br SOX SRY HMG Box is a
SOX2 (SRY HMG-Box 2) is a transcription factor that regulates oncogenic signaling pathways in different types CSCs. In human lung CSCs, a positive feedback loop between SOX2 and EGFR sustained high tumorigenic properties, and upregu-lation of SOX2 enhanced self-renewability and resistance of cancer MG132 to EGFR inhibitors.2,7 Silencing of SOX2 in mice bearing A549 xenograft tumors decreased the expression of
mediators of Wnt signaling in a subset of EGFR-mutant human lung adenocarcinoma cells constitutively expressing SOX2; treatment with PI3K/Akt inhibitors depleted SOX2 expression and hindered cell proliferation.8,9 And cooperation between SOX2, beta catenin and POU5F1 promoted insulin-like growth factor-1 (IGF-1)-mediated self-renewal and tumorigenicity
whereas inhibition of SOX2 abrogated cancer cell growth and metastasis.6,8,10
Currently, no drug is approved for SOX2 targeted therapy in cancer. However, evidence of the therapeutic value of SOX2 targeting has been demonstrated with cancer stem cell (CSC)-selective compounds (e.g. Salinomycin and all-trans retinoic acid),11,12 mTOR allosteric inhibitor (e.g. Rapamycin),13 experimental SOX2 immunotherapies (e.g. SOX2 DNA and peptides),14,15 and gene therapies (e.g. microRNA; miRNA, and small interfering RNA; siRNA).16,17 Importantly, molecular tools such as RNA provide opportunities for demonstrating the potential clinical benefits of SOX2-specific targeted therapies. The usefulness of these molecular tools, however, is often offset by challenges to their stability, tumor targeting, and efficient cellular uptake in vivo. The ideal delivery system for macromolecule therapies (e.g. siRNA) in lung cancer must be able to evade the effects of degrading enzymes and antibodies, as well as promote the specific targeting to the tumor cells and subsequent extravasation of the therapy.
Lipid nanocarriers have been shown to provide the versatility for ensuring ideal delivery characteristics for macromolecules in vivo.18–20 We previously reported the successful formulation and delivery of liposomes containing short hairpin RNA (shRNA) targeting Annexin A2 (AnxA2) in a mouse orthotopic lung tumor model.21 In the current study, SOX2 siRNA was formulated in a cationic lipoplex nanocarrier for targeted treatment of SOX2-enriched, H1650 CSC-derived lung tumors in mice.
Materials and methods
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanola-mine-N-[maleimide(polyethylene glycol)-2000] (DSPE-mPEG ) were obtained from Avanti Polar Lipids (Alabaster, AL). DMEM:F12 culture medium, fetal bovine serum (FBS), penicillin/streptomycin/neomycin (PSN) cocktail, nitrogen sup-plement (10×), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-tricarbocyanine iodide (DiR) were obtained from Life Technologies (Grand Island, NY). Laminin, poly-D-Lysine, fibroblast growth factor (FGF) and epidermal growth factor (EGF) were from Sigma Aldrich (St. Louis, MO). Matrigel® extracellular matrix was procured from BD Biosciences (Bedford, MA). Lipidure®-coat plates were kindly donated by NOF Corporation (Japan). Primary and secondary antibodies were from Cell Signaling Technology (Danvers, MA). Control and SOX2 siRNA were purchased from Santa Cruz Biotechnol-ogy (Santa Cruz, CA). The SOX2 siRNA (sc-38,408) consisted of a triplex with the following sequences (5′ → 3′ orientation):
(sc-38408A) sense: GAAUGGACCUUGUAUAGAUTT; anti-
sense: AUCUAUACAAGGUCCAUUCTT, (sc-38408B) sense:
GGACAGUUGCAAACGUGAATT; antisense: UUCACG
UUUGCAACUGUCCTT, and (sc-38408C) sense: GAAUCA GUCUGCCGAGAAUTT; antisense: AUUCUCGGCAGACU GAUUCTT. Cultrex® cell migration assay kit was obtained from Trevigen (Gaithersburg, MD). All other chemicals and reagents were of cell culture or reagent grade.
Human A549 and H1650 cells were procured from the American Type Culture Collection (ATCC) and sorted into main population (MP) and side population (SP) cells as previously described.7 MP cells were maintained in DMEM:F12 media, supplemented with FBS (10% v/v) and a PSN cocktail (2% v/v). SP cells were cultured in an ultra-low attachment culture flask coated with a thin film of simulated basement membrane layer consisting of laminin-bound poly-D-Lysine. Cells were refreshed with serum-free selective base media containing FGF (10 μg/mL), EGF (10 μg/mL), nitrogen supplement (1X), and PSN cocktail (2% v/v). Cells were maintained under standard culture conditions of 95% air and 5% CO2 at 37 °C.
Side population (SP) or main population (MP) cells from H1650 and A549 were plated in ultra-low attachment 96 well plates at a density of 10,000 cells/well in 100 μL medium (100,000 cells/mL) in serum free stem cell selective media and incubated for 10 days to form the spheres. The total number of spheres greater than 50 μm size was counted using a phase contrast microscope (Nikon Instruments). Results were present-ed as average number of spheres with standard deviation of triplicates of each experiment.