Activation of MEK/ERK and PI3K/Akt pathways by fibronectin requires integrin alphav-mediated ADAM activity in hepatocellular carcinoma: a novel functional target for gefitinib

Abstract

We have shown that the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839) inhibits the development of intrahepatic metastases of hepatocellular carcinoma CBO140C12, and EGFR transactivation by tumor necrosis factor-alpha is a possible target of gefitinib. In the present study, we focused on the fibronectin (FN)-dependent signaling pathway to further elucidate the antimetastatic activity of gefitinib in CBO140C12 cells. We initially observed that FN induced activation of extracellular signal-regulated kinase (ERK), p38 and Akt, as well as cell proliferation and CBO140C12 cell invasion. These responses were mediated by EGFR tyrosine kinase, because gefitinib inhibited these effects of FN. FN-induced ERK, p38 and Akt activation was partly blocked by the Arg-Gly-Asp (RGD)-pseudo-peptide FC-336, anti-alphav integrin antibody RMV-7, the broad-spectrum matrix metalloprotease inhibitor GM6001 and the broad spectrum a disintegrin and metalloprotease (ADAM) inhibitor TAPI-1. But these inhibitors had no effect on EGF-induced signaling pathways, suggesting that integrins and ADAM may be upstream components of EGFR in these responses. These results suggest that FN-induced activation of ERK, p38, Akt, cell proliferation and invasion was mediated, at least in part, via integrins, ADAM and EGFR, and that this FN-induced signaling pathway might be involved in the antimetastatic activity of gefitinib.

Figure 1

Inhibition of fibronectin (FN)‐induced cell proliferation and invasion by gefitinib. (a) CBO140C12 cells were seeded in culture medium containing 0.5% fetal bovine serum onto 50 µg/mL FN‐coated wells in the absence or the presence of 1 µM gifitinib. After a 48‐h incubation, proliferative activity was determined. These data are presented as percentage of the control for mean absorbance. (b) CBO140C12 cells were seeded onto the filters, which were precoated with Matrigel (5 µg) on the upper surface with or without FN (1 µg) on the lower surface, in the presence of 1 µM gefitinib. After a 12‐h incubation, the cells that had invaded into the lower surface were counted by crystal violet staining. Each column and bar represents the mean value ± SD of four wells. *P < 0.05.

Figure 2

Induction of extracellular signal‐regulated kinase (ERK), p38 and Akt phosphorylation by fibronectin (FN). (a,b) Time course of ERK, p38 and Akt phosphorylation. Serum‐starved CBO140C12 cells were stimulated with 50 µg/mL FN for the indicated time. (c) Dose‐dependent increase in ERK, p38 and Akt phosphorylation. Serum‐starved CBO140C12 cells were treated with the indicated concentration of FN for 10 min. Phospho‐ERK, phospho‐p38 and phospho‐Akt were determined by western blotting using phospho‐ERK (Thr202 and Tyr204), phospho‐p38 (Thr180, Tyr182) and phospho‐Akt (Ser473) antibodies, respectively.

Figure 3

Effects of p38, phosphatidylinositol‐3‐kinase (PI3K) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK) inhibitors on cell proliferation and invasion induced by fibronectin (FN). (a) CBO140C12 cells were seeded onto 50 µg/mL FN‐coated wells with culture medium containing 0.5% fetal bovine serum in the absence or presence of 10 µM SB203580, 20 µM LY294002 or 10 µM U0126. After a 48‐h incubation, proliferative activity was determined. These data are presented as percentage of the control for mean absorbance. (b) CBO140C12 cells were seeded onto the filters, which were precoated with Matrigel (5 µg) on the upper surface with or without coating with FN (1 µg) on the lower surface, in the presence of 10 µM SB203580, 20 µM LY294002 or 10 µM U0126. After a 12‐h incubation, the cells that had invaded into the lower surface were counted by crystal violet staining. Each column and bar represents the mean ± SD of four wells. *P < 0.05.

