Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (herceptin) on SKBR3 breast cancer cells

Abstract

The clinical usefulness of trastuzumab (Herceptin; Genentech, San Francisco, CA) in breast cancer treatment is limited by the rapid development of resistance. We previously reported that IGF-I signaling confers resistance to the growth-inhibitory actions of trastuzumab in a model system, but the underlying molecular mechanism remains unknown. We used SKBR3/neo cells (expressing few IGF-I receptors) and SKBR3/IGF-IR cells (overexpressing IGF-I receptor) as our experimental model. IGF-I antagonized the trastuzumab-induced increase in the level of the Cdk inhibitor p27Kipl. This resulted in decreased association of p27Kipl with Cdk2, restoration of Cdk2 activity and attenuation of cell-cycle arrest in G1 phase, all of which had been induced by trastuzumab treatment in SKBR3/IGF-IR cells. We also found that the decrease in p27Kipl induced by IGF-I was accompanied by an increase in expression of Skp2, which is a ubiquitin ligase for p27 Kipl, and by increased Skp2 association with p27Kipl. A specific proteasome inhibitor (LLnL) completely blocked the ability of IGF-I to reduce the p27Kipl protein level, while IGF-I increased p27 Kipl ubiquitination. This suggests that the action of IGF-I in conferring resistance to trastuzumab involves targeting of p27Kipl to the ubiquitin/proteasome degradation machinery. Finally, specific inhibitors of MAPK and PI3K suggest that the IGF-I-mediated reduction in p27 Kipl protein level by increased degradation predominantly involves the PI3K pathway. Our results provide an example of resistance to an antineoplastic therapy that targets one tyrosine kinase receptor by increased signal transduction through an alternative pathway in a complex regulatory network. © 2003 Wiley-Liss, Inc.