Biologic effects of CP-724,714, a selective HER-2/neu kinase inhibitor, on human breast cancer cells with variable expression of EGFR and HER-2

4556 HER-2/neu and the epidermal growth factor receptor (EGFR) are Type I receptor tyrosine kinase receptors which consist of an extracellular ligand binding domain, transmembrane domain, tyrosine kinase domain, and intracellular cytoplasmic tail. Amplification of the HER-2 proto-oncogene is found in ∼25% of human breast cancers and correlates with a poor prognosis. Elevated expression of EGFR has also been correlated with a more aggressive phenotype and is believed to play a role in the pathogenesis of many human epithelial malignancies as well. For these reasons, HER-2 and EGFR have been targeted for therapy in human cancers. Trastuzumab, a monoclonal antibody against HER-2 has single agent activity of ∼30% of patients, and prolongs survival in combination with chemotherapy in women whose breast cancers have amplification of HER2. CP-724,714 is a novel, selective, reversible, small molecule inhibitor of the HER2 tyrosine kinase. To evaluate its potential role in the treatment of human breast cancer, we tested its activity across a panel of >20 human breast cancer cell lines with variable levels of HER2 and EGFR as determined by Northern and Western analysis. On day 0, cells were seeded in 24-well plates with duplicate wells for each concentration of CP-724,714. On day 1 cells were treated with concentrations ranging from 10μ M to 0.312μ M in order to determine a dose response curve. On day 6, cells were trypsinized and counted with an automated cell counter. Experiments were repeated at least two times. We found that in 9 of 9 lines with HER2 amplification, CP-724,714 had significant activity in vitro with IC50s less than 5μ M and was more potent in inhibiting growth than 10 mcg/ml trastuzumab in most of these cell lines. An in vivo comparison is yet to be done. Of the 13 cell lines without HER2 amplification, CP-724,714 had an IC50 of less than 5μ M in only 3 cell lines, one of which has elevated levels of HER2 with multiple copies of chromosome 17 by FISH. Of note, CP-724,714 did not have significant activity against two cell lines with elevated expression of EGFR. In conclusion, CP-724,714 has significant activity in human breast cancer cell lines with elevated levels of HER2. CP-724,714 may also have activity in some breast cancers that do not have HER2 amplification. Studies to evaluate the molecular mechanism of growth inhibition are in progress.