HIF-1α Stabilization Research Articles

14-3-3σ-NEDD4L axis promotes ubiquitination and degradation of HIF-1α in colorectal cancer

A hypoxic microenvironment contributes to tumor progression, with hypoxia-inducible factor-1α (HIF-1α) being a critical regulator. We have reported that 14-3-3σ is negatively associated with HIF-1α expression; however, its role in hypoxia-induced tumor progression remains poorly characterized. Here we show that 14-3-3σ suppresses cancer hypoxia-induced metastasis and angiogenesis in colorectal cancer (CRC). 14-3-3σ opposes HIF-1α expression by regulating the protein stability of HIF-1α, thereby decreasing HIF-1α transcriptional activity and suppressing tumor progression. Mechanistic studies show that the 14-3-3σ-interacting protein neural precursor cell-expressed developmentally down-regulated 4-like (NEDD4L) is an E3 ligase that targets HIF-1α. 14-3-3σ promotes the binding of S448-phosphorylated NEDD4L to HIF-1α, thereby enhancing HIF-1α poly-ubiquitination and subsequent proteasome-mediated degradation. Consistent with this anti-tumorigenic function for 14-3-3σ, low 14-3-3σ expression levels correlate with poor CRC patient survival, and 14-3-3σ enhances the response of CRC to bevacizumab. These results reveal an important mechanism for 14-3-3σ in tumor suppression through HIF-1α regulation.

First evidence of the incorporation of daprodustat and other hypoxia-inducible factor stabilizers into equine hair by passive transfer based on segmental quantitative analysis

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0–2 cm) at 0.375–0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z-scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair.

Loss of USP22 alleviates cardiac hypertrophy induced by pressure overload through HiF1-α-TAK1 signaling pathway

Ubiquitin-specific protease 22 (USP22) is a member of the ubiquitin specific protease family (ubiquitin-specific protease, USPs), the largest subfamily of deubiquitinating enzymes, and plays an important role in the treatment of tumors. USP22 is also expressed in the heart. However, the role of USP22 in heart disease remains unclear. In this study, we found that USP22 was elevated in hypertrophic mouse hearts and in angiotensin II (Ang II)-induced cardiomyocytes. The inhibition of USP22 expression with adenovirus significantly rescued hypertrophic phenotype and cardiac dysfunction induced by pressure overloaded. Consistent with in vivo study, silencing by USP22 shRNA expression in vitro had similar results. Molecular analysis revealed that transforming growth factor-β-activating protein 1 (TAK1)-(JNK1/2)/P38 signaling pathway and HIF-1α was activated in the Ang II-induced hypertrophic cardiomyocytes, whereas HIF-1α expression was decreased after the inhibition of USP22. Inhibition of HIF-1α expression reduces TAK1 expression. Co-immunoprecipitation and ubiquitination studies revealed the regulatory mechanism between USP22 and HIF1α.Under hypertrophic stress conditions, USP22 enhances the stability of HIF-1α through its deubiquitination activity, which further activates the TAK1-(JNK1/2)/P38 signaling pathway to lead to cardiac hypertrophy. Inhibition of HIF-1α expression further potentiates the in vivo pathological effects caused by USP22 deficiency. In summary, this study suggests that USP22, through HIF-1α-TAK1-(JNK1/2)/P38 signaling pathway, may be potential targets for inhibiting pathological cardiac hypertrophy induced by pressure overload.

Oxygen-independent stabilization of HIF-2α in breast cancer through direct interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer.

Overexpression of SENP3 promotes PPAR-γ transcription through the increase of HIF-1α stability via SUMO2/3 and participates in molecular mechanisms of osteoporosis

