Hypoxia Growth Research Articles

Do air-breathing fish suffer branchial oxygen loss in hypoxic water?

In hypoxia, air-breathing fish obtain O2 from the air but continue to excrete CO2 into the water. Consequently, it is believed that some O2 obtained by air-breathing is lost at the gills in hypoxic water. Pangasionodon hypophthalmus is an air-breathing catfish with very large gills from the Mekong River basin where it is cultured in hypoxic ponds. To understand how P. hypophthalmus can maintain high growth in hypoxia with the presumed O2 loss, we quantified respiratory gas exchange in air and water. In severe hypoxia (PO2: ≈ 1.5 mmHg), it lost a mere 4.9% of its aerial O2 uptake, while maintaining aquatic CO2 excretion at 91% of the total. Further, even small elevations in water PO2 rapidly reduced this minor loss. Charting the cardiovascular bauplan across the branchial basket showed four ventral aortas leaving the bulbus arteriosus, with the first and second gill arches draining into the dorsal aorta while the third and fourth gill arches drain into the coeliacomesenteric artery supplying the gut and the highly trabeculated respiratory swim-bladder. Substantial flow changes across these two arterial systems from normoxic to hypoxic water were not found. We conclude that the proposed branchial oxygen loss in air-breathing fish is likely only a minor inefficiency.

Genetic variation in HIF-2α attenuates ventilatory sensitivity and carotid body growth in chronic hypoxia in high-altitude deer mice.

The gene encoding HIF-2α, Epas1, has experienced a history of natural selection in many high-altitude taxa, but the functional role of mutations in this gene is still poorly understood. We investigated the influence of the high-altitude variant of Epas1 in North American deer mice (Peromyscus maniculatus) on the control of breathing and carotid body growth during chronic hypoxia. We created hybrids between high- and low-altitude populations of deer mice to disrupt linkages between genetic loci so that the physiological effects of Epas1 alleles (Epas1H and Epas1L , respectively) could be examined on an admixed genomic background. In general, chronic hypoxia (4weeks at 12kPa O2 ) enhanced ventilatory chemosensitivity (assessed as the acute ventilatory response to hypoxia), increased total ventilation and arterial O2 saturation during progressive poikilocapnic hypoxia, and increased haematocrit and blood haemoglobin content across genotypes. However, the effects of chronic hypoxia on ventilatory chemosensitivity were attenuated in mice that were homozygous for the high-altitude Epas1 allele (Epas1H/H ). Carotid body growth and glomus cell hyperplasia, which was strongly induced in Epas1L/L mice in chronic hypoxia, was not observed in Epas1H/H mice. Epas1 genotype also modulated the effects of chronic hypoxia on metabolism and body temperature depression in hypoxia, but had no effects on haematological traits. These findings confirm the important role of HIF-2α in modulating ventilatory sensitivity and carotid body growth in chronic hypoxia, and show that genetic variation in Epas1 is responsible for evolved changes in the control of breathing and metabolism in high-altitude deer mice. KEY POINTS: High-altitude natives of many species have experienced natural selection on the gene encoding HIF-2α, Epas1, including high-altitude populations of deer mice. HIF-2α regulates ventilation and carotid body growth in hypoxia, and so the genetic variants in Epas1 in high-altitude natives may underlie evolved changes in control of breathing. Deer mice from controlled crosses between high- and low-altitude populations were used to examine the effects of Epas1 genotype on an admixed genomic background. The high-altitude variant was associated with reduced ventilatory chemosensitivity and carotid body growth in chronic hypoxia, but had no effects on haematology. The results help us better understand the genetic basis for the unique physiological phenotype of high-altitude natives.

HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

Фактор роста гипоксии как биомаркер у пациентов с макулярным отеком на фоне ретинальных венозных окклюзий

Фактор роста гипоксии как биомаркер у пациентов с макулярным отеком на фоне ретинальных венозных окклюзий

Neurotransmitter Modulation of Carotid Body Germinal Niche.

