Bruton's agammaglobulinemia tyrosine kinase (Btk) regulates TPA‑induced breast cancer cell invasion via PLCγ2/PKCβ/NF‑κB/AP‑1‑dependent matrix metalloproteinase‑9 activation

Abstract

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B‑lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen‑activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor‑κB (NF‑κB) activation, suggesting an anti‑metastatic effect of BTK inhibitors on MCF‑7 cells that leads to the downregulation of matrix metalloproteinase (MMP)‑9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti‑metastatic activity of BTK inhibitors was examined in MCF‑7 cells focusing on MMP‑9 expression in 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑stimulated MCF‑7 cells. The expression and activity of MMP‑9 in MCF‑7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX‑774 (10 µM)] significantly attenuated TPA‑induced cell invasion and migration in MCF‑7 cells and inhibited the activation of the phospholipase Cγ2/PKCβ signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP‑9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA‑induced MMP‑9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF‑κB/activator protein‑1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP‑9 expression in MCF‑7 cells.

Keywords: Bruton's agammaglobulinemia tyrosine kinase; matrix metalloproteinase‑9; protein kinase C; MCF‑7 cells.

Figure 1.

Effect of BTK inhibitors on the MCF-7 cells by TPA-induced MMP-9 expression. To examine the cytotoxicity of BTK inhibitors, cells were cultured in 96-well plates to 70% confluency and various concentrations of (A) ibrutinib or (B) CNX-774 were added to the cell culture and incubated for 24 h. The EZ-Cytox Enhanced Cell Viability Assay Kit was used to determine cell viability. The optical density of the control was considered 100%. Data represent the mean ± SEM of three independent experiments. ^#P<0.05 compared with 0 µM of BTK inhibitors at 24 h. MCF-7 cells were treated with BTK inhibitors in the presence or absence of TPA for 24 h. Cell lysates were analyzed by western blotting using an anti-MMP-9 antibody. The blot was re-probed with an anti-β-actin antibody to confirm equal loading. Conditioned medium was prepared and used for gelatin zymography (C). MMP-9 mRNA levels were analyzed by quantitative PCR and GAPDH was used as an internal control Data are the mean ± SEM of three independent experiments. ^#P<0.01 vs. untreated control; *P<0.01 vs. TPA. (D) The blot was re-probed with an anti-β-actin antibody to confirm equal loading.

Figure 2.

Effect of BTK inhibitors on TPA-induced intracellular signaling pathways in MCF-7 cells. The cells were pretreated with BTK inhibitors for 1 h and then stimulated with TPA. Cell lysates were analyzed by western blotting using anti-p-PLCγ2 and anti-PLC γ2 antibodies (A). MCF-7 cells were pretreated with PLC inhibitors for 1 h and then stimulated with TPA. Cell lysates were subjected to SDS-PAGE and analyzed by western blotting using an anti-MMP-9 antibody (B). Data are the mean ± SEM of three independent experiments. ^#P<0.01 vs. untreated control; *P<0.01 vs. TPA. The blot was reprobed with an anti-β-actin antibody to confirm equal loading.

Figure 3.

BTK inhibitors decrease TPA-induced activation of the PKC, MAPK, and IKK signaling pathways in MCF-7 cells. The cells were pretreated with BTK inhibitors for 1 h and then stimulated with TPA. The cell lysates were analyzed by western blotting of the PKCα, PKC δ, and PKCβ levels in the cytosolic and membrane fractions. The blot was reprobed with an antibody against Na k-ATPase to confirm equal loading (A). Cells (1×10⁶) were pretreated with BTK inhibitors and then stimulated with TPA for 15 min. Cell lysates were assessed by western blotting using antibodies against p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and their phosphorylated forms (B), and the levels of p-IκBα, IκBα, p-IKKαβ, IKKα, and IKKβ were determined (C). The blot was reprobed with an anti-β-actin antibody to confirm equal loading. Data are the mean ± SEM of three independent experiments. ^#P<0.01 vs. untreated control; *P<0.01 vs. TPA. The blot was reprobed with an anti-β-actin antibody to confirm equal loading.

Figure 4.

BTK inhibitors block TPA-induced NF-κB and AP-1 activation in MCF-7 cells. Cells (2×10⁶) were pretreated with BTK inhibitors and then stimulated with TPA for 3 h. Western blot analysis was performed to determine the nuclear levels of NF-κB (p65 and p50) and AP-1 (p-c-Jun). The blot was reprobed with an antibody against proliferating cell nuclear antigen to confirm equal loading (A). NF-κB-Luc or AP-1-Luc reporter and Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells were treated with BTK inhibitors and stimulated with TPA and the promoter activity of NF-κB and AP-1 was measured with dual luciferase reporter assays (B and C). Data are the mean ± SEM of three independent experiments. ^#P<0.01 vs. untreated control; *P<0.01 vs. TPA.

Figure 5.

BTK inhibitors suppress TPA-induced Matrigel invasion and migration of MCF-7 cells. The invasive ability of cells following treatment with BTK inhibitors and TPA was determined using a Matrigel invasion assay (magnification, ×5). BTK inhibitors block 12-O-TPA-induced Matrigel invasion of MCF-7 cells. Cells (3×10⁵) were seeded onto the upper chamber of Matrigel-coated wells and BTK inhibitors and TPA were placed in the bottom wells. After 24 h, the cells attached to the bottom of the filter were fixed, stained, and counted. The error bars are representative of three independent experiments performed in triplicate, ^#P<0.005 vs. untreated control; *P<0.005 vs. control + TPA.

Figure 6.

BTK siRNA inhibits TPA-induced MMP-9 expression and Matrigel invasion and migration of MCF-7 cells. BTK expression was significantly suppressed in MCF-7 cells transfected with BTK siRNA. BTK levels were analyzed by quantitative PCR with GAPDH as an internal control (A). Transfected cell lysates were analyzed by western blotting using an anti-MMP-9 antibody (B). Transfected cell lysates were subjected to Matrigel invasion and migration analyses (magnification, ×5) (C). The error bars are representative of three independent experiments performed in triplicate. ^#P<0.005 vs. control; *P<0.005 vs. control + TPA.

Figure 7.

Schematic signaling pathway of the effect of downregulation of BTK expression on TPA-induced MMP-9 expression in MCF-7 cells. Inhibition of BTK expression attenuates TPA-induced MMP-9 expression and metastasis by blocking NF-κB and AP-1 activation via the PLCγ2/PKC/MAPK and IKK signaling pathways. →, activated; ⊥ means inhibited.