RNA-based TWIST1 inhibition via dendrimer complex to reduce breast cancer cell metastasis

Abstract

Breast cancer is the leading cause of cancer-related deaths among women in the United States, and survival rates are lower for patients with metastases and/or triple-negative breast cancer (TNBC; ER, PR, and Her2 negative). Understanding the mechanisms of cancer metastasis is therefore crucial to identify new therapeutic targets and develop novel treatments to improve patient outcomes. A potential target is the TWIST1 transcription factor, which is often overexpressed in aggressive breast cancers and is a master regulator of cellular migration through epithelial-mesenchymal transition (EMT). Here, we demonstrate an siRNA-based TWIST1 silencing approach with delivery using a modified poly(amidoamine) (PAMAM) dendrimer. Our results demonstrate that SUM1315 TNBC cells efficiently take up PAMAM-siRNA complexes, leading to significant knockdown of TWIST1 and EMT-related target genes. Knockdown lasts up to one week after transfection and leads to a reduction in migration and invasion, as determined by wound healing and transwell assays. Furthermore, we demonstrate that PAMAM dendrimers can deliver siRNA to xenograft orthotopic tumors and siRNA remains in the tumor for at least four hours after treatment. These results suggest that further development of dendrimer-based delivery of siRNA for TWIST1 silencing may lead to a valuable adjunctive therapy for patients with TNBC.

Figure 1

Mechanism of dendrimer-mediated siRNA delivery and TWIST1 knockdown. (1) Negatively charged siRNA is electrostatically attracted to positive charges on the YTZ3-15 dendrimer, leading to the formation of 6–8 nm diameter micelles coated with siRNA. (2) These dendriplexes are administered to tumor cells. (3) Dendriplexes are taken up via macropinocytosis. (4) Dendriplexes are trafficked to late endosomes. (5) Due to the proton sponge effect, electrostatic interactions between the dendrimer and siRNA are disrupted and siRNA escapes from the disrupted endosome into the cytosol. (6) Once in the cytosol, siRNA recruits the endogenous RNAi machinery to degrade TWIST1 mRNA. Following TWIST1 knockdown, TWIST1 target gene expression is altered to reduce invasive capacity.

Figure 2

Stable and transient RNA-mediated TWIST1 knockdown in SUM 1315. (a) Western blotting demonstrated robust TWIST1 knockdown in both shTwistA and shTwistB lines. (b) qPCR confirmed TWIST1 knockdown at the mRNA level for both stable knockdown lines. Error bars represent standard deviation. (c) SUM 1315 cells expressing shTwistA or shTwistB exhibited decreased directional migration compared to those expressing shScram control in wound healing assays. Dashed lines indicate migratory front and were added manually. Images shown are representative data from experiments performed in triplicate. (d) Western blot demonstrated substantial TWIST1 knockdown in SUM 1315 cells transfected with siTwistA and siTwistB using Lipofectamine 2000 when compared to nontransfected (NTF), Lipofectamine 2000 alone (Lipo only), or control siRNA (siCtrl). (e) qPCR results mirrored those seen in the Western blot.

Figure 3

YTZ3-15 effectively delivers siRNA to SUM 1315 cells. (a) Left: nontransfected SUM 1315 cells had low background fluorescence. Right: more than 99% of YTZ3-15 transfected cells were positive for AlexaFluor 647-labeled siQ. (b) Fluorescent microscopy revealed that AlexaFluor 488-labeled siQ was taken up into cells within one day, and AlexaFluor signal was still detectable in cells at seven days after transfection. (c) Confocal images of SUM 1315 cells stably expressing eGFP + luc and transiently transfected with AlexaFluor 647-labeled siQ using YTZ3-15. LysoTracker dye revealed that siQ primarily colocalized with mid to late endosomes after 24-hour incubation with YTZ3-15 siRNA dendriplexes.

Figure 4

TWIST1 knockdown following YTZ3-15 delivery of siTwist decreases cell motility and downstream EMT marker expression. (a) Compared to siQ control (at seven days), siTwistA (TwA) and siTwistB (TwB) delivered via YTZ3-15 produced >90% TWIST1 knockdown at the mRNA level. Knockdown lasted seven days after transfection. (b) Compared to siQ control (at seven days), TwA and TwB delivered via YTZ3-15 produced knockdown of the TWIST1 targets N-Cadherin and Vimentin. N-Cadherin mRNA levels decreased by >40% after one day, and by approximately 90% after seven days. Vimentin mRNA was nearly undetectable after one day, and remained at <10% after seven days. (c) YTZ3-15 transfection of siTwistA decreased directional migration compared to siQ transfected cells (control) in wound healing assays. Dashed lines indicate migratory front and were placed manually. Images shown are representative data from experiments performed in triplicate. (d) Left: YTZ3-15 transfection of TwA or TwB resulted in >50% decrease in invasion of SUM 1315 cells through Matrigel. Cells were allowed to migrate for one day, following one day incubation with YTZ3-15-siRNA dendriplexes. Five fields per condition were imaged (representative images shown). Right: quantification of image data. Bars represent mean and standard deviation of five fields per condition.

Figure 5

YTZ3-15 concentrates in orthotopic breast cancer tumors in vivo. (a) Representative animals from the mice that received YTZ3-15 + siQ via intratumoral (IT) and intravenous (IV) injections. Control animals received IV injections of the dendriplexes but had no tumors. Mice receiving IT injections showed accumulation of siQ lasting at least 15 minutes post injection, whereas mice receiving IV injections showed little accumulation after 5 minutes. Control animals do not show accumulation of siQ due to the absence of tumors. (b) Ex vivo imaging of spleen, kidney, liver, and tumors (where applicable) from the three animals shown in Figure 5(a), demonstrating concentration of YTZ3-15 + siQ dendriplexes in the tumors but not in other organs. Images were obtained 240 mins after the administration (IT or IV) of YTZ3-15 + siQ. The units for the scale bars in this figure are photons/sec/cm²/steradian.