Analysis of HIF dependent transcriptional regulation resolved by transcriptional start site, using 5′ end-Seq technology
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Homo sapiens
SUBMITTER: Yoichiro Sugimoto
PROVIDER: E-MTAB-10688 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Changes in translational efficiency of mRNAs consequent on alteration of HIF and/or mTOR pathway activity in VHL-defective kidney cancer cell were analysed. High-resolution polysome profiling followed by sequencing of the 5′ ends of mRNAs (HP5) was used to measure translational efficiency of mRNAs resolved by their transcription start sites. HP5 was applied to VHL-defective kidney cancer cell lines with or without VHL reintroduction and/or mTOR inhibition with Torin 1 treatment. In addition, the contribution of HIF2A/EIF4E2 pathway was assessed using 786-O cells in which EIF4E2 was inactivated.
2022-05-14 | E-MTAB-10689 | biostudies-arrayexpress
Project description:Altered metabolism is an important part of malignant transformation of tumor cells. Oncogenic transformation may reprogram tumor metabolism and render tumor cells addicted to extracellular nutrients. Such nutrient addictions associated with oncogenic mutations may offer therapeutic opportunities; however, it remains difficult to predict these nutrient addictions. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear-cell renal cancer cells (ccRCC) with or without VHL upon the deprivation of individual amino acids. We identified that cystine deprivation triggered rapid programmed necrosis in VHL-deficient RCC, but not in their VHL-restored counterparts. Similar cystine addiction was also observed in VHL-deficient primary RCC tumors cells. Blockage of cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status. Therefore, metabolic differences due to cystine deprivation are not different enough to readily explain the distinct fate of life vs. death in VHL-deficient and restored cell.. Instead, we found that increased levels of TNFα associated with VHL loss in the VHL-deficient RCC force them to rely on intact RIPK1 to inhibit apoptosis. However, this pre-existing elevated TNFα in the VHL-deficient ccRCC renders these cells susceptible to the necrosis signaling triggered by cystine deprivation. In addition, we identified that cystine-deprived necrosis in VHL-deficient RCC depends on reciprocal amplification of the Src-p38-Noxa signaling and TNFα-RIP1/3-MLKL necrosis pathways that culminate in MLKL oligomerization and programmed necrosis. Together, our data reveal that the contextual cystine-addictions in VHL-deficient ccRCC is dependent on activating pre-existing oncogenic pathways to trigger programmed necrosis. RNA was extracted by RNAeasy kits (Qiagen) from the RCC4 Vec and VHL-reconstituted cells which were exposed to the control full DMEM or cystine deprived DMEM media for 6 hours (three replicates in each condition).
2016-07-01 | E-GEOD-60422 | biostudies-arrayexpress
Project description:Renal hypoxia is widespread in acute kidney injury (AKI) of various aetiologies. Hypoxia adaptation, conferred through the hypoxia-inducible factor (HIF), appears to be insufficient. Here we show that HIF activation in renal tubules through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO) protects from rhabdomyolysis-induced AKI. In this model, histological observations indicate that injury mainly affects proximal convoluted tubules, with 5% necrosis at d1 and 40% necrosis at d2. HIF-1alpha up-regulation in distal tubules reflects renal hypoxia. However, lack of HIF in proximal tubules suggests insufficient adaptation by HIF. AKI in VHL-KO mice leads to prominent HIF activation in all nephron segments, as well as to reduced serum creatinine, serum urea, tubular necrosis, and apoptosis marker caspase-3 protein. At d1 after rhabdomyolysis, when tubular injury is potentially reversible, HIF mediated protection in AKI is associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal as demonstrated by qPCR, pathway enrichment analysis and immunohistochemistry. Together, our data provide evidence for a HIF-orchestrated multi-level shift towards glycolysis as a major mechanism for protection against acute tubular injury. All experiments were carried out in transgenic mice in which selective renal tubular VHL knockout (VHL-KO) was inducible by doxycycline (Reference: Mathia S, Paliege A, Koesters R, Peters H, Neumayer HH, Bachmann S, Rosenberger C. Action of hypoxia-inducible factor in liver and kidney from mice with Pax8-rtTA-based deletion of von Hippel-Lindau protein. Acta Physiol (Oxf). 2013; 207(3):565-76.). Four groups of animals were used: 1) controls: untreated mice; 2) VHL-KO: injected with doxycycline (0.1 mg per 10 g body weight SC), 4 days prior to sacrifice; 3) AKI: rhabdomyolysis; 4) VHL-KO/AKI: doxycycline plus rhabdomyolysis. To induce AKI, 50% glycerol (0.05 ml per 10 g body weight) was injected IM into the left hind limb under isoflurane narcosis. Drinking water was withdrawn between 20 h prior and 24 h after glycerol injection.