Patients with type II diabetes are exposed to a high risk of osteoporosis. The present study sought to exploit the detailed mechanisms of the SENP3/HIF-1α/PPAR-γ axis in osteoporosis. A rat model of type II diabetic osteoporosis was established, followed by the isolation of bone marrow mononuclear macrophages (BMMs). Gain- and loss-of-function assays were conducted in rat models and BMMs from rat models, followed by the evaluation of SENP3, HIF-1α, and PPAR-γ expression and detection of osteoclast differentiation-related indexes. Next, the SUMOylated modification of HIF-1α and the regulation of SENP3 on SUMOylated modification level of HIF-1α were assessed using immunoprecipitation, and the binding of HIF-1α to the PPARγ promoter was identified with ChIP and dual-luciferase reporter assays. SENP3 and HIF-1α expression was down-regulated in tissues of type II diabetes-induced osteoporotic rats and BMMs, with high SUMOylated modification levels of HIF-1α. Mechanically, HIF-1α was modified by SUMO2/3. SENP3 suppressed SUMOylated modification of HIF-1α and enhanced HIF-1α stability. HIF-1α bound to the PPAR-γ promoter and facilitated PPAR-γ transcription. SENP3 overexpression restrained osteoblast differentiation in type II diabetes-induced osteoporotic rats and BMMs from rat models. SENP3 knockdown facilitated osteoclast differentiation in type II diabetes-induced osteoporotic rats and BMMs from rat models, which was neutralized by further HIF-1α overexpression. To sum up, SENP3 overexpression restrained osteoclast differentiation in type II diabetic osteoporosis by increasing HIF-1α stability and expression and thus promoting PPAR-γ expression via de-SUMOylation, which might expand the understanding of the mechanisms of type II diabetes combined with osteoporosis.

PDK1 promotes breast cancer progression by enhancing the stability and transcriptional activity of HIF-1α

Pyruvate dehydrogenase kinase 1 (PDK1) phosphorylates the pyruvate dehydrogenase complex, which inhibits its activity. Inhibiting pyruvate dehydrogenase complex inhibits the tricarboxylic acid cycle and the reprogramming of tumor cell metabolism to glycolysis, which plays an important role in tumor progression. This study aims to elucidate how PDK1 promotes breast cancer progression. We found that PDK1 was highly expressed in breast cancer tissues, and PDK1 knockdown reduced the proliferation, migration, and tumorigenicity of breast cancer cells and inhibited the HIF-1α (hypoxia-inducible factor 1α) pathway. Further investigation showed that PDK1 promoted the protein stability of HIF-1α by reducing the level of ubiquitination of HIF-1α. The HIF-1α protein levels were dependent on PDK1 kinase activity. Furthermore, HIF-1α phosphorylation at serine 451 was detected in wild-type breast cancer cells but not in PDK1 knockout breast cancer cells. The phosphorylation of HIF-1α at Ser 451 stabilized its protein levels by inhibiting the interaction of HIF-1α with von Hippel-Lindau and prolyl hydroxylase domain. We also found that PDK1 enhanced HIF-1α transcriptional activity. In summary, PDK1 enhances HIF-1α protein stability by phosphorylating HIF-1α at Ser451 and promotes HIF-1α transcriptional activity by enhancing the binding of HIF-1α to P300. PDK1 and HIF-1α form a positive feedback loop to promote breast cancer progression.

Mitochondrial UQCC3 controls embryonic and tumor angiogenesis by regulating VEGF expression

Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5-10.5days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

PRMT5 facilitates angiogenesis and EMT via HIF-1α/VEGFR/Akt signaling axis in lung cancer.

Abnormal angiogenesis is a critical factor in tumor growth and metastasis, and protein arginine methyltransferase 5 (PRMT5), a prominent type II enzyme, is implicated in various human cancers. However, the precise role of PRMT5 in regulating angiogenesis to promote lung cancer cell metastasis and the underlying molecular mechanisms are not fully understood. Here, we show that PRMT5 is overexpressed in lung cancer cells and tissues, and its expression is triggered by hypoxia. Moreover, inhibiting or silencing PRMT5 disrupts the phosphorylation of the VEGFR/Akt/eNOS angiogenic signaling pathway, NOS activity, and NO production. Additionally, inhibiting PRMT5 activity reduces HIF-1α expression and stability, resulting in the down-regulation of the VEGF/VEGFR signaling pathway. Our findings indicate that PRMT5 promotes lung cancer epithelial-mesenchymal transition (EMT), which might be possibly through controlling the HIF-1α/VEGFR/Akt/eNOS signaling axis. Our study provides compelling evidence of the close association between PRMT5 and angiogenesis/EMT and highlights the potential of targeting PRMT5 activity as a promising therapeutic approach for treating lung cancer with abnormal angiogenesis.

Targeting 2-Oxoglutarate-Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model.

