Abstract A29: The mechanism of differential effect of metformin on colorectal cancer stem cells: Metabolic alteration via glutamine metabolic pathway

Background/Aim: There have been reports about cancer stem cells (CSCs) suppressing effect of metformin in several cancers. However, the differential effect of metformin on CSCs and its mechanism has not been reported. We identified various effect of metformin in suppression of colorectal CSCs and its molecular mechanism in colorectal cancer (CRC) cell lines. Methods: Eight CRC cell lines including HT29 and SW620 cells were used. The CSC suppressing effects of metformin (10 mM), glutaminase inhibitor (compound 968, 10 μM), and combination of two drugs were measured by flow cytometry (CD133/44), and tumor sphere formation assay. AMPK and mTOR signaling were examined by Western blotting using anti-pAMPK and anti-pS6, and oxygen consumption rate was measured. The CSC inhibitory effects were also evaluated in xenografted nude mice of HT19 and SW620 cells, using immunohistochemical stain for CD133/CD44. Results: CSCs decreased after treatment of metformin in HT29, DLD-1, and LoVo, and HCT116 cells, but not in SW620, WiDr, Colo205, and Caco-2 cells. Treatment of metformin induced pAMPK activation and pS6 suppression in HT29 cells (metformin-sensitive cells), but not in SW620 cells (metformin-resistant cells). Oxygen consumption rate was higher in HT29 cells than in SW620 cells, and showed more prominent decrease after metformin treatment in HT29 cells. In addition, SW620 cells became sensitive to metformin in CSC-suppressing effect in glutamine-depleted media, but not in low glucose media. Considering the alternative energy source of glutamine, which escapes the effect of metformin on tumor cells, we found that combination of metformin and compound 968 induced CSC-suppressing effect in SW620 cells, and enhanced that effect in HT29 cells. Further, SW620 cells showed higher expression of glutaminase and glutamine transporter than HT29 cells. In xenograft model of HT29 and SW620 cells, combined treatment of metformin and 968 also showed the same results with in vitro experiments. Conclusion: The effect of metformin on CSCs of CRC cells is various depending on AMPK-mTOR pathway and glutamine metabolic pathway. Inhibition of glutamine pathway could enhance the CSC-suppressing effect of metformin, overcoming metformin-resistance. Citation Format: Kyoung Jin Lee, Jae Hyun Kim, Ji-Hee Kwon, Yoo Jung Seo, Tae Il Kim. The mechanism of differential effect of metformin on colorectal cancer stem cells: Metabolic alteration via glutamine metabolic pathway. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A29.