Loss of α-1,2-mannosidase MAN1C1 promotes tumorigenesis of intrahepatic cholangiocarcinoma through enhancing CD133-FIP200 interaction.

CD133 is widely used as a marker to isolate tumor-initiating cells in many types of cancers. The structure of N-glycan on CD133 is altered during the differentiation of tumor-initiating cells. However, the relationship between CD133 N-glycosylation and stem cell characteristics remains elusive. Here, we found that the level of α-1,2-mannosylated CD133 was associated with the level of stemness genes in intrahepatic cholangiocarcinoma (iCCA) tissues. α-1,2-mannosylated CD133+ cells possessed the characteristics of tumor-initiating cells. The loss of the Golgi α-mannosidase I coding gene MAN1C1 resulted in the formation of α-1,2-mannosylated CD133 in iCCA-initiating cells. Mechanistically, α-1,2-mannosylation promoted the cytoplasmic distribution of CD133 and enhanced the interaction between CD133 and the autophagy gene FIP200, subsequently promoting the tumorigenesis of α-1,2-mannosylated CD133+ cells. Analysis of iCCA samples showed that the level of cytoplasmic CD133 was associated with poor iCCA prognosis. Collectively, α-1,2-mannosylated CD133 is a functional marker of iCCA-initiating cells.

Sialylation-dependent interaction between PD-L1 and CD169 promotes monocyte adhesion to endothelial cells.

The monocyte adhesion to endothelial cells is an early step in chronic inflammation. Interferon-γ (IFN-γ) is regarded as a master regulator of inflammation development. However, the significance and mechanisms of IFN-γ in the monocyte adhesion to endothelial cells remains largely unknown. IFN-γ up-regulates PD-L1 on various types of cells. Here, we performed flow cytometry to examine the contribution of IFN-γ-induced PD-L1 expression on monocyte adhesion to endothelial cells. Up-regulation of PD-L1 by IFN-γ enhanced the adhesion of monocytes to endothelial cells. By immunoprecipitation and lectin blot, PD-L1 in endothelial cells interacted with CD169/Siglec 1 in monocytes depending on the α2,3-sialylation of PD-L1. ST3Gal family (ST3ß-galactoside α-2,3-sialyltransferase) was the major glycosyltransferase responsible for the α2,3-sialylation of membrane proteins. Down-regulation of ST3Gal4 by RNAinterference partially reduced the α2,3-sialylation of PD-L1 and the PD-L1-CD169 interaction. Finally, purified PD-L1 protein with α2,3-sialylation, but not PD-L1 protein without α2,3-sialylation, partially reduced IFN-γ-induced monocyte adhesion to endothelial cells. These findings provide evidence that the interaction between PD-L1 and CD169 promoted monocyte adhesion to endothelial cells and might elucidate a new mechanism of monocyte adhesion to endothelial cells.

The Interaction between DNMT1 and High-Mannose CD133 Maintains the Slow-Cycling State and Tumorigenic Potential of Glioma Stem Cell.

The quiescent/slow-cycling state preserves the self-renewal capacity of cancer stem cells (CSCs) and leads to the therapy resistance of CSCs. The mechanisms maintaining CSCs quiescence remain largely unknown. Here, it is demonstrated that lower expression of MAN1A1 in glioma stem cell (GSC) resulted in the formation of high-mannose type N-glycan on CD133. Furthermore, the high-mannose type N-glycan of CD133 is necessary for its interaction with DNMT1. Activation of p21 and p27 by the CD133-DNMT1 interaction maintains the slow-cycling state of GSC, and promotes chemotherapy resistance and tumorigenesis of GSCs. Elimination of the CD133-DNMT1 interaction by a cell-penetrating peptide or MAN1A1 overexpression inhibits the tumorigenesis of GSCs and increases the sensitivity of GSCs to temozolomide. Analysis of glioma samples reveals that the levels of high-mannose type N-glycan are correlated with glioma recurrence. Collectively, the high mannose CD133-DNMT1 interaction maintains the slow-cycling state and tumorigenic potential of GSC, providing a potential strategy to eliminate quiescent GSCs.

The binding of LDN193189 to CD133 C-terminus suppresses the tumorigenesis and immune escape of liver tumor-initiating cells.

The tumor-initiating cell (TIC) marker CD133 promotes TIC self-renewal and tumorigenesis through the tyrosine phosphorylation of its c-terminal domain. Therefore, finding compounds that target the phosphorylation of CD133 will provide an effective method for inhibiting TICs characteristics. Here, through small molecule microarray screening, compound LDN193189 was found to bind to the c-terminus of CD133 and influenced its tyrosine phosphorylation. LDN193189 inhibited the interaction between CD133 and p85, accompanied by a reduction in the self-renewal and tumorigenicity of liver TIC. In addition, LDN193189 inhibited the expression and transcription of Galectin-3 by reducing the tyrosine phosphorylation of CD133. Galectin-3 secreted by liver TICs inhibited the proliferation of activated CD8+ T cells by binding to PD-1. LDN193189 suppressed the immune escape ability of liver TICs by downregulating Galectin-3. Taken together, LDN193189 suppressed the tumorigenesis and immune escape of liver CSCs by targeting the CD133-Galectin-3 axis.

