Hypoxia-induced Gene Expression Research Articles

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

Lactiplantibacillus plantarum K8 lysates regulate hypoxia-induced gene expression

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.

A Luciferase Reporter Assay to Detect Cellular Hypoxia In Vitro.

Hypoxia is a hallmark of ischemic cardiovascular diseases and solid malignant tumors. Cellular hypoxia induces numerous physiological and pathological processes, including hematopoiesis, angiogenesis, metabolic changes, cell growth, and apoptosis. Hypoxia-inducible factor-1 (HIF-1) binds to hypoxia response elements (HREs) to selectively induce the expression of various genes in response to hypoxia. Therefore, HREs have been used to develop hypoxia-targeted gene therapy.More than 70 pairs of HREs and hypoxia-inducible genes have been identified. The hypoxia-induced gene expression levels vary among HRE sequences depending on the number of HRE copies and oxygen levels. Most known HREs have not yet been thoroughly studied. Recent studies have revealed that the HRE-mediated effects of hypoxia are cell line-dependent. Herein we describe an in vitro method to investigate gene activation levels and characteristics based on varying the copy number of HREs in response to cellular hypoxia. We explain how to clone HREs into luciferase reporter constructs in the sense, antisense, and dual directions to measure luciferase expression for functional analyses.

Glucose Regulates Glucose Transport and Metabolism via mTOR Signaling Pathway in Bovine Placental Trophoblast Cells.

It has been confirmed that improving the energy level of the diet contributed to the greater reproductive performance and birth weight of calves in periparturient dairy cows. To investigate the effect of glucose on nutrient transport during fetal development, the bovine placental trophoblast cells (BPTCs) were cultured in media with different glucose concentrations (1, 2, 4, 8, or 16 mg/mL). Subsequently, the BPTCs were cultured in media with 1, 8 mg/mL glucose and 8 mg/mL glucose plus 100 nmol/L rapamycin (the inhibitor of mTOR pathway). Compared with the 1 mg/mL glucose, the addition of 8 mg/mL glucose stimulated cell proliferation, upregulated the mRNA abundance of the glucose transporter GLUT1 and GLUT4, and increased the activity of glucose metabolism-related enzyme glucose-6-phosphate dehydrogenease (G6PD), lactate dehydrogenase (LDHA) and phosphoglycerate kinase 1 (PGK1), as well as adenosine-triphosphate (ATP) content (p < 0.05).Furthermore, compared with the treatment of 1 mg/mL glucose, adding 8 mg/mL of glucose-upregulated gene expression in the mTOR signaling pathway, including phosphatidylinositol3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase 2 (P70S6K) (p < 0.05).The supplementation of rapamycin downregulated the gene and protein expression of the mTOR signaling pathway, including mTOR, P70S6K, EIF4E-binding protein 1 (4EBP1), hypoxia-inducible factor 1-alpha (HIF-1α) and gene expression of glucose transporter upregulated by 8 mg/mL glucose (p < 0.05). Thus, these results indicated that the addition of 8 mg/mL glucose regulated the glucose transport and metabolism in BPTCs through the mTOR signaling pathway, thereby promoting the supply of nutrients to fetus.

CSIG-26. TARGETING HYPOXIC CANCER CELLS IN MEDULLOBLASTOMA

Abstract The goal of this study is to thoroughly assess the role of hypoxia-induced transcription factors (HIFs) in MB growth and examine HIF as a potential therapeutic target. We first examined patient MB tumor by IHC and identified scattered areas with expression of HIF1a and HIF2a. We then established orthotopic human MB xenografts in mice and found hypoxic areas throughout the tumors using pimonidazole detection and they expressed both HIF1a, and HIF2a. There are only few observations of hypoxic areas within experimental MB. To explore the impact of HIFs on MB growth, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We observed a decrease in cell growth, an increase in HIF1a and HIF2a protein expression, activation of HIF targets (Glut-1, VEGF), and a hypoxia-response element-driven luciferase reporter assay. To further examine the role of individual HIF isoforms, we employed CRISPR/Cas9 to knockout (KO) the HIF1a and/or HIF2a genes in ONS76 MB cells. The HIF reporter activity was partially canceled with single HIF-KO and completely abrogated with double KO and double KO reduced cell growth, indicating that both HIF-1 and HIF-2 are having roles for activating transcription and cell growth. Treatment of MB cells with 64B, an arylsulfonamide HIF/p300 inhibitor abrogated reporter activity by both HIF-1 and HIF-2 and reduced expression of hypoxia-induced genes. To investigate the potential of targeting HIF in vivo, we injected mice brains with wild-type and HIF1a/2a-KO ONS-76 cells and monitored their growth and survival. Mice injected with double KO HIF cells exhibited prolonged survival compared to those with parental cells (n = 5, p&amp;lt; 0.01). Further experiments to expand this approach with other MB cell line and test the anti-tumor effects of 64B in mice are ongoing. These findings support targeting HIFs as a novel therapeutic strategy in MB treatment.

