Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-Peptide analogue of insulin-like growth factor 1

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGFl receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen- sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS- 1 may serve as an alternative target to overexpressed IGFIR in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRSl siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRSl siRNA, of a serum-stabilized IRSl siRNA conjugated with our previously characterized peptide mimetic of IGFl, D-(CYSSer-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGFIR. Excess native IGFl blocked uptake. An IRSl siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRSl mRNA knockdown and IRSl protein knockdown were comparable for the IGF peptide and cholesterol conjugates. The unconjugated serum-stabilized IRSl siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.