Abstract 4406: Inducible knockdown of insulin receptor substrate I desensitizes ER alpha response to both agonist (estradiol) and antagonist (fulvestrant) in MCF-7L breast cancer cells

Abstract

Abstract Insulin receptor substrate (IRS) proteins are adaptor proteins phosphorylated by insulin and insulin-like growth factor (IGF) receptors. In addition to their roles in regulating IGF and insulin responses, IRS proteins function in other pathways in normal and malignant physiology. Our data suggested that IRS1 enhanced IGF/insulin stimulated proliferation while IRS-2 was important in regulating motility. Therefore IRS proteins could be potential therapeutic target for cancer therapy. We have previously shown that reduced IRS1 can hinder cancer cell growth and tumorigenicity stimulated by IGF-I, IGF-II and insulin. In this study, we evaluated whether inducible IRS1 suppression affected estrogen receptor-alpha (ER) responsiveness to ER agonist estradiol and antagonist fulvestrant. We evaluated several doxycycline inducible shRNA IRS1 constructs (Thermo Scientific pTRIPZ inducible Lentiviral IRS1 shRNA) expressed in the ER+ MCF-7L cell line. Doxcycline treatment reduced IRS1 mRNA and protein levels confirmed by qRT-PCR and immunoblot in several independently selected clones. We used a representative clone (3G5) to evaluate effects on ER function. After IRS1 inducible knockdown, estradiol (E2) stimulated growth was suppressed. To investigate if reduced IRS1 regulated E2 stimulated binding of ER to the pS2 estrogen response element (ERE), chromatin immunoprecipitation (ChIP) assays were performed. Compared to non-induced MCF-7L cells, inducible IRS1 suppression demonstrated significantly reduced binding of ER to this promoter. The pharmacologic antagonist of IRS proteins, NT157, also resulted in diminished ER binding to pS2 promoter. mRNA of several ER regulated genes, such as PGR, TFF1, etc, showed downregulation in response to E2 when IRS-1 was suppressed. We have shown that the pure steroidal ER antagonist fulvestrant activates EGFR signaling in an ER dependent manner through upregulation of EGFR ligands. However, inducible IRS1 suppression blocked this ER-dependent activation of EGFR phosphorylation by fulvestrant yet EGFR phosphorylation remained intact when stimulated by addition of EGF. These data suggest that regulation of EGFR ligands by fulvestrant also requires IRS-1 expression. We conclude that ER function is affected by IRS-1 expression. Thus, IRS-1 may also be a target in endocrine responsive and resistant breast cancers. Citation Format: Xihong Zhang, Sidhant Varma, Douglas Yee. Inducible knockdown of insulin receptor substrate I desensitizes ER alpha response to both agonist (estradiol) and antagonist (fulvestrant) in MCF-7L breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4406.