Expression of hypoxia-inducible genes is suppressed in altered gravity due to impaired nuclear HIF1α accumulation

Extravehicular activities, the backbone of manned space exploration programs, set astronauts into mild hypoxia. Unfortunately, microgravity aggravates threatening symptoms of hypoxia such as vision impairment and brain edema. Hypoxia-inducible factors (HIFs) sense cellular hypoxia and, subsequently, change the cells’ expression profile instantaneously by rapidly translocating—most likely cytoskeleton-dependently—into the nucleus and subsequently forming transcription complexes with other proteins. We tested the hypothesis that this fundamental process could be altered by sudden changes in gravitational forces in parabolic flights using a newly developed pocket-size cell culture lab that deoxygenizes cells within 15 min. Sudden gravity changes (SGCs 1g–1.8g–0g–1.8g–1g) during hypoxic exposure suppressed expression of the HIF1α-dependent genes investigated as compared with hypoxia at constant 1g. Normoxic cells subjected to SGCs showed reduced nuclear but not cytoplasmatic HIF1α signal and appeared to have disturbed cytoskeleton architecture. Inhibition of the actin-dependent intracellular transport using a combination of myosin V and VI inhibitors during hypoxia mimicked the suppression of the HIF1α-dependent genes observed during hypoxic exposure during SGCs. Thus, SGCs seem to disrupt the cellular response to hypoxia by impairing the actin-dependent translocation of HIF1α into the nucleus.

Global gene expression analysis of the turkey hen hypothalamo-pituitary-gonadal axis during the preovulatory hormonal surge

The preovulatory hormonal surge (PS) consists of elevated circulating luteinizing hormone (LH) and progesterone levels and serves as the primary trigger for ovarian follicle ovulation. Increased LH and progesterone, produced by the pituitary and the granulosa layer of the largest ovarian follicle (F1), respectively, result from hypothalamic stimulation and steroid hormone feedback on the hypothalamo-pituitary-gonadal (HPG) axis. The hypothalamus, pituitary, F1 granulosa, and granulosa layer of the fifth largest follicle (F5) were isolated from converter turkey hens outside and during the PS and subjected to RNA sequencing (n=6 per tissue). Differentially expressed genes were subjected to functional annotation using DAVID and IPA. A total of 12, 250, 1235, and 1938 DEGs were identified in the hypothalamus, pituitary, F1 granulosa, and F5 granulosa respectively (q<0.05, |fold change|>1.5, FPKM>1). Gene Ontology (GO) analysis revealed key roles for metabolic processes, steroid hormone feedback, and hypoxia induced gene expression changes. Upstream analysis identified a total of 4, 42, 126, and 393 potential regulators of downstream gene expression in the hypothalamus, pituitary, F1G, and F5G respectively, with a total of 63 potential regulators exhibiting differential expression between samples collected outside and during the PS (|z-score|>2). The results from this study serve to increase the current knowledge base surrounding the regulation of the PS in turkey hens. Through GO analysis, downstream processes and functions associated with the PS were linked to identified DEGs, and through upstream analysis, potential regulators of DEGs were identified for further analysis. Linking upstream regulators to the downstream PS and ovulation events could allow for genetic selection or manipulation of ovulation frequencies in turkey hens.

Cell Dome as an Evaluation Platform for Organized HepG2 Cells.

