A synergistic interaction between transcription factors nuclear factor-κB and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion

Abstract

Background: The transcription factor nuclear factor-κB (NF-κB) has been implicated in gastric cancer metastasis, but the underlying molecular mechanisms remain unclear. We investigated the role of the interaction between NF-κB and signal transducers and activators of transcription 3 (STAT3) in controlling metastatic potential of gastric cancer cells.

Methods: Immunohistochemistry for NF-κB p65 (RelA), phospho-Tyr705-STAT3 (pSTAT3), or matrix metalloproteinase 9 (MMP9) was performed on tissue array slides containing 255 gastric carcinoma specimens. NF-κB inhibition in SNU-638 and MKN1 gastric cancer cell lines were performed by transduction with a retroviral vector containing NF-κB repressor mutant of IκBα, and STAT3 was silenced by RNA interference. We also did luciferase reporter assay, double immunofluorescence staining and immunoblotting. Cell migration and invasion were determined by wound-healing assay and invasion assay, respectively.

Results: NF-κB and STAT3 were constitutively activated and were positively correlated (P=0.038) in gastric cancer tissue specimens. In cell culture experiments, NF-κB inhibition reduced STAT3 expression and activation, whereas STAT3 silencing did not affect NF-κB activation. Moreover, both NF-κB inhibition and STAT3 silencing decreased gastric cancer cell migration and invasion in a synergistic manner. In addition, both NF-κB activation and STAT3 activation were positively correlated with MMP9 in gastric cancer tissues (P=0.001 and P=0.022, respectively), decreased E-cadherin expression and increased Snail and MMP9 expressions in cultured cells.

Conclusion: NF-κB and STAT3 are positively associated and synergistically contribute to the metastatic potential of gastric cancer cells. Thus, dual use of NF-κB and STAT3 inhibitors may enhance the efficacy of the anti-metastatic treatment of gastric cancer.

Figure 1

Representative immunohistochemical features of NF-κB, pSTAT3 and MMP9 in human gastric cancer specimens. (A) NF-κB RelA (p65)-positive. (B) pSTAT3-positive. (C) MMP9-positive. (D) Negative control treated without the primary antibodies. Scale bars = 100 μm.

Figure 2

Effect of downregulation of NF-κB p65 on the STAT3 activation in gastric cancer cells and vice versa. (A-D) SNU-638 cells were infected with either MFG.IκBαM.IRES.puro (IκBαM) retroviral vector or empty (EGFP) vector. (A) The protein expressions of total NF-κB p65 (RelA), phospho-Ser536-NF-κB p65 (pRelA), total STAT3 and phosphor-Tyr705-STAT3 (pSTAT3) were determined by immunoblotting. (B) Cells were transiently co-transfected with pNF-κB-luciferase and a β-galactosidase vector, and effect of IκBαM overexpression on NF-κB transcriptional activity was determined by luciferase reporter assay. Each bar represents the mean ± SD (n = 6). * P < 0.05 versus control cells infected with an empty vector. (C) Cells were transiently co-transfected with pSTAT-luciferase and a β-galactosidase vector, and effect of IκBαM overexpression on STAT transcriptional activity was determined by luciferase reporter assay. Each bar represents the mean ± SD (n = 5). * P < 0.05 versus control cells infected with an empty vector. (D) 4’,6’-Diamidino-2-phenylindole staining (blue) and double immunofluorescence staining showing the nuclear colocalization of pRelA (red) and STAT3 (green). (E and F) SNU-638 cells were transiently transfected with control siRNA (siCtrl) or STAT3 siRNA (siSTAT3). (E) The effects of STAT3 silencing on the protein expressions of total STAT3, pSTAT3, total RelA and pRelA were determined by immunoblotting. (F) Luciferase reporter assay was performed to show the regulation of NF-κB activation by STAT3 after STAT3 silening. Each bar represents the mean ± SD (n = 6). * P < 0.05 versus control siRNA-transfected cells. (G) Immunoblotting for pRelA and pSTAT3 in MKN1 cells after IκBαM overexpression. (H) Immunoblotting for total STAT3 and pSTAT3 in MKN1 cells after STAT3 silencing.

Figure 3

Effect of IκBαM overexpression on metastatic potential of gastric cancer cells. SNU-638 (A-C) cells and MKN1(D and E) cells were infected with either IκBαM retroviral vector or an empty (EGFP) vector. (A and D) Confluent cell monolayers were wounded and cell migration was analyzed by wound-healing assay 48 h after scratching. (B and E) Cells were seeded in the upper chamber coated with Matrigel and invasion ability was measured 48 h after cell plating. Results were calculated as percentages relative to control cells infected with an empty vector. Each bar represents the mean ± SD (n = 4). * P < 0.05 versus control cells infected with an empty vector. (C) The protein expressions of E-cadherin, Snail, MMP9 and β-actin expressions were determined by immunoblotting.

Figure 4

Effect of STAT3 silencing on metastatic potential of gastric cancer cells. SNU-638 cells (A-C) and MKN1 cells (D and E) were transiently transfected with control siRNA (siCtrl) or STAT3 siRNA (siSTAT3). (A and D) Cell migration capacity was evaluated by wound-healing assay 48 h after scratching. (B and E) Cell invasion ability was measured 48 h after cell plating. Results were calculated as percentages relative to control siRNA-transfected cells. * P < 0.05 versus control cells (n = 4). (C) The protein expressions of E-cadherin, Snail, MMP9 and β-actin in SNU-638 cells were determined by immunoblotting after STAT3 silencing.

Figure 5

Synergistic effect of NF-κB and STAT3 on metastatic potential of gastric cancer cells. Stable SNU-638 cells infected with either IκBαM retroviral vector or an empty (EGFP) vector were transiently co-transfected with either control siRNA (siCtrl) or STAT3 siRNA (siSTAT3). (A) Whole cell lysates (WL) and nuclear extracts (NE) were obtained 24 h after co-transfection and protein levels for pRelA, pSTAT3 and β-actin or TFIIB were measured by immunoblotting. (B) Effect of interaction of NF-κB and STAT3 on cell migration capacity was evaluated by wound-healing assay 48 h after scratching. (C) Effect of interaction of NF-κB and STAT3 on cell invasion ability was measured by invasion assay 48 h after cell plating. Results were calculated as percentages relative to controls. Each bar represents the mean ± SD (n = 4). * P < 0.05 and ** P < 0.005. (D) The protein expressions of E-cadherin, Snail and β-actin in SNU-638 cells were determined by immunoblotting after co-transfection of IκBαM and siSTAT3.