Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.
Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.
D'Alessio
Amelia
A
Cell Biology and Biotherapy Unit, INT-Fondazione Pascale, 80131, Naples, Italy.
De Luca
Antonella
A
Maiello
Monica R
MR
Lamura
Luana
L
Rachiglio
Anna Maria
AM
Napolitano
Maria
M
Gallo
Marianna
M
Normanno
Nicola
N
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004357:Drug Synergism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D000077156:Gefitinib; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000077341:Lapatinib; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018797:Cell Cycle Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D066246:ErbB Receptors; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D000077156:Gefitinib; D047908:Intracellular Signaling Peptides and Proteins; D000077341:Lapatinib; D020928:Mitogen-Activated Protein Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011799:Quinazolines; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D050717:Retinoblastoma-Like Protein p130; D000068878:Trastuzumab; C436291:CDKN1B protein, human; C499517:FOXO3 protein, human; C493837:RBL2 protein, human; C512478:EGFR protein, human; C508053:ERBB2 protein, human