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of spheroids exhibit specific behaviors attributed to hypoxic conditions, a 3D cell culture system that allows the observation of such cells using conventional optical or fluorescence microscopes would be useful. In this study, HepG2 cells were cultured in "Cell Dome", a micro-dome in which cells are enclosed in a cavity consisting of a hemispherical hydrogel shell. HepG2 cells formed hemispherical cell aggregates which filled the cavity of Cell Domes on 18 days of culture and the cells could continue to be cultured for 29 days. The cells at the center of hemispherical cell aggregates were observed using a fluorescence microscope. The cells grew in Cell Domes for 18 days exhibited higher Pi-class Glutathione S-Transferase enzymatic activity, hypoxia inducible factor-1α gene expression, and higher tolerance to mitomycin C than those cultured in 2D on tissue culture dishes (* p &lt; 0.05). These results indicate that the center of the glass adhesive surface of hemispherical cell aggregates which is expected to have the similar environment as the center of the spheroids can be directly observed through glass plates. In conclusion, Cell Dome would be useful as an evaluation platform for organized HepG2 cells.

CSIG-35. TARGETING HYPOXIC CANCER CELLS IN MEDULLOBLASTOMA

Abstract New treatment approaches are urgently needed for children with medulloblastoma (MB) to augment survival and reduce the long-term side effects from the treatment. Malignant tumors often display hypoxic regions where hypoxia-induced transcription factors (HIFs) increase the expression of genes that promote tumor growth (e.g., metabolic adaptation and invasion/metastasis). Based on the known roles of HIF in many solid tumors, we hypothesized that Hypoxia/HIFs contribute to MB cell growth and targeting HIF proteins is a viable strategy for treating MB. To test our hypothesis, first, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We found increased expression of both HIF1a and HIF2a proteins and activation of a hypoxia-response element driven luciferase reporter assay. Treatment of the cells with 64B, an arylsulfonamide HIF inhibitor abrogated reporter activity and reduced expression of hypoxia-induced genes (GLUT-1, VEGF). Next, we used CRISPR/Cas9 to knockout singly or in combination HIF1a/HIF2a genes. The inhibition of HIF reporter activity by 64B was partially canceled in single HIF-KO cells and completely canceled in double HIFs-KO cells, showing both isoforms were active. Second, to investigate HIFs can be therapeutic targets, we established orthotopic human MB xenografts in mice. We first examined the tumor for the presence of hypoxic areas and used pimonidazole (Hypoxyprobe) injections followed by immunohistochemistry and found hypoxic areas throughout the tumors where tumor cells expressed HIF1a and/or HIF2a. Evaluation of human MB samples for HIF1/2 expression is also ongoing. Next, we plan on injecting mice with cells devoid of HIFs genes and examine their growth. We will also treat the mice with 64B to test whether it has anti-tumor effects and extends survival. In conclusion, these results indicate that HIF activation is part of the pathobiology of MB and HIF proteins may be a target for MB treatment.

Effects of Ion-Transporting Proteins on the Digestive System Under Hypoxia.

Hypoxia refers to a state of oxygen limitation, which mainly mediates pathological processes in the human body and participates in the regulation of normal physiological processes. In the hypoxic environment, the main regulator of human body homeostasis is the hypoxia-inducible factor family (HIF). HIF can regulate the expression of many hypoxia-induced genes and then participate in various physiological and pathological processes of the human body. Ion-transporting proteins are extremely important types of proteins. Ion-transporting proteins are distributed on cell membranes or organelles and strictly control the inflow or outflow of ions in cells or organelles. Changes in ions in cells are often closely related to extensive physiological and pathological processes in the human body. Numerous studies have confirmed that hypoxia and its regulatory factors can regulate the transcription and expression of ion-transporting protein-related genes. Under hypoxic stress, the regulation and interaction of ion-transporting proteins by hypoxia often leads to diseases of various human systems and even tumors. Using ion-transporting proteins and hypoxia as targets to explore the mechanism of digestive system diseases and targeted therapy is expected to become a new breakthrough point.

Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

HIF-1α-mediated augmentation of miRNA-18b-5p facilitates proliferation and metastasis in osteosarcoma through attenuation PHF2

Extensive evidence has explored the involvement of microRNAs (miRNAs) in osteosarcoma (OS). Limitedly, the concrete function of microRNA-18b-5p (miR-18b-5p) in OS remains unexplored and largely elusive. Here, we validated that miR-18b-5p significantly elevated in OS via analyzing the data from GEO database. The results showed that miR-18b-5p was overexpressed in human OS tissues and cell lines. The clinical evidence suggested that high level of miR-18b-5p was negatively correlated with the poor prognosis of OS. Meanwhile, miR-18b-5p upregulation facilitated the proliferation and metastasis of OS cells in vitro and in vivo. The mechanism exploration demonstrated that miR-18b-5p acted as a potential inhibitor of PHF2, a tumor suppressor gene, at post-transcriptional level. Moreover, hypoxia induced gene expression of miR-18b-5p was clarified to be transcriptionally mediated by HIF-1α. The clinicopathological analysis in samples of OS patients further supported that miR-18b-5p had a positive correlation with HIF-1α expression, and negative correlation with PHF2. Collectively, the present study uncovered a new molecular mechanism of OS tumorigenesis and development and miR-18b-5p might be a prognostic biomarker and potential therapeutic target for OS treatment.

Abstract 118: BACH1 is an oxygen-sensitive mediator of the hypoxia response in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive solid tumor characterized by a hypoxic phenotype that promotes metastatic progression and resistance to therapy. The canonical cellular response to hypoxia is largely driven by hypoxia inducible factors (HIFs) that stimulate transcription of genes related to angiogenesis, cancer stem cells, cell survival, and glucose and iron metabolism. There is growing evidence that several factors, in addition to HIFs, contribute to the tumor hypoxia response. Thus, identifying novel mechanisms that regulate this cellular response in addition to the HIFs is key to our understanding of hypoxia and could have direct clinical impact. High expression of BTB and CNC homology 1 (BACH1), a heme-regulated bZIP transcription factor, drives metastatic progression in various cancers including TNBC. While BACH1 has been shown to be induced under hypoxia in some cell lines, its regulation and role in hypoxia signaling, specifically with regards to cancer cells, is poorly understood. Here we report a novel regulatory pathway of the tumor hypoxia response in TNBC. We show that BACH1 is post-translationally prolyl-hydroxylated by the HIF prolyl-hydroxylases (PHDs) in an oxygen-dependent and NRF2/HMOX1-independent manner. Hypoxic exposure thus stabilizes BACH1 protein and enhances its DNA binding activity at loci of both canonical and hypoxia-specific downstream targets. We further show that BACH1 regulates the hypoxia response by promoting expression of both HIF-dependent and HIF-independent hypoxia-induced genes in TNBC cells. Our findings describe BACH1 as a novel oxygen-sensitive effector of the hypoxia response in TNBC that represents an alternative target for attenuating hypoxia-induced, pro-metastatic signaling and therapeutic resistance. Citation Format: Christopher M. Dann, Long C. Nguyen, Dongbo Yang, Joseph P. Wynne, Andrea Valdespino, Letícia Stock, Marsha R. Rosner. BACH1 is an oxygen-sensitive mediator of the hypoxia response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 118.

Abstract 951: BNIP3L/NIX promotes breast cancer growth and metastasis by inducing epithelial-mesenchymal transition

Abstract Mitophagy is induced in response to physiological stresses, including hypoxia, as an adaptive response that decreases mitochondrial mass to promote efficient use of limiting nutrients. BNIP3L/NIX is a mitophagy cargo receptor that integrates into the outer mitochondrial membrane where it interacts with processed LC3 at growing phagophore membranes to promote mitophagy. Our preliminary data shows that elevated BNIP3L/NIX expression in basal-like breast cancers correlates with worse overall patient survival. However, the functional significance of BNIP3L/NIX for growth and progression of breast cancer has not been determined. We generated a CRISPR/Cas9 KO BNIP3L/NIX 4T1 mouse mammary tumor model to determine how loss of BNIP3L/NIX affected tumor progression and metastasis. We observed that loss of BNIP3L/NIX decreased tumor cell growth and importantly attenuated metastasis to the lungs. By performing RNA-seq analysis, we found that these striking findings were associated with a decrease in molecular signatures expression of hypoxia-inducible genes and epithelial-mesenchymal transition (EMT) genes, including Twist and Vimentin. In addition, expression of Vimentin was complete ablated in 4T1 KO BNIP3L/NIX tumors in vivo and 4T1 KO BNIP3L/NIX tumor cells had significantly reduced ability to migrate and invade in vitro. Furthermore, CRISPR/Cas9-mediated deletion of HIF1α a in the 4T1 cell model reduced Vimentin expression in a manner dependent on BNIP3L/NIX expression. These results indicate that BNIP3L/NIX acts as an oncogene downstream of HIF1α in breast cancer by promoting expression of key EMT genes, stimulating tumor cell proliferation and migration, and that targeting BNIP3L/NIX might prove an effective strategy in the clinic. Citation Format: Damian E. Berardi, Kay F. Macleod. BNIP3L/NIX promotes breast cancer growth and metastasis by inducing epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 951.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages

