Abstract 5056: Signaling pathways critical for insulin receptor substrate (IRS)-mediated disruption of MCF-10A acini formation

Abstract Insulin receptor substrate (IRS)-1 and IRS-2 are important adaptor proteins in insulin-like growth factor-I receptor (IGF-IR) signaling. IRS-1 and IRS-2 are expressed during mammary gland development and also in human breast tumors. In order to study the role of IRS-1 and IRS-2 in breast tumorigenesis, we overexpressed both IRS proteins in immortalized, non-transformed human mammary epithelial MCF-10A cells. It has been shown that MCF-10A cells form uniform, empty-lumen acini in three dimensional (3D) culture. We previously showed that overexpression of either IRS-1 or IRS-2 in MCF-10A cells disrupted the acini in 3D culture and resulted in extensive proliferation and disrupted apical-basal polarity. In both IRS-1 and IRS-2 overexpressing MCF-10A cells, an increase in tyrosine phosphorylation and association with p85 of both IRS proteins indicated that IRS-mediated signaling was activated. siRNA knockdown of the overexpressed IRS proteins reversed the disruption of MCF-10A acini, confirming that this is indeed an IRS-related event. In order to identify the critical effectors in IRS-mediated disruption of MCF-10A polarity and growth, we used small molecule inhibitors to screen multiple kinase signaling pathways upstream and downstream of the IRS proteins. Inhibition of Rac or atypical PKC (aPKC) didn't affect MCF-10A cell acini formation, however it abolished the IRS-mediated disruption of MCF-10A acini formation. Consistent with this, knockdown of Rac and aPKC by siRNA suppressed IRS-mediated disruption in acini formation. Rac belongs to the family of GTPase that regulates cell motility. aPKC is a key component of the PAR polarity complex, and has previously been shown to be involved in IRS-signaling in adipocytes. While the signaling mechanism of Rac and aPKC in the IRSs-induced disruption of acini is still unknown, we found that another component in the PAR polarity complex Par3 may associate with IRS proteins and leads to dissociation of the polarity complex. Taken together, overexpression of IRS-1 and IRS-2 disrupts the MCF-10A acini in 3D culture in part through Rac and aPKC pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5056.