Dysregulation of host metabolism is a feature of lethal SARS-CoV-2 infection. Perturbations in α-ketoglutarate levels can elicit metabolic reprogramming through 2-oxoglutarate-dependent dioxygenases (2-ODDGs), leading to stabilization of the transcription factor HIF-1α. HIF1-α activation has been reported to promote antiviral mechanisms against SARS-CoV-2 through direct regulation of ACE2 expression (a receptor required for viral entry). However, given the numerous pathways HIF-1α serves to regulate it is possible that there are other undefined metabolic mechanisms contributing to the pathogenesis of SARS-CoV-2 independent of ACE2 downregulation. In this study, we used invitro and invivo models in which HIF-1α modulation of ACE2 expression was negated, allowing for isolated characterization of the host metabolic response within SARS-CoV-2 disease pathogenesis. We demonstrated that SARS-CoV-2 infection limited stabilization of HIF-1α and associated mitochondrial metabolic reprogramming by maintaining activity of the 2-ODDG prolyl hydroxylases. Inhibition of 2-ODDGs with dimethyloxalylglycine promoted HIF-1α stabilization following SARS-CoV-2 infection, and significantly increased survival among SARS-CoV-2-infected mice compared with vehicle controls. However, unlike previous reports, the mechanism by which activation of HIF-1α responses contributed to survival was not through impairment of viral replication. Rather, dimethyloxalylglycine treatment facilitated direct effects on host metabolism including increased glycolysis and resolution of dysregulated pools of metabolites, which correlated with reduced morbidity. Taken together, these data identify (to our knowledge) a novel function of α-ketoglutarate-sensing platforms, including those responsible for HIF-1α stabilization, in the resolution of SARS-CoV-2 infection and support targeting these metabolic nodes as a viable therapeutic strategy to limit disease severity during infection.

Gene Expression Under Combined Hypoxia And Acidosis In Chondrosarcoma

Chondrosarcomas are the second most common cause of bone cancer and are removed surgically with wide margins. On recurrence, they are resistant to chemo and radiation therapy and new treatment options are critically required. This tumor type produces hyaline cartilage, a cartilage normally formed under hypoxic and acidic environment due to lack of vasculature in cartilage. Paradoxically, chondrosarcomas arise in the well vascularized, oxygen rich environment of the bone. Hypoxia and acidosis are two stressors where the cellular effects are typically reported separately even though cells experience combined effects of hypoxia and acidosis. Given the mechanistic links between hypoxia and acidosis we hypothesized that gene expression profiles will be differentially changed when chondrosarcoma cells were exposed to individual compared to combined stressors. We investigated expression of four genes expressed during cartilage and cartilage tumor formation in primary chondrocytes and two grade II chondrosarcoma cell lines, SW1353 and JJ012. Two genes, PTH1R and SOX9 are known to respond to hypoxia and acidosis separately. Two genes, IDH1 and IDH2, are mutated in chondrosarcoma cell lines JJ012 and SW1353 respectively. These mutations confer a condition of false hypoxia on the cells through stabilization of HIF-1α. The result is chondrosarcoma cells metabolize glycolytically through aerobic glycolysis. How the cells respond to hypoxia and acidosis is of considerable interest as metabolically the cells are molecularly predisposed to these conditions. Our gene expression data found that combined hypoxia and extracellular acidosis alter gene expression compared to either stressor alone. Cells showed gene specific responses to stressors that were cell type specific likely indicating influence on gene expression regulatory sequences. The importance of this work is highlighting that conditions under which cells are investigated is crucial and should be considered when measuring cell response to in vitro treatment exposures.

Association of Apolipoprotein E4-related Microvascular Disease in the Alzheimer’s Disease Hippocampal CA1 Stratum Radiatum

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer’s disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-β (Aβ) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.

Mitochondrial reactive oxygen species modify extracellular vesicles secretion rate.

