Epithelial to Mesenchymal Transition (EMT) is mechanism involved in tumor progression and dissemination, with great potential for development of new targeted anti-cancer therapies. In this Final Degree Project we inhibited the main pathways involved in EMT, by small interference RNA (siRNA).
We used specific anti-AKT2 siRNA, to ...»»»»
Epithelial to Mesenchymal Transition (EMT) is mechanism involved in tumor progression and dissemination, with great potential for development of new targeted anti-cancer therapies. In this Final Degree Project we inhibited the main pathways involved in EMT, by small interference RNA (siRNA).
We used specific anti-AKT2 siRNA, to stop the EMT process in various breast cancer cell lines (MCF7, MDA-MB-468, and SKBR3). The downregulation of AKT2 mRNA and EMT markers was evaluated by gene expression study and was quantified by q-PCR. The effect on migration and proliferation was assessed by wound healing assay and proliferation assay. In addition, we tested lipid nanoparticles (LNP) internalization abilities by flow cytometry and confocal microscopy, since they are planned to be used for specific siRNA delivery in the near future. We have observed effective silencing of AKT2 in MDA-MB-468 and SKBR3 basal cell lines. As expected, this led to decrease in proliferation and migration capacity of these cells and decrease in expression of mesenchymal markers. In the luminal MCF7 cell line, the AKT2 silencing was not enough pronounced and had no effect in migration and proliferation of the cells. This could be due to more epithelial phenotype of this cells and low expression of endogenous AKT2 messenger RNA (mRNA) in this MCF7 cells. However, we have observed increase in EMT gene expression in Cancer Stem Cells (CSC) isolated from MCF7 cells. We have also observed substantial internalization of LNP into the cells. The targeted (CD44 antibody) LNP internalized faster than non-targeted LNP. CD44 was chosen as target because it is highly expressed in CSC. In conclusion, we proved that silencing of AKT2 leads to reversion of EMT process and could thus be used as anti-cancer therapy. More importantly, we showed that, CSC express EMT genes, suggesting that this approach (AKT2 silencing) could eliminate also these highly tumorigenic cells. In addition we have observed effective internalization of LNP targeted against CSC, which will lead to development of AKT2-siRNA loaded LNP in the near future.^^^^
