Abstract 500: Activation of 5’-flanking sequences of the NANOG pseudogene (NANOGP8) in prostate cancer cell lines increases NANOG RNA expression, sphere formation, and clonogenicity
Authors: Yin Hong , Zhang Kai , Glass Jonathan 
Published: June 2012

Abstract
In cancer cells, in contrast to normal embryonic stem cells, NANOG is transcribed from the NANOG pseudogene, NANOGP8. The over-expression of NANOGP8 has been documented to promote the proliferation of NIH3T3 cells, the invasion of prostate PC3 cells, and the development of prostate tumors. In this study, we hypothesized that NANOGP8 transcription could be controlled by a promoter-like region in its 5’-flanking sequences and cells with activation of these sequences should exhibit NANOGP8 function, namely sphere formation and increased cell growth.
To test this hypothesis, we first verified if the expressed NANOG in prostate cancer comes from NANOGP8. RT-PCR was conducted from RNA collected from the prostate cell lines PC3 and DU145 and primary culture of normal and cancerous prostate cells with primers spanning across both the whole coding region and the 3’UTR. The 1.6kb PCR products were sequenced verifying that the NANOG RNA was transcribed from NANOGP8 and not from NANOG in cancer cells and normal prostate cells. To examine if the 5’-flanking sequences of NANOGP8 have promoter-like function, we cloned a 5kb fragment of the 5’-flanking region of NANOGP8 from a PAC clone containing the whole NANOGP8 gene and inserted the fragment into the pEGFP vector to replace the CMV promoter. The EGFP expression plasmid driven by the 5’-flanking sequence was then transfected into PC3 and DU145 cells. After selection with G418, the EGFP positive cells were separated from EGFP negative cells by flow cytometry and expression of NANOG was analyzed with real-time RT-PCR in the two populations. The RNA expression of NANOG in EGFP positive DU145 and PC3 cells was 3.5 fold and 1.5 fold higher than in EGFP negative cells, respectively. In a DU145 stably transfected cell line the colony formation in the EGFP positive subset was 24% compared to 13.4% in the EGFP negative subset. Similarly, sphere formation in the EGFP positive DU145 cell subset was 40% more than that in the EGFP negative subset. Interestingly, in the spheres formed from the EGFP negative subset about 50% became EGFP positive and of the total spheres formed from both subsets, 82% were EGFP positive and only 18% of spheres were EGFP negative, suggesting that culture in serum-free medium without cell attachment promoted activation of the 5’-flanking region of NANOGP8. In conclusion, NANOG P8 is transcribed in both normal and cancerous prostate cells. The 5’-flanking sequence of NANOGP8 could play a role in expression of the NANOGP8 gene in prostate cancer cells and in regulation of stem-like properties of cancer stem cells. The mechanisms of the activation of the promoter-like 5’-flanking sequence of NANOGP8 is under investigation but could provide potential therapeutic targets.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2011-500