Comparative Molecular and Biological Characteristic of the Systemic Inflammatory Response in Adult and Old Male Wistar Rats with Different Resistance to Hypoxia.

Morphological, molecular, and biological features of the systemic inflammatory response induced by LPS administration were assessed in adult and old male Wistar rats with high and low resistance to hypoxia. In 6 h after LPS administration, mRNA expression levels of Hif1a, Vegf, Nfkb, and level of IL-1ß protein in old rats were higher than in adult rats regardless of hypoxia tolerance. The morphometric study showed that the number of neutrophils in the interalveolar septa of the lungs was significantly higher in low-resistant adult and old rats 6 h after LPS administration. Thus, in old male Wistar rats, systemic inflammatory response is more pronounced than in adult rats and depends on the initial tolerance to hypoxia, which should be considered when developing new approaches to the therapy of systemic inflammatory response in individuals of different ages.

Construction and validation of a hypoxia-related gene signature to predict the prognosis of breast cancer.

BACKGROUND: Among the most common forms of cancer worldwide, breast cancer posed a serious threat to women. Recent research revealed a lack of oxygen, known as hypoxia, was crucial in forming breast cancer. This research aimed to create a robust signature with hypoxia-related genes to predict the prognosis of breast cancer patients. The function of hypoxia genes was further studied through cell line experiments. MATERIALS AND METHODS: In the bioinformatic part, transcriptome and clinical information of breast cancer were obtained from The Cancer Genome Atlas(TCGA). Hypoxia-related genes were downloaded from the Genecards Platform. Differentially expressed hypoxia-related genes (DEHRGs) were identified. The TCGA filtered data was evenly split, ensuring a 1:1 distribution between the training and testing sets. Prognostic-related DEHRGs were identified through Cox regression. The signature was established through the training set. Then, it was validated using the test set and external validation set GSE131769 from Gene Expression Omnibus (GEO). The nomogram was created by incorporating the signature and clinicopathological characteristics. The predictive value of the nomogram was evaluated by C-index and receiver operating characteristiccurve. Immune microenvironment and mutation burden were also examined. In the experiment part, the function of the two most significant hypoxia-related genes were further explored by cell-line experiments. RESULTS: In the bioinformatic part, 141 up-regulated and 157 down-regulated DEHRGs were screened out. A prognostic signature was constructed containing nine hypoxia genes (ALOX15B, CA9, CD24, CHEK1, FOXM1, HOTAIR, KCNJ11, NEDD9, PSME2) in the training set. Low-risk patients exhibited a much more favorable prognosis than higher-risk ones (P < 0.001). The signature was double-validated in the test set and GSE131769 (P = 0.006 and P = 0.001). The nomogram showed excellent predictive value with 1-year OS AUC: 0.788, 3-year OS AUC: 0.783, and 5-year OS AUC: 0.817. Patients in the high-risk group had a higher tumor mutation burden when compared to the low-risk group. In the experiment part, the down-regulation of PSME2 inhibited cell growth ability and clone formation capability of breast cancer cells, while the down-regulation of KCNJ11 did not have any functions. CONCLUSION: Based on 9 DEHRGs, a reliable signature was established through the bioinformatic method. It could accurately predict the prognosis of breast cancer patients. Cell line experiment indicated that PSME2 played a protective role. Summarily, we provided a new insight to predict the prognosis of breast cancer by hypoxia-related genes.

Development of a Prognostic Model for Gastric Cancer Based on Apoptosis- and Hypoxia-Related Genes: Predictive Insights into Survival and Immune Landscape.

