Abstract 4903: 5’-flanking sequences of the NANOG pseudogene 8 (NANOGP8) plays roles in sphere formation, clonogenicity, and tumor growth in mice xenograft model of prostate cancer cells.
Authors: Yin Hong , Zhang Kai , Li Zhen , Glass Jonathan 
Published: August 2014

Abstract
The expression of NANOGP8 has been documented in several cancers and related to cell proliferation and tumor development. However, the regulatory pathways that allow for NANOGP8 rather than NANOG expression have not been investigated. In this study, we hypothesized that NANOGP8 transcription in prostate cancer cells could be controlled by its 5’-flanking sequences and that cells with activation of these sequences should exhibit NANOGP8 function in cell growth and tumor development.
We first examined whether or not the 5’-flanking sequences of NANOGP8 on Chromosome 15 have a 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 so as to replace the CMV promoter. The EGFP expression plasmid driven by the 5’-flanking sequence was then transfected into DU145 cells. After selection with G418, stable cell lines were established. The EGFP positive cells were separated from EGFP negative cells by flow cytometry, and the expression of NANOG was analyzed with real-time RT-PCR in the two populations. The RNA expression of NANOG in EGFP positive cells was 3.5 fold higher than in EGFP negative cells. The colony formation in the EGFP positive DU145 subset was 24% compared to 13.4% in the EGFP negative subset. Similarly, sphere formation in the EGFP positive DU145 subset was 40% higher than that in the EGFP negative subset. Interestingly, in the spheres formed from the EGFP negative subset, about 50% became EGFP positive. Of the total spheres formed from both subsets, 82% were EGFP positive and only 18% spheres were EGFP negative. These results suggest that sphere culture condition activated the 5’-flanking region of NANOGP8, in turn promoted sphere formation. To examine the role of activating 5’-flanking sequence of NANOGP8 in tumor initiation and development, stably transfected DU145 cells were injected subcutaneously on the flank of SCID/beige male mice at 200 cells /mouse. With EGFP positive cells, a tumor was found in all 14 mice (100%) after 15 weeks; while with EGFP negative cells, a tumor was found in 11 of 14 mice (78%). Analyses of tumor volume and weight showed that tumor volume and weight with EGFP positive cells was 5.3 and 7.5 fold higher than with EGFP negative cells, respectively. Further evaluation of EGFP expression in collected xenograph tumor revealed that all analyzed tumor samples from mice injected with EGFP negative cells became EGFP positive. In conclusion, the activation of 5’-flanking sequence of NANOGP8 could play a role in the regulation of the stem-like properties of cancer stem cells and prostate tumor initiation and development. The mechanisms of the activation of 5’-flanking sequence of NANOGP8 is under investigation but could provide potential therapeutic targets.
Citation Format: Hong Yin, Kai Zhang, Zhen Li, Jonathan Glass. 5’-flanking sequences of the NANOG pseudogene 8 (NANOGP8) plays roles in sphere formation, clonogenicity, and tumor growth in mice xenograft model of prostate cancer 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 4903. doi:10.1158/1538-7445.AM2013-4903