The carotid body (CB), a neural-crest-derived organ and the main arterial chemoreceptor in mammals, is composed of clusters of cells called glomeruli. Each glomerulus contains neuron-like, O2-sensing glomus cells, which are innervated by sensory fibers of the petrosal ganglion and are located in close contact with a dense network of fenestrated capillaries. In response to hypoxia, glomus cells release transmitters to activate afferent fibers impinging on the respiratory and autonomic centers to induce hyperventilation and sympathetic activation. Glomus cells are embraced by interdigitating processes of sustentacular, glia-like, type II cells. The CB has an extraordinary structural plasticity, unusual for a neural tissue, as it can grow several folds its size in subjects exposed to sustained hypoxia (as for example in high altitude dwellers or in patients with cardiopulmonary diseases). CB growth in hypoxia is mainly due to the generation of new glomeruli and blood vessels. In recent years it has been shown that the adult CB contains a collection of quiescent multipotent stem cells, as well as immature progenitors committed to the neurogenic or the angiogenic lineages. Herein, we review the main properties of the different cell types in the CB germinal niche. We also summarize experimental data suggesting that O2-sensitive glomus cells are the master regulators of CB plasticity. Upon exposure to hypoxia, neurotransmitters and neuromodulators released by glomus cells act as paracrine signals that induce proliferation and differentiation of multipotent stem cells and progenitors, thus causing CB hypertrophy and an increased sensory output. Pharmacological modulation of glomus cell activity might constitute a useful clinical tool to fight pathologies associated with exaggerated sympathetic outflow due to CB overactivation.

Characterization of a heme-protein responsive to hypoxia in Paracoccidioides brasiliensis

Oxygen is fundamental to the life of aerobic organisms and is not always available to Paracoccidioides cells. During the life cycle stages, reduced oxygen levels directly affect general metabolic processes and oxygen adaptation mechanisms may play a fundamental role on fungal ability to survive under such condition. Heme proteins can bind to oxygen and participate in important biological processes. Several fungi, including Paracoccidioides, express a heme-binding globin (fungoglobin - FglA) presumable to regulate fungal adaptation to hypoxia. However, the characterization of fungoglobin in Paracoccidioides spp. has not yet been performed. In this study, we predicted the structure of fungoglobin and determined its level of expression during hypoxic-mimetic conditions. Genomic screening revealed that the fungoglobin gene is conserved in all species of the Paracoccidioides genus. Molecular modeling showed biochemical and biophysical characteristics that support the hypothesis that FglA binds to the heme group and oxygen as well. The fungoglobin transcript and proteins are expressed at higher levels at the early treatment time, remaining elevated while oxygen is limited. A P. brasiliensis fglA knockdown strain depicted reduced growth in hypoxia indicating that this protein can be essential for growth at low oxygen. Biochemical analysis confirmed the binding of fungoglobin to heme. Initial analyzes were carried out to establish the relationship between FlglA and iron metabolism. The FglA transcript was up regulated in pulmonary infection, suggesting its potential role in the disease establishment. We believe that this study can contribute to the understanding of fungal biology and open new perspectives for scientific investigations.

Abstract 1877: Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway

Abstract The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In our previous study, we showed that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. In this study, we found that hypoxia also induces third generation EGFR TKI, osimertinib (AZD9291), resistance in the NSCLC cell line, H1975, which had developed resistance to first and second generation of EGFR TKIs by harboring second EGFR mutation, T790M. Following growth in hypoxia, hypoxia-induced gefitinib resistant HCC827 clones and hypoxic H1975 cells show increased N-cadherin expression and decreased E-cadherin expression, characteristics of an epithelial-mesenchymal transition (EMT), coordinated by an increased expression of ZEB1, an EMT activator. Mechanistically, we show that hypoxia induces increased fibroblast growth factor receptor 1 (FGFR1) expression in HCC827 and H1975 cells, and that knockdown of the FGFR1 by shRNA can attenuate hypoxia induced EGFR TKI resistance in both HCC827 and H1975 cells. We also found the upregulated expression of FGFR1 by hypoxia is mediated through the MAPK pathway to lead to EGFR TKI resistance. In keeping with this, inhibition of FGFR1 function by the selective small molecular inhibitor, BGJ398, attenuates hypoxia-induced EGFR TKI resistance and promotes upregulation of BIM levels. Similarly, inhibition of MEK activity by the inhibitor, trametinib, shows similar effects. In vivo in tumor xenografts in mice, BGJ398 treatment or trametinib treatment suppresses tumor growth and enhances AZD9291 response. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLCs. Citation Format: Yuhong Lu, Yanfeng Liu, Sebastian Oeck, Gary J. Zhang, Peter M. Glazer. Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1877.