Methylation and serum response factor mediated in the regulation of gene ARRDC3 in breast cancer.

Breast cancer poses a serious threat to women's life and health and many factors contribute to breast cancer including gene mutation and epigenetics. Gene ARRDC3 was usually repressed in breast cancer and methylation in promoter was reported to be involved in gene ARRDC3 expression regulation. To this end, the methylation status for gene ARRDC3 promoter was assayed by the Massarray quantitative method. The results indicated that different methylation level CpG sites including CpG_6, CpG_13.14, CpG_17.18, and CpG_25 existed between the tumor tissue and the adjacent normal tissue. In order to further verify whether methylation participated in gene ARRDC3 expression, three cell lines were treated with methylation inhibitor Aza-2'-deoxycytidine including A-375, HepG2, and MDA-MB-231. The results revealed that methylation inhibition observably increased ARRDC3 mRNA expression. Then we confirmed the effective length of promoter through the fluorescence report assay used for further analysis. The results showed that the 1746 bp length promoter produced the maximum fluorescence signal. To obtain the direct evidence that methylation in gene ARRDC3 promoter mediated in ARRDC3 expression regulation, the promoter plasmid was methylated by M.SssI enzyme and subjected to the fluorescence report assay. The results showed that methylation in the promoter markedly suppressed relative luciferase activity. In addition, the ecRNA was also analyzed for the methylation regulation and results illustrated that the ecRNA did not regulate ARRDC3 promoter methylation. However, several methylation CpG sites were found to be around CpG_25 site such as TGCATGG, TTGCAA, TTCGTA, and ATAGTT. These sites provide a good clue for further research in methylation for gene ARRDC3 expression regulation. Furthermore, the possible transcription factors involved in the ARRDC3 regulation were investigated by western blot, luciferase activity analysis and ChiP assay. These results documented that gene ARRDC3 expression was improved by SRF and that the methylation affected the interaction between the promoter and SRF. Lastly, the inhibition role of gene ARRDC3 on breast cancer was probed in vivo and in vitro and our results demonstrated that ARRDC3 could inhibit breast cancer growth through the STAT3 signal pathway. In summary, Gene ARRDC3 was inhibited by promoter methylation and was promoted by transcription factor SRF by binding the promoter region and the inhibition on breast cancer growth was exerted by ARRDC3 through STAT3 signal pathway.

IL-17A secreted from lymphatic endothelial cells promotes tumorigenesis by upregulation of PD-L1 in hepatoma stem cells.

BACKGROUND & AIMS: The microenvironment regulates hepatoma stem cell behavior. However, the contributions of lymphatic endothelial cells to the hepatoma stem cell niche remain largely unknown; we aimed to analyze this contribution and elucidate the mechanisms behind it. METHODS: Associations between lymphatic endothelial cells and CD133+ hepatoma stem cells were analyzed by immunofluorescence and adhesion assays; with the effects of their association on IL-17A expression examined using western blot, quantitative reverse transcription PCR and luciferase reporter assay. The effects of IL-17A on the self-renewal and tumorigenesis of hepatoma stem cells were examined using sphere and tumor formation assays. The role of IL-17A in immune escape by hepatoma stem cells was examined using flow cytometry. The expression of IL-17A in hepatoma tissues was examined using immunohistochemistry. RESULTS: CD133+ hepatoma stem cells preferentially interact with lymphatic endothelial cells. The interaction between the mannose receptor and high-mannose type N-glycans mediates the interaction between CD133+ hepatoma stem cells and lymphatic endothelial cells. This interaction activates cytokine IL-17A expression in lymphatic endothelial cells. IL-17A promotes the self-renewal of hepatoma stem cells. It also promotes their immune escape, partly through upregulation of PD-L1. CONCLUSION: Interactions between lymphatic endothelial cells and hepatoma stem cells promote the self-renewal and immune escape of hepatoma stem cells, by activating IL-17A signaling. Thus, inhibiting IL-17A signaling may be a promising approach for hepatoma treatment. LAY SUMMARY: The microenvironment is crucial for the self-renewal and development of hepatoma stem cells, which lead to the development of liver cancer. Lymphatic endothelial cells are an important component of this niche microenvironment, helping hepatoma stem cells to self-renew and escape immune attack, by upregulating IL-17A signaling. Thus, targeting IL-17A signaling is a potential strategy for the treatment of hepatoma.

Complex N-glycan promotes CD133 mono-ubiquitination and secretion.

CD133 is a widely used cell surface marker of cancer stem cells that plays an important role in tumor initiation and metastasis. Increasing evidence shows that CD133 is secreted to the extracellular space. However, the underlying mechanisms of CD133 secretion remain largely unknown. In this study, we report that secreted CD133 has a complex-type N-glycosylation and is modified by beta1,6GlcNAc N-glycan. We found that inhibition of CD133 complex-type N-glycosylation by swainsonine does not affect the membrane localization of CD133, but significantly reduces CD133 secretion and promotes its accumulation in early endosomes. Moreover, swainsonine reduces CD133 secretion by reducing its mono-ubiquitination and inhibiting the interaction between CD133 and Tsg101. These findings reveal a new mechanism of glycosylation-dependent secretion of CD133.