Gastric cancer (GC) is the fifth most prevalent malignancy worldwide, characterized by poor prognosis. Apoptosis is interacted with hypoxia in tumorigenesis. This study attempted to delineate potential value of apoptosis and hypoxia-related genes (AHRGs) in prognosis of gastric cancer. Differential expression analysis was performed on GC transcriptomic data from TCGA. Apoptosis-related genes (ARGs) and hypoxia-related genes (HRGs) were obtained from MSigDB, followed by intersecting them with differentially expressed genes (DEGs) in GC. A prognostic model was constructed using univariate, LASSO, and multivariate regression analyses. The model was validated using a Gene Expression Omnibus dataset. DEGs between risk groups were subjected to enrichment analysis. A nomogram was plotted by incorporating clinical information. Non-negative matrix factorization based on core prognostic genes from the multifactorial model was employed to cluster tumor samples. The subsequent analyses involved immunophenoscore, immune landscape, Tumor Immune Dysfunction and Exclusion (TIDE) score, and chemosensitivity for distinct subtypes. A prognostic model based on AHRGs was established, and its predictive capability was verified in external cohorts. Riskscore was determined as an independent prognostic factor, and it was used, combined with other clinical features, to plot a prognostic nomogram. Patients were clustered into cluster1 and cluster2 based on prognostic model genes. Cluster2 showed poorer prognosis and IPS scores, higher immune cell infiltration, immune function and TIDE scores than cluster1. Distinct therapeutic potential for various chemotherapeutic agents was observed between the two clusters. The developed AHRG scoring introduced a novel and effective avenue for predicting GC prognosis and identifying potential targets for further investigation.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

ERN1 knockdown modifies the hypoxic regulation of homeobox gene expression in U87MG glioblastoma cells.

OBJECTIVE.: Homeobox genes play an important role in health and disease including oncogenesis. The present investigation aimed to study ERN1-dependent hypoxic regulation of the expression of genes encoding homeobox proteins MEIS (zinc finger E-box binding homeobox 2) and LIM homeobox 1 family, SPAG4 (sperm associated antigen 4) and NKX3-1 (NK3 homeobox 1) in U87MG glioblastoma cells in response to inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioblastoma growth. METHODS.: The expression level of homeobox genes was studied in control (transfected by vector) and ERN1 knockdown U87MG glioblastoma cells under hypoxia induced by dimethyloxalylglycine (0.5 mM for 4 h) by quantitative polymerase chain reaction and normalized to ACTB. RESULTS.: It was found that hypoxia down-regulated the expression level of LHX2, LHX6, MEIS2, and NKX3-1 genes but up-regulated the expression level of MEIS1, LHX1, MEIS3, and SPAG4 genes in control glioblastoma cells. At the same time, ERN1 knockdown of glioblastoma cells significantly modified the sensitivity of all studied genes to a hypoxic condition. Thus, ERN1 knockdown of glioblastoma cells removed the effect of hypoxia on the expression of MEIS1 and LHX1 genes, but increased the sensitivity of MEIS2, LHX2, and LHX6 genes to hypoxia. However, the expression of MEIS3, NKX3-1, and SPAG4 genes had decreased sensitivity to hypoxia in ERN1 knockdown glioblastoma cells. Moreover, more pronounced changes under the conditions of ERN1 inhibition were detected for the pro-oncogenic gene SPAG4. CONCLUSION.: The results of the present study demonstrate that hypoxia affected the expression of homeobox genes MEIS1, MEIS2, MEIS3, LHX1, LHX2, LHX6, SPAG4, and NKX3-1 in U87MG glioblastoma cells in gene-specific manner and that the sensitivity of all studied genes to hypoxia condition is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling, and possibly contributed to the control of glioblastoma growth. A fundamentally new results of this work is the establishment of the fact regarding the dependence of hypoxic regulation of SPAG4 gene expression on ER stress, in particular ERN1, which is associated with suppression of cell proliferation and tumor growth.

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.

Lactate in breast cancer cells is associated with evasion of hypoxia-induced cell cycle arrest and adverse patient outcome.

Tumor hypoxia is a common microenvironmental factor in breast cancers, resulting in stabilization of Hypoxia-Inducible Factor 1 (HIF-1), the master regulator of hypoxic response in cells. Metabolic adaptation by HIF-1 results in inhibition of citric acid cycle, causing accumulation of lactate in large concentrations in hypoxic cancers. Lactate can therefore serve as a secondary microenvironmental factor influencing cellular response to hypoxia. Presence of lactate can alter the hypoxic response of breast cancers in many ways, sometimes in opposite manners. Lactate stabilizes HIF-1 in oxidative condition, as well as destabilizes HIF-1 in hypoxia, increases cellular acidification, and mitigates HIF-1-driven inhibition of cellular respiration. We therefore tested the effect of lactate in MDA-MB-231 under hypoxia, finding that lactate can activate pathways associated with DNA replication, and cell cycling, as well as tissue morphogenesis associated with invasive processes. Using a bioengineered nano-patterned stromal invasion assay, we also confirmed that high lactate and induced HIF-1α gene overexpression can synergistically promote MDA-MB-231 dissemination and stromal trespass. Furthermore, using The Cancer Genome Atlas, we also surprisingly found that lactate in hypoxia promotes gene expression signatures prognosticating low survival in breast cancer patients. Our work documents that lactate accumulation contributes to increased heterogeneity in breast cancer gene expression promoting cancer growth and reducing patient survival.

