The Factor Inhibiting HIF regulates T cell differentiation and anti-tumor efficacy
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ABSTRACT:
PROVIDER: PRJNA981153 | ENA |
REPOSITORIES: ENA
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Project description:T cells must adapt to variations in tissue microenvironments; these adaptations include the degree of oxygen availability. The hypoxia-inducible factor (HIF) transcription factors control much of this adaptation, and thus regulate many aspects of T cell activation and function. The HIFs are in turn regulated by oxygen-dependent hydroxylases: both the prolyl hydroxylases (PHDs) which interact with the VHL tumor suppressor and control HIF turnover, and the asparaginyl hydroxylase known as the Factor inhibiting HIF (FIH), which modulates HIF transcriptional activity. To determine the role of this latter factor in T cell function, we generated T cell-specific FIH knockout mice. We found that FIH regulates T cell fate and function in a HIF-dependent manner and show that the effects of FIH activity occur predominantly at physiological oxygen concentrations. T cell-specific loss of FIH boosts T cell cytotoxicity, augments T cell expansion in vivo, and improves anti-tumor activity in mice. Specifically inhibiting FIH in T cells may therefore represent a promising strategy for cancer immunotherapy.
2023-06-14 | GSE234381 | GEO
Project description:Factor inhibiting HIF (FIH) promotes lung cancer progression
| PRJNA914705 | ENA
Project description:Factor-inhibiting HIF (FIH) is an asparagine hydroxylase that acts on hypoxia-inducible factors (HIFs) to control cellular adaptation to hypoxia. FIH is expressed in several tumor types, but its impact in tumor progression remains largely unexplored. We observed that FIH was expressed on human lung cancer tissue. Deletion of FIH in mouse and human lung cancer cells resulted in an increased glycolytic metabolism, consistent with increased HIF activity. FIH-deficient lung cancer cells exhibited decreased proliferation. Analysis of RNA-Seq data confirmed changes in the cell cycle and survival and revealed molecular pathways that were dysregulated in the absence of FIH, including the upregulation of angiomotin (Amot), a key component of the Hippo tumor suppressor pathway. We show that FIH-deficient tumors were characterized by higher immune infiltration of NK and T cells compared with FIH competent tumor cells. In vivo studies demonstrate that FIH deletion resulted in reduced tumor growth and metastatic capacity. Moreover, high FIH expression correlated with poor overall survival in non-small cell lung cancer (NSCLC). Our data unravel FIH as a therapeutic target for the treatment of lung cancer.
2023-11-08 | GSE221505 | GEO
Project description:The aspariginyl-hydroxylase activity of Factor Inhibiting HIF (FIH) is a key regulator of the transcriptional activity of the hypoxia inducible factor. FIH also catalyses the hydroxylation of asparaginyl- and other-residues in ankyrin repeat domain (ARD) containing proteins, including Apoptosis stimulating of p53 (ASPP) protein family members. ASPP2 is reported to undergo a single FIH catalysed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing FIH can catalyse the unprecedented post-translational hydroxylation of both asparaginyl-residues in “VNVN” and related motifs of ankyrin repeat domains in apoptosis-stimulating of p53 (ASPP) proteins (i.e. ASPP1, ASPP2 and iASPP) and the related ASB11 and p18-INK4C proteins. The results extend the substrate scope of FIH catalysis and may have implications for its role in the hypoxic response and, ASPP protein function.
2022-08-11 | PXD017278 | Pride
Project description:Adoptive transfer of anti-tumor cytotoxic T cells is a novel form of cancer immunotherapy, and a key challenge is to ensure the survival and function of the transferred T cells. Immune cell survival requires adaptation to different micro-environments, and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation, and we undertook experiments to define structural determinants of HIF that would potentiate anti-tumor efficacy in cytotoxic T cells. We created retroviral vectors to deliver ectopic expression of HIF-1ɑ and HIF-2ɑ in mouse CD8+ T cells, together or individually, and with or without sensitivity to their oxygen-dependent inhibitors Von Hippel-Lindau (VHL) and Factor Inhibiting HIF (FIH). We found that HIF-2ɑ, but not HIF-1ɑ, drives broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. We further found that a specific mutation replacing the hydroxyl group acceptor site for FIH in the HIF-2ɑ isoform gives rise to the most effective anti-tumor T cells after adoptive transfer in vivo. Lastly, we show that co-delivering an FIH-insensitive form of HIF-2ɑ with an anti-CD19 chimeric antigen receptor greatly enhances cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments provide a means to increase the anti-tumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor. Method: OT-I CD8+ T cells were activated with 100 ng/ml OVA for 24 hours before retroviral transducton with with mouse HIF1 or mouse HIF2-expressing vectors, expanded in IL-2 for 5 days before being sorted on Thy-1.1 surface expression on a BD FACSAria Fusion cell sorter (BD Biosciences) directly into RLT Plus lysis buffer (Qiagen, #1053393). Total RNA was subjected to quality control with Agilent Tapestation according to the manufacturer’s instructions. To construct libraries suitable for Illumina sequencing the Illumina TruSeq Stranded mRNA Sample preparation protocol, which includes cDNA synthesis, ligation of adapters and amplification of indexed libraries, was used (Illumina, #20020594). The yield and quality of the amplified libraries were analysed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalised and combined and the pools were sequenced on the Nextseq 550 for a 50-cycle v2.5 sequencing run generating 75 bp single-end reads. Basecalling and demultiplexing was performed using CASAVA software with default settings generating Fastq files for further downstream mapping and analysis.
