Gene Expression profiles of colon from Hif-1α+/+, Hif-1αLSL/LSL
ABSTRACT: 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).
Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response. Global gene expression profiling in colon RNAs isolated from 7-week-old Hif-2αF/F (n=6, Shah 007) and Hif-2αΔIE (n=5, Shah 008).
Project description:To identify the precise molecular mechanisms that could contribute to the increase in colon carcinogenesis, microarray gene expression analysis was performed on colon RNA isolated from 5-week-old VhlF/F and VhlΔIE, VhlΔIE/Apcmin/+ and VhlF/F/Apcmin/+ mice. Hypoxia-inducible factor (HIF) is a key modulator of the transcriptional response to hypoxia and is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. Intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apcmin/+ intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and Hif-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue, however tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apcmin/+ mice in which HIF-2α was activated in the intestine. Consistent with this result, BrdU incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis demonstrated that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings demonstrate that a chronic increase in HIF-2α in the colon initiates pro-tumorigenic signaling which may have important implications in developing preventive and therapeutic strategies for colon cancer. Global gene expression profiling in colon RNAs isolated from 5-week-old VhlF/F (n=4, Shah 001), VhlF/F/Apcmin/+(n=3, Shah 003), VhlΔIE (n=3, Shah 002) and VhlΔIE/Apcmin/+ mice (n=5, Shah 004).
Project description:Analysis of Huh-7 hepatocarcinoma cell line depleted of NDRG3 or HIF-1α under hypoxic condition. HIF-1α and NDRG3 have distinct functions in hypoxia responses. Results provide insight into molecular basis of HIF-independent signaling in the development and progression of hypoxic tumors Gene expression profiles of Huh-7 cells stably expressing NDRG3-shRNA or HIF-1α-shRNA under normoxia were compared to gene expression profiles of Huh-7 stable cells under hypoxia for 6, 12 and 24 hours.
Project description:Transcription mediated by hypoxia inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in the activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compared the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element (HRE), siRNA targeted to HIF-1α, and echinomycin, a DNA binding natural product with a similar but less specific sequence preference to the polyamide. The polyamide affects a subset of hypoxia-induced genes that are consistent with the binding site preferences of the polyamide. For comparison, siRNA targeted to HIF-1α and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data shows how polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression. Keywords: Gene expression changes in cultured U251 cells after DFO-stimulation and various treatment conditions Overall design: Hypoxia-mimetic DFO (deferoxamine)-stimulated U251 cells that were treated with polyamide 1, HIF-1a siRNA, and echinomycin were compared to control cells that were also DFO-stimulated. Cells not stimulated with DFO were also compared to the DFO-stimulated controls. Three biological replicates were included for each treatment/condition.
Project description:Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. The oxygen-sensitive Hypoxia Inducible Factor (HIF) transcriptional regulators HIF-1α and HIF-2α are overexpressed in many human NSCLCs, and constitutive HIF-2α activity can promote murine lung tumor progression, suggesting that HIF proteins may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted Hif-1α or Hif-2α in an established KrasG12D-driven murine NSCLC model. Deletion of Hif-1α had no obvious effect on tumor growth, whereas Hif-2α deletion resulted in an unexpected increase in tumor burden that correlated with reduced expression of the candidate tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 as a direct HIF-2α target gene, and demonstrate that HIF-2α regulates Scgb3a1 expression and tumor formation in human KrasG12D-driven NSCLC cells. AKT pathway activity, reported to be repressed by Scgb3a1, was enhanced in HIF-2α deficient human NSCLC cells and xenografts. Finally, a direct correlation between HIF-2α and SCGB3a1 expression was observed in approximately 70% of human NSCLC samples analyzed. These data suggest that whereas HIF-2α overexpression can contribute to NSCLC progression, therapeutic inhibition of HIF-2α below a critical threshold may paradoxically promote tumor growth by reducing expression of tumor suppressor genes, including Scgb3a1. Overall design: RNA was isolated from tumors of experimental (Hif2alpha deficient) mice and control mice (seven for each group).
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.
Project description:Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin-remodeling, upon loss of the cardiolipin acyl transferase Tafazzin, mutes HIF-1a signaling in hypoxia. Tafazzin-deficiency does not affect posttranslational HIF-1a regulation but rather HIF-1a gene-expression, a dysfunction recapitulated in iPSCs-derived cardiomyocytes from Barth Syndrome patients with Tafazzin-deficiency. RNAseq analyses confirmed drastically altered signaling in Tafazzin mutant cells. In hypoxia, Tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-kB activation and concomitantly HIF-1a gene-expression. In agreement, Tafazzin-deficient mice hearts display reduced HIF-1a levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin-remodeling dampens HIF-1a signaling through inactivation of a non-canonical signaling pathway: Lack of NF-kB activation through reduced mitochondrial ROS production diminishes HIF-1a transcription. Overall design: Cells lacking tafazzin were subjected to hypoxia and compared to the ones at normoxia or isogenic WT cells either at hypoxia or nomoxia
Project description:Based on the results of numerous clinical and preclinical analyses, the transcription factor HIF-1a has been identified as an important tumor-promoting factor and is considered to be an attractive target for cancer therapy. To further deconstruct the molecular nature of HIF-1a’s role in tumorigenesis, we have applied lentiviral shRNA transduction to establish HIF-1a-deficient gastric cancer cells. Interestingly, functional analyses failed to show a significant growth defect of HIF-1a-deficient gastric cancer cells in vitro and in vivo. These observations led us to propose that stable inactivation of HIF-1a resulted in efficient compensation enabling cell growth and, ultimately, tumor progression in a HIF-1a-independent manner. To better understand the mechanisms that control this compensation, we performed transcriptomics of control (“scrambled” (SCR)) and HIF-1a-deficient (HIF) gastric cancer cells. Analysis of hypoxia-inducible factor-1alpha (HIF-1a)-deficient gastric cancer cells under normoxia. The transcription factor HIF-1a is a key regulator of oxygen homeostasis and has been identified as an important tumor-promoting factor. Results provide insight into the role of HIF-1a in gastric carcinogenesis. Overall design: Gastric cancer AGS cells were lentiviral and stably transduced with a small hairpin RNA targeting human HIF-1a or a control shRNA. RNA was extracted from control and HIF-1a-deficient cells. Each cell line was analyzed in triplicate for a total of six samples.
Project description:10 days old tumor spheroids were processed for RNA isolation using the Quiagen RNeasy Micro Kit and cDNA synthesis was done using the Ambion WT Expression Kit. Wt, HIF-1α k/d and HIF-2α k/d samples were compared to each other. Overall design: Samples from 4 different spheroid experiments of wt, HIF-1α knockdown and HIF-2α knockdown HepG2 cells, respectively, were compared with each other