Zebrafish mutants in egln1 display a hypoxic response and develop polycythemia.

AIMS: Prolyl hydroxylase domain 2 (PHD2), encoded by the Egln1 gene, serves as a pivotal regulator of the hypoxia-inducible factor (HIF) pathway and acts as a cellular oxygen sensor. Somatic inactivation of Phd2 in mice results in polycythemia and congestive heart failure. However, due to the embryonic lethality of Phd2 deficiency, its role in development remains elusive. Here, we investigated the function of two egln1 paralogous genes, egln1a and egln1b, in zebrafish. MAIN METHODS: The egln1 null zebrafish were generated using the CRISPR/Cas9 system. Quantitative real-time PCR assays and Western blot analysis were employed to detect the effect of egln1 deficiency on the hypoxia signaling pathway. The hypoxia response of egln1 mutant zebrafish were assessed by analyzing heart rate, gill agitation frequency, and blood flow velocity. Subsequently, o-dianisidine staining and in situ hybridization were used to investigate the role of egln1 in zebrafish hematopoietic function. KEY FINDINGS: Our data show that the loss of egln1a or egln1b individually has no visible effects on growth rate. However, the egln1a; egln1b double mutant displayed significant growth retardation and elevated mortality at around 2.5 months old. Both egln1a-null and egln1b-null zebrafish embryo exhibited enhanced tolerance to hypoxia, systemic hypoxic response that include hif pathway activation, increased cardiac activity, and polycythemia. SIGNIFICANCE: Our research introduces zebrafish egln1 mutants as the first congenital embryonic viable systemic vertebrate animal model for PHD2, providing novel insights into hypoxic signaling and the progression of PHD2- associated disease.

Construction of a ferroptosis and hypoxia-related gene signature in cervical cancer to assess tumour immune microenvironment and predict prognosis.

BACKGROUND: This study aimed to investigate the potential role of ferroptosis/hypoxia-related genes in cervical cancer to improve early management and treatment of cervical cancer. METHODS: All data were downloaded from public databases. Ferroptosis/hypoxia-related genes associated with cervical cancer prognosis were selected to construct a risk score model. The relationship between risk score and clinical features, immune microenvironment and prognosis were analysed. RESULTS: Risk score model was constructed based on eight signature genes. Drug prediction analysis showed that bevacizumab and cisplatin were related to vascular endothelial growth factor A. Risk score, as an independent prognostic factor of cervical cancer, had a good survival prediction effect. The two groups differed significantly in degree of immune cell infiltration, gene expression, tumour mutation burden and somatic variation. CONCLUSIONS: We developed a novel prognostic gene signature combining ferroptosis/hypoxia-related genes, which provides new ideas for individual treatment of cervical cancer.

Ferroptosis, hypoxia and immune regulation play important roles in cervical cancer progression. In this study, we developed a novel prognostic signature combining ferroptosis and hypoxia-related genes, which provides new ideas for individual treatment of cervical cancer patients. The risk score established by ferroptosis and hypoxia-related gene as an independent prognostic factor of cervical cancer has a good survival prediction effect. High and low risk groups showed significant differences in TIME, prognosis, biological metabolic pathway and tumour mutation burden. In addition, we found drugs associated with signature genes. In short, this study has laid a theoretical foundation for exploring the related molecular mechanisms and prognosis of cervical cancer. It also contributes to the exploration of clinical management and treatment.

High hypoxia status in pancreatic cancer is associated with multiple hallmarks of an immunosuppressive tumor microenvironment.

