Hypoxia-inducible Signaling Research Articles

Single-nuclei and bulk-tissue gene-expression analysis of pheochromocytoma and paraganglioma links disease subtypes with tumor microenvironment

Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors associated with autonomic nerves. Here we use single-nuclei RNA-seq and bulk-tissue gene-expression data to characterize the cellular composition of PCPG and normal adrenal tissues, refine tumor gene-expression subtypes and make clinical and genotypic associations. We confirm seven PCPG gene-expression subtypes with significant genotype and clinical associations. Tumors with mutations in VHL, SDH-encoding genes (SDHx) or MAML3-fusions are characterized by hypoxia-inducible factor signaling and neoangiogenesis. PCPG have few infiltrating lymphocytes but abundant macrophages. While neoplastic cells transcriptionally resemble mature chromaffin cells, early chromaffin and neuroblast markers are also features of some PCPG subtypes. The gene-expression profile of metastatic SDHx-related PCPG indicates these tumors have elevated cellular proliferation and a lower number of non-neoplastic Schwann-cell-like cells, while GPR139 is a potential theranostic target. Our findings therefore clarify the diverse transcriptional programs and cellular composition of PCPG and identify biomarkers of potential clinical significance.

HIF-1α-mediated autophagy and canonical Wnt/β-catenin signalling activation are involved in fluoride-induced osteosclerosis in rats

Fluoride (F) exposure can cause osteosclerosis, which is characterised by a high bone mass, but its mechanism is not fully illustrated. Here, we aimed to evaluate the effects of excessive F exposure on the bone lesion by treating female Sprague–Dawley rats with different concentrations of sodium fluoride (NaF) (0, 55, 110 and 221 mg/L) for 90 days and the corresponding concentrations of fluorine ion (0, 25, 50 and 100 mg/L, respectively). Histopathological results showed that excessive F exposure caused the enlargement of trabeculae and their integration into one large piece, growth plate thickening, articular cartilage impairment and bone collagen abnormality. Meanwhile, F promoted calcium deposition and bone mineralisation, and induced abnormal osteogenesis increased. The results of micro-computed tomography also confirmed that excessive F destroyed the bone microstructure and induced a high-bone-mass phenotype, consistent with the results of pathomorphology. Mechanistically, excessive amounts of F led to angiogenesis inhibition and HIF-1α signalling enhancement. Subsequently, F induced autophagy and canonical Wnt/β-catenin signalling pathway activation. Collectively, these results manifested that F enhanced the hypoxia inducible factor-1α signalling, which in turn triggered autophagy and canonical Wnt/β-catenin signalling activation, ultimately leading to osteosclerosis in the rats.

Hypoxia-inducible factor signaling in vascular calcification in chronic kidney disease patients.

Chronic kidney disease (CKD) affects approximately 15% of the adult population in high-income countries and is associated with significant comorbidities, including increased vascular calcifications which is associated with a higher risk for cardiovascular events. Even though the underlying pathophysiology is unclear, hypoxia-inducible factor (HIF) signaling appears to play a central role in inflammation, angiogenesis, fibrosis, cellular proliferation, apoptosis and vascular calcifications which is influenced by multiple variables such as iron deficiency anemia, serum phosphorus and calcium levels, fibroblast growth factor-23 (FGF-23) and Klotho. Along with the growing understanding of the pathology, potential therapeutic alternatives have emerged including HIF stabilizers and SGLT-2 inhibitors. The aim of this review is to discuss the role of HIF signaling in the pathophysiology of vascular calcification in CKD patients and to identify potential therapeutic approaches.

H2O2-induced oxidative stress improves meat tenderness by accelerating glycolysis via hypoxia-inducible factor-1α signaling pathway in postmortem bovine muscle.

Reactive oxygen species (ROS) affect meat quality through multiple biochemical pathways. To investigate the effect of ROS on postmortem glycolysis and tenderness of bovine muscle, ROS content, glycolytic potential, glycolysis rate-limiting enzyme activities, expression of hypoxia-inducible factor-1α (HIF-1α), phosphatidylinositol 3-kinase (PI3K), serine-threonine kinase (AKT), phosphorylated AKT (p-AKT), and tenderness were determined in the H2O2 group and control group. Results showed that the H2O2 group exhibited significantly higher ROS content within 48h, coupled with increased glycolytic potential, pH decline, hexokinase (HK), and phosphofructokinase activities (PFK) early postmortem. These were attributed to ROS-induced PI3K/AKT signaling pathway activation and resultant HIF-1α accumulation. Moreover, shear force in the H2O2 group reached the peak 12h earlier and decreased obviously after 24h, accompanied by a significantly higher myofibril fragmentation index (MFI). These findings suggested that ROS drive HIF-1α accumulation by activating PI3K/AKT signaling pathway, thereby accelerating glycolysis and tenderization of postmortem bovine muscle.

