Abstract 214: GLI-1 plays a pivotal role in Hedgehog pathway on self-renewal of pancreatic cancer stem cells.

Abstract

Abstract Background: Cancer stem cells (CSCs) have unique functions of self-renewal, pluripotency and hierarchical proliferation. CSCs are considered to play a crucial role in the recurrence of cancer and are assumed resistance to anticancer agents. Aberrant Hedgehog (Hh) pathway signaling has been implicated in the pathogenesis, self-renewal, and drug resistance of a growing number of solid malignancies. Sonic hedgehog (Shh) is expressed in PanIN and pancreatic adenocarcinoma, especially implicating as an early and late mediator of pancreatic cancer tumorigenesis. In this study, we focused on Hh pathway to regulate self-renewal of CD133high pancreatic CSCs. Materials and Methods:Cells: We established endogenous CD133high (over 90%) pancreatic cancer cells derived from Capan-1 cells. Consequently, CD133knock-down cells were established by the lentiviral transduction method. Reagents: 1) Cyclopamine inhibits the Hh pathway by interaction with the Hh signaling protein smoothened (SMO). 2) GANT61 blocks the GLI function which constitutes the final step in the Hh pathway and interferes with GLI-1 DNA binding in living cells. To analyze the Hh pathway: Stemness-related genes were analyzed by DNA microarray. Self-renewal signal activation was confirmed by fluorescent immunohistochemistry. To analyze Hh pathway, sphere assay and MTT assay were performed on sphere-forming abilities and cytostatic effects by Hh signal inhibitors, respectively. The migration assay was used to evaluate cell migratory ability. Results: 1) Sphere-forming cells in CD133high cells showed upregulation of BMP3, SOX2, GLI1, ALDH1A, et al. and downregulation of PTCH1, SMO, WNT3A, et al. by DNA microarray but the monolayer cells did not. 2) Cyclopamine could suppress cell growth and sphere formation of CD133high and CD133knock-down cells. GANT61 showed more suppressive effect than Cyclopamine. In addition, GANT61 treatment showed smaller spheroid than the cyclopamine treatment. These results suggest that Hh signal participates in proliferation and self-renewal of the pancreatic CSCs. 3) CD133high cells showed higher migratory ability than CD133knock-down cells. Because the expression of epithelial-mesenchymal transition (EMT)-associated transcription factors, such as Slug and snail, in CD133high cells are upregulated but not in CD133knock-down cells. Interestingly, Hh signal inhibitors suppressed the migratory ability of CD133high cells by the migration assay. Conclusion: GLI-1 acts more effectively on self-renewal of pancreatic CSCs through Hh pathway than Cyclopamine. Further study on GLI-1 inhibitor can lead to a new targeted therapy for invasion and metastasis of pancreatic cancer. Citation Format: Yumi Miyazaki, Qiang Ding, Makoto Yoshimitsu, Toru Obara, Koichirou Tsukasa, Shyuichiro Matsubara, Sonshin Takao. GLI-1 plays a pivotal role in Hedgehog pathway on self-renewal of pancreatic cancer stem 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 214. doi:10.1158/1538-7445.AM2013-214