2013-09-05 | E-GEOD-44925 | biostudies-arrayexpress
Project description:Background: PTEN loss contributes to the development of many cancers and is associated with both hepatocellular carcinoma and cholangiocarcinoma. The pathogenesis of these malignancies is unclear, but they are speculated to arise from common cellular origins. We explored the influence of secondary effects, like hypoxia signaling, through co-deletion of Pten and Vhl in a murine model.Methods: We used a CreER-linked keratin 18 mouse model to conditionally delete Pten, Vhl or both, evaluating the resultant tumors by histology and gene expression microarray. A cohort of human cholangiocarcinoma samples was evaluated for relationships between HIF-1a expression and clinical outcomes.Results: Both Pten deletion genotypes developed liver tumors, but with differing phenotypes. Pten deletion alone led to large, invasive tumors with widespread hepatosteatosis. Co-deletion of Pten and Vhl resulted in low tumor burden and reduced steatosis. Microarray analysis divided mouse tumors’ respective genotypes by gene expression. This gene expression profile grouped a human tumor cohort according to histologic type with the Pten deletion signature aligning with hepatocellular carcinoma, whereas the Pten; Vhl deletion signature associated with cholangiocarcinomas. In a human cholangiocarcinoma cohort, we observed correlation between HIF-1a expression and overall survival.Conclusions: Pten deletion leads to tumor formation and steatosis in mouse livers. Co-deletion of Vhl and Pten resulted in lower tumor burden with gene expression profiling suggesting a switch from hepatocellular expression features to an expression profile more consistent with cholangiocarinoma. A possible relation between HIF-1a expression and increased overall survival in human cholangiocarcinoma suggests that hypoxia signaling influences tumor phenotype. reference x sample
2014-10-31 | E-GEOD-55874 | biostudies-arrayexpress
Project description:Gene expression data from VHL teratomas comparing genes differentially expressed based on apoptotic response to tumor microenvironment. Abstract BACKGROUND: The risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations. METHODS: VHL deficient (Vhl -/- ) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided. RESULTS: The P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation-induced apoptosis. CONCLUSIONS: The TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response. Three genotypes of VHL teratomas were examined: Three replicate wildtype VHL, Three replicate R167Q VHL, and Four replicate P81S VHL (representing two replicates of two different P81S clones)
2013-09-01 | E-GEOD-37464 | biostudies-arrayexpress
Project description:Cytolytic activity by CD8+ cytotoxic T lymphocytes (CTL) is a powerful tactic in the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTL is progressively dampened during chronic infection - yet the environmental cues and molecular pathways controlling immune “exhaustion” remain unclear. We find CTL immunity is regulated by the central transcriptional response to hypoxia, mediated by the von-Hippel-Lindau/Hypoxia-Inducible-Factor (VHL/HIF) pathway. Deletion of VHL, the primary negative regulator of HIF, leads to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTL display enhanced control of persistent viral infection and neoplastic growth. We find HIF and oxygen influence expression of pivotal CTL transcription, effector and costimulatory-inhibitory molecules, which is relevant to strategies to promote viral and tumor clearance. To understand the role of the VHL/HIF pathway in regulating T cell responses to acute and persistant antigen in vivo, a mixture of ~10^4 WT and Vhl KO virus-specific CD8+ T cells (P14s) was transferred iv into uninfected WT host mice. After infection with either LCMV-Armstrong (acute viral infection) or LCMV-clone13 (persistent viral infection) we double-FACS isolated the responding P14 donor cells from pooled spleens from two sets of host mice to obtain duplicates for microarray for the four conditions, resulting in eight samples (2 WT Arm, 2 VHL KO Arm; 2 WT cl13, 2 VHL KO cl13) at 6 to 7 days post-infection. All conditions were sorted on KRLG1lo P14 cells. Note this was a mixed P14 transfer, so WT and KO cells were responding to infection in the same WT host mice to aid in normalizing effects such as antigen load and cytokine environment.
2013-09-29 | E-GEOD-50158 | biostudies-arrayexpress
Project description:VHL is a tumor suppressor gene involved in the oxygen-sensing pathway whose germline mutations predispose to distinct phenotypes. Heterozygous mutations predispose to von Hippel-Lindau disease characterized by the development of multiple tumors (including hemangioblastomas, renal cell carcinomas and pheochromocytomas)1-3. More recently, a specific VHL-R200W mutation was shown to be responsible for Chuvash Polycythemia in homozygous carriers whereas heterozygous individuals have no clinical manifestation4. We report here a family carrying, on the same allele, VHL mutations characteristics of the two types of disease (a Chuvash polycythemia-R200W mutation and a von Hippel-Lindau disease-R161Q mutation). Genotyping, modeling analysis and functional studies, including transcriptomic profile of the distinct mutants validated for the first time on direct HIF target genes, show a gradual capacity of the VHL mutants to regulate the hypoxia responsive pathway that correlate with the severity of the developed phenotype. Our study provide original results that illuminate genotype/phenotype correlations in von Hippel-Lindau disease.
2013-09-03 | E-MTAB-1269 | biostudies-arrayexpress
Project description:VHL is the most frequently mutated gene in ccRCC.It functions via the oxygen-dependent ubiquitin-mediated degradation of hypoxia-inducible factor (HIF). The stabilization and hyperactivation of HIF1 play essential roles in tumorigenesis. This is a TMT based proteomics experiment aimed to investigate the functions of VHL in ccRCC systematically.
2023-01-05 | PXD012665 | Pride
Project description:RCC cells (786-O) were transfected with VHL. The parental cell line should be compared to the transfectant (+ VHL) under nomoxia as well as under hypoxia conditions. We want to distinct the VHL-mediated gene expression from the hypoxia-mediated and study the influence of both on the cellular metabolism. RNA of VHL-negative 786-O RCC cell line and VHL-transfectants incubated during normoxia and hypoxia conditions were analysed by cDNA microarray.
2015-01-22 | E-GEOD-65168 | biostudies-arrayexpress
Project description:The human renal cancer cell line 786-0 was transfected with 3 vectors allowing the doxycycline-inducible expression of 1) the full length wild type sequence of VHL: 786-0 VHL WT, VHL+/+, 2) the R167Q mutant : 786-0 R167Q, VHL mutated, 3) the empty vector : 786-0 EV, VHL-/-. The aim of the study was to examine whether the VHL-R167Q mutation, which is associated with a high risk of developping clear cell renal carcinomas, could impact the plasticity of renal carcinoma cells.
2021-12-09 | E-MTAB-11168 | biostudies-arrayexpress