Lapatinib Suppresses RTK-Mediated Signaling through Multiple Signaling Pathways.

Abstract

Abstract Background: Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR and HER2 are validated therapeutic targets; but unfortunately, only a small percentage of patients with EGFR or HER2-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Lapatinib is a small molecule dual tyrosine kinase inhibitor that suppresses the activation of EGFR and HER2. Completely understanding the molecular mechanisms and protein targets involved in the effects of Lapatinib and other RTK inhibitors can help determine why efficacy varies. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are modulated by Lapatinib in mammary cancer.Methods and Materials: In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Lapatinib (150 mg/kg BW/day) by gavage for 7 days. This dose of lapatinib was effective in a prevention study (causing a greater than 80% inhibition of mammary cancers). At sacrifice, the mammary cancer was rapidly removed and fixed for immunohistochemistry or frozen for protein array analysis. Fixed samples were analyzed for phosphorylated EGFR (Tyr1177) and HER2 expression by incubating with specific primary antibodies to detect pEGFR (Tyr1177) or HER2 and secondary antibodies conjugated to a fluorescence dye. Samples were observed by confocal microscopy. Frozen tissues were subjected to Proteome Profiler™ Arrays (R&D, Minneapolis, MN) including phospho-MAP kinase, phospho-kinase, and apoptosis arrays.Results: The results indicated that Lapatinib very effectively suppressed the abundance of both HER2 and phosphorylated EGFR (Tyr1177) compared to tumors from untreated control rats. The array analyses allowed parallel determination of the effect of Lapatinib on the relative levels of protein phosphorylation and apoptosis. Interestingly, unlike Gefitinib (Iressa), which appears to target EGFR and EGFR signaling mediated primarily through ERK MAP kinases, Lapatinib seems to suppress EGFR/HER2 signaling through Src-related proteins, Lyn and Lck, leading to increased cytochrome c release. The complete signaling cascade involves a number of additional signaling molecules, including inhibition of p38δ and γ, JNKs, GSK3α and β, and increased phosphorylation and activation of p27 and p53 potentially leading to apoptosis or cell cycle arrest. A second pathway that might also have a role in the action of Lapatinib involves the suppression of RTK activation of STAT5α and STAT3 potentially leading to decreased cell proliferation.Conclusion: These results suggest that RTK inhibitors have a wide range of potential targets. More work is in progress to continue the dissection of the action of Lapatinib and other RTK inhibitors. Supported by NCI Contract Number HHSN-261200433009C - NO1-CN-55006-72. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1130.