We treated cell lines developed from breast tumors with the irreversible pan erb-B kinase inhibitor CI-1033, and demonstrated that sensitivity to the inhibitor is not closely correlated with its ability to inhibit the phosphorylation of the tyrosine kinase receptors present in the cell lines. We thus hypothesized that sensitivity to CI-1033 is related to the ability of the inhibitor to influence downstream signaling pathways involved in cell growth and survival. The ERBB-2 oncogene is amplified and over expressed in the SUM190 and SUM225 cell lines. These cells express constitutively activated erbB-2, and also have constitutively activated EGFR and erbB-3. Treatment of these cells with CI-1033 resulted in apoptosis. CI-1033 inhibited phosphorylation of AKT (p-AKT) in the SUM190 cells, and eliminated p-AKT in SUM225's, the cell line most sensitive to CI-1033. We next investigated the effects of CI-1033 on the SUM149 and SUM229 cell lines that over express constitutively activated EGFR. The SUM149 and SUM229 cells exhibited growth inhibition, but not apoptosis, after treatment with CI-1033. Phosphorylated AKT was not inhibited by CI-1033 in the SUM149 cells, and the SUM229 cells did not express detectable levels of p-AKT. Consequently, the AKT pathway did not appear to be pivotal in the SUM149 and SUM229 cells after treatment with CI-1033. We next investigated two other known survival pathways, the NFkB and MAPK pathways. The SUM149 cell line, which was more sensitive to CI-1033 then the SUM229 cell line, exhibited high levels of NFkB on electro mobility shift assay, and treatment with CI-1033 resulted in decreased NFkB activity levels. Treatment with CI-1033 decreased p-erk levels in the SUM149 cells, but not the SUM229 cells. Thus, in the EGFR over expressing cell lines, exposure to CI-1033 results in growth inhibition, and sensitivity to the inhibitor is best correlated with an ability to decrease NFkB. This suggests that although AKT is important for cell survival, growth arrest can occur after inhibition of other signaling pathways. These data confirm the importance of AKT activation for cell survival, and indicate that the ability of CI-1033 to cause apoptosis is correlated with its ability to inhibit activation of AKT.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]