Extracellular vesicle (EV) secretion rate is stimulated by hypoxia that causes increased reactive oxygen species (ROS) production by the mitochondrial electron transport chain (ETC) and hypoxia-induced factor (HIF)-1 signaling; however, their contribution to the increased EV secretion rate is unknown. We found that the EV marker secretion rate in our EV reporter cell line CD9truc-EGFP was unaffected by the HIF-1α stabilizer roxadustat; yet, ETC stimulation by dichloroacetic acid (DCA) significantly increased EV secretion. The DCA-induced EV secretion was blocked by the antioxidant TEMPO and rotenone, an inhibitor of the ETC's Complex I. Under hypoxic conditions, the limited oxygen reduction impedes the ETC's Complex III. To mimic this, we inhibited Complex III with antimycin A, which increased ROS-dependent EV secretion. The electron transport between Complex I and III is accomplished by coenzyme Q created by the mevalonate pathway and tyrosine metabolites. Blocking an early step in the mevalonate pathway using pitavastatin augmented the DCA-induced EV secretion, and 4-nitrobenzoate-an inhibitor of the condensation of the mevalonate pathway with tyrosine metabolites-increased ROS-dependent EV secretion. Our findings indicate that hypoxia-mimetics targeting the ETC modify EV secretion and that ROS produced by the ETC is a potent stimulus for EV secretion.

#3777 ACTIVATION OF HYPOXIA-INDUCIBLE FACTORS IN ERYTHROID PROGENITOR CELLS RESULTS IN DEFECTIVE ERYTHROPOIESIS

Abstract Background and Aims Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) activates HIF in renal pericytes and fibroblasts to promote erythropoietin production and erythropoiesis. However, the effects of HIF stabilization in the erythroid lineage are not clear. We aim to study the effects of erythroid lineage-specific stabilization of HIF on erythropoiesis by preclinical models. Method EporGFP-Cre/+;VhlF/F mice were used to achieve erythroid lineage-specific stabilization of HIF. Surface expression of TER-119, CD44, and forward scatter (FSC) were used to define erythroblast, reticulocyte, and red blood cell in the bone marrow. Expression of propidium iodide and surface annexin V were used to define apoptosis. Stress erythropoiesis was induced by subcutaneous administration of phenylhydrazine. Results Compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had decreased hematocrit, decreased percentage of erythroblasts in the bone marrow, and increased apoptosis of erythroblasts in the bone marrow under steady state. These phenotypes of defective erythropoiesis were normalized in mice harboring concomitant Vhl, Hif1a, and Hif2a deletion (EporGFP-Cre/+;VhlF/F;Hif1aF/F;Hif2aF/Fmice). Although macrophages in the bone marrow also express Epor, macrophage-specific Vhl deletion in either Tg(Csf1r-CreESR1);VhlF/F or Lyz2Cre/+;VhlF/F mice did not result in defective erythropoiesis. During stress erythropoiesis, compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had similar hematocrit, lower percentage of erythroblast in the bone marrow, and increased percentage of erythroblast in the spleen. Conclusion Vhl deletion in erythroid progenitor cells impairs erythropoiesis in murine bone marrow in the steady state. The phenotypes of defective erythropoiesis were partially reversed during stress erythropoiesis.

Nicotinamide Antagonizes Lipopolysaccharide-Induced Hypoxic Cell Signals in Human Macrophages.

Mechanisms to control the immune response are important to pathogen evasion and host defense. Gram-negative bacteria are common pathogens that can activate host immune responses through their outer membrane component, LPS. Macrophage activation by LPS induces cell signals that promote hypoxic metabolism, phagocytosis, Ag presentation, and inflammation. Nicotinamide (NAM) is a vitamin B3 derivative and precursor in the formation of NAD, which is a required cofactor in cellular function. In this study, treatment of human monocyte-derived macrophages with NAM promoted posttranslational modifications that antagonized LPS-induced cell signals. Specifically, NAM inhibited AKT and FOXO1 phosphorylation, decreased p65/RelA acetylation, and promoted p65/RelA and hypoxia-inducible transcription factor-1α (HIF-1α) ubiquitination. NAM also increased prolyl hydroxylase domain 2 (PHD2) production, inhibited HIF-1α transcription, and promoted the formation of the proteasome, resulting in reduced HIF-1α stabilization, decreased glycolysis and phagocytosis, and reductions in NOX2 activity and the production of lactate dehydrogenase A. These NAM responses were associated with increased intracellular NAD levels formed through the salvage pathway. NAM and its metabolites may therefore decrease the inflammatory response of macrophages and protect the host against excessive inflammation but potentially increase injury through reduced pathogen clearance. Continued study of NAM cell signals invitro and invivo may provide insight into infection-associated host pathologies and interventions.

HIF and MYC signaling in adrenal neoplasms of the neural crest: implications for pediatrics.

