Abstract 3738: Knockdown of Annexin A2 by liposomal formulation of Annexin A2 small hairpin RNA induces apoptosis and inhibits angiogenesis in lung cancer stem cells.
Authors: Andey Terrick A. , Marepally Srujan , Singh Pomila , Sachdeva Mandip S. 
Published: August 2014

Abstract
Recent studies support the existence of small populations of pluripotent stem-like cancer cells capable of self-renewal. These cancer stem cells (CSCs) are attributed to the resistance phenotype occurring in many tumors such as invasion and metastasis. Annexin A2 (Anxa2) overexpression is associated with invasion, metastasis and drug resistance in many cancers. We investigated the effect of Anxa2 knockdown by a liposomal (nanosome) formulation of Anxa2 shRNA (shAnxa2) on growth and metastasis in H1650 CSCs. H1650 CSCs were maintained on basement membrane-coated supports in DMEM:F12 media enriched with growth factors. Nanosomes were prepared by solvent evaporation and bath sonication. H1650 CSCs were transfected with lipoplexes of shAnxa2 and varying amounts of nanosome in serum-free media. After 4 hrs of incubation, the medium was replaced with regular media and knockdown of Anxa2 analyzed after 48 hrs by Western blot. Inhibition of H1650 CSC sphere formation over 7 days was investigated in lipidure® plates following treatment with lipoplexes for 4 hrs. CSCs were assessed for induction of apoptosis after treatment with lipoplexes (4 hrs) by acridine orange-ethidium bromide staining. The effect of Anxa2 lipoplexes on proliferation, migration and capillary-like tube formation was investigated in human primary pulmonary artery endothelial cells (HPAEC). The effect of Anxa2 lipoplexes on the expression of Anxa2, EGFR, VEGF, and NFkβ was evaluated by Western blot. Efficient loading of shAnxa2 in nanosomes was achieved with high transfection efficiency compared to lipofectamine. Anxa2 shRNA lipoplex was cytotoxic to H1650 CSC spheroids, inhibiting spheroid growth by 30-60% between day 3 and 7. Early and late apoptosis induction was observed after treatment with shAnxa2 with a 2-fold increase of cells undergoing apoptosis compared to shAnxa2-lipofectamine 2000 complex. Anxa2 knockdown (90%) was observed in H1650 CSCs parallel to decreases in EGFR, VEGF, and NFkβ expression as shown by Western blot. High loading and enhanced transfection of shAnxa2 was achieved with the nanosome formulation. Anxa2 knockdown was achieved in parallel with inhibition of H1650 CSC spheroid growth, angiogenesis and induction of apoptosis. These results altogether support evidence that Anxa2 could be a therapeutic target for treatment of non-small cell lung cancer and that nanosome formulation enhances delivery of shAnxa2 as an anticancer agent.
Citation Format: Terrick A. Andey, Srujan Marepally, Pomila Singh, Mandip S. Sachdeva. Knockdown of Annexin A2 by liposomal formulation of Annexin A2 small hairpin RNA induces apoptosis and inhibits angiogenesis in lung cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3738. doi:10.1158/1538-7445.AM2013-3738