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.
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(R)).


METHODS
MCF-7 cells were forced to overexpress HER2 following stable transduction with pBABE-HER2 retroviruses. The Human Phospho-MAPK Array Proteome Profilertrade mark (R&D Systems) was used to molecularly assess the effects of both the mono-HER2 inhibitor trastuzumab (Herceptintrade mark) 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 p38alpha/beta/gamma/delta 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 (Thr(421)/Ser(424)) and, as a consequence, p70S6K1 activity measured by its phospho-Thr(389) levels was abolished. The mTOR inhibitor rapamycin was found to supraadditively increase lapatinib efficacy in MCF-7/HER2 cells [ approximately 10-fold enhancement; combination index (CI(50)) = 0.243 < 1.0 = additivity, P < 0.001] but not in p70S6K1 gene-amplified MCF-7 parental cells ( approximately 1.3-fold enhancement; CI(50) = 0.920 congruent with 1.0 = additivity). Lapatinib-resistant MCF-7/HER2-Lap10 cells, which are capable of growing in the continuous presence of 10 microM 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; CI(50) = 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.
Vazquez-Martin
A
A
Catalan Institute of Oncology, Girona, Catalonia, Spain.
Oliveras-Ferraros
C
C
Colomer
R
R
Brunet
J
J
Menendez
J A
JA
D000970:Antineoplastic Agents / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000188:drug therapy*; D045744:Cell Line, Tumor; D004351:Drug Resistance / Q000502:physiology*; D005260:Female; D006801:Humans; D000077341:Lapatinib; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D011494:Protein Kinases / Q000378:metabolism; D020543:Proteome; D040901:Proteomics; D011799:Quinazolines / Q000627:therapeutic use*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D000077341:Lapatinib; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D020543:Proteome; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1