Abstract P6-12-05: Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells

Abstract

Abstract Insulin receptor substrate (IRS) proteins are adaptor proteins downstream of insulin-like growth factor (IGF) and insulin receptors. IRS proteins play a role in cancer biology, with IRS1 function linked to mitogenesis and survival and IRS2 associated with metastasis. Moreover, this family of adaptor proteins is also involved in the signal transduction of many other transmembrane receptors. Therefore IRS proteins could be potential therapeutic targets for cancer therapy. To test the function of IRS-1, we created a doxycycline inducible IRS1 shRNA and stably transfected MCF-7L breast cancer cells. The level of IRS1 protein knockdown varied within different clones from 50 to 95% reduction compared to non-induced cells. IRS2 mRNA and protein levels were not significantly affected. We chose a high (3G5) and intermediate (3A7) IRS1 knockdown clones for further study. Doxycycline treated 3G5 and 3A7, showed partial inhibition of IGF-I, IGF-II and insulin stimulated phosphorylation of IRS1(Tyr1222) and AKT. pErk1,2 were slightly increased and pIGF-IR was unchanged compared to doxycycline non-treated cells. IRS-2 tyrosine phosphorylation was stimulated when IRS-1 was suppressed and may account for the persistent downstream cell signaling. Anti IGFIR antibody dalotuzumab (20µg/ml) effectively inhibited cell signaling stimulated by IGF-I, IGF-II and insulin in both doxycycline treated and non-treated cells. There was no obvious difference in cell signaling pattern between plus and minus doxycycline treated cells within 24 hour time course. In monolayer cell growth assays, IGF-I, IGF-II, insulin and estradiol growth stimulation were significantly inhibited when IRS-1 was suppressed in 3G5 cells. In 3A7 cells, which have a lower level of IRS-1 suppression, growth stimulation was moderately inhibited. In anchorage independent growth assays stimulated by IGF-I, colony number and size were inhibited by doxycycline-induced IRS-1 suppression with a reduction of 80% in 3G5 cells and 50% reduction in 3A7 cells. These results show that growth of MCF-7L cells are dependent on IRS-1 expression. Reduced IRS1 can hinder cancer cell growth and tumorigenicity even when signaling was not impaired potentially due to compensation by other adaptor proteins. We conclude that suppression of IRS-1 may inhibit several growth regulatory pathways in breast cancer and could serve as a potential drug target. Citation Format: Xihong Zhang, Sidhant Varma, Douglas Yee. Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-05.