Cellular and molecular characterization of the altered metabolism in RCC
ABSTRACT: 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.
Project description:The aim of the present study was to investigate the potential role of HIF-2a in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-O with mutated VHL was resistant to NK-mediated lysis as compared to the VHL corrected cell line (WT7). This resistance was independent of immunological synapse formation and NK ligand expression but was found to be reliant on HIF-2a stabilization in 786-0 cells. In order to elucidate the molecular basis of HIF-2a-induced impairment of NK-mediated killing, we performed global gene analysis using DNA microarray. Three candidate genes (ANGPTL4, ADM and ITPR1) were selected on the basis of their fold change and their involvement in cell death and survival. SiRNA targeting of these genes revealed that only ITPR1 silencing resulted in a significant increase of 786-O susceptibility to NK-mediated lysis. Using gene silencing of HIF2-a and chromatin immunoprecipitation assay, we showed for the first time that ITPR1 was a direct novel target of HIF2-a. Notably, a strong and significant correlation was found between ITPR1and HIF2-a staining in RCC patients. Transcriptional profiling of ITPR1silenced RCC cells indicated that several important genes involved in cell survival and apoptosis were differentially regulated. Using ITPR1 silenced Renca cells, we further found that ITPR1 was involved in the control of tumor progression. Moreover ITPR1 targeting combined with NK depletion significantly enhanced tumor growth as compared to ITPR1 knocked down Renca xenografts. Our data provide insights into the link between HIF-2a, ITPR1-related pathway and natural immunity and suggest a role of the HIF2/ITPR1 axis in regulating RCC cell survival.
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.
Project description:While early stages of clear cell renal cell carcinoma (ccRCC) are curable, survival outcome for metastatic ccRCC remains poor. The purpose of the current study was to apply a new individualized bioinformatics analysis (IBA) strategy to these transcriptome data in conjunction with Gene Set Enrichment Analysis of the Connectivity Map (C-MAP) database to identify and reposition FDA-approved drugs for anti-cancer therapy. We demonstrated that one of the drugs predicted to revert the RCC gene signature towards normal kidney, pentamidine, is effective against RCC cells in culture and in a RCC xenograft model. Most importantly, pentamidine slows tumor growth in the 786-O human ccRCC xenograft mouse model. To determine which genes are regulated by pentamidine in a human RCC cell line, 786-O, we treated these cells with pentamidine and performed transcriptional profiling analysis. We used microarrays to determine the set of genes regulated by pentamidine in 786-O renal cell cancer cells. Total RNA was isolated from 786-O cells treated with 25 μM pentamidine or vehicle control (DMSO) for 6 hours and hybridized to Affymetrix microarrays.
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.
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).
Project description:Renal cell carcinoma (RCC) accounts for around 2-3% of all adult neoplasms and its incidence is increasing. RCC is largely resistant to standard cancer treatment regimens including chemotherapy and radiation therapy, whilst immunotherapy modestly provides benefit to only a small subset of patients. As a result, prognosis for RCC patients with advanced disease is very poor, with a 5-year survival rate of less than 10%. An early event in the development of familial and up to 90% of sporadic RCC is the loss and/or mutation/methylation of the von Hippel-Lindau (VHL) tumour suppressor gene. The tumor suppressor role of VHL relies primarily on its ability to target members of the hypoxia-inducible factors (HIF) for proteosomal degradation, however it has also been implicated in other biological processes linked to oncogenesis, such as the extracellular matrix assembly and the regulation of actin cytoskeleton, that are less clearly defined. Targeting the VHL/HIF pathway may dramatically improve RCC treatment, as demonstrated by an increasing number of novel agents that have shown promising activity in recent clinical trials6. Identification of other possible proteins regulated by VHL that may be involved in tumour suppression will improve our understanding of RCC pathobiology and also provide novel disease biomarkers or potential targets. A number of our studies have identified changes in glycosylation in RCC, at least some of which appear to alter as a result of changes in the VHL gene. Lectin binding immunohistochemistry shows altered reactivity with a number of lectins in the malignant tissue compared with the normal tissue. Comparison of plasma membrane protein enriched fractions from human RCC cells stably transfected with control vector or with wild type VHL led to the identification of VHL-associated changes in the forms of several plasma membrane proteins which we beelive to be due to differential glycosylation. For one of these we have now shown that it is differentially O-glycosylated and this has been confimed in cell lysates and patient tissue by Western blotting. A parallel approach using 2D-PAGE analysis of RCC cell line conditioned medium (CM) has also identified a tumour-specific glycoform of a secretory protein that appears to undergo differential N-glycosylation in RCC and this has been confirmed in analysis of some matched primary tumour/normal pairs. These findings are potentially very interesting, as these proteins could serve as prognostic or diagnostic markers, or even as targets for improving RCC therapy. We are therefore keen to explore the underlying mechanism of differential glycosylation that may be associated to VHL dependent gene expression. As there are an immense number of enzymes involved in the conjugation of N- and particularly O-glycans to proteins, the glyco-microarray is an ideal tool to elucidate this further. For chip analyses we have three cell lines generated from human RCC cells (RCC4, 786-0 and UMRC2) that have been stably transfected with control vector or with wild type VHL i.e. 6 cell lines in total. Whole RNA was extracted from each cell line in 3 replicates per line.
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)
Project description:VHL loss is the most common genetic alteration event in ccRCC. VHL loss stabilizes hypoxia-inducible factor-2 alpha (HIF2a). We compared the changes in transcriptomics profiles after VHL restoration or HIF2a siRNA knockdown in 786-O cells. Overall design: RNA-seq profiles of ccRCC cell line 786-O with and without wild-type VHL restoration, with non-targeting control or pooled siRNA against HIF2a
Project description:This study was performed to understand the gene expression changes that accompany treatment of renal cell carcinoma (RCC) with vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) therapy. Human RCC cell lines were implanted into the flanks of nude beige mice, allowed to reach 12mm in long axis, and then treated with TKIs (sunitinib or sorafenib). Tumors were excised at 2 timepoints (prior to any therapy and at the 20mm endpoint of the study) and gene expression analysis was performed. Sunitinb or sorafenib were administered to mice bearing either 786-O or A498 xenografts. Mice were sacrificed and tumors excised for RNA extraction at pretreatment size of 12mm, or at 20mm, the mandated maximum tumor size allowed at our institution.
Project description:The clinical response to sunitinib in advanced renal-cell carcinoma (RCC) is frequently limited in magnitude and duration due to drug resistance, but the underlying mechanisms remain elusive. To discover mechanisms of resistance, we developed drug-resistant cell lines and have their gene expressions profiled and compared. Results provide insight into the molecular mechanisms underlying sunitinib-resistance. Overall design: RCC cell lines 786-O and ACHN, which were initially sensitive to Erlotinib treatment, are injected subcutaneously into the flanks of nude mice. Mice were treated with sunitinib orally (40 mg/kg/day) in a standard 4 weeks on, 2 weeks off treatment schedule. After one cycle of treatment, RCC cells dissociated from xenografts were transplanted into secondary mice followed with the same sunitinib-treatment. RCC cells are finally obtained from the 3rd generation xenografts. 7Su3rd is a shortcut of 786-O cells with sunitinib-resistance from the 3rd generation. ACSu3rd is a shortcut of ACHN cells with sunitinib-resistance from the 3rd generation.Gene expression profiles of acquired resistant RCC cells 7Su3rd, ACSu3rd were compared to their parental counterpart 786-O and ACHN, respectively. We designed this experiment to screen for lncRNA which is required for sunitinib-resistance.