The JAM-B/c-src/MMP9 pathway is associated with progression and regulates the invasion of pancreatic cancer

Abstract

Junctional adhesion molecule B (JAM-B) is a multifunctional transmembrane protein that plays an important role in tumor progression. JAM-B is significantly upregulated in gastric cancer, melanoma cell metastasis and oral squamous cell carcinoma. JAM-2 may also function as a putative tumor suppressor in the progression and metastasis of colorectal cancer. The inconsistency of the results in different cancers has led to uncertainty regarding the role of JAM-B in carcinogenesis. For this purpose, the expression levels of JAM-B in pancreatic cancer (PanCa) tissues were associated with T stage and lymph node involvement with significant differences. A relatively high expression of JAM-B was found in PanCa cell lines by immunohistochemistry and western blot analysis. By cell transfection, JAM-B was silenced in tumor cell lines to determine cell invasion and migration abilities. Scratch wound assays and Transwell assays revealed that shJAM-B significantly decreased Panc-1 cell migration and invasion. Experiments were also conducted using a subcutaneous PanCa nude mouse model. A significant difference in tumor diameter at the injection site was found between the control group and the JAM-B low expression group. The expression levels of c-Src and MMP9 were also significantly reduced compared to that in the control group by immunohistochemistry. In conclusion, our results suggest that JAM-B secreted by cancer cells can promote progression and invasion in PanCa by upregulating the c-Src signal and related downstream proteins.

Keywords: JAM-B; c-src; invasion; pancreatic cancer; progression.

Figure 1

Expression of JAM-B in pancreatic cancer (PanCa) tissues and its association with patient progression. (A) Expression of JAM-B in stage I, II, III and IV PanCa. Arrowheads show the cells of interest. (B) Relative protein levels of JAM-B are shown as bar diagrams (*P<0.05, compared with stage I).

Figure 2

Expression of JAM-B in pancreatic cancer (PanCa) cell lines, and the effect of the silencing JAM-B on cell signal pathway. (A)Western blot was used to detect the protein level of JAM-B and JAM-C in 5 PanCa (BxPC-3, Panc-1, AsPC-1, SW1990 and CFPAC-1 cell lines. (B) Western blot analysis with IMAGE J was used to detect the protein level of JAM-B and JAM-C in 5 PanCa cells. The expression of each protein in cells was determined following normalization with a loading control, GAPDH. (C) Transfection efficiency of JAM-B in PanCa cell lines following transfection with JAM-B shRNA at the protein level determined by western blot analysis and the downstream pathway was also analyzed in BxPC-3 and Panc-1. (D and E) Western blot analysis with IMAGE J was used to detect the protein level of JAM-B and the downstream pathway. The expression of each protein in cells was determined following normalization with a loading control, GAPDH. Relative protein levels of JAM-B are shown as bar diagrams (*P<0.05, compared with control and NSC).

Figure 3

Effects of silencing JAM-B on cell migration in PanCa cell lines. (A) Cell scratch wound assay was carried out to detect the effects of the silencing of JAM-B on the migration of PANC-1at 0h and 72h. (B) Relative migration levels of PANC-1 are shown as bar diagrams (*P<0.05, compared with control and Nsc). (C) Cell scratch wound assay was carried out to detect the effects of the silencing of JAM-B on the migration of BxPC-3 at 0h and 48h. (D) Relative migration levels of BxPC-3 are shown as bar diagrams (*P<0.05, compared with control and Nsc).

Figure 4

Effects of silencing JAM-B on cell invasion in PanCa cell lines. (A) Transwell assay was performed to detect the effects of the silencing of JAM-B on invasion of PANC-1 and BxPC-3. Arrowheads show the cells of interest. (B) Relative invasion cells are shown as bar diagrams in PANC-1. (C) Relative invasion cells are shown as bar diagrams in BxPC-3. Data are shown as the means ± SD from 3 independent experiments. (*P<0.05, compared with control and Nsc).

Figure 5

Effects of silencing JAM-B on cell invasion in PanCa subcutaneous nude mice model. (A and B) The tumor diameters increased more slowly in the JAM-B low expression groups than that in Nsc group in PANC-1. (C and D) The tumor diameters increased more slowly in the JAM-B low expression groups than that in Nsc group in BxPC-3. (*P<0.05, compared with NSC).

Figure 6

Relationship of JAM-B-c-src-MMP9 axis and pancreatic cancer progression in vivo. (A) Immunohistochemical staining of JAM-B, c-src and MMP9 were used to detect the protein level of PANC-1 and BxPC-3. (B and C) Immunohistochemical staining analysis with IMAGE J was used to detect the protein level of JAM-B and the downstream pathway of PANC-1(B) and BxPC-3(C). Relative protein levels of JAM-B are shown as bar diagrams (*P<0.05, compared with control and Nsc).