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Gene expression profiling of FUT8 knockdown CL1-5 cells

ABSTRACT: Overexpression of fucosyltransferase 8 (FUT8) is found in many cancers including liver, ovarian, thyroid, colorectal and non-small cell lung cancers. Unlike other FUTs which are functionally redundant, FUT8 is the only enzyme responsible for the alpha1,6-linked fucosylation (core fucosylation) by adding fucose to the innermost GlcNAc residue of an N-linked glycan. A growing body of evidence indicates that core fucosylation is important for regulating protein functions, such as EGFR, TGF beta receptor and integrins. To understand the downstream molecular events in response to the global alteration of core fucosylation during cancer progression, microarray analysis was employed to profile the changes in gene expression following FUT8 silencing. The genes significantly (2-fold, P<0.01) changed in CL1-5/shFUT8 cells were selected for functional annotations using a Gene Ontology database. The result revealed that many genes involved in cell adhesion, motility, growth, angiogenesis, and inflammation were under the control of core fucosylation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE42405 | GEO | 2012/12/07

SECONDARY ACCESSION(S): PRJNA181308

REPOSITORIES: GEO

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Project description:Most patients with triple negative breast cancer (TNBC) fail to respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression remain elusive. Here, we identify aberrant B7H3 glycosylation and found N-glycosylation of B7H3 at NXT motif sites are responsible for its protein stability and immunosuppression in TNBC tumors. Mechanistically, fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression could be physiologically significant and clinically relevant in TNBC patients. Notably, combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These suggest targeting FUT8-B7H3 axis can be a promising strategy for improving anti-tumor immune responses in TNBC patients. To obtain the direct evidence that B7H3 is N-glycosylated in TNBC cells, we analysed the peptides of purified human B7H3 protein from B7H3-WT re-expressed and B7H3-8NQ re-expressed MDA-MB-231 cell lines by Nanoscale liquid chromatography coupled to tandem MS (nano LC-MS/MS). The result showed that there were eight N-glycosylation sites (Asn positions 91, 104, 189, 215, 309, 322, 407, and 433) in B7H3-WT cells, but not in B7H3-8NQ cells, as determined by Asn to Asp conversion after PNGase F treatment. As B7H3 contains a nearly exact tandem duplication of the IgV-IgC domain, there were four pairs of N-glycosylation sites identified through identical peptide sequence, including N91 and N309, N104 and N322, N189 and N407, and N215 and N433, in each of the IgV-IgC domains. Together, the results indicate that B7H3 is exclusively N-glycosylated at these four pairs of glycosylation sites in TNBC cells.

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Gene expression profiling of FUT8 knockdown CL1-5 cells

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