Hypoxia-Inducible Factors Signaling in Osteogenesis and Skeletal Repair.

Sufficient oxygen is required to maintain normal cellular and physiological function, such as a creature’s development, breeding, and homeostasis. Lately, some researchers have reported that both pathological hypoxia and environmental hypoxia might affect bone health. Adaptation to hypoxia is a pivotal cellular event in normal cell development and differentiation and in pathological settings such as ischemia. As central mediators of homeostasis, hypoxia-inducible transcription factors (HIFs) can allow cells to survive in a low-oxygen environment and are essential for the regulation of osteogenesis and skeletal repair. From this perspective, we summarized the role of HIF-1 and HIF-2 in signaling pathways implicated in bone development and skeletal repair and outlined the molecular mechanism of regulation of downstream growth factors and protein molecules such as VEGF, EPO, and so on. All of these present an opportunity for developing therapies for bone regeneration.

The Study on the Action Mechanism of the Jinyingzi (Rosae Laevigatae Fructus)–Qianshi (Euryales Semen) Couplet Herbs on Membranous Nephropathy Based on Network Pharmacology

Abstract Objective Our objective was to explore the action mechanism of the Jinyingzi (Rosae Laevigatae Fructus)–Qianshi (Euryales Semen) couplet herbs in the treatment of membranous nephropathy (MN) based on network pharmacology. Methods The active ingredients and targets of Jinyingzi (Rosae Laevigatae Fructus) and Qianshi (Euryales Semen) were screened by systematic pharmacology database and analysis platform. Online Human Mendelian Genetic database and GeneCards database were used to retrieve MN-related targets. The active ingredient-related targets and MN disease targets were introduced into Venny 2.1, and Wayne diagram was drawn. The intersection targets were the potential targets of the Jinyingzi (Rosae Laevigatae Fructus)–Qianshi (Euryales Semen) couplet herbs in the treatment of MN. The protein interaction network of potential targets was constructed, and the core targets were screened with String platform. Metascape platform was used for functional enrichment analysis of gene ontology (GO) and pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG). The “herb-active ingredient-target-pathway” networks were drawn by using Cytoscape software, and the key components, targets, and signaling pathways were screened. Results A total of 8 active ingredients and 193 related targets in Jinyingzi (Rosae Laevigatae Fructus) and Qianshi (Euryales Semen) were screened out; a total of 1,621 targets of MN disease and 105 potential targets for the treatment of MN were obtained in the treatment with Jinyingzi (Rosae Laevigatae Fructus)–Qianshi (Euryales Semen) couplet herbs; 40 core targets were screened by protein–protein interaction network topology analysis; a total of 1,978 results were obtained by GO function enrichment analysis, and 206 signal pathways were obtained by KEGG pathway enrichment analysis and screening. The network topology analysis of “herb-active ingredient-target-pathway” showed that the key components included quercetin, kaempferol, β-sitosterol, etc.; the key targets included protein kinase Bα (AKT), mitogen-activated protein kinase 1 (MAPK1), B-cell lymphoma-2 (BCL2), prostaglandin-endoperoxide synthase 2 (PTGS2), etc.; the key pathways included advanced glycation end product/receptor of AGE signaling pathway, phosphatidyl inositol 3-kinase (PI3K)/AKT signaling pathway, MAPK signaling pathway, hypoxia-inducible factor-1 signaling pathway, Ras signaling pathway, nuclear factor kappa-B (NF-κB) signaling pathway, Toll-like receptors signaling pathway, p53 signaling pathway and vascular endothelial growth factor signaling pathway in the late stage of diabetic complications. Conclusion The Jinyingzi (Rosae Laevigatae Fructus)–Qianshi (Euryales Semen) couplet herbs can regulate PI3K/AKT, MAPK, NF-κB signaling pathways in MN by targeting proteins of AKT1, MAPK8, PTGS2 through key components of quercetin, β-sitosterol, and kaempferol, so as to inhibit the overexpression of inflammatory factors in renal tissues, regulate inflammatory response, and improve renal function.

