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

Abstract

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 IGF1 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 IGF1R 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 IRS1 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 IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 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.

Improving siRNA Bio-Distribution and Minimizing Side Effects

“…In this sense, oligoconjugation represents a strategy to improve the delivery specificity: it is in principle possible to link to the siRNA cell-specific ligand such as aptamers, antibodies, sugar molecules, vitamins, and hormones [82][83][84][85][86] to confer cell specificity to siRNA delivery systems. Specific interaction between a ligand and its cellular membrane receptor can enhance siRNA cellular uptake by means of receptor-mediated endocytosis.…”

Bioconjugates for targeted delivery of therapeutic oligonucleotides

“…siRNAs, ASOs or SSOs have been conjugated with peptides designed to bind to specific receptors, for example, integrin αvβ3 [26, 33, 51, 52], bombesin receptor [53], and insulin-like growth factor 1 receptors [54, 55]. Conjugates of ONs with integrin-targeting RGD peptide have been the most studied peptide-ON conjugate.…”

DNA and RNA derivatives to optimize distribution and delivery

“…Furthermore, conjugated peptides that are fragments of known protein ligands have also served as effective ligands for receptor-mediated endocytosis, such as octreotide, an octapeptide mimic of somatostatin [165, 166], JB3, a tridecapeptide mimic of insulin-like growth factor 1 [167], JB9, a tetrapeptide mimic of insulin-like growth factor 1 [135, 148, 168, 169], or DAMGO, an enkephalin analog [149]. …”