E7080, a multi-targeted tyrosine kinase inhibitor suppresses tumor cell migration and invasion

Abstract

Background: E7080 is an orally active multi-targeted kinase inhibitor whose targets include vascular endothelial growth factor receptors (VEGFR), fibroblast growth factor receptor (FGFR) and platelet derived growth factor receptors (PDGFR). It has been shown to inhibit tumor angiogenesis by targeting endothelial cells. A number of the targets of E7080 are also expressed on tumor cells and here we have looked at the direct effects of E7080 on tumor cell behavior.

Methods: Using a panel of human tumor cell lines we determined the effect of E7080 on cell proliferation, migration and invasion. Inhibition of FGFR and PDGFR signaling in the cells was measured.

Results: E7080 had little effect on tumor cell proliferation. However, it blocked migration and invasion at concentrations that inhibited FGFR and PDGFR signaling. Knock-down of PDGFR-β in U2OS osteosarcoma cells also inhibited cell migration which, could not be further inhibited in the presence of E7080. Furthermore, E7080 could not inhibit the migration of a PDGFR negative cell line.

Conclusion: E7080 does not significantly affect tumor cell proliferation but can inhibit their migration and invasion at concentrations that both inhibit its known targets and are achievable clinically.

Figure 1

Effect of E7080 on tumor cell proliferation. Dose-response curves for E7080 in a panel of cell lines. Results are shown from representative experiments for each cell line in a series of at least 3 where values are the mean of 4 replicates.

Figure 2

Inhibition of PDGFR-β and FGFR-1 signaling by E7080. (A) DX3 and (B) U2OS cells were treated with increasing concentrations of E7080, then stimulated with PDGF-BB. Lysates were prepared and the PDGFR-β immunoprecipitated and the immune complexes resolved by SDS-PAGE and probed with an anti-phosphotyrosine (pTyr) antibody. Membranes were then stripped and reprobed with an anti-PDGFR-β antibody. (C) DX3 and (D) U2OS cells were treated with increasing concentrations of E7080, then stimulated with bFGF. Lysates were prepared and FRS2 immunoprecipitated and the immune complexes then resolved by SDS-PAGE and probed with an anti-pTyr antibody. Membranes were then stripped and reprobed with an anti-FRS2 antibody. (E) HUVECs were treated with increasing concentrations of E7080, then stimulated with VEGF-A. Lysates were prepared and the VEGFR-2 immunoprecipitated and the immune complexes then resolved by SDS-PAGE and probed with an anti-pTyr antibody. Membranes were then stripped and reprobed with an anti-VEGFR-2 antibody.

Figure 3

Inhibition of cell migration by E7080. (A) Individual DX3 and U2OS cell movements were captured over 16 hours in the presence or absence of E7080 and then tracked and plotted using Image J. Representative plots show 8 cells for each condition. (B) Accumulated and euclidean distances travelled and velocity were quantified from at least 20 cells per condition and plotted as mean + s.e.m., p values, comparing treated cells with controls, all <0.01, Paired t-test. (C) Representative images (x10) of wound healing assay in U2OS cells. Cells were grown to confluency, then wounded and wound closure in the presence or absence of E7080 followed over 16 hours. (D) % wound closure was calculated after 16 hours from 8 images. Values are mean + s.e.m., p values comparing treated cells with controls were calculated using Paired t-test.

Figure 4

Inhibition of DX3 cell invasion by E7080. (A) Invasion of DX3 cells into Matrigel in the presence or absence of E7080 was measured: DX3 cells were stained with calcien AM and images taken every 15 μm. (B) Quantification of invasion is shown for a representative experiment in a series of three. Values are mean + s.e.m. of two transwells with 4 fields per transwell. p values comparing treated cells with controls were calculated using Paired t-test. (C) The fluorescent intensity of DX3 cells on the upper side of the Transwell was measured in untreated and E7080 treated cells following staining with calcein AM. Results are mean + s.e.m from a representative experiment in a series of three.

Figure 5

PDGFR-β is required for U2OS cell migration. (A) Cell lysates were prepared following transfection of U2OS cells with PDGFR-β or non-targeting siRNA oligonucleotides and PDGFR-β immunoprecipitated. The immune complexes were resolved by SDS-PAGE and probed with an anti-PDGFR-β antibody. (B) Wound healing assays with U2OS cells were carried out in the presence or absence of E7080 and/or PDGFR-β siRNA oligonucleotides. Values are mean + s.e.m. from a representative experiment in a series of three, p values comparing all with control were calculated using Paired t-test. (C) Wound healing assay in DU145 cells treated with E7080. Values are mean + s.e.m. from a representative experiment in a series of three.