Clonal ZEB1-Driven Mesenchymal Transition Promotes Targetable Oncologic Antiangiogenic Therapy Resistance.

Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy. SIGNIFICANCE: Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1498/F1.large.jpg.

Abstract 312: Hypoxia induces EGFR inhibitor resistance in lung cancer cells via fibroblast growth factor receptor 1 (FGFR1) by promoting epithelial-mesenchymal transition (EMT)

Abstract The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In this study, we show that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. We also found that hypoxia induces resistance to the third generation EGFR TKI, osimernitib, in the NSCLC cell line, H1975, which developed resistance to first and second EGFR TKI resistance by harboring a second EGFR mutation, T790M. Following growth in hypoxia, HCC827 and H1975 cells show increased N-cadherin expression with downregulation of E-cadherin, characteristics of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, we show that hypoxia increases fibroblast growth factor receptor 1 (FGFR1) expression in both HCC827 and H1975 cells, accompanied by down regulated BIM expression. ZEB1, an EMT activator, was also increased by hypoxia in HCC827 H1975 cells. Knockdown of the FGFR1 by shRNA can attenuate gefitinib resistance in HCC827 cells and osimerinitib resistance in H1975 cells, respectively. Inhibition of FGFR1 function by the small molecular inhibitor, ponatinib, resensitizes hypoxia-induced resistant HCC827 clones to gefitinib treatment, and also attenuates osimerinitib resistance in hypoxic H1975 cells. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 inhibitors may offer an attractive therapeutic strategy for NSCLCs. Citation Format: Yuhong Lu, Gary J. Zhang, Peter M. Glazer. Hypoxia induces EGFR inhibitor resistance in lung cancer cells via fibroblast growth factor receptor 1 (FGFR1) by promoting epithelial-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 312.

PO-276 Histone acetylation readers BET in hypoxia adaptation in triple negative breast cancer (TNBC)

IntroductionHypoxia (low oxygen) is associated with worst prognosis and therapy resistance in most solid tumours. Hypoxia occurs in ~50% of breast cancer, most frequently in triple negative breast cancer (TNBC), the most aggressive subtype. Hypoxia drives tumour aggressiveness through HIF, which activates genes that promote adaptation to hypoxia, including CA9, VEGFA, LOX, that promote respectively pH regulation, angiogenesis and metastasis. Epigenetic factors mediate gene expression changes that occur in pathological conditions, like hypoxia. BET proteins read histone-acetylation and recruit transcription factors. BET inhibition has anti-tumour effects. Thus we investigated BET proteins as regulators of TNBC hypoxic adaptation.Material and methodsCell lines were investigated in 2D in normoxia and hypoxia (0.1%–1% O2), spheroids and as orthotopic xenografts with BET inhibitors (BET-i: JQ1, iBET151 and iBET762). The impact of BET-i on transcriptional regulation and hypoxic phenotypes was investigated using a variety of molecular and cell biology techniques.Results and discussionsBET-i reduced growth in hypoxia and normoxia in vitro (2D and 3D) and in vivo where xenograft vascularisation was reduced (reduced CD31 staining). JQ1 modulated 44% of hypoxia-induced genes (including CA9, VEGFA and LOX), but did not alter HIF expression. Ongoing investigations have identified that hypoxia increases H4ac and H3K27ac, associated with transcription and BET protein binding, in the promoters of HIF targets CA9, VEGFA and LOX. BET-i also reduced HIF binding to the CA9 promoter. Silencing of individual BET proteins (BRD 2, 3 and 4) downregulates CA9 in hypoxia, while only BRD4 knockdown reduces VEGFA expression. The knockdown of individual BET proteins reduced colony formation in hypoxia and normoxia, but not as comprehensively as JQ1, suggesting there could be a compensatory role of the BET proteins.ConclusionWe identified histone acetylation and BET proteins play an important role in tumour adaptation to hypoxia. This provides evidence to support the development of BET-i as a therapeutic option for hypoxic TNBC. Acknowledgements: This research is funded by Breast Cancer Now.