Preclinical findings reveal the pharmacological targets of ferulic acid in the treatment of traumatic brain injury.

Traumatic brain injury (TBI) is characterized by cellular damage and inflammation in lesioned brain tissue. Ferulic acid has been shown to have a melioration effect on neurological functions. However, the active pharmacological effects and the underlying mechanisms of ferulic acid against TBI remain unclear. On the basis of network pharmacology and molecular docking methodology, this study aimed to investigate the beneficial effects of ferulic acid in treating TBI, and characterized the detailed biotargets and mechanisms of these actions. The identified core targets were validated via in silico simulation. We identified 91 overlapping targets associated with ferulic acid and TBI. In-silico simulation analysis validated the putative core targets of tumor protein p53, mitogen-activated protein kinase (MAPK) 1, and estrogen receptor 1. The Gene Ontology-enriched annotations and findings were largely associated with cell proliferation, apoptosis, and inflammation in nerve cells. Additional Kyoto Encyclopedia of Genes and Genomes enrichment analysis unmasked the pharmacological pathways of ferulic acid in treating TBI, including the MAPK signaling pathway and hypoxia-inducible factor-1 signaling pathway. Bioinformatic analyses and findings provide a new preclinical strategy for revealing the core targets and network pathways of ferulic acid in treating TBI. Moreover, some bioinformatic findings were computationally validated in silico for exhibiting the neuroprotective action of ferulic acid against TBI.

Comparative study between effectiveness of low molecular weight heparin injection and unfractionated heparin in terms of morbidity and outcome in COVID-19 cases

Background: The novel coronavirus 2019 presented a variety of disease spectrums that range from asymptomatic to sepsis with multi-organ dysfunction and death. One of the mechanisms being a hypercoagulable state with micro and macro-circulatory thrombosis is found in critically ill COVID-19 patient with ARDS with pathology consistent with a vascular occlusive aetiology of respiratory failure rather than more classic finding of ARDS. Venous thromboembolism, MI, acute peripheral arterial thrombosis is seen in COVID-19. Endothelial cells dysfunction induced by infection causing excess thrombin generation, fibrinolysins shutdown, hypoxia inducible transcription factor dependent signalling pathways, increasing blood viscosity leading to Hypercoagulable state. Early application of anticoagulant therapy in severe COVID-19 was suggested for improving outcome in patients with elevated d-dimer. Aim of study was comparing effectiveness of LMWH with unfractionated heparin in outcome of COVID-19.Methods: This retrospective observational study including 400 patients meeting inclusion criteria were divided 2 groups out of which 200 patients were treated with inj. LMWH and other 200 were treated with injection heparin and various parameters were compared. A probability value (p value) of less than or equal to 0.05 was considered statistically significant.Results: Death rate is seen lower with LMWH in critically ill patients with high discharge rate with minimal lab parameter derangement and high efficacy to lower down inflammatory markers (LDH, ferritin, CRP, D-dimer, ESR) in comparison to unfractionated heparin.Conclusions: Early use of LMWH may be beneficial for the outcome.

An Integrative Multi-Omics Analysis Reveals MicroRNA-143 as a Potential Therapeutic to Attenuate Retinal Angiogenesis.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

Metabolomic and transcriptomic study to understand changes in metabolic and immune responses in steers under heat stress.

Heat stress (HS) damages livestock by adversely affecting physiological and immunological functions. However, fundamental understanding of the metabolic and immunological mechanisms in animals under HS remains elusive, particularly in steers. To understand the changes on metabolic and immune responses in steers under HS condition, we performed RNA-sequencing and proton nuclear magnetic resonance spectroscopy-based metabolomics on HS-free (THI value: 64.92 ± 0.56) and HS-exposed (THI value: 79.13 ± 0.56) Jersey steer (n = 8, body weight: 559.67 ± 32.72 kg). This study clarifies the metabolic changes in 3 biofluids (rumen fluid, serum, and urine) and the immune responses observed in the peripheral blood mononuclear cells of HS-exposed steers. This integrated approach allowed the discovery of HS-sensitive metabolic and immunological pathways. The metabolomic analysis indicated that HS-exposed steers showed potential HS biomarkers such as isocitrate, formate, creatine, and riboflavin (P < 0.05). Among them, there were several integrative metabolic pathways between rumen fluid and serum. Furthermore, HS altered mRNA expression and immune-related signaling pathways. A meta-analysis revealed that HS decreased riboflavin metabolism and the expression of glyoxylate and dicarboxylate metabolism-related genes. Moreover, metabolic pathways, such as the hypoxia-inducible factor-1 signaling pathway, were downregulated in immune cells by HS (P < 0.05). These findings, along with the datasets of pathways and phenotypic differences as potential biomarkers in steers, can support more in-depth research to elucidate the inter-related metabolic and immunological pathways. This would help suggest new strategies to ameliorate the effects of HS, including disease susceptibility and metabolic disorders, in Jersey steers.

