RNA interference-mediated silencing of Foxo3 in antigen-presenting cells as a strategy for the enhancement of DNA vaccine potency

Abstract

The transcription factor Forkhead box O3 (Foxo3) has a critical role in suppressing the expansion of antigen-specific effector T-cell populations; hence, Foxo3 is a potential target for enhancing the antitumor immunity of cancer vaccines. In this report, we evaluated the potential of RNA interference (RNAi)-mediated silencing of Foxo3 in antigen-presenting cells as an adjuvant for HER2/neu DNA cancer vaccines. Bicistronic plasmids expressing the N-terminal extracellular domain of human HER-2/neu and the Foxo3 short hairpin RNA (hN'-neu-Foxo3 shRNA) or the scrambled control (hN'-neu-scramble shRNA) were subcutaneously injected into mice by gene gun administration to elicit antitumor immunity against p185neu-overexpressing MBT-2 bladder tumor cells. We found that mice treated with hN'-neu-Foxo3 shRNA showed greater reductions in tumor growth and longer survival times than mice treated with hN'-neu-scramble shRNA, indicating that the silencing of Foxo3 enhanced the antitumor efficacy of the HER-2/neu cancer vaccine. Cytotoxicity analyses further revealed that the Foxo3 shRNA-enhanced antitumor effect was associated with significant increases in the number of functional CD8 + T cells and in the levels of cytotoxic T lymphocytes activity. Interleukin-6 was induced by hN'-neu-Foxo3 shRNA treatment but did not have a critical role in the antitumor effect of the hN'-neu-Foxo3 shRNA vaccine. Moreover, in vivo lymphocyte depletion analyses confirmed that the antitumor efficacy of the hN'-neu-Foxo3 shRNA vaccine depended on functional CD8 + T cells. Finally, Foxo3 suppression was shown to markedly improve the effect of the HER-2/neu DNA vaccine in limiting the growth and lung metastases of MBT-2 cells. Overall, these results support RNAi-mediated silencing of Foxo3 as an effective strategy to enhance the therapeutic antitumor effect of HER-2/neu DNA vaccines against p185neu-positive tumors.

Unravelling the role of fatty acid metabolism in cancer through the FOXO3-FOXM1 axis

“…The levels of another key adipokine IL-6 are also regulated by FOXO3. IL-6 is increased after Foxo3 silencing or Sirt1-mediated FOXO3 inhibition (Wang et al, 2011 andWang et al, 2013), suggesting that FOXO3 represses IL-6 expression. Moreover, IL-6 signalling pathway inhibition has been shown to be related to FOXO3 activation, up-regulating the pro-apoptotic FOXO3 target protein Bim in myeloma cancer cells (Shen et al, 2013;Essafi et al, 2005).…”

Clitocybe nudaActivates Dendritic Cells and Acts as a DNA Vaccine Adjuvant

“…Mouse DCs were generated from bone marrow as previously described [23]. Bone marrow (BM) cells were flushed from the femurs and tibias of C57BL/6 mice, lyzed red blood cells with ammonium chloride, and then washed with PBS.…”

“…The protocol for OVA-specific T-cell activation was modified from our previous report [23, 24]. Briefly, immature DCs were pulsed with 2  μ g/mL OVA 257–264 (OVAP 1 ) or OVA 323–339 (OVAP 2 ) (synthesized by Echo Chemical Co., Taiwan) in the presence of LPS or WE-CN (100  μ g/mL) for 24 hr.…”

“…Immature DCs were stimulated with 100  μ g/mL WE-CN, and whole cell lysates were prepared at the indicated time points as previously described [23]. The protease inhibitors leupeptin (Sigma-Aldrich, St. Louis, MO, USA) and aprotinin (Sigma-Aldrich, St. Louis, MO, USA) were used at a concentration of at 10  μ g/mL in all steps.…”

“…The protocol for detection of antigen-specific CD8 + /IFN-gamma + T lymphocytes was modified from our previous report [23, 26]. After the last DNA vaccination for three days, RBC lysis buffer (eBioscience)-lysed, single-cell spleen suspensions (2 × 10 6 cells/well) from mice from the different vaccination groups were pulsed for 18 hr with a peptide pool composed of 10  μ g/mL each of peptide 362–370 (EFAGKKI) (BioBasic, Canada) and peptide 404–412 (EEITGYLYI) (BioBasic, Canada) of the human HER-2/neu sequence.…”