Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2

Abstract

HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2′deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at −966 bp in the 5′UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

Haplotypes of the HLA-G 3’ Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially

“…Moreover and surprisingly, UTR1, which is theoretically associated with higher HLA-G expression, exists in the HLA-G- cell line (HLA-G mRNA relative to JEG-3: ~1.10 −4 ) exclusively, whilst UTR-2 and UTR-4, which are theoretically associated with intermediate HLA-G expression, occur in M8 and U251MG, respectively. We have previously showed that the M8 HLA-G promoter is strongly methylated [36] and that demethylation treatment upregulates HLA-G transcription in M8 [16, 36] and U251MG cells [37, 49]. This result first suggests that the HLA-G gene is functional in these cell lines especially because U251MG-treated cells may produce HLA-G protein [37, 49].…”

“…We have previously showed that the M8 HLA-G promoter is strongly methylated [36] and that demethylation treatment upregulates HLA-G transcription in M8 [16, 36] and U251MG cells [37, 49]. This result first suggests that the HLA-G gene is functional in these cell lines especially because U251MG-treated cells may produce HLA-G protein [37, 49]. Second, in the present work we have tested the impact of HLA-G 3’UTR polymorphism irrespective of modifications in chromatin, and we have focused on post-transcriptional mechanisms only.…”

Hypoxic Modulation of HLA-G Expression through the Metabolic Sensor HIF-1 in Human Cancer Cells

“…Recently, the impact of hypoxic stress in HLA-G expression in U251MG glioblastoma cell line was explored [32]. In this study, under hypoxia, the U251MG cells (HLA-G − ) induced significant HLA-G transcriptional activity compared to untreated cells, but with negative protein detection.…”

“…The authors found by in silico analysis, a new HIF-1 binding site located in the HLA-G coding sequence at exon 2, 281 base pairs after start nucleotide [32]. This novel +281 HRE comprehends two hypoxia binding sites (HBS), one at +281 bp (sense, 5′-ACGTG-3′) and one at +291 bp (antisense, 5′-CACGC-3′) positions [32].…”

Regulation of immunity and inflammation by hypoxia in immunological niches

“…Two mechanisms that involve HIF activity and provide immune privilege in the placenta are immune invisibility (where fetal cells are invisible to the mother’s immune system) and immune blockade (where the mother’s immune system is blocked in the placenta). The placenta avoids immune attack mostly through trophoblast expression of the non-classical class I histocompatibility antigen HLA-G, a HIF1α-dependent gene product that protects against attack by natural killer cells 95,96 . Immune blockade in the placenta is provided by hypoxia-dependent regulation of factors such as programmed cell death 1 ligand 1 (PDL1), which inactivates or induces cell death in infiltrating T cells 95 .…”