BackgroundHuman immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression...

Cornelian cherry (Cornus mas L.) fruit extract improves growth performance, disease resistance, and serum immune-and antioxidant-related gene expression of common carp (Cyprinus carpio)

This study evaluated the use of cornelian cherry (Cornus mas L.) fruit extract on the growth performance, immune responses, and disease resistance in common carp (Cyprinus carpio). A total of 192 fish (initial body weight of 18.34 ± 1.8 g) were randomly distributed in 12 tanks with 16 fish per tank (4 treatments with 3 replications) and fed with diets containing cornelian cherry extract (CCE11CCE (cornelian cherry extract)) at the rate of 0, 0.25, 0.5 and 1%. The final weight of common carp fed CCE at 0.5% was significantly higher than fish fed 0 and 0.25% doses (p < 0.05). In addition, weight gain and specific growth rate were increased, and feed conversion ratio decreased in fish-fed diets containing 0.5 and 1% CCE compared to other groups. The challenge test with A. hydrophila revealed that common carp-fed CCE had a lower mortality rate than the control. The lysozyme activity was markedly higher in common carp fed 0.5% of cornelian cherry extract than in control. The total immunoglobulin and total protein showed higher values in fish fed 0.5 and 1% than the control. The catalase gene showed higher mRNA levels in fish fed 0.5 and 1% than the control. Glutathione reductase gene expression was markedly upregulated in common carp fed 1% of CCE than the control and 0.25% groups. The glutathione S-transferase gene showed higher mRNA levels in fish fed 0.5 and 1% than the control and 0.25% groups. The superoxide dismutase gene expression was markedly higher in common carp fed 0.5% of CCE than in control. The CC-chemokine and interleukin-10 showed higher mRNA levels in fish fed 0.5% than the remaining groups. Hypoxia-inducible factor 1-alpha gene expression was markedly upregulated in common carp fed 1% of CCE than the control and 0.25% groups. The heat shock 70 kDa protein and interleukin-1 beta genes showed higher mRNA levels in fish fed 0.25, 0.5, and 1% than the control. In conclusion, the supplementation of CCE at 0.5–1% is recommended to enhance the growth performance and relieve the impacts of A. hydrophila infection in common carp.

Slc38a9 Deficiency Induces Apoptosis and Metabolic Dysregulation and Leads to Premature Death in Zebrafish

Eukaryotic cells control nutritional homeostasis and determine cell metabolic fate through a series of nutrient transporters and metabolic regulation pathways. Lysosomal localized amino acid transporter member 9 of the solute carrier family 38 (SLC38A9) regulates essential amino acids’ efflux from lysosomes in an arginine-regulated fashion. To better understand the physiological role of SLC38A9, we first described the spatiotemporal expression pattern of the slc38a9 gene in zebrafish. A quarter of slc38a9−/− mutant embryos developed pericardial edema and died prematurely, while the remaining mutants were viable and grew normally. By profiling the transcriptome of the abnormally developed embryos using RNA-seq, we identified increased apoptosis, dysregulated amino acid metabolism, and glycolysis/gluconeogenesis disorders that occurred in slc38a9−/− mutant fish. slc38a9 deficiency increased whole-body free amino acid and lactate levels but reduced glucose and pyruvate levels. The change of glycolysis-related metabolites in viable slc38a9−/− mutant fish was ameliorated. Moreover, loss of slc38a9 resulted in a significant reduction in hypoxia-inducible gene expression and hypoxia-inducible factor 1-alpha (Hif1α) protein levels. These results improved our understanding of the physiological functions of SLC38A9 and revealed its indispensable role in embryonic development, metabolic regulation, and stress adaption.