Inhibiting Interleukin-6/Signal Transducers and Activators of Transduction-3/Hypoxia-Inducible Factor-1α Signaling Pathway Suppressed the Growth of Infantile Hemangioma.

This study aims to evaluate the expression of interleukin 6 (IL-6) in patients with infantile hemangioma (IH) and investigate the role of the IL-6/signal transducers and activators of transduction-3 (STAT3)/hypoxia-inducible factor-1α (HIF-1α) pathways in the progression of IH. Serum samples were obtained from the patients with IH and normal infants to measure IL-6 expression. Hemangioma-derived stem cells (HemSCs) were transfected with small interfering RNA (siRNA) targeting IL-6, HIF-1α, or STAT3. Then, cell viability and wound healing assays were conducted. After that, the HemSC tumor mouse model was established. The in vivo anticancer effect of the IL-6 inhibitor was investigated. The patients with IH had much higher IL-6 levels compared with the healthy controls (p = 0.005). HemSCs transfected with IL-6 siRNA had significantly lower viability and migration rates than normal HemSCs. HemSCs transfected with STAT3 siRNA or HIF-1α siRNA had similar tendencies. On tumor-bearing mice, the IL-6 inhibitor treatment significantly delayed tumor growth. Compared with the control group, caspase-3 was significantly increased in the IL-6 inhibitor group (p < 0.05), whereas Ki-67 was decreased in the IL-6 inhibitor group (p < 0.05). In the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the IL-6 inhibitor group had much higher apoptosis rates than the controls (p < 0.05). Our findings indicate that inhibiting the IL-6/STAT3/HIF-1α signaling pathways could suppress IH growth.

A phase 1b/2 study of batiraxcept (AVB-S6-500) in combination with cabozantinib in patients with advanced or metastatic clear cell renal cell (ccRCC) carcinoma who have received front-line treatment (NCT04300140).

4511 Background: AXL is up-regulated by hypoxia-inducible factor-1 signaling in both VHL-deficient and hypoxic tumor cells and plays a critical role in the metastatic phenotype of ccRCC. Batiraxcept is a recombinant fusion protein containing an extracellular region of human AXL combined with the human immunoglobulin G1 heavy chain (Fc), demonstrating highly potent, specific AXL inhibition. Methods: Batiraxcept at doses of 15 and 20 mg/kg, plus cabozantinib 60 mg daily, was evaluated using a 3+3 dose escalation study design. The primary objective was safety; secondary and exploratory objectives included identification of the recommended phase 2 dose (RP2D), overall response rate (ORR), and duration of response (DOR). Correlation of serum soluble AXL (sAXL)/GAS6 with ORR was evaluated. Key eligibility criteria include previously treated (2L+) ccRCC patients; prior treatment with cabozantanib was not allowed. sAXL/GAS6 was evaluated at baseline. Results: Data as of 4-February-2022, Phase 1b enrolled 26 patients, 16 patients treated with 15 mg/kg and 10 patients with 20 mg/kg dose of batiraxcept. Baseline characteristics: median age 60 (40-81); male 22 (85%); median prior line of therapy 1 (1-5); IMDC risk group of favorable 6 (23%); prior VEGF inhibitor 15 (58%); 100% with prior immunotherapy. At median follow up of 4.9 months, 92% (n=24) patients remained on the study. No dose limiting toxicities were observed at either 15 mg/kg or 20 mg/kg dose. Batiraxcept and cabozantinib related adverse events (AEs) occurred in 17 subjects (65%). Most common related AE include decreased appetite 31% (n=8), diarrhea and fatigue 23% (n=6). Grade 3 related AEs occurred in 4 patients (15%) including diarrhea, thromboembolism, hypertension, small bowel obstruction, and thrombocytopenia (n=1, 4% each) being most common. No grade 4 or 5 related AEs were observed. The ORR was 46% (n=12, partial response [PR]; Table). No patients had primary progressive disease. Among the patients who had baseline sAXL/GAS6 ratio of ≥ 2.3, the ORR was 67% (12/18). Regardless of baseline sAXL/GAS6 ratio, 3-month DOR was 100%; and 6-month progression free survival was 79%. Batiraxcept PK levels were similar across both doses and GAS6 levels suppressed through the dosing period. Conclusions: Batiraxcept plus cabozantinib is well tolerated. The RP2D of batiraxcept was identified as 15 mg/kg. Early efficacy signals were observed including 100% DOR at 3 months. Baseline sAXL/GAS6 may serve as a potential biomarker to enrich the population. Clinical trial information: NCT04300140. [Table: see text]

