Exosomal O-GlcNAc transferase from esophageal carcinoma stem cell promotes cancer immunosuppression through up-regulation of PD-1 in CD8+ T cells.

Esophageal carcinoma stem cells (ECSCs) are responsible for the initiation and therapy-resistance of esophageal cancer. Nutrient sensor O-GlcNAc transferase (OGT) promoted the growth and metastasis of cancer cells. However, the contributions of OGT to the tumorigenesis of ECSCs remain largely uncover. In the present study, as compared to matched non-stem cancer cells, the expression of OGT was higher in ALDH+ ECSCs. Knock down of OGT by lentivirus system reduced the self-renewal capacities and tumorigenicity of ALDH+ ECSCs. In addition, OGT in exosome derived from ALDH+ ECSCs was taken up by neighboring CD8+ T cells and increased the expression of PD-1 in CD8+ T cells. Down-regulation of OGT increased the apoptosis of ALDH+ ECSCs induced by CD8+ T cells, which could be blocked by overexpression of PD-1 in CD8+ T cells. Together, OGT in exosome from ECSCs protects ECSCs from CD8+ T cells through up-regulation of PD-1.

O-GlcNAc transferase activates stem-like cell potential in hepatocarcinoma through O-GlcNAcylation of eukaryotic initiation factor 4E.

O-GlcNAcylation catalysed by O-GlcNAc transferase (OGT) is a reversible post-translational modification. O-GlcNAcylation participates in transcription, epigenetic regulation, and intracellular signalling. Dysregulation of O-GlcNAcylation in response to high glucose or OGT expression has been implicated in metabolic diseases and cancer. However, the underlying mechanisms by which OGT regulates hepatoma development remain largely unknown. Here, we employed the lentiviral shRNA-based system to knockdown OGT to analyse the contribution of OGT in hepatoma cell proliferation and stem-like cell potential. The sphere-forming assay and western blot analysis of stem-related gene expression were used to evaluate stem-like cell potential of hepatoma cell. We found that the level of total O-GlcNAcylation or OGT protein was increased in hepatocellular carcinoma. OGT activated stem-like cell potential in hepatoma through eukaryotic initiation factor 4E (eIF4E) which bound to stem-related gene Sox2 5'-untranslated region. O-GlcNAcylation of eIF4E at threonine 168 and threonine 177 protected it from degradation through proteasome pathway. Expression of eIF4E in hepatoma was determined by immunostaining in 232 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of eIF4E expression with prognosis. High glucose promoted stem-like cell potential of hepatoma cell through OGT-eIF4E axis. Collectively, our findings indicate that OGT promotes the stem-like cell potential of hepatoma cell through O-GlcNAcylation of eIF4E. These results provide a mechanism of HCC development and a cue between the pathogenesis of HCC and high glucose condition.

ß1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells.

Malignant glioblastoma multiforme is one of the most aggressive human cancers, with very low survival rates. Recent studies have reported that glioma stem-like cells transdifferentiate into endothelial cells, indicating a new mechanism for tumor angiogenesis and potentially providing new therapeutic options for glioblastoma treatment. Glioma malignancy is strongly associated with altered expression of N-linked oligosaccharide structures on the cell surface. We have previously reported that ß1,4-galactosyltransferase V (ß1,4GalTV), which galactosylates the GlcNAcß1-6Man arm of the branched N-glycans, is highly expressed in glioma and promotes glioma cell growth in vitro and in vivo However, the mechanism by which ß1,4GalTV stimulates glioma growth is unknown. Here we demonstrate that short hairpin RNA-mediated ß1,4GalTV knockdown inhibits the tumorigenesis of glioma stem-like cells and reduces their transdifferentiation into endothelial cells. We also found that ß1,4GalTV overexpression increased glioma stem-like cell transdifferentiation into endothelial cells and that this effect required ß1,4GalTV galactosylation activity. Moreover, ß1,4GalTV promoted ß1,4-galactosylation of Notch1 and increased Notch1 protein levels. Of note, ectopic expression of activated Notch1 rescued the inhibitory effect of ß1,4GalTV depletion on glioma stem-like cell transdifferentiation. In summary, our findings indicate that ß1,4GalTV stimulates transdifferentiation of glioma stem-like cells into endothelial cells by activating Notch1 signaling. These detailed insights shed important light on the mechanisms regulating glioma angiogenesis.

The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration.

CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells.

Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth.

The membrane glycoprotein CD133 is a popular marker for cancer stem cells and contributes to cancer initiation and invasion in a number of tumor types. CD133 promotes tumorigenesis partly through an interaction between its phosphorylated Y828 residue and the PI3K regulatory subunit p85, and the interaction with ß-catenin. Although CD133 glycosylation is supposed to be associated with its function, the contribution of N-glycosylation to its functions remains unclear. Here we analyzed the exact site(s) of N-glycosylation in CD133 by mass spectrometry and found that all eight potential N-glycosylation sites of CD133 could be indeed occupied by N-glycans. Loss of individual N-glycosylation sites had no effect on the level of expression or membrane localization of CD133. However, mutation at glycosylation site Asn548 significantly decreased the ability of CD133 to promote hepatoma cell growth. Furthermore, mutation of Asn548 reduced the interaction between CD133 and ß-catenin and inhibited the activation of ß-catenin signaling by CD133 overexpression. Our results identified the characteristics and function of CD133 glycosylation sites. These data could potentially shed light on molecular regulation of CD133 by glycosylation and enhance our understanding of the utility of glycosylated CD133 as a target for cancer therapies.

Activation of PI3K/Akt pathway by CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells.

The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.