Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model

Abstract

The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

Role of Annexin A2 in the EGF-induced epithelial-mesenchymal transition in human CaSki cells

“…However, this data also suggested that this inhibition coincides with enhanced EGF-induced cell migration and downstream signaling via JNK and Akt, which may explain why ANXA2 knockdown increases lung metastasis formation in mice (31).…”

Simultaneous delivery of anti-miR21 with doxorubicin prodrug by mimetic lipoprotein nanoparticles for synergistic effect against drug resistance in cancer cells

“…In terms of tumor-targeted delivery, a suitable delivery system is required that helps this combination escape from the recognition of macrophage systems, penetrate into tumor tissues, and accumulate in their intracellular sites of action. In recent years, significant progress in combination therapy has been made with cationic nanoparticles formed with liposomes, 26,27 polymers, 28,29 micelles, 30,31 and dendrimers. 29 Among these, endogenous lipoproteins have attracted growing attention, due to their advantages, such as ease of formulation, excellent biocompatibility, and clinical potential.…”

Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor

“…The enhanced sialylation of malignant cells [23] benefits the idea of using cationic Ch to target cancer cells and may be the reason behind its occasionally observed anticancer properties [24]. The previously observed ability of the positively charged molecules and particles to localize in lungs [25–28] may also be a key to explaining the pronounced lung-targeting potential of HAp/Ch-PLGA particles [19]. …”