Background
B Lymphocyte Stimulator (BLyS), a key member of the tumor necrosis factor superfamily, binds to three receptors: B-cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI), and B cell-activating factor receptor (BAFF-R). BLyS promotes survival of splenic immature transitional and mature B cells [
1]. Over-expression of BLyS has been associated with multiple myeloma (MM) [
2], Systemic lupus erythematosus (SLE) [
3] and B cell lymphoma [
4]. It has also been reported that this ligand/receptor dyad plays a critical role in the growth and survival of malignant plasma cells and B cells [
5]. Recent studies in ductal breast cancer patients have suggested a role of BLyS in the development of breast cancer. But its molecular mechanisms remain to be elucidated [
6].
Hypoxia plays a significant role in the pathogenesis of heart disease, cancer, neuron death, etc. [
7]. Inflammatory factors have been shown to be transcriptional regulated by hypoxia induced factor-1α (HIF-1α) or NF-kappa B in hypoxic conditions [
8]. The expression of BLyS is up-regulated by hypoxia, while the mechanism is still uncertain. We hypothesized that HIF-1α or NF-kappa B pathway might be responsible for the up-regulation. In addition, the inflammatory factors such as TNF-α, IL-1α lead to increased cancer cell migration [
9]. Therefore, the human breast cancer cell migration in response to BLyS and possible molecular mechanisms were explored in this study.
Methods
Cell line and cell culture
Breast cancer cell lines MDA-MB-435, MDA-MB-231 and MDA-MB-468 and B cell line Ramos were purchased from Cell Bank of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MDA-MB-435 cells and Ramos cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco, Grand Island, NY) and MDA-MB-231 cells and MDA-MB-468 cells were cultured in L-15 (Gibco, Grand Island, NY), containing 10% fetal bovine serum (Gibco, Grand Island, NY). The cells were used from three to six passages.
Materials
Anti-human BLyS and anti-human TACI antibodies were obtained from R&D Systems (Minneapolis, MN). Anti-human BAFF-R and anti-human BCMA antibodies were purchased from Abcam Inc (Cambridge, MA). Anti-Lamin B, anti-NF-kappa B p65 antibodies and donkey anti-goat secondary antibodies were obtained from Santa-Cruz (Santa Cruz, CA). Anti-Akt, anti-p-Akt (Ser 473), anti-p38 MAPK, anti-p-p38 MAPK (Tyr 182), anti-HIF-1α antibodies and goat anti-rabbit secondary antibodies were obtained from Cell Signaling (Beverly, MA) Anti-β-actin antibody was obtained from Sigma (St. Louis, MO). Goat anti-mouse peroxidase-conjugated antibody was from Sigma (St. Louis, MO). RevertAid™ first strand cDNA Synthesis Kit, TurboFect™ in vitro transfection reagent and restriction enzymes Kpn I and Xho I were purchased from Fermentas (Shenzhen, China), Dual-luciferase assay system, pGL3-basic (promoterless) luciferase vector and pRL-SV40 plasmid were obtained from Promega (San Francisco, California, USA). API-1, SB 202190, PX 12 and Caffeic acid phenethyl ester (CAPE) were from Tocris (Bristol, UK). Recombinant human BAFF was purchased from R&D system (Minneapolis, MN). SYBR Premix Ex Taq II and pMD® 18-T Vector were purchased from TAKARA (Dalian, China). DNA purification kit, QIAprep spin miniprep kit and QIAquick gel extraction kit were purchased from Qiagen (Shanghai, China).
Migration assay
Cell migration assay were performed in a double chamber transwell (Corning) with polycarbonate membranes (8.0 μm pore size). 8 × 104 cells were added to the upper chamber, treated with or without specific antagonists. Different concentrations of BLyS were added to the lower chamber. 1% FBS was used as a negative control. After incubation at 37 for 8 h in hypoxic or normoxic conditions, migrated cells were stained and counted in five randomly selected fields.
Quantitative real-time PCR
Total RNA was extracted using a Trizol reagent (Invitrogen Corporation, Grand Island, NY, USA) and was reversed to cDNA using RevertAid™ first strand cDNA Synthesis Kit according to the manufacturer's instructions. All primers were synthesized by Sangon Biotech (Shanghai, China) or TAKARA (Dalian, China). The primers used in Q-PCR are listed as follow: BLyS (GenBank, NM_006573.4) 5'- CGT GCC GTT CAG GGT CCA G-3' (forward) and 5'-TCG AAA CAA AGT CAC CAG ACT CAA T-3' (reverse); β-actin (GenBank, AF035119) 5'-CTC CTC CTG AGC GCA AGT ACT C-3' (forward) and 5'-CGG ACT CGT CAT ACT CCT GCT-3' (reverse). The gene levels in the resultant cDNAs were examined using the detection system (TAKARA) with SYBR-green as fluorescent dye enabling real time detection of PCR products according to the manufacturer's protocol. The relative expression levels of the genes were determined against β-actin levels in the samples.
Western blotting analysis
Total cell lysates were prepared in RIPA buffer supplemented with protease inhibitors. The proteins were fractionated by 8%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane (Bio-Rad). The membranes were probed with primary antibodies and then probed with relative secondary antibody. β-actin was used as a loading control.
