Synergy of Epidermal Growth Factor Receptor Kinase Inhibitor AG1478 and ErbB2 Kinase Inhibitor AG879 in Human Colon Carcinoma Cells Is Associated with Induction of Apoptosis

Abstract

Previous studies have shown that constitutive activation of epidermal growth factor receptor (EGFR) and ErbB2 by elevated autocrine transforming growth factor-A (TGF-A) expression plays an important role in colon cancer progression. Coexpression of EGFR and ErbB2 is found in a subset of colon cancers and may cooperatively promote cancer cell growth and survival, as heterodimerization is known to provide for diversification of signal transduction. In this study, the EGFR-selective tyrosine kinase inhibitor (TKI) AG1478 inhibited cell growth of an aggressive human colon carcinoma cell line, FET6AS26X, which harbors constitutively activated EGFR after stable transfection with TGF-A cDNA. However, AG1478 failed to induce apoptosis in FET6AS26X cells at concentrations sufficient for cell growth inhibition and complete suppression of EGFR phosphorylation. Similarly, AG879, a selective ErbB2 TKI, was incapable of inducing apoptosis in FET6AS26X cells at concentrations sufficient to inhibit cell growth and ErbB2 phosphorylation. To test the hypothesis that targeting both ErbB family members would show better efficacy than targeting the single receptors, combinations of inhibitors at fixed ratios of 1:1, 5:1, and 10:1 of AG1478 and AG879, respectively, were compared with single drugs for inhibition of cell growth. All combinations resulted in synergistic effects as indicated by combination index analysis. Synergistic inhibition was associated with induction of apoptosis as reflected by poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Annexin V staining. Finally, Western blot analysis showed significant inhibition of phosphorylation of both EGFR and ErbB2 by the combination treatment. These data suggest that the strategy to target both EGFR and ErbB2 simultaneously might result in more efficient inhibition of tumor growth than to target single receptor alone. (Cancer Res 2005; 65(13): 5848-56)

NLRR1 Enhances EGF-Mediated MYCN Induction in Neuroblastoma and Accelerates Tumor Growth In Vivo

“…To verify our experimental data that EGF induces MYCN, we used VEGF as positive control, which was reported to be induced by EGF (28). AG1478 is a specific inhibitor for EGFR, and is sufficient to block EGFR-mediated activation of ERK (29,30). We have confirmed the effect of the optimum concentration of AG1478 to inhibit EGF-mediated phosphorylation of EGFR in the cells (Supplementary Fig.…”

“…PVDF membranes were then blocked with TBS containing 5% nonfat dry milk and 0.1% Tween 20 at room temperature for 1 hour. After blocking, the membranes were incubated at 4 C overnight with anti-MYCN (Ab-1, Oncogene), anti-EGFR (Rockland), antiactin (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)Sigma), and other antibodies against ERK1/2, phospho-ERK, IGF1R, phospho IGFR1, Akt, phospho-Akt, and phospho-EGFR were purchased from Cell Signaling Technology. After incubation with primary antibodies, membranes were incubated with horseradish peroxidase-coupled goat anti-mouse or anti-rabbit IgG secondary antibody (Cell Signaling Technology) for 1 hour at room temperature.…”

A Novel Mechanism of Resistance to Epidermal Growth Factor Receptor Antagonism In vivo

“…This suggests that EGFR inhibition with the kinase inhibitor CI-1033 does not result in increased apoptosis. Data from our group using a combination of selective EGFR and ErbB2 inhibitors or a dual EGFR/ERBb2 selective inhibitor with FETa and other cell lines, shows that when both members of the ErbB family are targeted in combination, the effects on activation status, apoptosis, and inhibition of growth are synergistic in vitro (31,32).…”

PTHrP increases xenograft growth and promotes integrin α6β4 expression and Akt activation in colon cancer

“…Integrin β4 signaling also promotes ErbB2-mediated cell proliferation in a mammary tumor model [32]. Since ErbB2 exerts pro-survival effects in colon carcinoma cell lines [33,34], the effects of PTHrP on colon cancer cell proliferation in vitro and on xenograft growth in vivo may be mediated via the integrin β4/ErbB2 pathway.…”