TARGETED DELETION OF SMAD4 FORCES MALIGNANT TRANSFORMATION OF ACTIVE KRAS-INDUCED DYSPLASTIC CELLS IN GASTRIC CARCINOGENESIS

Background: Ras pathway activation in gastric chief cells induces a sequential cascade from normal to metaplasia and dysplasia, a key stage of developing gastric cancer. Despite of various gene mutations in cancer, it is unclear impacts of additional gene mutations in precancerous dysplasia induced by Kras activation. Smad4 is known as one of the most frequently mutated gene in TGF-β pathway in gastric cancer. We therefore performed a genetic manipulation of the Smad4 gene using the CRISPR/Cas9 technology in mouse dysplastic cells induced by Kras activation in gastric chief cells and determined whether the loss of Smad4 can lead to dysplasia evolution to cancer cells.
Methods: Previously, we established dysplastic organoid lines (Meta4) from active Kras induced mouse stomachs. To delete Smad4 gene in dysplastic cells, we utilized CRISPR/Cas9 to target the Smad4 MH2 domain, which is required for Smad4 homo- or hetero- interaction, and established an isogenic line (Meta4-Smad4KO). Both 3D and Air-Liquid Interface (ALI) cultures and RNA-sequencing were conducted to compare altered cellular behaviors and characteristics. We also generated a novel mouse model, GIF-CreERT2-mApple;LSL-Kras;Smad4^fl/fl (GApple-Kras-Smad4KO) to simultaneously induce active Kras expression and Smad4 gene deletion in chief cells. Tamoxifen was injected intraperitoneally to mice at 6 weeks of age and histological examinations were performed.
Results: In 3D cultures, Meta4-Smad4KO organoids showed increases in organoid size and budding formation, indicating enhanced proliferation activity and cell behavior changes. While the Meta4 cells displayed a single layer of mucinous cells positive for MUC5AC or TFF3 in ALI condition, the Meta4-Smad4KO cells displayed multilayering structures with an increase in Ki67 and TROP2 positive cells. The Meta4-Smad4KO cells also increased the CTTN expression, which is important for cancer cell migration, and acquired enhanced cell migration activities in Boyden chamber assay. Transcriptomic profiling identified global changes in gene expression. In particular, genes associated with cell junction, metabolism and Myc signaling pathway were changed in Meta4-Smad4KO cells. The GApple-Kras-Smad4KO mice (2/4) developed adenoma with dysplasia, 6 months after the tamoxifen injection. We confirmed the increased neoplastic feature with increased in nucleus/cytosol ratio, elongated nucleus morphology and dysplastic marker expression including CLDN7, CEACAM5 and TROP2 in the adenoma.
Conclusion: Loss of Smad4 in Kras induced dysplastic cells promoted cell lineage evolution to cancerous cells and induced adenoma development with dysplasia. These results suggest that Smad4 is responsible for dysplasia evolution to cancer during gastric carcinogenesis.