Abstract 2911: Gene knockout of NANOG and NANOGP8 mediated by CRISPR/Cas9 system decreases the malignant potential of prostate cancer cells
Authors: Kawamura Norihiko , Nimura Keisuke , Kawashima Atsunari , Ujike Takeshi , Nagahara Akira , Fujita Kazutoshi , Uemura Motohide , Kaneda Yasufumi , Nonomura Norio 
Published: July 2016

Abstract
Introduction & objectives :
NANOG is an essential transcription factor for self-renewal and pluripotency of embryonal stem cells. Recently, it has been reported that NANOG is expressed in various somatic cancers, including prostate cancer, and drive tumor development, and that increased NANOG expression in human prostate cancer tissues is correlated with an increased Gleason score. NANOG (hereinafter NANOG1) has many pseudogenes, and only the NANOGP8 pseudogene encodes the full-length NANOG1 protein with high sequence similarity. NANOGP8 is reported to be expressed in most types of cancer as a primary contributor of NANOG mRNA expression and to increase the malignant potential. However, the proportion of NANOG protein expression that comes from NANOG1 and NANOG8 in cancer cells is not known because of the high similarity between them. Therefore, a causal role of NANOG1 and NANOGP8 in prostate cancer cells is not clear.
Materials & methods :
We established NANOG1-/- and NANOGP8-/- prostate cancer cell lines from DU145 cells using CRISPR/Cas9, and also we established NANOG1-rescued cells and NANOGP8-rescued cells from each NANOG knockout cells for rescue experiments. We examined cancer properties associated with malignant potential in these cells and DU145 cells, including self-renewal, sphere-formation, migration, drug resistance and tumorigenic potential.
Results :
Colony formation assays were performed to examine the role of NANOG1 and NANOGP8 in self-renewal. The colony-forming capacity of NANOG1-/- and NANOGP8-/- cells was decreased compared to parental cells. Similary, the sphere-forming capacity of NANOG1-/- and NANOGP8-/- cells decreased to approximately 50% compared to the parental cells. Wound-healing assays were performed to examine the migration capacity, and migration was decreased in NANOG1-/- and NANOGP8-/- cells by 40-60%. MTS assays 48 hours after docetaxel administration were performed to evaluate the effect of NANOG1 and NANOGP8 on drug sensitivity. NANOG1-/- and NANOP8-/- cells showed increased sensitivity to docetaxel. NANOG1 and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG1- and NANOGP8-rescued cell lines.
Conclusion :
These results indicate that both NANOG1 and NANOGP8 proteins are expressed in prostate cancer cell lines, and both genes equally contribute to the high malignant potential of prostate cancer cells.
Citation Format: Norihiko Kawamura, Keisuke Nimura, Atsunari Kawashima, Takeshi Ujike, Akira Nagahara, Kazutoshi Fujita, Motohide Uemura, Yasufumi Kaneda, Norio Nonomura. Gene knockout of NANOG and NANOGP8 mediated by CRISPR/Cas9 system decreases the malignant potential of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2911.