Pediatric neural crest-derived adrenal neoplasms include neuroblastoma and pheochromocytoma. Both entities are associated with a high degree of clinical heterogeneity, varying from spontaneous regression to malignant disease with poor outcome. Increased expression and stabilization of HIF2α appears to contribute to a more aggressive and undifferentiated phenotype in both adrenal neoplasms, whereas MYCN amplification is a valuable prognostic marker in neuroblastoma. The present review focuses on HIF- and MYC signaling in both neoplasms and discusses the interaction of associated pathways during neural crest and adrenal development as well as potential consequences on tumorigenesis. Emerging single-cell methods together with epigenetic and transcriptomic analyses provide further insights into the importance of a tight regulation of HIF and MYC signaling pathways during adrenal development and tumorigenesis. In this context, increased attention to HIF-MYC/MAX interactions may also provide new therapeutic options for these pediatric adrenal neoplasms.

Hypoxia-inducible factor activation promotes osteogenic transition of valve interstitial cells and accelerates aortic valve calcification in a mice model of chronic kidney disease.

Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model. HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.

LncRNA CDKN2B-AS1 interacts with LIN28B to exacerbate sepsis-induced acute lung injury by inducing HIF-1α/NLRP3-mediated pyroptosis.

Sepsis-associated acute lung injury (ALI) is a life-threatening condition in intensive care units with high mortality. LncRNAs have been confirmed to participate in the underlying pathogenesis of septic ALI. This study investigated the biological functions of lncRNA CDKN2B-AS1 in septic ALI and its potential mechanism.BEAS-2B cells were challenged with lipopolysaccharide (LPS) and mice were subjected to caecal ligation and puncture (CLP) to induce septic ALI in vitro and in vivo. The expression levels of CDKN2B-AS1, LIN28B, HIF-1α, and pyroptosis-related molecules were assessed by qRT-PCR or Western blotting. The production of IL-1β and IL-18 was detected by ELISA. BEAS-2B cell pyroptosis was examined by flow cytometry. The interaction between LIN28B and CDKN2B-AS1/HIF-1α was validated by RIP and RNA pull-down assays. Colocalization of CDKN2B-AS1 and LIN28B was observed by FISH. ALI was determined by HE staining, the lung wet-to-dry (W/D) weight ratio, inflammatory cell numbers, and total protein concentration in bronchoalveolar lavage fluid (BALF). Caspase-1 expression in the lung tissues was examined by immunohistochemical staining.CDKN2B-AS1 was upregulated in BEAS-2B cells after LPS stimulation. CDKN2B-AS1 knockdown inhibited pyroptosis in LPS-exposed BEAS-2B cells in vitro and the lung tissues of septic mice in vivo. Mechanistically, CDKN2B-AS1 interacted with LIN28B to enhance HIF-1α stability. Rescue experiments showed that HIF-1α overexpression counteracted the inhibitory effect of sh-CDKN2B-AS1 on LPS-induced pyroptosis. CDKN2B-AS1 bound to LIN28B to trigger NLRP3-mediated pyroptosis by stabilizing HIF-1α, which promoted sepsis-induced ALI. CDKN2B-AS1 might be a novel therapeutic target for this disease.

Korean Red Ginseng-Induced SIRT3 Promotes the Tom22-HIF-1α Circuit in Normoxic Astrocytes.

Astrocytes play a key role in brain functioning by providing energy to neurons. Increased astrocytic mitochondrial functions by Korean red ginseng extract (KRGE) have been investigated in previous studies. KRGE administration induces hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in astrocytes in the adult mouse brain cortex. VEGF expression can be controlled by transcription factors, such as the HIF-1α and estrogen-related receptor α (ERRα). However, the expression of ERRα is unchanged by KRGE in astrocytes of the mouse brain cortex. Instead, sirtuin 3 (SIRT3) expression is induced by KRGE in astrocytes. SIRT3 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that resides in the mitochondria and maintains mitochondrial homeostasis. Mitochondrial maintenance requires oxygen, and active mitochondria enhance oxygen consumption, resulting in hypoxia. The effects of SIRT3 on HIF-1α-mediated mitochondria functions induced by KRGE are not well established. We aimed to investigate the relationship between SIRT3 and HIF-1α in KRGE-treated normoxic astrocyte cells. Without changing the expression of the ERRα, small interfering ribonucleic acid targeted for SIRT3 in astrocytes substantially lowers the amount of KRGE-induced HIF-1α proteins. Reduced proline hydroxylase 2 (PHD2) expression restores HIF-1α protein levels in SIRT3-depleted astrocytes in normoxic cells treated with KRGE. The translocation of outer mitochondrial membranes 22 (Tom22) and Tom20 is controlled by the SIRT3-HIF-1α axis, which is activated by KRGE. KRGE-induced Tom22 increased oxygen consumption and mitochondrial membrane potential, as well as HIF-1α stability through PHD2. Taken together, in normoxic astrocytes, KRGE-induced SIRT3 activated the Tom22-HIF-1α circuit by increasing oxygen consumption in an ERRα-independent manner.

