Search

Fumarate is a tricarboxylic acid cycle metabolite whose intracellular accumulation is linked to inflammatory signaling and development of cancer. In this study, we demonstrate that endogenous fumarate accumulation upregulates surface expression of the immune stimulatory NK group 2, member D (NKG2D) ligands ULBP2 and ULBP5. In agreement with this, accumulation of fumarate by the therapeutic drug dimethyl fumarate (DMF) also promotes ULBP2/5 surface expression. Mechanistically, we found that the increased ULBP2/5 expression was dependent on oxidative stress and the antioxidants N -acetylcysteine and glutathione (GSH) abrogated ULBP2/5 upregulated by DMF. Fumarate can complex with GSH and thereby exhaust cells of functional GSH capacity. In line with this, inhibition of GSH reductase (GR), the enzyme responsible for GSH recycling, promoted ULBP2/5 surface expression. Loss of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) associates with a malignant form of renal cancer characterized by fumarate accumulation and increased production of reactive oxygen species, highlighting fumarate as an oncometabolite. Interestingly, FH-deficient renal cancer cells had low surface expression of ULBP2/5 and were unresponsive to DMF treatment, suggesting that the fumarate-stimulating ULBP2/5 pathway is abrogated in these cells as an immune-evasive strategy. Together, our data show that ULBP2/5 expression can be upregulated by accumulation of fumarate, likely by depleting cells of GSH antioxidant capacity. Given that DMF is an approved human therapeutic drug, our findings support a broader use of DMF in treatment of cancers and inflammatory conditions., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Soluble ULBP2 is a marker for poor prognosis in several types of cancer. In this study we demonstrate that both soluble and cell surface-bound ULBP2 is transported via a so far unrecognized endosomal pathway. ULBP2 surface expression, but not MICA/B, could specifically be targeted and retained by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties. The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest that the endosomal transport pathway constitutes a novel therapeutic target for ULBP2-producing tumors., (Copyright © 2014 by The American Association of Immunologists, Inc.)

NKG2D ligand surface expression is important for immune recognition of stressed and neotransformed cells. In this study, we show that surface expression of MICA/B and other NKG2D ligands is dependent on N-linked glycosylation. The inhibitor of glycolysis and N-linked glycosylation, 2-deoxy-D-glucose (2DG), potently inhibited surface expression of MICA/B after histone deacetylase inhibitor treatment; the inhibition occurred posttranscriptionally without affecting MICA promoter activity. Transient overexpression of MICA surface expression was also inhibited by 2DG. 2DG blocks N-linked glycosylation of MICA/B by a reversible mechanism that can be alleviated by addition of d-mannose; this does not, however, affect the inhibition of glycolysis. Addition of d-mannose restored MICA/B surface expression after 2DG treatment. In addition, specific pharmacological or small interfering RNA-mediated targeting of glycolytic enzymes did not affect MICA/B surface expression, strongly suggesting that N-linked glycosylation, and not glycolysis, is essential for MICA/B surface expression. Corroborating this, tunicamycin, a selective inhibitor of N-linked glycosylation, abolished MICA/B surface expression without compromising activation of MICA promoter activity. NK cell-mediated killing assay and staining with a recombinant NKG2D-Fc fusion protein showed that all functional NKG2D ligands induced by histone deacetylase inhibitor treatment were abolished by 2DG treatment and fully reconstituted by further addition of d-mannose. Our data suggest that posttranslational N-linked glycosylation is strictly required for NKG2D ligand surface expression. Cancer and infection often result in aberrant glycosylation, which could likely be involved in modulation of NKG2D ligand expression. Our data further imply that chemotherapeutic use of 2DG may restrict NKG2D ligand surface expression and inhibit secretion of immunoinhibitory soluble NKG2D ligands.

We found that propionic acid secreted from propionibacteria induces expression of the NKG2D ligands MICA/B on activated T lymphocytes and different cancer cells, without affecting MICA/B expression on resting peripheral blood cells. Growth supernatant from propionibacteria or propionate alone could directly stimulate functional MICA/B surface expression and MICA promoter activity by a mechanism dependent on intracellular calcium. Deletion and point mutations further demonstrated that a GC-box motif around -110 from the MICA transcription start site is essential for propionate-mediated MICA promoter activity. Other short-chain fatty acids such as lactate, acetate, and butyrate could also induce MICA/B expression. We observed a striking difference in the molecular signaling pathways that regulate MICA/B. A functional glycolytic pathway was essential for MICA/B expression after exposure to propionate and CMV. In contrast, compounds with histone deacetylase-inhibitory activity such as butyrate and FR901228 stimulated MICA/B expression through a pathway that was not affected by inhibition of glycolysis, clearly suggesting that MICA/B is regulated through different molecular mechanisms. We propose that propionate, produced either by bacteria or during cellular metabolism, has significant immunoregulatory function and may be cancer prophylactic.

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.