Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover
2020; Elsevier BV; Volume: 295; Issue: 41 Linguagem: Inglês
10.1074/jbc.ra120.014887
ISSN1083-351X
AutoresKatie L. Alexander, Carolina Serrano, Asmi Chakraborty, Marie Nearing, Leona N. Council, Arnoldo Riquelme, Marcelo Garrido, Susan L. Bellis, Lesley E. Smythies, Phillip D. Smith,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoProgrammed 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. 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. Epithelial cell homeostasis in the gastrointestinal mucosa is maintained by the finely tuned balance between cell proliferation and apoptosis. This balance is disrupted transiently in mucosal injury and permanently in mucosal cancer cells, which acquire ineffective apoptosis. 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The glycosyltransferase ST6Gal-I has been shown to contribute to multiple cell processes, including apoptosis, differentiation, and survival (6Schultz M.J. Swindall A.F. Bellis S.L. Regulation of the metastatic cell phenotype by sialylated glycans.Cancer Metastasis Rev. 2012; 31 (22699311): 501-51810.1007/s10555-012-9359-7Crossref PubMed Scopus (186) Google Scholar, 7Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76 (27216178): 3978-398810.1158/0008-5472.CAN-15-2834Crossref PubMed Scopus (71) Google Scholar). ST6Gal-I is located in the Golgi, where it catalyzes the addition of α2,6-linked sialic acids to N-glycans on glycoproteins destined via the trans-Golgi network for the plasma membrane. Homeostatic epithelial cell death receptors tumor necrosis receptor 1 (TNFR1) and Fas are key glycoproteins targeted by ST6Gal-I for addition of α2,6 sialic acids (α2,6 sialylation). The α2,6 sialylation of TNFR1 does not block ligand binding but prevents TNFR1 internalization, thereby blocking TNF-induced apoptosis and subsequent cell turnover (8Liu Z. Swindall A.F. Kesterson R.A. Schoeb T.R. Bullard D.C. Bellis S.L. ST6Gal-I regulates macrophage apoptosis via α2-6 sialylation of the TNFR1 death receptor.J. Biol. Chem. 2011; 286 (21930713): 39654-3966210.1074/jbc.M111.276063Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 9Holdbrooks A.T. Britain C.M. Bellis S.L. 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Notably, gastric tissue from uninfected children and children with H. pylori chronic gastritis, who rarely develop gastric cancer (27Harting M.T. Blakely M.L. Herzog C.E. Lally K.P. Ajani J.A. Andrassy R.J. Treatment issues in pediatric gastric adenocarcinoma.J. Pediatr. Surg. 2004; 39 (15300556): e8-e1010.1016/j.jpedsurg.2004.04.043Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 28Harris P.R. Wright S.W. Serrano C. Riera F. Duarte I. Torres J. Peña A. Rollán A. Viviani P. Guiraldes E. Schmitz J.M. Lorenz R.G. Novak L. Smythies L.E. Smith P.D. Helicobacter pylori gastritis in children is associated with a regulatory T-cell response.Gastroenterology. 2008; 134: 491-49910.1053/j.gastro.2007.11.006Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 29Subbiah V. Varadhachary G. Herzog C.E. Huh W.W. Gastric adenocarcinoma in children and adolescents.Pediatr. 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These findings extend the epithelioid tumors that express ST6Gal-I to gastric adenocarcinoma. To determine the role of ST6Gal-I in gastric epithelial cell biology and gastric adenocarcinoma pathogenesis, we established gastric epithelial stem cell organoids and organoid-derived monolayers to model antral stem cells and epithelium, respectively. Biopsies of normal human gastric antrum were digested and enriched for stem cells through culture in the presence of 50% l-WRN conditioned media (BASIC-CM) (32Miyoshi H. Stappenbeck T.S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.Nat. Protoc. 2013; 8 (24232249): 2471-248210.1038/nprot.2013.153Crossref PubMed Scopus (288) Google Scholar). Upon passaging, individual epithelial stem cells proliferated to form spheroid organoids lined by a single layer of cells with the epithelial apical surface directed toward the lumen (Fig. 2A). The stem cell organoids expressed high levels of mRNA for the canonical stem cell biomarker LGR5 and decreased levels of the mucin gene MUC5AC, present in differentiated goblet cells, compared with the biopsy tissue from which the organoids were derived (Fig. 2B). The gastric epithelial stem cell organoids also expressed both transmembrane tight junction protein zonula occludens 1 (ZO-1) and epithelial cell adhesion/signaling molecule E-cadherin (Fig. 2C). Epithelial stem cells generated from normal gastric antrum stained strongly for ST6Gal-I (Fig. 2D) and potently bound Sambucus nigra agglutinin (SNA) (Fig. 2E), which binds specifically to the sialic acid attached to a terminal galactose in an α2,6 linkage, confirming functional ST6Gal-I protein activity. To determine whether ST6Gal-I is associated with stemness, we examined the expression of ST6Gal-I in decreasing concentrations of stem cell factor–rich media. Gastric epithelial organoid expression of LGR5 and ST6GAL1 mRNA progressively decreased in the presence of diminishing levels of stem cell growth factor availability (Fig. 2, F and G), indicating that stem cell expression of LGR5 and ST6GAL1 was dependent, at least in part, on Wnt factor signaling. To generate differentiated gastric epithelium, gastric organoids were trypsinized and the individual stem cells plated on glass slides thinly coated with