Notch and mTORC1 Regulate Growth of Human Gastric Cancer Cells and Human Gastric Organoids
Authors: Wong Gabriela , Demitrack Elise S , Samuelson Linda C 
Published: November 2023

BackgroundThe Notch pathway regulates stem cell proliferation in the mouse and human stomach. We have previously shown that in mouse, Notch activation promotes proliferation of LGR5+ antral stem cells and that inhibition of mTORC1 signaling attenuates this increase in stem cell proliferation, although there was no effect of mTORC1 inhibition on basal cell proliferation. The goal of this study was to test Notch and mTORC1 regulation of growth of human gastric cancer (GC) cells and human organoids established from normal or GC tissue.MethodsGene expression analysis of Notch pathway components in human GC cell lines and paired non‐cancer and GC tissue samples was performed using qRT‐PCR. Human GC cell lines AGS, MKN45 and NCI‐N87 were treated with vehicle (DMSO), DAPT (Notch inhibition), rapamycin (mTORC1 inhibition) or both inhibitors for 5 consecutive days. Cell growth was measured each day using the CellTiter 96 Proliferation Assay. Established human organoids from paired non‐cancer and GC tissue were treated with the inhibitors, and organoid size was quantified after 5 days of treatment using ImageJ software.ResultsGene expression analysis revealed that NOTCH1 and NOTCH2 are the most abundant Notch receptors in both non‐cancer and GC tissue, and that both receptors were more highly expressed in GC tissue samples. NOTCH1 and NOTCH2 were also the predominant receptors in AGS and MKN45 GC cell lines. JAG1 was the most abundant Notch ligand expressed in the human stomach and in GC cell lines, and was significantly increased in GC tissue compared to paired normal tissue. Notch inhibition reduced growth of MKN45 and NCI‐N87, but AGS cell growth was not significantly affected. Similarly, mTORC1 inhibition with rapamycin significantly reduced growth of all 3 GC cell lines, and growth was further reduced by combined pathway inhibition, suggesting that both Notch and mTORC1 are required to sustain GC cell growth. Notch inhibition reduced the growth of human organoids derived from paired non‐cancer and GC tissue, although organoids derived from GC tissue required 10‐fold increased DAPT for significant growth inhibition. Interestingly, treatment of both non‐cancer and GC‐derived human organoids with rapamycin led to increased organoid size and increased epithelial cell proliferation, which contrasts with the decreased cell growth observed in GC cell lines.ConclusionNotch pathway receptors and ligands are expressed in human gastric cells, with increased levels observed in GC. Furthermore, Notch signaling is required to sustain growth and proliferation of both normal human gastric stem cells and GC cells. mTORC1 regulation of human gastric cells was variable, with pathway inhibition decreasing growth of GC cell lines and increasing growth of both normal and GC organoids. These findings suggest that while Notch is a fundamental pathway regulating gastric cell proliferation, mTORC1 function is context specific. Thus Notch and possibly mTORC1 may provide useful therapeutic targets for GC treatment.Support or Funding InformationAPS Undergraduate Research Excellence Fellowship; AACR‐Debbie's Dream Foundation Career Development Award; NIH P01‐DK062041; NCI P50‐CA13081; AGA Funderburg Research Award