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. 2020 Oct 9;295(41):14153-14163.
doi: 10.1074/jbc.RA120.014887. Epub 2020 Aug 6.

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover

Katie L Alexander  1 , Carolina A Serrano  2 , Asmi Chakraborty  3 , Marie Nearing  1 , Leona N Council  4 , Arnoldo Riquelme  5   6 , Marcelo Garrido  7 , Susan L Bellis  3 , Lesley E Smythies  8 , Phillip D Smith  8
Affiliations

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover

Katie L Alexander et al. J Biol Chem. .

Abstract

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

Keywords: apoptosis; cancer biology; epithelial cell; gastric cancer; glycosylation; sialic acid; stem cells; stem cellsα-26-sialyltransferase 1 (ST6Gal-I); β-galactoside α2,6-sialyltransferase 1 (ST6GAL1).

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
Epithelial ST6Gal-I expression is progressively up-regulated in the successive stages of gastric premalignancy and gastric cancer. A, gastric antral biopsies from 39 subjects with the indicated histological finding were analyzed for the number of ST6Gal-I+ cells per HPF in 5 randomly selected fields per donor tissue. B and C, gastric adenocarcinoma and noninvolved tissue from the same donor (confirmed by pathologist L. N. C.) were (B) analyzed for ST6Gal-I by immunohistochemistry (representative image, 20×; n = 6; scale bar, 50 μm) and (C) enumerated for ST6Gal-I+ cells per HPF in 10 randomly selected fields per donor tissue. D, sequential sections from gastric adenocarcinoma (representative image, 20×; n = 3; scale bar, 50 μm) were stained for ST6Gal-I–positive and Sox9-positive cells using immunohistochemistry. E, 42 gastric antral biopsies were examined for mRNA expression of ST6GAL1 and SOX9 by quantitative real-time PCR and expressed as -fold change relative to GAPDH expression. (A and C, significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
Figure 2.
Figure 2.
ST6Gal-I is a novel biomarker of gastric epithelial stem cells. A, epithelial stem cells derived from normal gastric antrum (gastric organoids) cultured in Matrigel supplemented with BASIC-CM for 5 days (see “Experimental procedures”) showed a progressive increase in the formation, number and size of stem cell organoids (representative images, 10×; n = 6; scale bar, 200 μm). B, matching gastric biopsies and organoids were analyzed for LGR5 and MUC5AC expression by real time qPCR. C, gastric organoids were isolated on day 3 and examined by immunofluorescence after staining with antibodies to ZO-1 (Alexa Fluor 594) or E-cadherin (FITC) and counterstained with DAPI (representative organoids, 20×; n = 3; scale bar, 50 μm). D, gastric organoids were stained with an antibody to ST6Gal-I (FITC) in addition to phalloidin (Alexa Fluor 594) and DAPI (representative donor; n = 3) (40×; scale bar, 100 μm). E, gastric organoids were stained with SNA lectin (blue) (see “Experimental procedures”) using flow cytometry. F and G, organoids were cultured 3–4 days in decreasing concentrations of l-WRN CM in BASIC-CM and analyzed for (F) LGR5 and (G) ST6GAL1 gene expression (n = 3). B, F, and G, significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
Figure 3.
Figure 3.
ST6Gal-I is not expressed in normal differentiated gastric epithelium. A, epithelial cell monolayers derived from gastric organoids (see “Experimental procedures”) were examined for confluence by microscopy on days 1, 2, and 4 (representative images, 10×; n = 3; scale bar, 100 μm, 20 μm for inset). B, mRNA from day 0 (organoids) and day 2 (monolayers) was analyzed for MUC5AC expression by qPCR (n = 3). C, confluent epithelial cell monolayers derived from gastric organoids were stained for ZO-1 (Alexa Fluor 594) and E-cadherin (FITC) using antibodies and nuclei (DAPI) using immunofluorescence (representative staining, 40×; n = 3; scale bar, 50 μm). D, gastric organoid–derived monolayers (day 2) were stained with an antibody for ST6Gal-I (FITC) as well as phalloidin (Alexa Fluor 594) and DAPI (representative donor; n = 3) (20×; scale bar, 50 μm). E and F, gastric organoids (day 0) and organoid-derived epithelial monolayers (days 1–4) from a representative donor were analyzed for (E) LGR5 and (F) ST6GAL1 gene expression and (E and F, insets) from three donors analyzed on day 2. G, monolayers were generated as in (A) and on day 2 the media was kept at 5% l-WRN (monolayer media) or changed to 50% l-WRN (organoid media, BASIC-CM) for 24 h and analyzed for LGR5 and ST6GAL1 gene expression (n = 3–5). B, E, and F, significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
Figure 4.
Figure 4.
ST6Gal-I overexpression in gastric epithelial organoids is maintained in organoid-derived monolayers and inhibits TNF-mediated apoptosis. A, epithelial stem cell organoids derived from normal gastric antrum (Normal) with ST6Gal-I knockdown (KD) or ST6Gal-I overexpressed (OE) or empty vehicle control (EV) (see “Experimental procedures”) were analyzed on days 3–4 for ST6GAL1 gene expression (n = 4). B, organoids were stained with an ST6Gal-I antibody (FITC), phalloidin (Alexa Fluor 594) and DAPI (representative images; n = 3; scale bar, 50 μm). C, normal, KD, and OE gastric organoids (day 0) and their derived epithelial monolayers (days 1–4) were analyzed for ST6GAL1 gene expression. D, normal, KD and OE epithelial monolayers (day 2) were stained with an ST6Gal-I antibody (FITC) as well as phalloidin (Alexa Fluor 594) and DAPI (monolayers from a representative donor (n = 3; 20×; scale bar, 50 μm). E, ST6Gal-I normal, KD or OE organoid-derived monolayers were analyzed for surface TNFR1 (FITC) by flow cytometry (representative data shown, n = 4). F, mRNA from day 2 monolayers was analyzed for TNFRSF1 expression by qPCR (n = 7). G, ST6Gal-I normal, OE, and KD organoid-derived monolayers (n = 5–10 in four independent experiments) were treated with TNF 50 ng/ml for 24 h or media and evaluated for caspase 3/7 activity by CaspaseGlo 3/7 luminescence assay. A–C significance: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
Figure 5.
Figure 5.
Impact of ST6Gal-I expression on gastric cancer cell apoptosis. A and B, gastric organoids and organoid-derived epithelial monolayers from gastric adenocarcinoma (red line) and normal gastric mucosa (black line) were analyzed on day 0 (organoids) and days 1–4 (epithelial monolayers) for (A) LGR5 and (B) ST6GAL1 gene expression (each n = 4). C, gastric organoids (day 0) and organoid-derived epithelial monolayers (day 2) from normal gastric tissue or gastric cancer were analyzed for SOX9 gene expression (each n = 3). D, epithelial cell monolayers generated from normal antrum-derived (top panel) and gastric adenocarcinoma–derived (bottom panel) organoids were analyzed on day 2 by immunofluorescence by staining with an ST6Gal-I antibody (FITC), phalloidin (Alexa Fluor 594) and DAPI (representative donor, 20×; n = 4; scale bar, 50 μm). E, gastric cancer organoid–derived epithelial monolayers were pretreated with or without A. ureafaciens neuraminidase and assayed for caspase 3/7 activity (n = 4–8 in three separate gastric cancer-derived monolayers). F, gastric organoid–derived epithelial monolayers were analyzed for SNA expression using flow cytometry with (red) or without (green) A. ureafaciens neuraminidase pretreatment. A–C, E, significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.
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