Effects of Nanomaterial-Delivered Protein Kinase A on Human Breast Cancer Stem Cells

Abstract

The cancer stem cell (CSC) theory has been proposed as a viable explanation for the resistance of cancer cells to treatments such as radiotherapy and chemotherapy in clinical setting. Emerging studies indicate that epithelial-to-mesenchymal transition (EMT) plays a crucial role in the dedifferentiation of differentiated cancer cells into CSCs. In particular, residual cancer cells that are resistant to radiotherapy and chemotherapy often escape from the treatments via an EMT process and subsequently metastasize to other organs. Thus, EMT-related molecular targeted therapies have been one of the promising areas of oncology research. Although EMT-targeting therapy has shown promise against CSCs in preclinical studies, many of these treatments failed in clinical trials with adverse side effects or without significant efficacy. Here we propose a direct enzyme transfer strategy, using lipid-nanomaterial based transfection as a delivery platform for guiding functional EMT-inhibiting enzymes directly into breast cancer cells, providing a cost-effective solution as well as examining the biological effects on human breast cancer stem cells (BCSCs). Recent evidence has shown that protein kinase A (PKA) evokes mesenchymal human mammary epithelial cells that undergo a mesenchymal-to-epithelial transition (MET), a process of epigenetic modification of tumor-initiating cells (TICs), promoting their differentiation that leads to loss of tumor-initiating ability. Therefore, PKA is used as the model enzyme for investigating encapsulation of biomacromolecules (functional enzymes) within lipid nanoparticles (lipofectamine CRISPRMAX). To evaluate the effects of the lipofectamine-PKA complexes, the complexes were introduced into mesenchymal breast cancer cells; then functional experiments were performed, including chemoresistance assay, mammosphere assay, and flow cytometry to test whether the CSC- and/or EMT-related properties were suppressed through the delivery of the complexes. Based on the function determination, we further investigated at the molecular level by immunofluorescence to determine whether the EMT phenotype was repressed in cancer cells. Our results show that guiding CRISPRMAX-PKA complexes into breast cancer cells before administration of paclitaxel (PTX) increases the sensitivity to PTX, while introducing PKA does not affect the sensitivity to doxorubicin (DOX). The size of mammospheres (MSs) is significantly reduced after treatment with the complexes. Furthermore, we find the mesenchymal marker α-SMA is downregulated after CRISPRMAX-PKA treatment. Taken together, these results suggest that our direct enzyme transfer strategy by lipid-nanomaterials is successful and supports the notion that PKA enzyme delivery into breast cancer cells could inhibit EMT to some extent as well as CSCs in breast cancer.