Focal adhesion kinases crucially regulate TGFβ-induced migration and invasion of bladder cancer cells via Src kinase and E-cadherin

Abstract

Focal adhesion kinase (FAK) is a non-receptor protein-tyrosine kinase that is triggered off by special extracellular signals such as some growth factors and integrins. FAK is found in cell-matrix attachment sites and implicated in cell migration, invasion, movement, gene expression, survival and apoptosis. In this study, we aimed to investigate whether FAK plays a role in invasion and migration of bladder cancer cells. Using an FAK-specific small interfering RNA (siRNA) and an FAK inhibitor PF-228, we found that inhibition of FAK tyrosine phosphorylation or knockdown of FAK suppressed invasion and migration of bladder cancer cells. Src is an important mediator of FAK-regulated migratory and invasive activity. Tyrosine phosphorylation of Src and FAK is mutually dependent and plays a key role in transforming growth factor beta (TGFβ)-induced invasion and migration. E-cadherin acts downstream of FAK and is a critical negative regulator in FAK-regulated invasion and migration of bladder cancer cells. These findings imply that FAK is involved in oncogenic signaling of invasion and migration, which can be a novel therapeutic target to treat patients with bladder cancer.

Keywords: E-cadherin; Src kinase; bladder cancer; focal adhesion kinase; migration/invasion; transforming growth factor beta.

Figure 1

FAK was knocked down by a selected siRNA in bladder cancer cells. Notes: T24 and 5637 cells were transfected with a selected siRNA. (A and B) Expression of FAK protein was measured by Western blotting. (C) Cell invasion and migration were measured by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; siRNA, small interfering RNA; TGFβ, transforming growth factor beta; conRNA, control RNA.

Figure 2

Effect of TGFβ on FAK phosphorylation and migratory and invasive potential of bladder cancer cells. Notes: T24 and 5637 cells were treated with 5 ng/mL TGFβ. (A and B) Expression of Src, pSrc, FAK, pFAK and E-cadherin was measured by Western blotting. (C) Cell invasion and migration were examined by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; TGFβ, transforming growth factor beta.

Figure 3

Suppression of FAK phosphorylation reduces TGFβ-induced migration and invasion of bladder cancer cells. Notes: T24 and 5637 cells were treated with 5 ng/mL TGFβ and 10 μM PF-228. (A and B) Expression of Src, pSrc, FAK and pFAK was examined by Western blotting. (C) Cell invasion and migration were measured by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; TGFβ, transforming growth factor beta.

Figure 4

The role of Src in FAK-regulated migratory and invasive activity of bladder cancer cells. Notes: T24 and 5637 cells were treated with 10 μM PP2 or siRNA against Src. (A, B, D and E) Expression of Src, pSrc, FAK and pFAK was measured by Western blotting. (C and F) Cell invasion and migration were examined by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; siRNA, small interfering RNA; TGFβ, transforming growth factor beta; conRNA, control RNA.

Figure 4

The role of Src in FAK-regulated migratory and invasive activity of bladder cancer cells. Notes: T24 and 5637 cells were treated with 10 μM PP2 or siRNA against Src. (A, B, D and E) Expression of Src, pSrc, FAK and pFAK was measured by Western blotting. (C and F) Cell invasion and migration were examined by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; siRNA, small interfering RNA; TGFβ, transforming growth factor beta; conRNA, control RNA.

Figure 5

The role of E-cadherin in FAK-regulated migratory and invasive activity of bladder cancer cells. Notes: T24 and 5637 cells were treated with siRNA against E-cadherin or FAK. (A–D) Expression of E-cadherin, FAK and pFAK was measured by Western blotting. (E and F) Cell invasion and migration were measured by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; siRNA, small interfering RNA; TGFβ, transforming growth factor beta; E-cad, E-cadherin; conRNA, control RNA.

Figure 5

The role of E-cadherin in FAK-regulated migratory and invasive activity of bladder cancer cells. Notes: T24 and 5637 cells were treated with siRNA against E-cadherin or FAK. (A–D) Expression of E-cadherin, FAK and pFAK was measured by Western blotting. (E and F) Cell invasion and migration were measured by Transwell assays. Scale bar, 200 μm. Abbreviations: FAK, focal adhesion kinase; siRNA, small interfering RNA; TGFβ, transforming growth factor beta; E-cad, E-cadherin; conRNA, control RNA.