Non-viral (polymeric) delivery of combinational siRNAs against cell cycle and phosphatase proteins to prevent metastasis in breast cancer

摘要

Introduction: Conventional breast cancer therapies have significant limitations that warrant development of new therapies. Control of breast cancer growth by RNA interference (RNAi) using small interfering RNA (siRNA) has been a promising approach in recent years. The siRNA-mediated silencing of a unique or over-expressed cell cycle proteins could lead to better control of tumor growth without affecting normal tissues. The major hurdle in the treatment of most cancers is metastasis. Several lines of evidence have confirmed the role of protein-tyrosine phosphatases in metastasis, so that they could serve as suitable targets to curb metastasis. We hypothesize that dual siRNA delivery against a cell cycle protein (to decrease tumor cell growth) and a phosphatase protein (to decrease cell migration) may have a drastic impact to treat metastatic breast cancer. This study was designed to explore the possibility of non-viral (polymeric) delivery of siRNA for dual purpose of inhibition of cell growth and migration in vitro. Methods and Results: We initially confirmed the feasibility of delivering an siRNA against CDC20 (IDT, Coralville, IA), a key protein in cell cycle regulation using MDA-MB-435 breast cancer cells (a non-metastasizing cell-line) to curb uncontrolled growth of cells in vitro and in vivo. Here, we performed siRNA delivery studies using metastasizing breast cancer cell-line MDA-MB-231. To deliver CDC20 siRNA effectively to MDA-MB-231, we synthesized a library of lipid-substituted polyethylenimines (PEI) from 1.2 kDa PEI and evaluated their efficacy. PEI substituted with linoleic acid (PEI-LA; Figure 1) was found to be the most effective delivery agent based on inhibition of MDA-MB-231 cell growth. To increase the stability of siRNA/PEI-LA complexes, hyaluronic acid (HA) was used as an additive or coating on complexes131. Under selected conditions, HA additive was less toxic and inhibited the cell growth significantly higher compared to complexes without HA. While surface charge was significantly decreased by the HA, the size of the particles remained unchanged. The stability of complexes in fresh rat serum was increased when prepared with HA compared to without HA. To identify siRNAs that were effective against cell migration, we screened an siRNA library against 267 phosphatases (Ambion) for inhibition of cell growth (MTT assay) and migration (scratch assay). MDA-MB-231 cells were more responsive to siRNA against phosphatases compared to MDA-MB-435. Based on the library screening, siRNAs against PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 decreased the migration of MDA-MB-231 cells significantly. These identified targets were then validated in vitro using individually prepared siRNAs (IDT). The combinational siRNA therapy has successfully decreased the growth as well as migration of MDA-MB-231 in vitro (Figure 2). MDA-MB-231 cells were more responsive to HA additive complexes in scratch assay compared to HA coating. Conclusions: This study confirmed the importance of a particular cell cycle protein (CDC20) and several novel phosphatase targets to reduce metastasis of breast cancer. The lipid-substituted polymers enabled the desired dual delivery, where specific siRNAs inhibited both cell growth as well as migration. The non-viral delivery system described here could serve as a viable platform for delivery of multiple siRNAs against critical targets.