Introduction: Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is a particularly lethal disease that is often diagnosed late and is refractory to most forms of treatment. Tumour hypoxia is a key hallmark of PDAC and is purported to contribute to multiple facets of disease progression such as treatment resistance, increased invasiveness, metabolic reprogramming, and immunosuppression. Methods: We used the Buffa gene signature as a hypoxia score to profile transcriptomics datasets from PDAC cases. We performed cell-type deconvolution and gene expression profiling approaches to compare the immunological phenotypes of cases with low and high hypoxia scores. We further supported our findings by qPCR analyses in PDAC cell lines cultured in hypoxic conditions. Results: First, we demonstrated that this hypoxia score is associated with increased tumour grade and reduced survival suggesting that this score is correlated to disease progression. Subsequently, we compared the immune phenotypes of cases with high versus low hypoxia score expression (HypoxiaHI vs. HypoxiaLOW) to show that high hypoxia is associated with reduced levels of T cells, NK cells and dendritic cells (DC), including the crucial cDC1 subset. Concomitantly, immune-related gene expression profiling revealed that compared to HypoxiaLOW tumours, mRNA levels for multiple immunosuppressive molecules were notably elevated in HypoxiaHI cases. Using a Random Forest machine learning approach for variable selection, we identified LGALS3 (Galectin-3) as the top gene associated with high hypoxia status and confirmed its expression in hypoxic PDAC cell lines. Discussion: In summary, we demonstrated novel associations between hypoxia and multiple immunosuppressive mediators in PDAC, highlighting avenues for improving PDAC immunotherapy by targeting these immune molecules in combination with hypoxia-targeted drugs.

Deficiency in SLC25A15, a hypoxia-responsive gene, promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: The role of solute carrier family 25 member 15 (SLC25A15), a critical component of the urea cycle, in hepatocellular carcinoma (HCC) progression remains poorly understood. This study investigated the impact of SLC25A15 on HCC progression and its mechanisms. METHODS: We systematically investigated the function of SLC25A15 in HCC progression using large-scale data mining and cell, animal, and organoid models. Furthermore, we analyzed its involvement in reprogramming glutamine metabolism. RESULTS: SLC25A15 expression was significantly decreased in HCC tissues, and patients with low SLC25A15 levels had a poorer prognosis. Hypoxia-exposed HCC cells or tissues had lower SLC25A15 expression. A positive correlation between HNF4A, a transcription factor suppressed by hypoxia, and SLC25A15 was observed in both HCC tissues and cells. Modulating HNF4A levels altered SLC25A15 mRNA levels. SLC25A15 upregulated SLC1A5, increasing glutamine uptake. The reactive metabolic pathway of glutamine was increased in SLC25A15-deficient HCC cells, providing energy for HCC progression through additional lipid synthesis. Ammonia accumulation due to low SLC25A15 levels suppressed the expression of OGDHL (oxoglutarate dehydrogenase L), a switch gene that mediates SLC25A15 deficiency-induced reprogramming of glutamine metabolism. SLC25A15-deficient HCC cells were more susceptible to glutamine deprivation and glutaminase inhibitors. Intervening in glutamine metabolism increased SLC25A15-deficient HCC cells' response to anti-PD-L1 treatment. CONCLUSION: SLC25A15 is hypoxia-responsive in HCC, and low SLC25A15 levels result in glutamine reprogramming through SLC1A5 and OGDHL regulation, promoting HCC progression and regulating cell sensitivity to anti-PD-L1. Interrupting the glutamine-derived energy supply is a potential therapeutic strategy for treating SLC25A15-deficient HCC. IMPACT AND IMPLICATIONS: We first demonstrated the tumor suppressor role of solute carrier family 25 member 15 (SLC25A15) in hepatocellular carcinoma (HCC) and showed that its deficiency leads to reprogramming of glutamine metabolism to promote HCC development. SLC25A15 can serve as a potential biomarker to guide the development of precision therapeutic strategies aimed at targeting glutamine deprivation. Furthermore, we highlight that the use of an inhibitor of glutamine utilization can enhance the sensitivity of low SLC25A15 HCC to anti-PD-L1 therapy.

Network pharmacology prediction, molecular docking and in vitro experiment explored the potential mechanism of Gaoyuan'an capsule in improving hypoxia tolerance.

