Abstract 435: Reduction of the invasive phenotype of SNAI overexpressing human breast cancer cells by peptide aptamer-mediated inhibition of SNAI protein functions

Abstract

Abstract Two members of the SNAI superfamily of zinc-finger transcriptional repressors: SNAI1 (also known as SNAIL) and SNAI2 (also known as SLUG) are implicated in the induction of aggressiveness and metastatic phenotypes in human breast cancer cells. SNAI1 and SNAI2 are very similar in their amino acid sequences at the C-terminal zinc-finger domains, but the N-terminal repressor domain is somewhat different. SNAI2 has two essential sub-domains, SNAG and SLUG domains, required for its repressor activity. The SNAG domain is also important in SNAI1. We have developed a recombinant protein containing thioredoxin, a tri-repeat of SNAG and SLUG domains of SNAI2 and a membrane translocation motif (MTM; AAVLLPVLLAAP) for the protein to be targeted to the nucleus. We expressed and purified this protein from E. coli using the His-patch thiofusion expression system (Invitrogen). When delivered to MDA-MB-231 and BT549 cells, this aptamer inhibited the functions of the SNAI proteins. The effect of SNAI protein knockdown by the peptide aptamer on the transformed phenotypes of these cells was evaluated by investigating the following parameters: (i) Measurement of doubling time and saturation density by plating cells and performing daily counts. (ii) Evaluation of cellular proliferation by measuring incorporation of 5′-bromodeoxyuridine into logarithmically growing cells. (iii) Determination of anchorage-independent growth in soft agar assays and analysis of the number and size of colonies formed. (iv) Evaluation of the cellular migration in scratch assays and in modified Boyden Chamber/transwell migration assays (directional migration). (v) Measurement of cellular invasion in vitro by using Matrigel-coated chambers. (vi) Assessment of the sensitivity to anoikis by plating cells on poly-HEMA coated plates and determination of cells undergoing apoptosis with propidium iodine staining or annexin labeling and flow cytometric analysis. (vii) Performing 3D culture assays to analyze invasiveness, proliferation or matrix degradation. All these data indicated reduction of the invasive phenotypes of the SNAI protein over expressing highly aggressive and metastatic human breast cancer cells by the peptide aptamer. Wrapsome-mediated targeted delivery of this aptamer alone or with other drugs may be used to reduce the progression of breast cancer. Supported by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. 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 435.