Figure 4

Inhibitory effect of gefitinib on extracellular signal‐regulated kinase (ERK), p38 and Akt phosphorylation induced by fibronectin (FN). Serum‐starved CBO140C12 cells were treated for 15 min with the indicated concentration of gefitinib, followed by stimulation with 50 µg/mL FN for 10 min. Phospho‐ERK, phospho‐p38 and phospho‐Akt were determined by western blotting using phospho‐ERK (Thr202 and Tyr204), phospho‐p38 (Thr180, Tyr182) and phospho‐Akt (Ser473) antibodies, respectively.

Figure 5

Involvement of integrins in the fibronectin (FN)‐induced signaling pathway. (a) Effect of FC‐336. Serum‐starved CBO140C12 cells were treated for 30 min with FC‐336 (FC) (5 mg/mL) and gefitinib (Gf) (5 µM), followed by stimulation with 50 µg/mL FN for 10 min. (b) Effect of anti‐αv integrin subunit monoclonal antibody RMV‐7. Serum‐starved CBO140C12 cells were treated for 10 min with RMV‐7, followed by stimulation with 50 µg/mL FN for 10 min. (c) Effect of FC‐336 and RMV‐7. Serum‐starved CBO140C12 cells were treated for 30 min with FC‐336 (FC) (5 mg/mL), RMV‐7 and gefitinib (Gf) (1 µM), followed by stimulation with 50 ng/mL EGF for 5 min. Cell lysates were subjected to western blotting with phosphor‐EGFR (Tyr‐1068), phospho‐ERK (Thr202 and Tyr204), phospho‐p38 (Thr180, Tyr182) and phospho‐Akt (Ser473) antibodies, respectively.

Figure 6

Involvement of a disintegrin and metalloprotease (ADAM) on fibronectin (FN)‐induced signaling pathways and proliferation. (a) Serum‐starved CBO140C12 cells were treated for 30 min with GM6001 (GM) (10 µM), TAPI‐1 (TAPI) (10 µM) and GM6001‐negative control (GM‐N) (10 µM), and then stimulated with 50 µg/mL FN for 10 min. Cell lysates were subjected to western blotting with phospho‐ERK (Thr202 and Tyr204), phospho‐p38 (Thr180, Tyr182) and phospho‐Akt (Ser473) antibodies, respectively. (b) CBO140C12 cells were seeded onto 50 µg/mL FN‐coated wells with culture medium containing 0.5% FBS in the absence or presence of GM6001 (GM) (10 µM) or TAPI‐1 (TAPI) (10 µM). After a 48‐h incubation, proliferative activity was determined. These data are presented as percentage of the control for mean absorbance. Each column and bar represents the mean ± SD of four wells. *P < 0.05.

Figure 7

Extracellular signal‐regulated kinase (ERK) activation and Akt activation by fibronectin (FN) are mutually independent. Serum‐starved CBO140C12 cells were treated for 30 min with LY294002 (LY) (20 µM) and U0126 (10 µM), and then with FN (50 µg/mL) for 10 min. Cell lysates were subjected to western blotting with antiphospho‐ERK, p38 and Akt antibodies, and reprobed with anti‐ERK, p38 and Akt antibodies, respectively.

Figure 8

Proposed mechanism of gefitinib on inhibitory effect of intrahepatic metastasis in CBO140C12 cells. Extracellular fibronectin (FN) binds to integrins, which is partly consist with αv subunit, and induces the activation of a disintegrin and metalloproteases (ADAM) on the cell surface. Subsequent release of epidermal growth factor (EGF)‐like ligands by ADAM results in p38, ERK and Akt activation, followed by induction of cell proliferation and invasion. This pathway might be a novel functional target of gefitinib (solid line). However, p38 could also be activated by an EGF receptor (EGFR)‐independent pathway (dotted line).