Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.

Abstract

Abstract Abstract #2130 Co-expression of EGFR and ErbB-2 occurs in a subset of primary breast carcinoma, and it is associated with worse prognosis. We previously demonstrated that treatment of breast cancer cells that express both receptors with a combination of the EGFR tyrosine kinase inhibitor gefitinib (G) and the anti-ErbB-2 monoclonal antibody herceptin (H) results in a synergistic anti-tumor effect. Such combination failed to show significant activity in a phase I/II clinical trial. However, patients enrolled in this trial were not selected for expression of EGFR. We explored the molecular mechanisms involved in the synergism of G and H in breast cancer cells. Treatment of both SK-BR-3 and BT-474 cells with a combination of G+H produced a more significant reduction of AKT and p42/p44-MAPK (MAPK) activation as compared with treatment with a single agent. The combination also produced a more prolonged blockade of both signalling pathways. In agreement with these findings, treatment with G+H induced a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle as compared with treatment with a single drug. A sub-G0/G1 peak, suggestive of apoptosis, was evident in cells treated with the combination but not with G or H. The combination of G+H produced a more significant increase in the levels of p27kip1 and of hypophosphorylated forms of pRb2, and a more significant decrease in the levels of Cyclin D1 and survivin as compared with a single agent. Analysis of p27kip1 protein stability, in presence of the protein synthesis inhibitor cycloheximide, revealed that both G and the combination of G+H significantly increased the stability of p27kip1, with the combination showing higher effects. Furthermore, breast cancer cells treated with the combination expressed higher levels of p27kip1 mRNA as compared with cells treated with a single drug. The combination also induced a significant increase in the nuclear levels of the FKHRL-1 transcription factor that was not observed in cells treated with G or H alone. Treatment of breast cancer cells with the dual EGFR/ErbB-2 inhibitor lapatinib produced an increase in the levels of p27kip1 and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin comparable to that observed following treatment with the combination of G+H. In addition, an increase in p27kip1 mRNA and in the nuclear levels of FKHRL-1 was induced in breast cancer cells by treatment with lapatinib. These findings suggest that the synergistic anti-tumor effects deriving from EGFR and ErbB-2 blockade are mediated by significant alterations in the expression of cell cycle regulatory proteins, including transcriptional and post-transcriptional regulation of p27kip1 expression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2130.