Breast cancer remains a leading cause of cancer related deaths for women in the United States with the principal cause of mortality being metastatic disease. Mortality rates associated with metastatic breast cancer tumors are significantly elevated compared to localized disease because the most common metastatic sites (brain, bone marrow, and lungs) are difficult to target either with chemotherapy or surgical intervention. Therefore reduction of tumor cell dispersion is a key component to a reduction in mortality rates among patients with breast cancer. Epithelial-Mesenchymal Transition (EMT) is the process by which cancer cells downregulate proteins associated with cell to cell adhesion (e.g. E-cadherin) resulting in cells that are able to detach from neighboring cells, invade adjacent tissue, and eventually enter the circulatory system or lymphatics. Many breast cancers are known to commandeer the EMT process, thus allowing the cells to metastasize beyond the primary tumor. The process of EMT is tightly regulated by the transcription factor Twist1, which is often overexpressed in breast cancer. Therefore, Twist1 serves as an excellent therapeutic target whose downregulation would result in fewer metastatic cancer cells and correspondingly reduce cancer mortality. Twist1 is also a desirable target because it is almost nonexistent in adult tissues and thus its silencing would have minimal side effects, especially compared to many of the non-specific cancer treatments used today.
Our lab has elected to use an RNA-based mediated gene silencing approach to decrease the expression levels of Twist1 in a highly invasive breast cancer cell line (SUM 1315). Due to their fragile nature, siRNA molecules are often difficult to deliver at therapeutic levels either in vitro or in vivo. We have overcome these delivery and degradation limitations through the optimization and use of Poly (amidoamine) (PAMAM) dendrimers. By complexing the siRNA with the PAMAM dendrimers not only protects the RNA molecules, but also facilitates their uptake into the tumor cells. We hypothesize that suppression of the activity of Twist1 via dendrimer-delivered Twist1 siRNA will inhibit metastatic behavior of aggressive breast cancer cells. Here we demonstrate successful delivery of Alexa Fluor® 488 labeled siRNA using two different dendrimers (Generation 5 and a modified Generation 3) with transfection efficiency results exceeding those of Lipofectamine 2000 and with far less toxicity to cells. We also show up to a 90% reduction (lasting at least 7 days) in Twist1 expression using Western Blot and qPCR analysis. Along with the knock down of Twist1, here we also demonstrate a significant knockdown of EMT proteins N-Cadherin and Vimentin. Functionally, luciferase promoter assays of Twist1 targets were also reduced. Furthermore, we were able to appreciate a significant phenotypic decrease in the invasive nature of the breast cancer cells using migration/invasion assays. Taken together these results demonstrate successful knockdown of Twist1 using siRNA dendrimer complexes resulting in an altered cellular phenotype and function. These data will serve as a foundation for optimization of future in vivo experiments with both orthotopic and metastatic breast cancer models.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-08.