Hypoxie et régulation de l'epxression de la molécule HLA-G dans le glioblastome

HLA-G est une molécule HLA de classe I non classique jouant un rôle clé dans la tolérance immunitaire. Son expression est détectée dans les tumeurs cancéreuses participant aux mécanismes d'échappement tumoral. Nous analysons l'expression de HLA-G dans des biopsies de glioblastome où elle est détectée dans 60% des échantillons et est associée à un taux de survie plus réduit à long terme. Les mécanismes de régulation de son expression sont analysés dans les lignées cellulaires de gliome U251MG, U138MG et D247MG. L'expression du gène est induite par un traitement déméthylant à la 5-aza-2'déoxycytidine et se trouve augmentée par lïFN-y ou par un microenvironnement hypoxique. Son niveau d'expression est dépendant d'un état permissif de la chromatine au promoteur et de la variation de l'expression des composantes de la machinerie de présentation des antigènes. Par ailleurs, nous montrons que l'expression de HLA-G est régulé* par le facteur de transcription HIF-1 en situation mimant l'hypoxie via un site HRE ( Hypoxia Responsive Elément ) localisé à +274 pb dans l'exon 2 du gène, et qu'elle est dépendante du polymorphisme des régions 5'NT (SNP -966 A/G localisé dans un site HRE) et 3' NT (haplotypes UTR-5 et -7 plus réprimés par les miARNs endogènes). Nous observons également par microarrays que l'expression de miARNs putatifs ciblant HLA-G est modulée en situation d'hypoxie. HLA-G devrait donc être prise en compte dans le développement de nouvelles thérapies anti-tumorales considérant que son expression est la résultante de l'état de la chromatine au locus HLA-G, du microenvironnement tumoral, de l'expression de miARNs endogènes et du polymorphisme des régions 5' et 3'NT.HLA-G is a non classical MHC class I molecule involved in immune tolerance processes. Its expression has been described in various cancers favoring their immune escape. We analyzed HLA-G expression in glioblastoma biopsies, and found that HLA-G is expressed in 60% of the samples and is associated with reduced long term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG and U138MG. HLA-G expression is induced by 5-aza-2'-deoxycytidine demethylating treatment and enhanced by IFN-y and the hypoxic microenvironment. It is also dependent on the permissive state of the chromatin at the promoter and on the variation of expression of several antigen presenting machinery components. Under hypoxia mimicking conditions, we demonstrate that HLA-G expression is regulated by HIF-1 via a HRE (Hypoxia Responsive Element) located at +274 bp in exon 2. We also describe the role of HLA-G polymorphism in the 5'UTR (SNP -966 A/G located in a HRE) and 3'UTR (expression more repressed by endogenous miRNAs with haplotypes UTR-5 and -7) in the regulation of its expression. Moreover, we observe by microarray analysis that the expression of HLA-G putative miRNAs is modulated in hypoxia. HLA-G should thus be taken into account in the development of new anti-tumor therapies considering that its expression is a result of the chromatin state at the HLA-G locus, the tumor microenvironment, the endogenous miRNA expression and the polymorphism of both 5' and 3'UTR.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Transcriptional and Posttranscriptional Regulations of the HLA-G Gene

HLA-G has a relevant role in immune response regulation. The overall structure of the HLA-G coding region has been maintained during the evolution process, in which most of its variable sites are synonymous mutations or coincide with introns, preserving major functional HLA-G properties. The HLA-G promoter region is different from the classical class I promoters, mainly because (i) it lacks regulatory responsive elements for IFN-gamma and NF-kappa B, (ii) the proximal promoter region (within 200 bases from the first translated ATG) does not mediate transactivation by the principal HLA class I transactivation mechanisms, and (iii) the presence of identified alternative regulatory elements (heat shock, progesterone and hypoxia-responsive elements) and unidentified responsive elements for IL-10, glucocorticoids, and other transcription factors is evident. At least three variable sites in the 3' untranslated region have been studied that may influence HLA-G expression by modifying mRNA stability or microRNA binding sites, including the 14-base pair insertion/deletion, +3142C/G and +3187A/G polymorphisms. Other polymorphic sites have been described, but there are no functional studies on them. The HLA-G coding region polymorphisms might influence isoform production and at least two null alleles with premature stop codons have been described. We reviewed the structure of the HLA-G promoter region and its implication in transcriptional gene control, the structure of the HLA-G 3' UTR and the major actors of the posttranscriptional gene control, and, finally, the presence of regulatory elements in the coding region

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

International audienceHLA-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.

The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5' and 3' regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3' untranslated region (3'UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3'UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3'UTR haplotypes in healthy individuals and reinforce that 3'UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G

Human Leukocyte Antigen-G Is Frequently Expressed in Glioblastoma and May Be Induced in Vitro by Combined 5-Aza-2′-Deoxycytidine and Interferon-γ Treatments

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Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.

<p>Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.</p

Significant differences in the modulation of the expression of the luciferase reporter gene considering either the cell type or the status of HLA-G expression, with 1Fter-2R and 1Fter-4R constructions and haplotypes combined.

<p>Points indicate the mean and vertical bars indicate the 95% confidence interval. The number of experiments considering each cell line or the status of HLA-G expression is indicated between parentheses. MW: Mann-whitney test; KW: Kruskal-Wallis test. (A) Modulation of luciferase gene expression in each cell line separately. The decrease in the luciferase gene activity in U251MG (HLA-G-) and FON cells (HLA-G+) was not statistically different. (B) Modulation of luciferase gene expression in the HLA-G- (M8 + U251MG) cells <i>vs</i> HLA-G + (FON + JEG-3) cells combined.</p

Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.

<p>Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.</p