Ganetespib (STA-9090) augments sorafenib efficacy via necroptosis induction in hepatocellular carcinoma: Implications from preclinical data for a novel therapeutic approach

Sorafenib, a multikinase inhibitor, is a first-line treatment for advanced hepatocellular carcinoma, but its long-term effectiveness is limited by the emergence of resistance mechanisms. One such mechanism is the reduction of microvessel density and intratumoral hypoxia caused by prolonged sorafenib treatment. Our research has demonstrated that HSP90 plays a critical role in conferring resistance to sorafenib in HepG2 cells under hypoxic conditions and N-Nitrosodiethylamine-exposed mice as well. This occurs through the inhibition of necroptosis on the one hand and the stabilization of HIF-1α on the other hand. To augment the effects of sorafenib, we investigated the use of ganetespib, an HSP90 inhibitor. We found that ganetespib activated necroptosis and destabilized HIF-1α under hypoxia, thus enhancing the effectiveness of sorafenib. Additionally, we discovered that LAMP2 aids in the degradation of MLKL, which is the mediator of necroptosis, through the chaperone-mediated autophagy pathway. Interestingly, we observed a significant negative correlation between LAMP2 and MLKL. These effects resulted in a reduction in the number of surface nodules and liver index, indicating a regression in tumor production rates in mice with HCC. Furthermore, AFP levels decreased. Combining ganetespib with sorafenib showed a synergistic cytotoxic effect and resulted in the accumulation of p62 and inhibition of macroautophagy. These findings suggest that the combined therapy of ganetespib and sorafenib may offer a promising approach for the treatment of hepatocellular carcinoma by activating necroptosis, inhibiting macroautophagy, and exhibiting a potential antiangiogenic effect. Overall, continued research is critical to establish the full therapeutic potential of this combination therapy.

Targeting metabolism by B-raf inhibitors and diclofenac restrains the viability of BRAF-mutated thyroid carcinomas with Hif-1α-mediated glycolytic phenotype

BackgroundB-raf inhibitors (BRAFi) are effective for BRAF-mutated papillary (PTC) and anaplastic (ATC) thyroid carcinomas, although acquired resistance impairs tumour cells’ sensitivity and/or limits drug efficacy. Targeting metabolic vulnerabilities is emerging as powerful approach in cancer.MethodsIn silico analyses identified metabolic gene signatures and Hif-1α as glycolysis regulator in PTC. BRAF-mutated PTC, ATC and control thyroid cell lines were exposed to HIF1A siRNAs or chemical/drug treatments (CoCl2, EGF, HGF, BRAFi, MEKi and diclofenac). Genes/proteins expression, glucose uptake, lactate quantification and viability assays were used to investigate the metabolic vulnerability of BRAF-mutated cells.ResultsA specific metabolic gene signature was identified as a hallmark of BRAF-mutated tumours, which display a glycolytic phenotype, characterised by enhanced glucose uptake, lactate efflux and increased expression of Hif-1α-modulated glycolytic genes. Indeed, Hif-1α stabilisation counteracts the inhibitory effects of BRAFi on these genes and on cell viability. Interestingly, targeting metabolic routes with BRAFi and diclofenac combination we could restrain the glycolytic phenotype and synergistically reduce tumour cells’ viability.ConclusionThe identification of a metabolic vulnerability of BRAF-mutated carcinomas and the capacity BRAFi and diclofenac combination to target metabolism open new therapeutic perspectives in maximising drug efficacy and reducing the onset of secondary resistance and drug-related toxicity.