Heterogeneity in Pathogenicity-related Properties and Stress Tolerance in Aspergillus fumigatus Clinical Isolates.

Stress responses and pathogenicity have been extensively studied in Aspergillus fumigatus, the main causative pathogen of life-threatening aspergillosis. The heterogeneity in this pathogen's biology has recently attracted increasing attention. In the present work, we used 16 clinically isolated strains to investigate several properties relevant to the pathogenicity of A. fumigatus, namely, gliotoxin production, elastase activity, hypoxia growth, adaptation to iron-limiting conditions, and growth upon nitrosative, oxidative, and high osmotic stresses. The range of phenotypes was diverse across the strains, with gliotoxin production and elastase activity being negatively correlated at an intermediate index (R=-0.4717). Notably, there were strains that showed extraordinary high production of gliotoxin or elastase activity and hypersensitivity to nitrosative or oxidative stresses. Clustering analysis showed that the 7 potentially pathogenicity-related phenotypes were not correlated with the genetic sub-group or pathotype. These results contribute to the growing awareness of the genetic and phenotypic diversity in A. fumigatus isolates.

GPR133 Promotes Glioblastoma Growth in Hypoxia.

GPR133 Promotes Glioblastoma Growth in Hypoxia.

TMIC-16NOX GENERATED REACTIVE OXYGEN SPECIES PROMOTES GLIOBLASTOMA GROWTH AND SURVIVAL THROUGH INACTIVATION OF PTEN

TMIC-16. NOX GENERATED REACTIVE OXYGEN SPECIES PROMOTES GLIOBLASTOMA GROWTH AND SURVIVAL THROUGH INACTIVATION OF PTEN Kirsten Ludwig and Harley Kornblum; University of California, Los Angeles, Los Angeles, CA, USA PTEN is inactivated in 40% of glioblastoma (GBM), however almost all GBM’s have increased Akt activity, regardless of PTEN status. Treatment with exogenous reactive oxygen species (ROS) inactivates PTEN through oxidation, suggesting that a similar event may occur in the hypoxic tumor environment. Recent data indicate that low levels of ROS are required for cell maintenance and stem cell renewal while moderate increases may promote tumorigenesis. Much of the endogenous ROS is produced by NADPH oxidase (NOX), amembraneboundcomplexofproteins,activatedunderhypoxic conditions. This raises the possibility that the tumor microenvironment may inactivate PTEN through means other than mutation or deletion. We investigated the role of NOX generated ROS in primary cultures of patient-derived GBM cells across the spectrum of GBM subtypes. Endogenous ROS levels correlated with proliferation; those with higher ROS had higher proliferation rates. Placement of cells in low oxygen levels (1%) enhanced proliferation, an effect that was reversed by treatment with antioxidants and the NOX inhibitor Apocynin, supporting the hypothesis that NOX-induced ROS promotes GBM growth. The effects of hypoxia-induced NOX activation were dependent upon PTEN, as PTEN knockdown or PTEN null cells exhibited no enhanced growthwhenplaced inhypoxicconditions.Furthermore,pharmacologicblockade of AKT prevented enhanced growth in hypoxia. A role for PTEN was further demonstrated by the finding that it was oxidatively inactivated in GBM cells grown under hypoxic conditions. Finally, pharmacological inhibition of NOX rescued radiation-induced activation of Akt resulting in increased radio-sensitivity. Taken together, these data support the hypothesis that the relativelyhypoxic tumorenvironment activatesNOX, resulting in functional inactivation of PTEN andenhanced GBMproliferation andsurvival. Thesefindings represent a novel opportunity for therapeutic intervention in a subset of GBM. Neuro-Oncology 17:v221–v225, 2015. doi:10.1093/neuonc/nov236.16 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.

Hypoxia promotes uveal melanoma invasion through enhanced Notch and MAPK activation.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.

