Breast cancer, the second leading cause of female deaths worldwide, is usually treated with cytotoxic drugs, accompanied by adverse side-effects, development of chemoresistance and relapse of disease condition. Survival and proliferation of the cancer cells are greatly empowered by over-expression or over-activation of growth factor receptors and anti-apoptotic factors. Identification of these key players that cross-talk to each other, and subsequently, knockdown with their respective siRNAs in a synchronous manner could be a promising approach to precisely treat the cancer. Since siRNAs demonstrate limited cell permeability and unfavorable pharmacokinetic behaviors, pH-sensitive nanoparticles of carbonate apatite were employed to efficiently carry the siRNAs in vitro and in vivo. By delivering selective siRNAs against the mRNA transcripts of the growth factor receptors, such as ER, ERBB2 (HER2), EGFR and IGFR, and anti-apoptotic protein, such as BCL2 in human (MCF-7 and MDA-MB-231) and murine (4T1) breast cancer cell lines, we found that ESR1 along with BCL-2, or with ERBB2 and EGFR critically contributes to the growth/survival of the cancer cells by activating the MAPK and PI-3 kinase pathways. Furthermore, intravenous delivery of the selected siRNAs aiming to suppress the expression of ER/BCL2 and ER/ERBB2/EGFR groups of proteins led to a significant retardation in tumor growth in a 4T1-induced syngeneic mouse model.

While several treatment strategies are applied to cure breast cancer, it still remains one of the leading causes of female deaths worldwide. Since chemotherapeutic drugs have severe side effects and are responsible for development of drug resistance in cancer cells, gene therapy is now considered as one of the promising options to address the current treatment limitations. Identification of the over-expressed genes accounting for constitutive activation of certain pathways, and their subsequent knockdown with specific small interfering RNAs (siRNAs), could be a powerful tool in inhibiting proliferation and survival of cancer cells. In this study, we delivered siRNAs against mRNA transcripts of over-regulated cell adhesion molecules such as catenin alpha 1 (CTNNA1), catenin beta 1 (CTNNB1), talin-1 (TLN1), vinculin (VCL), paxillin (PXN), and actinin-1 (ACTN1) in human (MCF-7 and MDA-MB-231) and murine (4T1) cell lines as well as in the murine female Balb/c mice model. In order to overcome the barriers of cell permeability and nuclease-mediated degradation, the pH-sensitive carbonate apatite (CA) nanocarrier was used as a delivery vehicle. While targeting CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 resulted in a reduction of cell viability in MCF-7 and MDA-MB-231 cells, delivery of all these siRNAs via carbonate apatite (CA) nanoparticles successfully reduced the cell viability in 4T1 cells. In 4T1 cells, delivery of CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 siRNAs with CA caused significant reduction in phosphorylated and total AKT levels. Furthermore, reduced band intensity was observed for phosphorylated and total MAPK upon transfection of 4T1 cells with CTNNA1, CTNNB1, and VCL siRNAs. Intravenous delivery of CTNNA1 siRNA with CA nanoparticles significantly reduced tumor volume in the initial phase of the study, while siRNAs targeting CTNNB1, TLN1, VCL, PXN, and ACTN1 genes significantly decreased the tumor burden at all time points. The tumor weights at the end of the treatments were also notably smaller compared to CA. This successfully demonstrates that targeting these dysregulated genes via RNAi and by using a suitable delivery vehicle such as CA could serve as a promising therapeutic treatment modality for breast cancers.

Specific gene knockdown facilitated by short interfering RNA (siRNA) is a potential approach for suppressing the expression of ion channels and transporter proteins to kill breast cancer cells. The overexpression of calcium ion channels and transporter genes is seen in the MCF-7 breast cancer cell line. Since naked siRNA is anionic and prone to nuclease-mediated degradation, it has limited permeability across the cationic cell membrane and short systemic half-life, respectively. Carbonate apatite (CA) nanoparticles were formulated, characterized, loaded with a series of siRNAs, and delivered into MCF-7 and 4T1 breast cancer cells to selectively knockdown the respective calcium and magnesium ion channels and transporters. Individual knockdown of TRPC6, TRPM7, TRPM8, SLC41A1, SLC41A2, ORAI1, ORAI3, and ATP2C1 genes showed significant reduction (p < 0.001) in cell viability depending on the cancer cell type. From a variety of combinations of siRNAs, the combination of TRPC6, TRPM8, SLC41A2, and MAGT1 siRNAs delivered via CA produced the greatest cell viability reduction, resulting in a cytotoxicity effect of 57.06 ± 3.72% (p < 0.05) and 59.83 ± 2.309% (p = 0.09) in 4T1 and MCF-7 cell lines, respectively. Some of the combinations were shown to suppress the Akt pathway in Western Blot analysis when compared to the controls. Therefore, CA-siRNA-facilitated gene knockdown in vitro holds a high prospect for deregulating cell proliferation and survival pathways through the modulation of Ca²⁺ signaling in breast cancer cells.

For cells and tissues, cell–cell and cell–extracellular matrix adhesion is important for proliferation, differentiation, and response to mechanical stimuli. This adhesion is provided by various cell adhesion molecules (CAMs). However, in breast cancer, depending upon the type and stage, this adherence is dysregulated where the expression of these cell adhesion molecules is either overregulated or unregulated, triggering essential oncogenic pathways. Thus, to control the invasiveness of tumor cells, and reduce metastasis, regulating the homophilic and heterophilic interaction of these molecules and controlling the essential cell pathways is important. In this study, we targeted critical CAMs- integrins to regulate their aberrated behavior via siRNAs delivery. However, as due to charge repulsion and propensity to be degraded by nucleases prior to reaching the target site, naked siRNAs are unable to cross plasma membrane, use of a suitable carrier vehicle is essential. Thus, we employed carbonate apatite (CA), to deliver the selected siRNAs targeting integrin αv, α6, β1, β3, β4, β5, and β6 subunits to various breast cancer cell lines and 4T1-breast cancer induced murine model. Delivery of individual integrin siRNAs complexed with CA nanoparticles (NPs) reduced cell viability and caused decrease in tumor burden. To check the gene knockdown effects on phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and extracellular signal–regulated kinases/mitogen-activated protein kinase (ERK/MAPK) pathways, Western blot analysis was performed, revealing downregulation of the signaling molecules. Thus, CA-facilitated gene therapy targeting various integrins could poise potential therapeutic modality against breast cancer.