Immunofluorescence
For BLyS and its three receptors staining in cells, MDA-MB-435, MDA-MB-231, MDA-MB-468 cells and Ramos cells were seeded on coverslips and cultured in 5% CO2 incubator. At 12 h after subculture, the plate with Ramos cells was centrifuged at 1, 000 rpm for 10 min and all the cells were fixed in 4% paraformadehyde for 10 min, washed and incubated with anti-BLyS antibody, anti-BAFF-R antibody, anti-BCMA antibody and anti-TACI antibody (1:100 dilution in 1% BSA-PBS). The cells were then incubated with relative FITC-conjugated secondary antibody (1:1000 dilution in 1% BSA-PBS) for 1 h at room temperature and with Hoechst 33342 for 30 minutes. The processed cells were mounted and fluorescence microscopy images were taken from five random fields in each slide using an inverted microscope (Olympus IX 71, Japan).
Plasmid construction, transient transfection and luciferase assays
pGL3-Basic luciferase vector, a plasmid of luciferase-reporter for human BLyS promoter (GenBank, NT_009952.14, -1082 to +118), was used to prepare the reporter constructs. DNA was extracted from MDA-MB-435 cells. BLyS promoter was amplified by PCR using following primers: 5'- GCG GTA CCA AGC CTG GGT CTG GAG TTC T-3' (forward) and 5'- GCC TCG AGC CTT TCT GCC TTT CTG CAT C-3' (reserve). Cloned fragments were recovered and ligated into pGL3-basic luciferase vector. DNA transfectants were prepared using QIAprep spin miniprep kit. Cells were cultured in 24-well plates to 70-80% of confluence, and then transfected with 1 μg of pGL3-Basic/BP or pGL3-Basic. Plasmid pRL-SV40 Renilla luciferase reporter (20 ng) was used as internal control. Supernatant was removed after 24 h and the cells were subsequently treated with CAPE for 12 h. Cell extracts were prepared and analyzed for luciferase activity using Dual-luciferase reporter assay system. Luciferase activity was expressed as relative luciferase activity (RLA).
Statistical analyses
The results are presented as the mean ± SD where applicable. Data were analyzed using GraphPad Prism 5.0 and the Student's t-test to determine the level of significance. Statistical difference was accepted at p < 0.05. (GraphPad Prism 5.0 was used to perform statistical analysis.)
Discussion
We initially demonstrated that hypoxia modulated the expressions of BLyS and its receptors in human breast cancer cell lines. Our data also indicated enhanced breast cancer cell migration in response to BLyS in vitro. BlyS, an immunopotentiator, might be a potential therapeutic target in breast cancer treatment base on this study, but care should be taken for using immunopotentiator in cancer treatment. Cancer tissues consist of large amounts of mesenchymal cells including fibroblasts, endothelial cells, adipocytes as well as inflammatory cells. As we know, inflammatory cells are a major source of BLyS, suggesting that BLyS may act as a connection between inflammatory cells and cancer cells. Furthermore, growing evidences show that cancer can evolve from chronic inflammation [
16]. Inflammation often accompanies cancer and recruits inflammatory cells which release plenty of inflammatory factors [
17]. In addition, cancer-associated fibroblasts mediate cancer-enhancing inflammation [
18]. Despite the relationship between inflammation and cancer is still poorly understood, it is believed that inflammatory cells are not the "street sweeper" in cancer tissues all along, but may trigger cancer progression [
19]. Many other processes, such as EMT, are involved in the transition from inflammation to cancer [
20]. It is prospected that an advanced breast cancer treatment could be developed if this field is much deeply explored.
Previous study reported that NF-kappa B played a key role in the transition from inflammation to cancer [
21]. Cancer with NF-kappa B activity usually shows increased resistance to chemotherapy [
22]. Furthermore, NF-kappa B is required for the expressions of many inflammatory genes [
23]. Curcumin inhibited BLyS expression by decreasing the nuclear translocation of p65 in B lymphocyte cell lines [
10]. Regarding HIF-1α, its protein level is extremely low in normoxic conditions. HIF-1α protein accumulates under hypoxia and regulates the target genes [
8]. Interestingly, NF-kappa B also activates angiogenesis encoding genes HIF-1α and VEGF [
24,
25]. Mobility of cancer cells and cytokines productions are altered by hypoxia. All of these alterations will finally lead to angiogenesis, matrix degradation and metastasis in cancer. Cancer cells adapt to hypoxia for survival [
26].
It is reported that BLyS suppresses the progression of several kinds of tumors and plays an important role in the development of immune system diseases [
27]. However, our results showed an enhanced migratory in response to BLyS. Several reports support the critical roles of Akt and p38 MAPK in cancer cell survival, migration, apoptosis and anti-apoptosis [
28,
29]. Previous research indicated that BLyS led to rapid phosphorylations of Akt in B cells [
30]. Our studies suggested that phosphorylations of Akt were essential for BLyS-enhanced cell migration in vitro.
Conclusion
In conclusion, the results found that BLyS caused the enhanced migration of human breast cancer cells, while BLyS was up-regulated by hypoxia. However, further studies are required to confirm the mechanisms of BLyS action and reveal the relationship between inflammation and breast cancer progression.
Acknowledgements
This work was supported by the Standardized Platform Construction and Scientific Application in New Technologies for New Drug Screening (No.2009ZX09302-002), the Study of Saponin Monomer of Dwarf Lilyturf Tuber (DT-13): A new Natural Anti-metastatic Drug Candidate (No.2009ZX09103-308) and the Research on Anti-tumor metastasis effectof YS-1 (No.81071841)
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JZ proposed the study and wrote the first draft. LS and SSL modified the draft. RPZ contributed to the design of the study. LQZ and DDF helped analyzed the data. LC, JL and WTS aided with manuscript preparation. LYZ and STY provided the necessary funding. All authors read and approved the final manuscript.