Effect of chronic exposure to tyrosine kinase inhibitors on chemotherapy-induced DNA damage in the SKBR3 breast cancer cell line.
Authors: J Bhosle, A Makris, JA Hartley, D Hochhauser
Journal: Cancer Research (2009)

Abstract Abstract #1039 Introduction: The HER family of receptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) is critical in the regulation of cell growth, proliferation and survival. Several studies have linked over-expression of HER1 and HER2 with resistance to chemotherapy and radiation. Clinical trials combining the HER tyrosine kinase inhibitors (TKI) gefitinib (GEF), an inhibitor of HER1, and lapatinib (LAP), a dual inhibitor of HER1 and HER2, with chemotherapeutic agents have not shown synergy comparable to that found in preclinical models. We investigated the effects of chronic treatment of breast cancer cells with TKI inhibitors on DNA damage and repair resulting from chemotherapy and ionising radiation (IR).&amp;#x2028; Material and Methods: Western blotting was used to examine the expression of phosphorylated HER receptors, AKT and MAPK in the SKBR3 and BT474 human breast cancer cell lines. Etoposide (ET), doxorubicin (DOX) and IR induced DNA strand breaks (SB) were measured in the SKBR3 cell line using the single cell gel electrophoresis (comet) assay. Cisplatin-induced (PT) interstrand crosslinks (ICL) were measured with the modified alkaline comet assay. Cells were pre-treated with LAP or GEF for 1 hour or 48 hours, and TKI was replaced at 24-hourly intervals. Cell cycle analysis and intracellular DOX were measured by flow cytometry.&amp;#x2028; Results: In the SKBR3 and BT474 cell lines pHER1, pHER2, pHER3, pAKT and pMAPK were inhibited within 1 hour of exposure to LAP or GEF. However, after 48 hours, pHER3 and pAKT signalling was detected in cells treated with GEF, but not LAP. pHER1, pHER2 and pMAPK were inhibited for at least 72 hours with both drugs. Cell cycle analysis on the SKBR3 cell line showed that 48 hours exposure to either TKI reduced the number of cells in S (12.4±1.1% to 3.2±0.3%), and G2 (16.0±0.55% to 7.1±1.0%) phases of the cell cycle. There was no alteration in intracellular DOX levels at 1 hour or following 48 hours exposure to either TKI. SB were induced by ET, DOX and IR, and ICL by PT, in cells exposed to GEF or LAP for 1 hour. Exposure for 48 hours did not alter the number of ICL or SB induced by PT or IR. In contrast there was a 30-90% reduction in SB following ET treatment (depending on drug concentration) in cells treated with TKI for 48 hours compared with 1 hour treatment. The effect of 48 hours exposure to TKI on DOX-induced SB was more marked with &amp;gt;90% reduction, even at high DOX concentrations. Higher concentrations of ET resulted in increased SB, but increasing concentration of DOX did not alter the level of detectable SB.&amp;#x2028; Discussion: These results suggest why clinical trials of combinations of HER TKI with chemotherapy agents fail to show the magnitude of synergy predicted in preclinical studies. Whilst the cytostatic properties of both LAP and GEF may explain the reduction in DNA damage after 48 hours, this does explain why SB are absent in DOX-treated cells. Further studies investigating kinetics of DNA repair following chemotherapy or radiation in combination with TKI are on-going. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1039.