BACKGROUND: Tibetan medicine Gaoyuan'an capsule (GYAC) is widely used to prevent pulmonary edema at high altitude, but the specific mechanism has not been explored. In this study, we analyzed the mechanism of GYAC in hypoxia tolerance, and provided a new idea for the prevention and treatment of altitude disease. METHODS: The effective components and corresponding targets of GYAC were screened out by the Chinese herbal medicine network database, and the key targets of hypoxia tolerance were retrieved by Genecards, OMIM and PubMed database. Cytoscape 3.7.2 was used to construct GYAC ingredient-target-hypoxia tolerance-related target network. GO function annotation and KEGG enrichment analysis were performed to predict the pathways in which target genes may be involved, and molecular docking was used to verify the binding ability of the compound to target genes. In vitro, the above results were further verified by molecular experiment. RESULTS: We found that GYAC can improve hypoxia tolerance by regulating various target genes, including IL6, IFNG, etc. The main regulatory pathways were HIF-1 signaling pathway. Molecular docking showed that the affinity between luteolin and target genes (IL6, IFNG) were better. In vitro, we observed that hypoxia can inhibit cell viability and promote apoptosis of H9C2 cell. And hypoxia can promote the expression of LDH. After the addition of luteolin, the decrease of cell viability, the increase of cell apoptosis, LDH release and the decrease of mitochondrial membrane potential were inhibited. Besides, inflammatory related factors (IL-6, IL-10, IL-2, IFNG and VEGFA) expression were also inhibited hypoxic cell models. CONCLUSIONS: The results of network pharmacology and molecular docking showed that luteolin, a monomeric component of GYAC, played a role in hypoxia tolerance through a variety of target genes, such as IL6, IFNG. What's more, we have discovered that luteolin can reduce the inflammatory response in cardiac myocytes, thereby alleviating mitochondrial damage, and ultimately enhancing the hypoxia tolerance of H9C2 cardiomyocytes.

Hypoxia promotes metastasis by relieving miR-598-3p-restricted glycolysis in gastric cancer.

The activation of glycolysis, particularly in the context of reprogrammed energy metabolism, is increasingly recognized as a significant characteristic of cancer. However, the precise mechanisms by which glycolysis is promoted in metastatic gastric cancer cells under normal oxygen conditions remain poorly understood. MicroRNAs (miRNAs) play a crucial role in the development of malignant phenotypes in gastric cancer. Nevertheless, our understanding of the specific involvement of miRNAs in hypoxia-induced metabolic shifting and the subsequent metastatic processes is limited. Hypoxia-induced downregulation of miR-598-3p mechanistically leads to the upregulation of RMP and IGF1r, thereby promoting glycolysis. Either overexpression of miR-598-3p or R406 treatment effectively suppresses the metastasis of gastric cancer cells both in vitro and in vivo. Collectively, the depletion of miR-598-3p alters glucose metabolism from oxidative phosphorylation to glycolysis, thereby exacerbating the malignancy of gastric cancer cells. The present findings indicate a potential target for the development of therapeutics against gastric cancers with increased miR-598-3p expression.

Histone H3Y99sulf regulates hepatocellular carcinoma responding to hypoxia.

Histone H3 tyrosine-99 sulfation (H3Y99sulf) is a recently identified histone mark that can cross-talk with H4R3me2a to regulate gene transcription, but its role in cancer biology is less studied. Here, we report that H3Y99sulf is a cancer-associated histone mark that can mediate hepatocellular carcinoma (HCC) cells responding to hypoxia. Hypoxia-stimulated SNAIL pathway elevates the expression of PAPSS2, which serves as a source of adenosine 3'-phosphate 5'-phos-phosulfate for histone sulfation and results in upregulation of H3Y99sulf. The transcription factor TDRD3 is the downstream effector of H3Y99sulf-H4R3me2a axis in HCC. It reads and co-localizes with the H3Y99sulf-H4R3me2a dual mark in the promoter regions of HIF1A and PDK1 to regulate gene transcription. Depletion of SULT1B1 can effectively reduce the occurrence of H3Y99sulf-H4R3me2a-TDRD3 axis in gene promoter regions and lead to downregulation of targeted gene transcription. Hypoxia-inducible factor 1-alpha and PDK1 are master regulators for hypoxic responses and cancer metabolism. Disruption of the H3Y99sulf-H4R3me2a-TDRD3 axis can inhibit the expression and functions of hypoxia-inducible factor 1-alpha and PDK1, resulting in suppressed proliferation, tumor growth, and survival of HCC cells suffering hypoxia stress. The present study extends the regulatory and functional mechanisms of H3Y99sulf and improves our understanding of its role in cancer biology.

