Abstract 1711: Notch and mTORC1 regulate human gastric stem cell function during normal tissue homeostasis and gastric cancer

Abstract

Abstract Notch and mTOR pathway activation have been observed in human gastric cancers. We have recently shown that chronic Notch activation induces gastric stem cell proliferation and tissue expansion in mouse via gland fission, which can be attenuated by mTORC1 inhibition. These findings suggest that these two pathways may synergize to promote gastric tumorigenesis. Here we tested the requirement of Notch and mTOR signaling for maintenance of human stem cells from control and gastric cancer tissues. Gastric glands were isolated from control (non-cancer) and human gastric cancer tissues, as well as mouse antrum to initiate organoid cultures. Established organoids were treated with vehicle, the Notch inhibitor DAPT, the mTORC1 inhibitor rapamycin, or both, every other day for 5 days, to test epithelial-specific requirements of each pathway for stem cell maintenance. Growth was determined by measuring organoid size, and proliferation was assessed by EdU incorporation. Gastric cancer cell lines AGS, MKN45 and NCI-N87 were treated with pathway inhibitors once per day for 5 days, with cell growth measured each day. Gene expression analysis of Notch pathway components was performed by qRT-PCR. In both human and mouse stomach, Notch1 and Notch2 were the main Notch receptors expressed in tissue and organoids. Notch inhibition with DAPT significantly inhibited growth of both human and mouse gastric organoids; however organoids derived from human gastric cancer tissue required 10-fold increased DAPT for significant growth inhibition. Surprisingly, mTORC1 inhibition with rapamycin significantly increased organoid size and epithelial proliferation of mouse organoids, as well as both control and tumor-derived human organoids, suggesting that active mTORC1 signaling restricts human stem cell proliferation. Combination treatment of human organoid cultures with both pathway inhibitors attenuated the rapamycin-induced increase in organoid size and proliferation. In contrast, treatment of gastric cancer cell lines with DAPT, rapamycin or both inhibitors significantly reduced cell growth compared to vehicle, suggesting that blocking Notch or mTORC1 in gastric cancer cells can be used to reduce cancer cell growth. In conclusion, Notch and mTORC1 signaling regulate mouse and human gastric stem cell function although through opposite mechanisms, with Notch stimulating human stem cell proliferation and mTORC1 inhibiting proliferation. Additionally, mTORC1 may regulate human gastric stem cell proliferation via a different mechanism than gastric cancer cells. Further studies testing the mechanism of Notch and mTORC1 interactions to regulate human gastric stem cell maintenance will inform our understanding of therapeutic strategies for gastric cancer. NIH F32-DK093349; NIH UL1TR000433; NIH P01-DK062041 and NCI P50-CA130810 Citation Format: Elise S. Demitrack, Gail B. Gifford, Gabriela Wong, Linda C. Samuelson. Notch and mTORC1 regulate human gastric stem cell function during normal tissue homeostasis and gastric cancer. [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 1711.