Physiology of Plant Responses to Water Stress and Related Genes: A Review

Drought and waterlogging seriously affect the growth of plants and are considered severe constraints on agricultural and forestry productivity; their frequency and degree have increased over time due to global climate change. The morphology, photosynthetic activity, antioxidant enzyme system and hormone levels of plants could change in response to water stress. The mechanisms of these changes are introduced in this review, along with research on key transcription factors and genes. Both drought and waterlogging stress similarly impact leaf morphology (such as wilting and crimping) and inhibit photosynthesis. The former affects the absorption and transportation mechanisms of plants, and the lack of water and nutrients inhibits the formation of chlorophyll, which leads to reduced photosynthetic capacity. Constitutive overexpression of 9-cis-epoxydioxygenase (NCED) and acetaldehyde dehydrogenase (ALDH), key enzymes in abscisic acid (ABA) biosynthesis, increases drought resistance. The latter forces leaf stomata to close in response to chemical signals, which are produced by the roots and transferred aboveground, affecting the absorption capacity of CO2, and reducing photosynthetic substrates. The root system produces adventitious roots and forms aerenchymal to adapt the stresses. Ethylene (ETH) is the main response hormone of plants to waterlogging stress, and is a member of the ERFVII subfamily, which includes response factors involved in hypoxia-induced gene expression, and responds to energy expenditure through anaerobic respiration. There are two potential adaptation mechanisms of plants (“static” or “escape”) through ETH-mediated gibberellin (GA) dynamic equilibrium to waterlogging stress in the present studies. Plant signal transduction pathways, after receiving stress stimulus signals as well as the regulatory mechanism of the subsequent synthesis of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzymes to produce ethanol under a hypoxic environment caused by waterlogging, should be considered. This review provides a theoretical basis for plants to improve water stress tolerance and water-resistant breeding.

Abstract P5-05-04: Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes

Abstract MetAP2 inhibitors have shown clinical anti-tumor activity, but CNS side effects and poor drug-like properties have generally limited their development. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) polymer backbone. This conjugation approach is intended to improve safety (i.e., limit CNS penetration) while also improving biodistribution and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 recently completed a phase I safety trial in late-stage cancer patients (NCT02743637) and was well-tolerated with no apparent CNS side effects. In prior models of breast cancer, SDX-7320 significantly inhibited the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (i.e., “metabo-oncology”) and also synergized with the PI3Kα inhibitor alpelisib/Piqray® in ER+/Her2- MCF-7 xenografts. The objectives of these experiments were to determine the effect of SDX-7320 on the growth of Her2+ xenografts alone or in combination with the Akt/mTOR inhibitor capivasertib/AZD-5363 and to determine if SDX-7320 could attenuate hyperglycemia induced by capivasertib/AZD-5363. Female athymic, nude mice (9 weeks of age) were surgically implanted with slow-release estrogen pellets (90-day, 0.72 mg, inter-scapular) and three days later 2 x 107 BT474 cells (in Matrigel/RPMI-1640, 50/50, v/v) were injected into the fourth mammary gland. When the group mean tumor volume exceeded 100 mm3, treatment with test agents commenced. SDX-7320 (sc/q4d, 6, 12 mg/kg) exerted significant, dose-dependent inhibition of BT-474 tumor growth (TGI of 61 and 88% respectively), whereas capivasertib/AZD-5363 (po, qd, 100, 200 mg/kg) exhibited significant anti-tumor activity only at 200 mg/kg (54% TGI). The combination of SDX-7320 (12 mg/kg) plus capivasertib/AZD-5363 (200 mg/kg) produced additive effects in that a greater number of mice had tumor regression relative to mice treated with either SDX-7320 or capivasertib/AZD-5363 alone. Tumor tissue from a subset of treatment groups (Vehicle, SDX-7320-12 mg/kg, SDX-7320-12 mg/kg + AZD-5363-200 mg/kg, and AZD-5363-200 mg/kg) was snap frozen and stored at -80oC. PolyA+ RNA was isolated, cDNA libraries were constructed and RNASeq was conducted (range of 20-30 reads per million (RPM) base pairs). Analysis of RNASeq data showed that SDX-7320 alone significantly attenuated the expression of hypoxia-induced genes as well as genes related to the innate immune system. Based on these results, we conclude that SDX-7320 affected tumor growth by limiting the ability of tumors to adapt to a hypoxic microenvironment and by altering the innate tumor-immune microenvironment. In normal male, C57Bl/6 mice, capivasertib/AZD-5363 (200 mg/kg, po) elevated blood glucose after a single dose. Pretreatment with either one dose (24 hours before the Akt/mTOR inhibitor) or multiple doses (Q4D beginning 14 days prior to Akt/mTOR inhibitor) of SDX-7320 (8 mg/kg) significantly inhibited hyperglycemia induced by capivasertib/AZD-5363. In summary SDX-7320 significantly inhibited the growth of BT-474 xenografts, alone and in combination with capivasertib/AZD-5363, which was associated with suppression of hypoxia-induced genes, as well as genes of the innate immune system. In addition, SDX-7320 improved the safety profile of capivasertib/AZD-5363 by attenuating Akt/mTOR inhibitor-induced hyperglycemia, providing further support for the clinical exploration of SDX-7320 in combination with Akt/mTOR inhibitors in Her2+ breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Mark Kalinich, Jonathan Wang, Sara Little, Bradley Carver, James Shanahan. Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-04.