Tissue-of-origin for cancers determines HIF-1 activation induced phenotypic heterogeneity.

Hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular response to hypoxia, and is activated in many cancers contributing to many steps in the metastatic cascade by acting as a key transcription co-regulator for a large number of downstream genes. Presence of hypoxia within a tumor is spatially nonuniform, and can also by dynamic. Further, although HIF-1 is primarily stabilized and activated by lack of molecular O2, its stability is also affected by other factors present in the tumor microenvironment. HIF-1 also crosstalks with other transcription factors in co-regulating gene expression. Consequently, it is nontrivial to predict the gene expression patterns in cells in response to hypoxia, or HIF-1 activation. Additionally, cancers originating from tissue origins with different basal level of partial oxygen tension may activate HIF-1 at different threshold of hypoxia. We analyzed large published single cell RNAseq data for colorectal, lung, and pancreatic cancers to investigate the phenotypic outcome of HIF-1 activation in cancer cells. We found that cancers from tissues with different partial O2 tension levels exhibit HIF-1 activation at different stages of metastasis, and phenotypically respond differently to HIF-1 activation, likely by contextual co-option of different transcription factors. We experimentally confirmed these predictions by using cell lines representative of colorectal, lung, and pancreatic cancers, finding that while hypoxia enhances growth of colorectal cancer, it induces increased invasion of lung, and pancreatic cancers. Our analysis suggest that HIF-1 activation may act as a rheostat regulating downstream gene expression towards phenotypic outcomes differently in various cancers.

Gestational Intermittent Hypoxia Enhances Mammary Stem Cells and Alters Tumor Phenotype in Adult Female Offspring.

An adverse perinatal environment can increase long-term cancer risk, although the precise nature of associated perinatal triggers remain unknown. Sleep apnea is a common condition during pregnancy, characterized by recurrent cessations in breathing during sleep, and the potential consequences of sleep apnea during pregnancy as it relates to breast cancer risk in offspring have not been explored. To model sleep apnea, Sprague-Dawley dams were exposed during gestation to nightly intermittent hypoxia (GIH) or normoxia (GNx), and the mammary glands of female offspring were examined. GIH offspring demonstrated increased epithelial stem and progenitor cell populations, which are associated with diminished transforming growth factor beta (TGFß) activity. Elevations in adipose tissue stem cells in the mammary gland were also identified in GIH offspring. In aging females, mammary tumors formed in GIH offspring. These tumors displayed a dramatic increase in stroma compared to tumors from GNx offspring, as well as distinct patterns of expression of stem cell-related pathways. Together, these results suggest that exposure to sleep apnea during pregnancy leads to lasting changes in the mammary glands of female offspring. Increased stem and progenitor cell populations as a result of GIH exposure could enhance long-term breast cancer risk, as well as alter the clinical behavior of resulting breast tumors.

Genetically Encoded Reporters to Monitor Hypoxia.

Hypoxia resulting from an imbalance of oxygen availability and consumption defines a metabolic cellular state with a profound impact on developmental processes, tissue maintenance, and the development of pathologies. Fluorescence imaging using genetically encoded reporters enables hypoxia and oxygen imaging with cellular resolution. Thereby unrestricted visualization of hypoxic cells and regions essentially relies on the availability of oxygen-independent fluorescent proteins like UnaG, isolated from the Japanese freshwater eel. Here, we describe the application of recently developed members of a UnaG-based hypoxia reporter family to visualize oxygenation patterns by in vitro live-cell imaging and during the ex vivo analysis of intracranial xenografted tumors. Thus, the generation of stably transfected transgenic tumor cell lines, the in vitro calibration of the genetically encoded sensors, the surgical procedures for orthotopic xenografting of tumors in mice, and workflows for the respective sample preparation and microscopy are outlined.