In vivo hyperoxia induces hypoxia-inducible factor-1α overexpression in LNCaP tumors without affecting the tumor growth rate

Hypoxia is a recognized cause for solid tumors malignancy and resistance, probably via hypoxia-induced overexpression of the hypoxia-inducible factor (HIF)-1α, major modulator of the cell response to oxygen deprivation. Although hyperoxia, the opposite condition, may represent a key issue to assess this paradigm, its effect on tumor growth and HIF-1α expression remains unclear. To test whether hyperoxia and hypoxia have divergent effects, and to better focus into the role of HIF-1α in vivo, athymic mice xenografted with LNCaP cells were exposed for 28 days to atmospheres containing 10, 21 or 30% O2. Whereas the xenografts grew twice faster in hypoxia, their growth rates in hyperoxia and normoxia were similar. To analyze the involved molecular mechanisms, we performed various assays in xenograft tissues. Faster xenografts growth in hypoxia was associated with higher phosphorylation of protein kinase B (Akt) and higher expression of Ki67, both related with pro-survival and cell proliferation pathways. By contrast, the expression level of HIF-1α was similar in normoxia and hypoxia, but paradoxically twice higher in hyperoxia. The protein level of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was also higher in hyperoxia, suggesting marked cell response to redox imbalance. Whereas both the vascular-endothelial growth factor (VEGF) and its receptor VEGF-R2 were overexpressed in hyperoxia, the tissue hemoglobin content was not increased, despite a slight reduction in vascularization. As a whole, this data indicates that the xenografts growth rate was independent of HIF-1α expression level, suggesting that in an in vivo setting alternative more effective proliferative paths associated with the cell response to the redox imbalance may override the paths linked to HIF-1α signaling.

Chloroquine-mediated cell death in metastatic pancreatic adenocarcinoma through inhibition of autophagy.

Cells in the interior of pancreatic tumors are believed to undergo continual autophagy to maintain homeostasis during unregulated growth in hypoxia caused by impaired vascularity. We hypothesize that treating metastatic cells with chloroquine, an inhibitor of autophagy, in hypoxia will decrease cell viability and induce cell death.MiaPaCa2 (non-metastatic) and S2VP10 (metastatic) cell lines were treated with 25 and 50 µM chloroquine for 24 and 48 hours in normoxia and hypoxia (5-10% O₂). Viability was measured using ATPlite™. After treatment, the cell stress was analyzed, and protein was lysed and quantified using the Bradford assay. Autophagy-associated protein levels were determined by Western blot.S2VP10 cells treated for 48 hours with 50 μM chloroquine in hypoxia had 24% viability compared to normoxia control, with loss of 10% viability caused by low O₂ alone. MiaPaCa2 cells under these conditions had 60% viability compared to normoxia control, with loss of 25% viability caused by low O₂ alone. Analysis of cell stress pathways and dosimetry of Western blot data suggest that chloroquine inhibits the autophagy pathway in the metastatic S2VP10 cells.Autophagy blockage with chloroquine or similar-acting drugs may serve as a viable therapy for highly metastatic pancreatic cancers.

Methylseleninic acid elevates REDD1 and inhibits prostate cancer cell growth despite AKT activation and mTOR dysregulation in hypoxia

Methylseleninic acid (MSeA) is a monomethylated selenium metabolite theoretically derived from subsequent β-lyase or transamination reactions of dietary Se-methylselenocysteine that has potent antitumor activity by inhibiting cell proliferation of several cancers. Our previous studies showed that MSeA promotes apoptosis in invasive prostate cancer cells in part by downregulating hypoxia-inducible factor HIF-1α. We have now extended these studies to evaluate the impact of MSeA on REDD1 (an mTOR inhibitor) in inducing cell death of invasive prostate cancer cells in hypoxia. In both PTEN+ and PTEN− prostate cancer cells we show that MSeA elevates REDD1 and phosphorylation of AKT along with p70S6K in hypoxia. Furthermore, REDD1 induction by MSeA is independent of AKT and the mTOR inhibition in prostate cancer cells causes partial resistance to MSeA-induced growth reduction in hypoxia. Our data suggest that MSeA induces REDD1 and inhibits prostate cancer cell growth in hypoxia despite activation of AKT and dysregulation of mTOR.MSeA elevates REDD1 and AKT to promote cell death in invasive prostate cancer cells in hypoxia.

Zinc Finger Transcription Factors Displaced SREBP Proteins as the Major Sterol Regulators during Saccharomycotina Evolution

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.