SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells

Abstract

Src family kinase activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective Src family kinase inhibitor, on human cancer cells derived from breast cancer patients to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC50 of approximately 250 nmol/L, which was also the IC50 for inhibition of cellular Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cell-to-cell adhesion and membrane localization of beta-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Crk-associated substrate (p130Cas), with an IC50 similar to inhibition of cellular Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer.

Figure 1

SKI-606 induces cell aggregation and decreases cell motility. A) MDA-MB-231 and MDA-MB-468 cell lines were cultured in the presence of 1 µM SKI-606 or 0.01% DMSO control for 96 h. Photomicrographs were taken under brightfield illumination (4x magnification). B) Wound assay performed on the MDA-MB-231 cell line treated with 0, 0.01, 0.03, 0.1, 0.3 and 1µM of SKI-606 and allowed to heal for 48 h before fixation and image capture (brightfield illumination, 4x magnification). 0.01% DMSO was present in each assay well. C) Summary of migration speeds assessed via VTLM. Breast cancer cell line migration was monitored in a denuded area of a confluent cell monolayer. Speed value is shown as mean±S.E. of three independent experiments. *p<0.05 (Wilcoxon test). D) MDA-MB-435s human breast cancer cells were grown in soft agar or matrigel in the presence of 0.01% DMSO or 1 µM SKI-606 for 4–6 days and photomicrographs were taken under brightfield illumination (4x and 10x magnification).

Figure 2

SKI-606 inhibits cell invasion but not proliferation. A) Cells were seeded in an invasion chamber in the presence of 0.0025% DMSO vehicle control or 250 nM SKI-606 and allowed to invade the chamber towards cell-specific conditioned medium for 48 h. Photomicrographs of stained invasive cells were taken under brightfield illumination (4x magnification). B) Invasion potential of breast cancer cell lines treated with SKI-606. Values represent the % of invasive cells compared to the 0.0025% DMSO vehicle control treated cells and are shown as mean±S.E. of three independent experiments. *p<0.05 (one-sided t-test versus a 0% invasion reduction). C) Breast cancer patient-derived cell lines were assessed for proliferation upon treatment with 0.01% DMSO or 1 µM SKI-606 for 48 h using the MTS metabolic assay. Values are shown as mean±S.E. of three independent experiments, representing the % reduction in absorbance values of cells treated with SKI-606 relative to untreated cells. No statistically significant differences were observed using the Wilcoxon test. D) MDA-MB-231 cells were assessed for proliferation upon treatment with 0.01% DMSO or 0.1, 0.3, 1µM SKI-606 for 4 and 6 days using the MTS metabolic assay. Values are shown as mean±S.E. of three independent experiments, representing the % reduction in absorbance values of cells treated with SKI-606 relative to untreated cells.

Figure 3

SKI-606 inhibits the invasive properties of Src, Yes and Fyn null mouse embryo fibroblasts following reintroduction of c-Src. A) Wound healing assay using Src, Yes, Fyn null cells (SYF−/−) and SYF−/− cells with reintroduced c-Src (SYF-Src), treated with 0.01% DMSO vehicle control or increasing concentrations of SKI-606 and allowed to migrate for 48 h. B) Cells were seeded in serum-free medium in the top layer of an invasion chamber in the presence or absence of 0.01% DMSO, 250 nM or 1 µM SKI-606 and allowed to invade the chamber towards cell-specific conditioned medium for 48 h. Photomicrographs were taken under brightfield illumination (4x magnification). C) Invasion potential of SYF−/− and SYF-Src cells treated with 0.0025% DMSO or 250 nM SKI-606. Values represent the % of invasive cells compared to DMSO vehicle control SYF-Src treated cells and are shown as mean±S.E. of three independent experiments. *p<0.009 (one-sided t-test versus SYF-Src cells with 100% invasion potential).

Figure 4

SKI-606 causes rapid and prolonged inhibition of Src/FAK/Pyk2/p130^Cas phosphorylation. Western blot analysis of MDA-MB-468 whole cell extracts. Cells were treated with SKI-606 or 0.01% DMSO at the indicated concentrations for times up to A) 3 h or B) 48 h prior to extraction. Immunoblots were probed as indicated with antibodies to phospho-Src (pY419), phospho-FAK (pYpY576/577 or pY925), phospho-Pyk2 (pY580), phospho-p130^Cas (pY410), phospho-Stat3 (pY705), phospho-Akt (pS473), phospho-MAPK p44/42, beta-catenin, or PARP. Blots were re-probed with total anti-Src, anti-FAK, or anti-Stat3 antibodies, as indicated.

Figure 5

Immunofluorescence analysis of FAK and beta-catenin in cells treated with SKI-606. A) MDA-MB-231 cells were treated with 0.01% DMSO or 1 µM SKI-606 for 48 h then fixed, probed with antibodies to phospho-FAK (pY576/pY577), FAK, phospho-Stat3 (pY705) or beta-catenin as indicated, and stained with DAPI. Images were captured under fluorescence at 40x magnification. B) MCF-7 cells were treated with 0.01% DMSO or 1 µM SKI-606 for 48 h then fixed, probed with antibodies to beta-catenin antibodies or E-cadherin antibodies and stained with DAPI. Images were captured under fluorescence at 40x magnification.

Figure 6

Model for mechanism of action of SKI-606 in human breast cancer cells. SKI-606 inhibits Src kinase activity and thereby disrupts phosphorylation of the Src/FAK/p130^Cas mutli-protein complex. SKI-606 also increases beta-catenin localization to the membrane and leads to loss of phosphorylated Stat3 from focal adhesion sites. The biological consequences of SKI-606 treatment are enhanced cell-to-cell adhesion, as well as reduced cell migration and invasion.