Annals of Oncology Advance Access originally published online on February 17, 2008
Annals of Oncology 2008 19(6):1097-1109; doi:10.1093/annonc/mdm589
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breast cancer |
Low-scale phosphoproteome analyses identify the mTOR effector p70 S6 kinase 1 as a specific biomarker of the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (Tykerb®) in human breast carcinoma cells
1 Catalan Institute of Oncology, Girona, Catalonia
2 Girona Biomedical Research Institute, Girona, Catalonia
3 Department of Medical Oncology, Dr Josep Trueta University Hospital of Girona, Girona, Catalonia
4 MD Anderson International, Madrid, Spain
* Correspondence to: Dr J. A. Menendez, Catalan Institute of Oncology, Girona, Catalonia, Spain. Tel: +34-972-225-834 ext. 2579; Fax: +34-972-217-344; E-mail: jmenendez{at}ico.scs.es
Background: Discovery of key proliferative and/or survival cascades closely linked to the biological effects of human epidermal growth factor receptor (HER) 1 (erbB-1) and/or HER2 (erbB-2) inhibitors may identify a priori mechanisms responsible for the development of acquired resistance in breast cancer disease. Here, we took advantage of a semiquantitative protein array technology to identify intracellular oncogenic kinases that distinctively correlate with breast cancer cell sensitivity/resistance to the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (Tykerb®).
Materials and methods: MCF-7 cells were forced to overexpress HER2 following stable transduction with pBABE-HER2 retroviruses. The Human Phospho-MAPK Array Proteome ProfilerTM (R&D Systems) was used to molecularly assess the effects of both the mono-HER2 inhibitor trastuzumab (HerceptinTM) and the dual-HER1/HER2 inhibitor lapatinib on 21 different oncogenic kinases. A model of acquired resistance to lapatinib (MCF-7/HER2-Lap10 cells) was established by chronically exposing MCF-7/HER2 cells to increasing concentrations of lapatinib for >10 months.
Results: Treatment of MCF-7/HER2 cells with either trastuzumab or lapatinib similarly impaired HER2-enhanced activation status (i.e. phosphorylation) of the mitogen-activated protein kinases, c-Jun N-terminal kinases 1–3 and p38
/β/
/
and of the serine/threonine kinases AKT, glycogen synthase kinase-3, p90 ribosomal s6 kinase1/2, and mitogen- and stress-activated protein kinase1/2. Trastuzumab was less effective than lapatinib at blocking extracellular-signal regulated kinase (ERK) 1/2 and, notably, it failed to deactivate the mammalian target of rapamycin (mTOR) effector p70S6K1. Conversely, lapatinib treatment caused a drastic decrease in the phosphorylation of p70S6K1 at ERK1/2-regulated sites (Thr421/Ser424) and, as a consequence, p70S6K1 activity measured by its phospho-Thr389 levels was abolished. The mTOR inhibitor rapamycin was found to supraadditively increase lapatinib efficacy in MCF-7/HER2 cells [
10-fold enhancement; combination index (CI50) = 0.243 < 1.0 = additivity, P < 0.001] but not in p70S6K1 gene-amplified MCF-7 parental cells (1.3-fold enhancement; CI50 = 0.920 
1.0 = additivity). Lapatinib-resistant MCF-7/HER2-Lap10 cells, which are capable of growing in the continuous presence of 10 µM lapatinib without significant effects on cell viability, notably exhibited a lapatinib-insensitive hyperphosphorylation of p70S6K1. Rapamycin cotreatment suppressed p70S6K1 hyperactivation and synergistically resensitized MCF-7/HER2-Lap10 cells to lapatinib (>20-fold increase in lapatinib-induced cytotoxicity; CI50 = 0.175 < 1.0 = additivity).
Conclusions: Serine–threonine kinase p70S6K1, a marker for mTOR activity that regulates protein translation, constitutes a specific biomarker for the biological effects of the dual-HER1/HER2 inhibitor lapatinib. The clinical implications of our data are that the efficacy of lapatinib might be enhanced with therapies that target the mTOR pathway. Rapamycin analogues such as CCI-779 (Temsirolimus) and RAD001 (Everolimus) may warrant further clinical evaluation to effectively delay or prevent the development of acquired resistance to lapatinib in HER2-positive breast cancer patients.
Key words: breast cancer, HER2, lapatinib, mTOR, proteomics, rapamycin, tyrosine kinases
Received for publication August 25, 2007. Revision received October 22, 2007. Accepted for publication December 6, 2007.
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