Proteomics-based analysis of potential therapeutic targets in patients with peritoneal dialysis-associated peritonitis

BackgroundPeritoneal dialysis-associated peritonitis (PDAP) is the most common complication in peritoneal dialysis patients. We propose screening for characteristic expressed proteins in the dialysate of PDAP patients to provide clues for the diagnosis of PDAP and its therapeutic targets. MethodsDialysate samples were collected from patients with a first diagnosis of PDAP (n = 15) and from patients who had not experienced peritonitis (Control, n = 15). Data-independent acquisition (DIA) proteomic analysis was used to screen for differentially expressed proteins (DEPs). Co-expression networks were constructed via weighted gene co-expression network analysis (WGCNA) for detection of gene modules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for functional annotation of DEPs and gene modules. Hub proteins were validated using the parallel reaction monitoring (PRM) method. ResultsA total of 142 DEPs in the dialysate of PDAP patients were identified. 70 proteins were upregulated and 72 proteins were downregulated. GO and KEGG analysis showed that DEPs were mainly enriched in cell metabolism, glycolysis/glycogenesis and hypoxia-inducible factor-1 signaling pathway. Subsequently, a co-expression network was constructed and four gene modules were detected. Myeloperoxidase (MPO) and myeloperoxidase (HP) were the key proteins of the blue and turquoise modules, respectively. Additionally, PRM analysis showed that the expression of MPO and HP was significantly upregulated in the PDAP group compared to the non-peritonitis group, which was consistent with our proteomics data. ConclusionMPO and HP were differentially expressed in the dialysate of PDAP patients and may be potential diagnostic and therapeutic targets for PDAP.

Kynurenine-PARP-1 Link Mediated by MicroRNA 210 May Be Dysregulated in Pulmonary Hypertension.

BackgroundDysregulation of microRNAs is associated with pulmonary hypertension. The present study aimed to determine the alterations in microRNA and microRNA expressions and their role in signaling pathways and investigate the relationship with serum levels of apelin, kynurenine, and endocan in pulmonary hypertension.MethodsThe study design was prospective and single-centered. The study included 32 consecutive treatment-naive patients with precapillary pulmonary hypertension and 55 age and sex-matched healthy controls. All subjects underwent right heart catheterization. mRNA expressions of hypoxia-inducible factor-1 alpha, hypoxia-inducible factor-2 alpha, signal transducer and activator of transcription-3, fibroblast growth factor-2, fibroblast growth factor receptor-1, and poly-ADP-ribose polymerase-1 and microRNA expressions of miRNA-210, miRNA-130a, miRNA-424, miRNA-204, and miRNA-223 were determined by RT-PCR. Concentrations of kynurenine, apelin, and endocan were analyzed by ELISA.ResultsmRNA expressions of hypoxia-inducible factor-2 alpha, signal transducer and activator of transcription-33, and FGF-2 were increased; miRNA-210 and miRNA-130a were increased; miRNA-223 and miRNA-204 were decreased in pulmonary hypertension. Apelin and kynurenine concentrations were decreased in pulmonary hypertension. There were positive correlations: hypoxia-inducible factor-2 alpha-miRNA-424, Apelin-miRNA-424, kynurenine-miRNA-210, signal transducer and activator of transcription-3-PVR, miRNA-210-right atrial pressure, and kynurenine-right atrial pressure. There were negative correlations: poly-ADP-ribose polymerase-1-miRNA-210 and poly-ADP-ribose polymerase-1-right atrial pressure. On multiple logistic regression analyses, miRNA-130a and Apelin were independent risk factors for PH.ConclusionsWe report a novel relationship between the kynurenine and poly-ADP-ribose polymerase-1 signaling pathways that could be mediated by miRNA-210. We also report a connection between the Apelin and hypoxia-inducible factor-2 alpha signaling pathways that could be mediated by miRNA-424. Reduced levels of Apelin and elevated levels of miRNA-130a are associated with pulmonary hypertension. We also find that elevated levels of signal transducer and activator of transcription-3, miRNA-210, and kynurenine and reduced levels of poly-ADP-ribose polymerase-1 correlate with more severe hemodynamics.