Matrigel in the presence of reduced Wnt availability (5% l-WRN CM, monolayer media) (Fig. 3A). The epithelial stem cells formed monolayers that up-regulated MUC5AC mRNA compared with the organoids (Fig. 3B), indicating differentiated gastric epithelium. Epithelial stem cell monolayers expressed ZO-1 and E-cadherin (Fig. 3C) but exhibited very low levels of ST6Gal-I protein (Fig. 3D). Indeed, the differentiation of gastric organoids into epithelial monolayers resulted in sharp decreases in mRNA expression of both LGR5 and ST6GAL1 (Fig. 3, E and F). Further, re-culturing established monolayers in stem cell growth factor complete media (50% l-WRN CM) did not induce significant up-regulation of either LGR5 or ST6GAL1 (Fig. 3G). These data suggest ST6Gal-I is a potential biomarker of human antral gastric epithelial stem cells but not differentiated epithelium and that Wnt factor signaling contributes to ST6Gal-I expression. We next investigated the function of epithelial ST6Gal-I on homeostatic apoptosis. Epithelial stem cell organoids from healthy antral mucosa were transfected with an shRNA interference vector (7Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76 (27216178): 3978-398810.1158/0008-5472.CAN-15-2834Crossref PubMed Scopus (71) Google Scholar) targeting ST6GAL1 to knock down ST6Gal-I or transduced with an ST6GAL1 lentiviral vector (7Schultz M.J. Holdbrooks A.T. Chakraborty A. Grizzle W.E. Landen C.N. Buchsbaum D.J. Conner M.G. Arend R.C. Yoon K.J. Klug C.A. Bullard D.C. Kesterson R.A. Oliver P.G. O'Connor A.K. Yoder B.K. et al.The tumor-associated glycosyltransferase ST6Gal-I regulates stem cell transcription factors and confers a cancer stem cell phenotype.Cancer Res. 2016; 76 (27216178): 3978-398810.1158/0008-5472.CAN-15-2834Crossref PubMed Scopus (71) Google Scholar) to overexpress ST6Gal-I. ST6GAL1 mRNA knockdown and overexpression were confirmed in the organoids at both the mRNA and protein level (Fig. 4, A and B). Epithelial stem cells successfully formed organoids in the presence of ST6Gal-I knockdown (Fig. 4B), indicating ST6Gal-I was not essential for organoid formation. Importantly, forced overexpression of ST6Gal-I in gastric organoids resulted in sustained ST6Gal-I expression in the organoid-derived epithelial monolayers at both the mRNA and protein level (Fig. 4, C and D). TNFR1 participates in a wide variety of downstream signaling pathways, including homeostatic apoptosis, and is ubiquitously expressed in most tissues (33Wajant H. Pfizenmaier K. Scheurich P. Tumor necrosis factor signaling.Cell Death Differ. 2003; 10 (12655295): 45-6510.1038/sj.cdd.4401189Crossref PubMed Scopus (1758) Google Scholar). TNFR1 is packaged in the Golgi and trafficked via the trans-Golgi network to the cell surface, where it engages with its ligand TNF (34Bradley J.R. Thiru S. Pober J.S. Disparate localization of 55-kd and 75-kd tumor necrosis factor receptors in human endothelial cells.Am. J. 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Chem. 2018; 293 (29233887): 1610-162210.1074/jbc.M117.801480Abstract Full Text Full Text PDF PubMed Google Scholar). Interrogating this key function of ST6Gal-I, we next determined the impact of ST6Gal-I overexpression on the ability of epithelial monolayers derived from healthy gastric antrum to undergo apoptosis. Stable transduction of the organoids with a lentiviral vector containing the ST6GAL1 gene caused increased levels of surface TNFR1 (p = 0.03) in the epithelial cell monolayers (Fig. 4E), consistent with impaired receptor internalization and continued TNFR1 trafficking to the epithelial cell surface (40Schneider-Brachert W. Heigl U. Ehrenschwender M. Membrane trafficking of death receptors: Implications on signalling.Int. J. Mol. Sci. 2013; 14 (23852022): 14475-1450310.3390/ijms140714475Crossref PubMed Scopus (34) Google Scholar, 41Cendrowski J. Mamińska A. Miaczynska M. Endocytic regulation of cytokine receptor signaling.Cytokine Growth Factor Rev. 2016; 32 (27461871): 63-7310.1016/j.cytogfr.2016.07.002Crossref PubMed Scopus (54) Google Scholar). Importantly, the levels of TNFRSF1 gene expression in ST6Gal-I overexpressing and knockdown monolayers, as well as monolayers derived from normal gastric organoids, were similar (Fig. 4F), indicating the increased surface expression of TNFR1 in the ST6Gal-I overexpressing monolayers was indeed because of reduced receptor internalization and not increased TNFRSF1 mRNA expression. To determine the impact of increased ST6Gal-I expression on TNF-induced epithelial cell apoptosis, we treated epithelial monolayers generated from normal and ST6Gal-I knockdown or overexpressing gastric stem cell organoids with TNF for 24 h and measured caspase activity. Caspase activity was increased in both the normal monolayers and the monolayers derived from ST6Gal-I knockdown organoids (Fig. 4G), indicating susceptibility to TNF-induced apoptosis. In contrast, caspase activity was not significantly augmented by TNF treatment of ST6Gal-I overexpressing monolayers (Fig. 4G). Thus, increased epithelial ST6Gal-I expression leads to the suppression of TNF-mediated apoptosis, likely contributing to enhanced epithelial cell longevity in vivo. To determine the impact of high-level ST6Gal-I expression on epithelial cell function in gastric cancer, we generated epithelial organoids from gastric adenocarcinoma, derived differentiated epithelial cell monolayers, and a
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