Suppressed Cellular Senescence Mediated by T-box3 in Aged Gastric Epithelial Cells may Contribute to Aging-related Carcinogenesis

Abstract

Aging is a risk factor for cancers in various organs. Recent advances in the organoid culturing system have made it viable to investigate the influence of aging utilizing these mini organs. In this study, we aimed to examine the implications of aging for gastric carcinogenesis. Gastric organoids established from aged mice grew larger, proliferated vigorously, and survived longer than that from young mice. Because Wnt/β-catenin signaling was intensified in the aged organoids and because removal of Wnt-related factors diminished their proliferation, we investigated for Wnt target gene that contributed to enhanced proliferation and discovered that the aged organoids expressed the transcription factor T-box3 (Tbx3), which has been reported to suppress cellular senescence. Indeed, cellular senescence was suppressed in the aged organoids, and this resulted from enhanced G₂-M transition. As for the mechanism involved in the intensified Wnt/β-catenin signaling, we identified that Dickkopf3 (Dkk3) expression was reduced in the aged organoids due to methylation of the Dkk3 gene. Finally, the expression of TBX3 was enhanced in human atrophic gastritis and even more enhanced in human gastric cancers. In addition, its expression correlated positively with patients' age. These results indicated that the emergence of antisenescent property in aged gastric organoids due to enhanced Tbx3 expression led to accelerated cellular proliferation and organoid formation. Because the enhanced Tbx3 expression seen in aged gastric organoids was also observed in human gastric cancer tissues, this Dkk3-Wnt-Tbx3 pathway may be involved in aging-related gastric carcinogenesis.

Significance: This work provides an insight into the mechanism involved in aging-related gastric carcinogenesis through studies utilizing organoids established from young and aged murine stomachs.

FIGURE 1

Aged gastric organoids exhibit increased cellular proliferation and enhanced Wnt/β-catenin signaling. A, Representative images of young and aged gastric organoids on days 0 and 4. Scale bar: 500 μm. B, The numbers of young and aged gastric organoids larger than 50 μm in diameter on days 0, 2, and 4 (n = 3). C, Cell viability assay of young and aged gastric organoids performed on day 4. D, Survival of the young and aged gastric organoids (n = 3). E, Representative images of young and aged gastric organoids cultured in the presence (WRC+) and the absence (WRC−) of Wnt3a, R-Spondin1, and Chir99021. Scale bar: 500 μm. F, Cell viability assay of young and aged gastric organoids cultured in WRC+ and WRC− medium performed on day 4 (n = 3). G, TCF reporter activity of young and aged gastric organoids (n = 3). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 2

Cellular senescence is suppressed in aged gastric organoids. A, GSEA for (left) G₂–M checkpoint-related genes and (right) senescence-related genes. B, The expression of genes related to cellular proliferation and cell cycle in young and aged gastric organoids (n = 3). C, The expression of senescence-inducing genes in young and aged gastric organoids (n = 3). D, Immunofluorescence for genes related to cellular proliferation and senescence in young and aged gastric organoids (n = 5). E-cad: E-cadherin. E, Representative images of SABG assay for young and aged gastric organoids. Scale bar: 50 μm. F, The ratio of senescent cells in young and aged gastric organoids assessed by SABG assay (n = 3). G, The expression of SASP-related genes in young and aged gastric organoids (n = 3). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 3

Aged gastric organoids express Tbx3. A, The expression of Wnt target genes in young and aged gastric organoids (n = 3). B, Immunofluorescence for Tbx3 in young and aged gastric organoids (n = 5). E-cad: E-cadherin. C,Mki67 and Tbx3 expression in the presence (WRC+) and the absence (WRC−) of Wnt3a, R-Spondin1, and Chir99021 in aged organoids (n = 3). D, Western blot analysis for TBX3 and β-actin of AGS cells transfected with empty vector or TBX3-expressing plasmid. E, MTS assay for TBX3-overexpressing AGS cells (n = 3). F, Representative images of SABG assay for TBX3-overexpressing AGS cells. Scale bar: 50 μm. G, The ratio of senescent cells in TBX3-overexpressing AGS cells assessed by SABG assay (n = 3). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 4

Negligible expression of Dkk3 in aged gastric organoids induces enhanced organoid-forming capacity. A, The expression of niche factors in the gastric mucosae of young and aged mice (n = 3). B, The expression of SASP-and senescence-related genes in the gastric mucosae of young and aged mice (n = 3). C, The expression of genes involved in Wnt/β-catenin signaling in young and aged gastric organoids (n = 3). D, Representative images of aged gastric organoids cultured in the presence and the absence of recombinant Dkk3 protein. Scale bar: 500 μm. E, The numbers of aged gastric organoids larger than 100 μm in diameter cultured in the presence and the absence of recombinant Dkk3 protein (n = 3). F, A schema showing the location of CpG islands and the primers designed for methylation-specific PCR. G, Methylation-specific PCR for genomic DNA extracted from young and aged gastric organoids (n = 3). n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIGURE 5

TBX3 is expressed in human atrophic gastritis and gastric cancer tissues. A, IHC for TBX3, Ki67, and DKK3 in human oxyntic glands, atrophic gastritis, and gastric cancers. Scale bar: 100 μm. TBX3 positivity (B), Ki67 positivity (C), and DKK3 score (D) in human oxyntic glands, atrophic gastritis, and gastric cancers (n = 40). E, Correlation of patients’ age and TBX3 positivity (r = 0.483, P = 0.008). F, Correlation of patients’ age and DKK3 score (r = −0.762, P < 0.001). ***, P < 0.001.