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype.

Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.

Inhibition of carbonic anhydrases IX/XII by SLC-0111 boosts cisplatin effects in hampering head and neck squamous carcinoma cell growth and invasion

BackgroundHypoxic tumor microenvironment (TME) contributes to the onset of many aspects of the cancer biology associated to the resistance to conventional therapies. Hypoxia is a common characteristic and negative prognostic factor in the head and neck squamous carcinomas (HNSCC) and is correlated with aggressive and invasive phenotype as well as with failure to chemo- and radio-therapies. The carbonic anhydrase isoenzymes IX and XII (CA IX/XII), regulators of extra and intracellular pH, are overexpressed in TME and are involved in adaptative changes occurring in cancer cells to survive at low O2. In this study, we aim to investigate in HNSCC cells and murine models the possibility to target CA IX/XII by the specific inhibitor SLC-0111 to potentiate the effects of cisplatin in hampering cell growth, migration and invasion. Furthermore, we analyzed the signal pathways cooperating in acquisition of a more aggressive phenotype including stemness, epithelial-mesenchymal transition and apoptotic markers.MethodsThe effects of cisplatin, CA IX/XII specific inhibitor SLC-0111, and the combinatorial treatment were tested on proliferation, migration, invasion of HNSCC cells grown in 2D and 3D models. Main signal pathways and the expression of stemness, mesenchymal and apoptotic markers were analyzed by western blotting. Molecular imaging using NIR-Annexin V and NIR-Prosense was performed in HNSCC xenografts to detect tumor growth and metastatic spread.ResultsHNSCC cells grown in 2D and 3D models under hypoxic conditions showed increased levels of CA IX/XII and greater resistance to cisplatin than cells grown under normoxic conditions. The addition of CA IX/XII inhibitor SLC-0111 to cisplatin sensitized HNSCC cells to the chemotherapeutic agent and caused a reduction of proliferation, migration and invasiveness. Furthermore, the combination therapy hampered activation of STAT3, AKT, ERK, and EMT program, whereas it induced apoptosis. In HNSCC xenografts the treatment with cisplatin plus SLC-0111 caused an inhibition of tumor growth and an induction of apoptosis as well as a reduction of metastatic spread at a higher extent than single agents.ConclusionOur results highlight the ability of SLC-0111 to sensitize HNSCC to cisplatin by hindering hypoxia-induced signaling network that are shared among mechanisms involved in therapy resistance and metastasis.

Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist-Treated Prostate Cancers.

Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.

Fracture repair by IOX2: Regulation of the hypoxia inducible factor-1α signaling pathway and BMSCs

The treatment of fracture delayed union and nonunion has become a challenging problem. Hypoxia inducible factor-1α (HIF-1α) is reported to be a key factor in fracture healing, and is degraded by hydroxylation of prolyl hydroxylase (PHDs) under normal oxygen. Small molecules could inhibit the activity of PHDs, stabilize HIF-1α protein, regulate the expression of downstream target genes of HIF-1α, and make the body adapt to hypoxia. The migration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the most promising candidate for the treatment of fracture nonunion. Here we reported that IOX2, an HIF-1α PHD inhibitor, markedly improved the proliferation and migration of BMSCs by upregulating intracellular Ca2+ and concomitant decreasing reactive oxygen species (ROS) in vitro, and facilitated the repair of bone fracture by increasing the number of BMSCs and cartilage formation in vivo. No significant influence of IOX2 on the proliferation and migration of BMSCs after silencing of the HIF-1α. Together, our findings indicated that IOX2 promoted the proliferation and migration of BMSCs via the HIF-1α pathway and further accelerated fracture healing. These results provide a deeper understanding of the mechanism by which HIF promotes fracture healing.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma

The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

The Antarctic Weddell seal genome reveals evidence of selection on cardiovascular phenotype and lipid handling

The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes were SUMO2 and EP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.