The endothelial-enriched lncRNA LINC01235 regulates hypoxia response via HIF-3a

Abstract Background Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Hypoxia induces significant changes in cardiovascular control mechanisms potentially resulting in pathophysiology. Recently, an increasing number of long non-coding RNAs (lncRNAs) was reported to participate in the regulation of Hypoxia-inducible factors (HIF). Analysis of single-cell RNA-sequencing of human Abdominal Aortic Aneurysms pinpointed the endothelial-enriched lncRNA LINC01235. LINC01235 was previously correlated with tumour progression in gastric cancer and worse patient prognosis in breast cancer. Globally, the role of LINC01235 in the cardiovascular system remains unknown. Purpose The objective of this study is to unravel the function of LINC01235 in endothelial cells (ECs). Methods and results LINC01235 levels were elevated in human umbilical vein ECs (7.66 fold, p&amp;lt;0.05), human aortic ECs (16.84 fold, p&amp;lt;0.05) and human dermal microvascular ECs (639.73 fold, p&amp;lt;0.05) over other human cardiovascular cells like vascular smooth muscle cells, aortic fibroblasts and cardiomyocytes. Severe hypoxia (0.2% O2 for 24h) reduced LINC01235 expression significantly (0.33 fold, p&amp;lt;0.05). SiRNA-mediated LINC01235 silencing in HUVECs (0.12, p&amp;lt;0.05) resulted in decreased proliferation (0.76 fold, p&amp;lt;0.05) and vascular endothelial growth factor A (VEGFA)-stimulated angiogenic sprouting (0.39 fold, p&amp;lt;0.05). Loss of LINC01235 did not affect apoptosis, metabolism or barrier function. Analysis of RNA-sequencing data revealed that many hypoxia-responsive genes were downregulated after knockdown of LINC01235 (siCtrl vs. siLINC01235). These included HIF-3α (0.24 fold, p&amp;lt;5.86e-28) as a potential key regulator of the cellular feedback to hypoxia. Phenotypically, knockdown of HIF3A using siRNAs (0.07 fold, p&amp;lt;0.05) resulted in decreased proliferation (0.82 fold, p&amp;lt;0.05) and VEGFA-stimulated angiogenic sprouting (0.50 fold, p&amp;lt;0.05). Accordingly, hypoxia response and LINC01235 knockdown exhibit a negative correlation based on transcriptomics data (R=−0.157, p&amp;lt;2.2e-16), further emphasizing a role of LINC01235 in hypoxia response. Conclusion In summary, the EC-enriched lncRNA LINC01235 is likely required for the suppression of hypoxia-induced gene expression under normoxic conditions potentially mediated by HIF-3α. Functionally, loss of LINC01235 decreased proliferation and VEGFA-stimulated angiogenic sprouting without an effect on cell death, metabolism or barrier integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG - TRR267