Activation of focal adhesion kinase enhances the adhesion and invasion of pancreatic cancer cells via extracellular signal-regulated kinase-1/2 signaling pathway activation

Abstract

Background: Interaction with integrin and focal adhesion kinase (FAK) regulates the cancer cell adhesion and invasion into extracellular matrix (ECM). In addition, phosphorylation of FAK correlates with the increase of cell motility and invasion. Adhesion and spreading of cancer cells on a variety of ECM proteins, including collagen type IV (Coll IV), leads to an increase in tyrosine phosphorylation and activation of FAK. In this study, we investigated the mechanism of activation of FAK and its downstream extracellular signal-regulated kinase (ERK)-1/2 signaling following stimulation by interleukin (IL)-1alpha and adhesion to ECM with subsequent enhancement of pancreatic cancer cell adhesion and invasion.

Results: In immunoblotting analysis, all three pancreatic cancer cell lines (AsPC-1, BxPC-3, and Capan-2) expressed the protein of FAK and beta1 integrin. Enhancement of FAK protein association with beta1 integrin when cells were plated on Coll IV was more increased by stimulation with IL-1alpha. Preincubation with anti-beta1 integrin antibody and FAK siRNA transfection inhibited the association of FAK with beta1 integrin of pancreatic cancer cells. FAK phosphorylation was observed by adhesion to Coll IV, furthermore, stronger FAK phosphorylation was observed by stimulation with IL-1alpha of pancreatic cancer cells adhered to Coll IV in time-dependent manner. Genistein, a tyrosine kinase inhibitor, markedly inhibited the FAK phosphorylation. IL-1alpha stimulation and Coll IV adhesion enhanced the activation of Ras, as evidenced by the increased Ras-GTP levels in pancreatic cancer cells. Activation of Ras correlated with the phosphorylation of ERK. While not statistical affecting the apoptosis of pancreatic cancer cells, IL-1alpha-induced adhesion and invasion on Coll IV were inhibited with FAK gene silencing by siRNA, beta1 integrin blocking, and inhibition of FAK phosphorylation. PD98059, a MEK inhibitor, also inhibited IL-1alpha-induced enhancement of adhesion and invasion in pancreatic cancer cells.

Conclusion: Our results demonstrated that activation of FAK is involved with the aggressive capability in pancreatic cancer through Ras/ERK signaling pathway. Based on our results, we suggest that the modification of IL-1, FAK, and integrins functions might be a novel therapeutic approach to aggressive spread of pancreatic cancer.

Figure 1

Expression of FAK and β₁ integrin in pancreatic cancer cells. (A) FAK and β₁ integrin protein expression in pancreatic cancer cell lines was determined in whole cell lysates by Western blotting analysis. Fifty micrograms of total cell lysates was separated on 10 % SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with antibodies against FAK and β₁ integrin. The β-actin Western blot served as a loading control. (B) Knockdown of FAK expression by siRNA was confirmed by immunoblotting in all three pancreatic cancer cells. Re-probing with an anti-β-actin antibody served as a control.

Figure 2

FAK and β₁ integrin subunit interaction in AsPC-1, BxPC-3, and Capan-2 cells. After incubating on Coll IV with 10 ng/ml IL-1α or 0.5 μg/ml anti-β₁ integrin antibodies for 24 h, cells were added (2 × 10⁵cells/well) to each well and incubated at 37°C and 5% CO₂ for 30 min. After removing unattached cells, total cell lysates were immunoprecipitated with antibodies against β₁ integrin subunit. Samples were resolved in 10 % SDS-PAGE gel under nonreducing conditions and transferred to polyvinylidene difluoride membranes. Membranes were then probed with anti-FAK or anti-β₁ integrin antibodies. Samples from cells incubated on 3 % BSA were served as control.

Figure 3

Phosphorylation of FAK in AsPC-1, BxPC-3, and Capan-2 cells. After incubating on Coll IV with 10 ng/ml IL-1α and/or 60 μM Genistein for 24 h, cells were added (2 × 10⁵ cells/well) to each well and incubated at 37°C and 5 % CO₂ for 15, 30, or 60 min. After removing unattached cells, cells were collected from each time point and lysed by lysis buffer and immunoprecipated with FAK antibody as described in Methods and materials. Effect of IL-1α and Genistein on Coll IV mediated phosphorylation of FAK was demonstrated. IP, immunoprecipitation; WB, Western blot.

Figure 4

Involvement of FAK phosphorylation and integrin signaling with adhesion and invasion of pancreatic cancer cells. (A) AsPC-1, BxPC-3, and Capan-2 cells were incubated with 10 ng/ml IL-1α, with 10 ng/ml IL-1α and 60 μM Genistein, with 10 ng/ml rIL-1α and 25 μM PD98059, or with 10 ng/ml rIL-1α and equivalent amounts of DMSO vehicle for 24 h. FAK siRNA transfected cells were cultured with 10 ng/ml IL-1α for 24 h. After incubating for 30 min with/without anti-β₁ antibody, cell adhesion assay was performed at 37°C for 30 min. Statistical significance was tested by one-way analysis of variance and post hoc test (Turkey Kramer multiple comparisons). The p-values indicate statistical significance between data in controls and each treatment. Bars indicate the s.d.*: p < 0.05. (B) After incubation for 30 min with/without antibody against β₁ integrin, AsPC-1, BxPC-3, and Capan-2 cells were cultured with 10 ng/ml IL-1α, with 10 ng/ml IL-1α and 60 μM Genistein, with 10 ng/ml rIL-1α and 25 μM PD98059, or with 10 ng/ml rIL-1α and equivalent amounts of DMSO vehicle for 24 h in the inner chamber coated with Coll IV. FAK siRNA transfected cells were cultured with 10 ng/ml IL-1α for 24 h in the inner chamber. Statistical significance was tested by one-way analysis of variance and post hoc test (Turkey Kramer multiple comparisons). The p-values indicate statistical significance between data in controls and each treatment. Bars indicate the s.d. *: p < 0.05.

Figure 5

Involvement of FAK and β₁ integrin subunit with the activation of Ras and ERK pathway in pancreatic cancer cells. Examination of Ras and downstream ERK activation was performed as described in Materials and Methods. Cell lysates were prepared according to the instructions provided in the Ras Activation Assay Kit, and affinity precipitation of GTP-bound Ras was performed using GST-tagged Raf-RBD. Levels of pull-downed Ras (Ras-GTP) were determined by anti-Ras immunoblotting.(A) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α for 15, 30, or 60 min. The time-dependent Ras activation and ERK1/2 phosphorylation by adhesion to Coll IV was demonstrated. Detection of total ERK 1/2 levels served as a loading control.(B) AsPC-1, BxPC-3, and Capan-2 cells were serum starved for 24 h and then attached to Coll IV with 10 ng/ml IL-1α in the presence or absence of inhibitory antibodies against β₁ integrin subunit for 30 min. FAK siRNA transfected pancreatic cancer cells were attached to Coll IV with 10 ng/ml IL-1α. Effects of IL-1α, FAK gene silencing, and β₁ integrin blocking on the activation of Ras/ERK signaling pathway in pancreatic cancer cells were demonstrated. Detection of total ERK 1/2 levels served as a loading control.