Abstract 4385: Identification of the crucial microRNA, miR-1246 related to the chemoresistance and stemness in pancreatic cancer for new targeting therapy

Abstract

Abstract Recently there have been several reports that the presence of cancer stem cells (CSCs) was related to the high refractoriness and infiltration in pancreatic cancer. Considering that microRNAs (miRNAs) are number-limited, functional nucleotides, those should be beneficial for efficient diagnostic and therapeutic approaches of pancreatic cancer. To identify the miRNAs, which play a critical role in the chemoresistance related to pancreatic CSCs, we performed sphere formation assay, as a tool for concentrating CSCs. It can enrich CSCs successfully in several types of cancer including pancreatic cancer. We use Panc1 cells and MiaPaCa2 cells as pancreatic cancer cell lines and established gemicitabine (GEM) resistant clones (Panc1-GRs) and Panc1 parental spheroid cells which were cultured in ES medium with EGF and FGF and ultra-low attachment plate (Panc1-P-Sp). The miRNA profilings between Panc1-GRs and Panc1-P-Sp were compared each other and miR-1246 was selected as a candidate miRNA related to the chemoresistance in pancreatic CSCs. In vitro the drug sensitivity of Panc1 and MiaPaCa2 was changed by miR-1246 transfection and the sphere formation ability of Panc1 was increased by pre-miR-1246 transfection. In pancreatic cancer cell line, Panc1, miR-1246 was up-regulated notably in the spheroid cells. This study revealed that it was related to not only chemoresistance but also stemness feature in pancreatic CSCs. In vivo it was confirmed that the miR-1246 could increase the tumorigenicity and reduced the drug sensitivity in NOD/SCID mice in which Panc1 cells were injected. Subsequently we focused on the molecular targets regulated by miR-1246. Because they link with elucidation for the mechanism through which miR-1246 adds chemoresistance to Panc1 cells. Using Target Scan, which can predict the candidates, 178 genes were selected. 7 genes related to malignant potential, REPS2, CCNG2, GRHL1, UNC5B, NEO1 and ZFP36L1 from previous reports were chosen. Among them, we focused on CCNG2 for further analysis. siRNA for CCNG2 was used to validate its involvement in the resistance to gemcitabine. Knockdown of CCNG2 was confirmed by western blot analysis. The MTT assay demonstrated that transfection of siCCNG2 enhanced the resistance of Panc1-P to gemcitabine but did not change the sphere formation ability. In conclusion, we demonstrated in the present study that miR-1246 inhibited the anti-cancer effect of gemcitabine in pancreatic cancer cells and that CCNG2 mediated this effect. The response to gemcitabine in Panc1 cells was controlled by genetic manipulation of miR-1246 and CCNG2. In addition, in vivo examination revealed that miR-1246 could change the drug sensitivity of pancreatic cancer cell tumor. Considered together, the results suggest that the anti-sense of miR-1246 could be a new therapy targeting CSCs in pancreatic cancer. Citation Format: Shinichiro Hasegawa, Ishii Hideshi, Hidetoshi Eguchi, Shogo Kobayashi, Hiroshi Wada, Naoki Hama, Yoshito Tomimaru, Kawamoto Koichi, Masamitsu Konno, Hisataka Ogawa, Shimpei Nishikawa, Yoshihiro Kano, Yoshihiro Kano, Takahito Fukusumi, Atsushi Hamabe, Takenori Nishimura, Kunihiko Hinohara, Taroh Satoh, Noriko Gotoh, Hiroaki Nagano, Yuichiro Doki, Masaki Mori. Identification of the crucial microRNA, miR-1246 related to the chemoresistance and stemness in pancreatic cancer for new targeting therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4385. doi:10.1158/1538-7445.AM2014-4385