USA).
Cell proliferation assay
MDA-MB-468 cells were stably transfected with pc. DNA3.1-S100A7 or pc. DNA3.1. Then the cells were seeded at a density of 5 × 103 cells per well on 96-well plates in growth medium supplemented with 10% serum and cultured in a humidified chamber at 37°C for up to 3 days. Viable cells were identified using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 200 ul sterile MTT dye (5 mg/ml, Sigma, USA) was added. After 4 hours incubation at 37°C in 5% CO2, the MTT medium mixture was removed and 200 ul of dimethyl sulfoxide (DMSO) was added to each well, as was reported. Absorbance was measured at 540 nm using a multi-well spectrophotometer (Thermo Electron, Andover, USA). All experiments were carried out in triplicate.
Determination of cell mobility
The invasiveness of MDA-MB-468 cells was tested after transfection as previously described. The cells (1 × 106/mL) were added to the upper wells coated with Matrigel (1 mg/mL; Collaborative Research, Inc., Boston, MA, USA) with serum-free medium containing 25 ug/mL fibronectin as a chemoattractive agent in the lower wells. After a 24-hour incubation period, cells that migrated through the filters into the lower chamber were counted by the number of cells on the lower side of the membrane in five random fields after staining with Hema-3 kit(Fisher Scientific (Nepean, Canada).
In vitro cell invasion assay using Matrigel
Cell invasion assays were performed using Transwell membrane filter inserts with 8-mm pore size (Corning Costar, Cambridge, MA, USA). The upper surface of the Transwell membrane was coated with 250 mg/ml of growth factor-reduced Matrigel matrix(Becton Dickinson, Bedford, MA, USA) overnight at 4°C, rehydrated once with 0.1% BSA in (D)MEM for 1 hour at room temperature, and then placed in the upper compartment of six-well tissue culture plates.
MDA-MB-468 cells in different conditions were removed from tissue culture flasks by a short exposure to 5 mM ethylenediaminetetraacetic acid (EDTA) and washed once in PBS. Then 2 × 105 cells in serum-free medium containing 0.1% BSA were added to each Transwell chamber and allowed to migrate toward the underside of the membrane for 24 hours in the lower chamber as a chemoattractant. After the cells were fixed in 3.5% paraformaldehyde, cells on the upper surface of the membrane were removed by wiping with a cotton swab, and membranes were mounted onto glass slides. The relative number of invasion was determined by counting the number of invading EGFP(enhanced green fluorescent protein)-positive cells. The number of invading cells transfected with empty vector was assigned a value of 1.0 in each experiment. Twenty random fields/membrane were counted for each assay. Each determination represents the average of three separate experiments.
Statistical analysis
One-way analysis of variance was used to compare means. The level of statistical significance was set at P <0.05, and all statistical calculations were carried out using SPSS.11 software (SPSS Inc., Chicago, IL, USA).
Discussion
S100A7 is a member of the S100 family of proteins, which have been associated with preinvasive ductal carcinoma in situ (DCIS). Persistent expression of S100A7 occurs in some invasive cancers and is associated with poor prognostic factors [
1]. Persistent S100A7 expression also occurs in a subset of invasive breast carcinomas and is linked to worse clinical outcome [
5]. S100A7 has been shown to be overexpressed in breast cancers at sites of necrosis in tumor tissues [
1,
7], as well as in the nasal fluid during allergic inflammatory reactions [
17]. Although a number of putative functions have been proposed for S100A7, its biological role, particularly in breast cancer, remains to be defined. S100A7 has been reported to increase NF-κB activity levels in MDA-MB-468 cells [
7], and NF-κB-regulated genes have been documented to be involved in cellular proliferation and invasion along with tumor related angiogenesis and lymphangiogenesis [
18] but not much is known about the relation between S100A7 and NF-κB as well as its role in NF-κB-induced signaling.
In this study, we characterized the tumor-enhancing effects of S100A7 in MDA-MB-468 breast cancer cells.
We have observed that S100A7 overexpression by cDNA transfection increased tumor cell invasion and promoted proliferation but down-regulation of S100A7 by siRNA decreased cell invasion and inhibited proliferation. S100A7 overexpression increased NF-κB DNA binding activity and NF-κB, MMP-9 and VEGF expression levels and activity, but down-regulation of S100A7 by siRNA decreased NF-κB DNA binding activity and NF-κB, MMP-9 and VEGF expression levels and activity. Taken together, these results further support the view that the downregulation of S100A7 could be an effective approach for the inactivation of NF-κB and down-regulation of its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and proliferation.
NF-κB activation has also been reported to be associated with metastatic phenotype and to regulate the expression of a variety of important genes in some cellular responses, including metastasis related genes such as VEGF and MMP-9 [
19‐
27]. Because NF-κB plays important roles in many cellular processes, studies on the interaction of NF-κB activation with other cell signal transduction pathways, including the S100A7 pathway, have received increased attention in recent years. S100A7 has also been reported to crosstalk with the NF-κB pathway [
7]. S100A7 strongly induces NF-κB promoter activity and induces NF-κB DNA-binding activity [
7]. Activation of NF-κB leads to up-regulation of several downstream target genes, including MMP-9 and VEGF. Thus, the downregulation of S100A7 results in lower NF-κB activity and its downstream targets. Therefore, it is possible that S100A7-induced cell invasion is partly due to activation of the NF-κB pathway.
In the present study, we showed that S100A7 overexpression increased NF-κB expression and NF-κB DNA-binding activity and concomitantly increased the expression and activation of MMP-9 and VEGF. However, downregulation of S100A7 decreased NF-κB expression and NF-κB DNA-binding activity and concomitantly inhibited the expression and activation of MMP-9 and VEGF.
Because we observed that S100A7 overexpression promoted MMP-9 expression, we tested the effects of S100A7 on the invasion of MDA-MB-468 cells. We found that S100A7 overexpression promoted cell invasion of MDA-MB-468 cells, and down-regulation of S100A7 inhibited the cell invasion of MDA-MB-468 cells. These results are consistent with MMP-9 data, showing that down-regulation of S100A7 could inhibit cancer cell invasion partly through downregulation of MMP-9 and vice versa.
There was a trend toward an association between expression of VEGF and distant metastasis [
28]. In this study, consistent with our invasion data, we found a significant reduction in the expression of VEGF by down-regulation of S100A7 and a significant increase in the expression of VEGF by up-regulation of S100A7. It is well accepted that the expression of MMP-9 and VEGF is regulated by NF-κB [
20‐
28]. Down-regulation of S100A7 inhibited the NF-κB reporter gene and gene products, such as those involved in cell invasion and
vice versa. Based on our results, we speculate that one possible mechanism by which S100A7 induces invasion is due to activation of NF-κB DNA binding activity, which leads to up-regulation of NF-κB target genes, such as MMP-9 and VEGF.
On the basis of previous results that S100A7 has been shown to enhance tumorigenicity in ERα(−) cells, our study also shows that S100A7 overexpression promoted proliferation; however, S100A7 silencing inhibited proliferation in the MDA-MB-468 cells. On the basis of our results, we propose a hypothetical pathway by which S100A7 may promote cell proliferation of MDA-MB-468 cells partly through the NF-κB signaling pathway. However, further in-depth studies are needed to investigate the precise molecular mechanism regarding the cause and effect relationship between S100A7 and NF-κB.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LHM and YQF conceived and designed the study and participated in the part of the experimental work. WL and ZKJ participated in the experimental work and helped draft the manuscript. WXG participated in the analysis and interpretation of the data. CZ and YQF participated in the interpretation of data and revising of the manuscript. All authors read and approved the final manuscript.