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.

Hypoxia-induced miR-181a-5p up-regulation reduces epirubicin sensitivity in breast cancer cells through inhibiting EPDR1/TRPC1 to activate PI3K/AKT signaling pathway.

Breast carcinoma (BC) ranks as a predominant malignancy and constitutes the second principal cause of mortality among women globally. Epirubicin stands as the drug of choice for BC therapeutics. Nevertheless, the emergence of chemoresistance has significantly curtailed its therapeutic efficacy. The resistance mechanisms to Epirubicin remain not entirely elucidated, yet they are conjectured to stem from diminished tumor vascular perfusion and resultant hypoxia consequent to Epirubicin administration. In our investigation, we meticulously scrutinized the Gene Expression Omnibus database for EPDR1, a gene implicated in hypoxia and Epirubicin resistance in BC. Subsequently, we delineated the impact of EPDR1 on cellular proliferation, motility, invasive capabilities, and interstitial-related proteins in BC cells, employing methodologies such as the CCK-8 assay, Transwell assay, and western blot analysis. Our research further unveiled that hypoxia-induced miR-181a-5p orchestrates the regulation of BC cell duplication, migration, invasion, and interstitial-related protein expression via modulation of EPDR1. In addition, we identified TRPC1, a gene associated with EPDR1 expression in BC, and substantiated that EPDR1 influences BC cellular dynamics through TRPC1-mediated modulation of the PI3K/AKT signaling cascade. Our findings underscore the pivotal role of EPDR1 in the development of BC. EPDR1 was found to be expressed at subdued levels in BC tissues, Epirubicin-resistant BC cells, and hypoxic BC cells. The overexpression of EPDR1 curtailed BC cell proliferation, motility, invasiveness, and the expression of interstitial-related proteins. At a mechanistic level, the overexpression of hypoxia-induced miR-181a-5p was observed to inhibit the EPDR1/TRPC1 axis, thereby activating the PI3K/AKT signaling pathway and diminishing the sensitivity to Epirubicin in BC cells. In summation, our study demonstrates that the augmentation of hypoxia-induced miR-181a-5p diminishes Epirubicin sensitivity in BC cells by attenuating EPDR1/TRPC1 expression, thereby invigorating the PI3K/AKT signaling pathway. This exposition offers a theoretical foundation for the application of Epirubicin in BC therapy, marking a significant contribution to the existing body of oncological literature.

HMGB1 promotes mitochondrial transfer between hepatocellular carcinoma cells through RHOT1 and RAC1 under hypoxia.

Mitochondrial transfer plays an important role in various diseases, and many mitochondrial biological functions can be regulated by HMGB1. To explore the role of mitochondrial transfer in hepatocellular carcinoma (HCC) and its relationship with HMGB1, field emission scanning electron microscopy, immunofluorescence, and flow cytometry were used to detect the mitochondrial transfer between HCC cells. We found that mitochondrial transfer between HCC cells was confirmed using tunnel nanotubes (TNTs). The transfer of mitochondria from the highly invasive HCC cells to the less invasive HCC cells could enhance the migration and invasion ability of the latter. The hypoxic conditions increased the mitochondrial transfer between HCC cells. Then the mechanism was identified using co-immunoprecipitation, luciferase reporter assay, and chromatin immunoprecipitation. We found that RHOT1, a mitochondrial transport protein, promoted mitochondrial transfer and the migration and metastasis of HCC cells during this process. Under hypoxia, HMGB1 further regulated RHOT1 expression by increasing the expression of NFYA and NFYC subunits of the NF-Y complex. RAC1, a protein associated with TNTs formation, promoted mitochondrial transfer and HCC development. Besides, HMGB1 regulated RAC1 aggregation to the cell membrane under hypoxia. Finally, the changes and significance of related molecules in clinical samples of HCC were analyzed using bioinformatics and tissue microarray analyses. We found that HCC patients with high HMGB1, RHOT1, or RAC1 expression exhibited a relatively shorter overall survival period. In conclusion, under hypoxic conditions, HMGB1 promoted mitochondrial transfer and migration and invasion of HCC cells by increasing the expression of mitochondrial transport protein RHOT1 and TNTs formation-related protein RAC1.