FSMP-13. HYPOXIC REGULATION OF LACTATE DEHYDROGENASE GENES (LDHA/B) IN T98 GLIOBLASTOMA MULTIFORME CELLS

Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a poor prognosis. GBM cells exhibit extensive metabolic alterations that enhance survival and proliferation in the mixed normoxic-hypoxic tumor microenvironment. Lactate dehydrogenase (LDH) enzymes are critical mediators of the normoxic to hypoxic transition in cells. Two LDH genes (A/B) encode monomers that combine to form five isoenzymes (LDH1-5) with different properties for pyruvate to lactate interconversion. Hypoxic induction of LDHA in all cells appears to occur via HIF-1 mediated transcription. However, little is known about hypoxic regulation of LDHB in cancer. We report on hypoxic regulation of LDHA/B in T98G, a rare cell line that has both normal and neoplastic features. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Relative expression of LDH isoforms 1-5 was assessed using native gel electrophoresis. Expression of the LDHA and LDHB genes was measured using qRT-PCR. LDHA-dominant isoforms (4/5) were detected in T98G cells subjected to normoxia and hypoxia via gel electrophoresis, however, LDHB-dominant isoforms (1/2) were not. The LDHA/B-equimolar isoform (3) was decreased in T98G cells subjected to hypoxia. LDHA gene expression was over two-fold greater than LDHB in normoxia (p = .00256 by one-tailed Mann-Whitney U test), and over nine-fold greater in hypoxia (p = .00256). LDHA:LDHB expression in hypoxia compared to normoxia was significantly different (p = .00256). LDHA expression increased three-fold in hypoxia (p = .00256), while LDHB expression decreased 0.3-fold in hypoxia (p = .03288). We document LDHB dysregulation in T98G cells as the gene is minimally responsive to oxygen. Therapeutic strategies aimed at promoting LDHB expression may complement inhibition of LDHA and reduce GBM survival in hypoxia.

Investigating Glioblastoma Response to Hypoxia

Glioblastoma (GB) is the most common and deadly type of primary malignant brain tumor with an average patient survival of only 15–17 months. GBs typically have hypoxic regions associated with aggressiveness and chemoresistance. Using patient derived GB cells, we characterized how GB responds to hypoxia. We noted a hypoxia-dependent glycolytic switch characterized by the up-regulation of HK2, PFKFB3, PFKFB4, LDHA, PDK1, SLC2A1/GLUT-1, CA9/CAIX, and SLC16A3/MCT-4. Moreover, many proangiogenic genes and proteins, including VEGFA, VEGFC, VEGFD, PGF/PlGF, ADM, ANGPTL4, and SERPINE1/PAI-1 were up-regulated during hypoxia. We detected the hypoxic induction of invasion proteins, including the plasminogen receptor, S100A10, and the urokinase plasminogen activator receptor, uPAR. Furthermore, we observed a hypoxia-dependent up-regulation of the autophagy genes, BNIP-3 and DDIT4 and of the multi-functional protein, NDRG1 associated with GB chemoresistance; and down-regulation of EGR1 and TFRC (Graphical abstract). Analysis of GB patient cohorts’ revealed differential expression of these genes in patient samples (except SLC16A3) compared to non-neoplastic brain tissue. High expression of SLC2A1, LDHA, PDK1, PFKFB4, HK2, VEGFA, SERPINE1, TFRC, and ADM was associated with significantly lower overall survival. Together these data provide important information regarding GB response to hypoxia which could support the development of more effective treatments for GB patients.

The Expression of Decidual Protein Induced by Progesterone (DEPP) Is Controlled by Three Distal Consensus Hypoxia Responsive Element (HRE) in Hypoxic Retinal Epithelial Cells

Hypoxia affects the development and/or progression of several retinopathies. Decidual protein induced by progesterone (DEPP) has been identified as a hypoxia-responsive gene that may be part of cellular pathways such as autophagy and connected to retinal diseases. To increase our understanding of DEPP regulation in the eye, we defined its expression pattern in mouse and human retina and retinal pigment epithelium (RPE). Interestingly, DEPP expression was increased in an age-dependent way in the central human RPE. We showed that DEPP was regulated by hypoxia in the mouse retina and eyecup and that this regulation was controlled by hypoxia-inducible transcription factors 1 and 2 (HIF1 and HIF2). Furthermore, we identified three hypoxia response elements (HREs) about 3.5 kb proximal to the transcriptional start site that were responsible for hypoxic induction of DEPP in a human RPE cell line. Comparative genomics analysis suggested that one of the three HREs resides in a highly conserved genomic region. Collectively, we defined the molecular elements controlling hypoxic induction of DEPP in an RPE cell line, and provided evidence for an enrichment of DEPP in the aged RPE of human donors. This makes DEPP an interesting gene to study with respect to aging and age-related retinal pathologies.

The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis

ABSTRACTGlucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

The UTX Tumor Suppressor Directly Senses Oxygen to Control Chromatin and Cell Fate

Mammalian cells express multiple 2-oxoglutarate (OG)-dependent dioxygenases, including many chromatin regulators. The oxygen affinities, and hence oxygen sensing capabilities, of the 2-oxoglutarate (OG)-dependent dioxygenases reported to date vary widely. Hypoxia can affect chromatin, but whether this reflects a direct effect on chromatin-modifying dioxygenases, or indirect effects caused by the hypoxic-induction of the HIF transcription factor or the endogenous 2-OG competitor 2-hydroxyglutarate (2-HG), is unclear. Here we report that hypoxia induces a HIF- and 2-HG-independent histone modification signature consistent with KDM inactivation. We also show that the H3K27 histone demethylase KDM6A (also called UTX), but not its paralog KDM6B, is oxygen-sensitive. KDM6A loss, like hypoxia, prevented H3K27me3 erasure and blocked differentiation. Conversely, restoring H3K27me3 homeostasis in hypoxic cells reversed these effects. Therefore, oxygen directly affects chromatin regulators to control cell fate.One Sentence SummaryKDM6A demethylase activity is diminished under hypoxic conditions and causes changes in gene expression programs that govern cell fate.