2021-02-16 | GSE166756 | GEO
Project description:Adoptive transfer of anti-tumor cytotoxic T cells is a novel form of cancer immunotherapy, and a key challenge is to ensure the survival and function of the transferred T cells. Immune cell survival requires adaptation to different micro-environments, and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation, and we undertook experiments to define structural determinants of HIF that would potentiate anti-tumor efficacy in cytotoxic T cells. We created retroviral vectors to deliver ectopic expression of HIF-1ɑ and HIF-2ɑ in mouse CD8+ T cells, together or individually, and with or without sensitivity to their oxygen-dependent inhibitors Von Hippel-Lindau (VHL) and Factor Inhibiting HIF (FIH). We found that HIF-2ɑ, but not HIF-1ɑ, drives broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. We further found that a specific mutation replacing the hydroxyl group acceptor site for FIH in the HIF-2ɑ isoform gives rise to the most effective anti-tumor T cells after adoptive transfer in vivo. Lastly, we show that co-delivering an FIH-insensitive form of HIF-2ɑ with an anti-CD19 chimeric antigen receptor greatly enhances cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments provide a means to increase the anti-tumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor. Method: OT-I CD8+ T cells were activated with 100 ng/ml OVA for 24 hours before retroviral transducton with with mouse HIF1 or mouse HIF2-expressing vectors, expanded in IL-2 for 5 days before being sorted on Thy-1.1 surface expression on a BD FACSAria Fusion cell sorter (BD Biosciences) directly into RLT Plus lysis buffer (Qiagen, #1053393). Total RNA was subjected to quality control with Agilent Tapestation according to the manufacturer’s instructions. To construct libraries suitable for Illumina sequencing the Illumina TruSeq Stranded mRNA Sample preparation protocol, which includes cDNA synthesis, ligation of adapters and amplification of indexed libraries, was used (Illumina, #20020594). The yield and quality of the amplified libraries were analysed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalised and combined and the pools were sequenced on the Nextseq 550 for a 50-cycle v2.5 sequencing run generating 75 bp single-end reads. Basecalling and demultiplexing was performed using CASAVA software with default settings generating Fastq files for further downstream mapping and analysis.
2021-02-16 | GSE166757 | GEO
Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-1α in colon, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-1α+/+, Hif-1αLSL/LSL mice. Background & Aims: The progression and growth of solid tumors leads to a state where tumors outgrow their capacity for efficient oxygenation and nutrient uptake and an increase in tumor hypoxia. Tumor hypoxic response is mediated by hypoxia-inducible factor (HIF)-1a and HIF-2a. These transcription factors regulate a battery of genes that are critical for tumor oxygenation, tumor metabolism, and cell proliferation and survival. Therefore, inhibitors of HIF have been sought for as anti-neoplastic agents in several different kinds of cancers. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1a is beneficial, and can reduce intestinal inflammation. The efficacy of pharmacological agents that chronically activate HIF-1a are decreased due to the tumorigenic potential of HIF. However, recent advance in understanding HIF signaling have identified mechanisms, which could allow for isoform specific activators. Activation of HIF-2a increases colon carcinogenesis and progression in mouse models. However, the role of chronic HIF-1a activation is unclear in the progression in colon cancer. The present data demonstrates that activation of HIF-1a in epithelial cells does not increase colon carcinogens or progression in two mouse models of colon cancer, and provides the proof of principle that HIF-1a activation maybe safe as therapies for inflammatory bowel disease. Global gene expression profiling in colon RNAs isolated from 6- to 8-week-old Hif-1α+/+ (n=5, Shah 019) and Hif-1αLSL/LSL (n=5, Shah 020).
2014-05-14 | E-GEOD-55915 | biostudies-arrayexpress
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
2014-05-14 | E-GEOD-57613 | biostudies-arrayexpress
Project description:Dasatinib enhances anti-tumor efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion
| PRJNA637236 | ENA
Project description:Increased levels of hypoxia and hypoxia inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged anti-angiogenic therapy of tumors can delay tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the anti-vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene set enrichment analysis of microarrays from pre-treatment biopsies found the Gene Ontology category “Response to hypoxia” was upregulated in poor responders, and hierarchical clustering based on 140 hypoxia-responsive genes separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. We thus examined Dox treatment in 4 sarcoma cell lines, and found Dox at low concentrations (1-10 uM) blocked HIF-1α induction of VEGF-A by 84-97%, while inhibition of other HIF-1α-target genes including CA9, c-Met and FOXM1 was variable. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 and metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, primarily via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α-target genes which may promote resistance to anti-angiogenic and other therapies. Metronomic Dox can block HIF-1α activation of target genes and works synergistically with anti-VEGF therapy to inhibit sarcomas. Pre-treatment biopsies were collected from 16 human sarcoma. The gene expression analysis was performed using Illumina platform.
2011-08-29 | E-GEOD-31715 | biostudies-arrayexpress