Targeting embryonic transcription factors to affect cancer stem cells and systemic therapy.

Abstract

11573 Background: NANOG is an embryonic transcription factor essential for reprogramming cells. The retrogene NANOGP8 is expressed in human cancers, not in normal tissues. NANOGP8 modulates stemness of human colorectal carcinoma (CRC) cells and can replace NANOG as a cancer stem cell (CSC) driver. Our hypothesis is that NANOGP8 is a therapeutic target to inhibit CSC and enhance systemic therapy. Methods: Ability of single CRC cells to form spheres in serum free medium or to survive exposure to chemotherapy (topotecan – TOPO) or targeted therapy (Afatanib) in a 72 hr in vitro survival assay measured CSC. NANOG was measured in a tissue microarray of 298 primary CRC by quantitative immunofluorescence assay (qIFA). NANOGP8 expression was inhibited by lentivirus or oncolytic chimeric adenovirus Ad5/3 expressing shRNA to NANOGP8 (LVshNP8-1 or AdNP8, respectively). Sub cutis CRC xenografts grown in NOD/SCID mice were injected once intralesionally with LVshNP8-1 or AdNP8. Results: 3-62% of cells within 6 human CRC lines are CSC by spherogenicity. LVshNP8-1not only inhibits spherogenicity but also reduces expression of OCT4 and SOX2, the size of the side population and tumor growth in vivo. Overexpression of NANOGP8 rescues CSC when LV shRNA inhibits expression of NANOGs. qIFA identifies both NANOG proteins since antibody cannot separate the 2 proteins that only differ by 2 of 305 amino acids. NANOG was detected in 65% of primary CRC. NANOG expression is associated with a HR of 2.04 (95% CI 1.18-3.75, P = 0.009) in a Cox PH model where TNM stage and vascular invasion are significant (P < 0.0001) but age, grade, race, gender are not. Exposure to TOPO, but not afatinib, at IC50 increases NANOG gene expression. LVshNP8-1 and AdNP8 increase caspase-dependent cytotoxicity of TOPO and afatanib at their IC50 to > 90%. Single intralesional injection of LVshNP8-1 into 0.7 – 1.0 cm CRC xenografts induces transient inhibition of growth but a single injection of Ad5/3 with shRNA to NANOGP8 causes a sustained 40-50% inhibition of growth in 0.8 – 1.0 CRC xenografts for 30+ days Conclusions: NANOG and NANOGP8 are prognostic factors that drive CSC in CRC. Inhibition of NANOG/NANOGP8 gene expression by gene therapy may augment therapy of cancer and inhibit CSC function.