HIF-1α Protein Levels Research Articles

HIF1A protein expression is correlated with clinical features in gastric cancer: an updated systematic review and meta-analysis

To elucidate the correlation of HIF1A with clinicopathologic characteristics in patients with gastric cancer (GC), we conducted a systematic review and meta-analysis. We searched PubMed, Embase and Web of Science for studies on GC and HIF1A, covering studies published until January 31st, 2022. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) for clinical characteristics based on high and low HIF1A protein levels. We used random-effects and fixed-effects meta-analysis methods to determine mean effect sizes of ORs and evaluated publication heterogeneity with τ2, I2, and Q values. Additionally, we generated funnel plots to inspect publication bias. Our meta-analysis included 20 publications with 3416 GC patients to estimate the association between high or low HIF1A expression and clinical characteristics. Positive HIF1A expression was significantly associated with T stage progression (OR: 2.46; 95% CI 1.81–3.36; P < 0.01), TNM stage progression (OR: 2.50; 95% CI 1.61–3.87; P < 0.01), lymph node metastasis (OR: 2.06; 95% CI 1.44–2.94; P < 0.01), undifferentiated status (OR: 1.83; 95% CI 1.45–2.32; P < 0.01), M stage progression (OR: 2.34; 95% CI 1.46–3.77; P < 0.01), Borrmann stage progression (OR: 1.48; 95% CI 1.02–2.15; P = 0.04), larger tumor size (OR: 1.27; 95% CI 1.06–1.52; P < 0.01), vascular invasion (OR: 1.94; 95% CI 1.38–2.72; P < 0.01), and higher vascular endothelial growth factor (VEGF) protein expression (OR: 2.61; 95% CI 1.79–3.80; P < 0.01) in our meta-analysis. GC Patients highly expressing HIF1A protein might be prone to tumor progression, poorly differentiated GC cell types, and a high VEGF expression.

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)—a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein – a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

The Expression of Hypoxia-Inducible Factor-1 Alpha (HIF-1α) as a Potential Target for Hypoxic Therapy in COVID-19 Cases

Background: The cellular response to hypoxia is characterized by the stabilization of the HIF-1α protein, which will form a transcription factor that activates genes for low oxygen adaptation. Positive cases of COVID-19 are often characterized by hypoxia, especially in patients with comorbidities. This hypoxic condition will mediate drug resistance or weaken the patient's condition, resulting in lengthening treatment time and even causing death. This study aims to analyze the cellular expression of the HIF-1α gene in patients confirmed positive for COVID-19 with the alpha, beta, delta, and omicron variants. Methods: The research design used was observational, with parameters measuring HIF-1 mRNA expression (RT-PCR, protein (ELISA), and its correlation. The samples used were nose-throat swabs from COVID-19 patients. Results: The expression of HIF-1α mRNA and protein levels produced in variants of COVID-19 patients was compared with controls (times; ng/mL): alpha (0.53; 18.08), beta (0.92; 19.98), delta (0.65; 25.83), and omicron (0.73; 19.85) (Kruskal-Wallis test, p &lt;0.01). The correlation between mRNA expression of HIF-1α and HIF-1α proteins was found to be significantly negative (Pearson Correlation test R = -0.589). Conclusion: In this study, it was found that a decrease in HIF-1α mRNA expression (the result of transcription) increased the levels of HIF-1α protein (the result of translation) and stable HIF-1α expression. Moreover, cellular hypoxia occurred in patients with confirmed positive for COVID-19, with the alpha, beta, delta, and omicron variants. This study can be used as a basis for therapy to control viruses that cause hypoxia in the future. result: The expression of HIF-1α mRNA and protein levels produced in variants of COVID-19 patients compared with controls (times; ng/mL): alpha (0.53; 18.08), beta (0.92; 19.98), delta (0.65 ; 25.83), omicron (0.73; 19.85) (Kruskal Wallis test, p &lt;0.01); The correlation between mRNA expression of HIF-1α and HIF-1α proteins obtained strong negative results (Pearson Correlation test R = -0.589 ).

Role of hypoxia-mediated pyroptosis in the development of extending knee joint contracture in rats

Joint contracture is one of the common diseases clinically, and joint capsule fibrosis is considered to be one of the most important pathological changes of joint contracture. However, the underlying mechanism of joint capsule fibrosis is still controversial. The present study aims to establish an animal model of knee extending joint contracture in rats, and to investigate the role of hypoxia-mediated pyroptosis in the progression of joint contracture using this animal model. 36 male SD rats were selected, 6 of which were not immobilized and were used as control group, while 30 rats were divided into I-1 group (immobilized for 1 week following 7 weeks of free movement), I-2 group (immobilized for 2 weeks following 6 weeks of free movement), I-4 group (immobilized for 4 weeks following 4 weeks of free movement), I-6 group (immobilized for 6 weeks following 2 weeks of free movement) and I-8 group (immobilized for 8 weeks) according to different immobilizing time. The progression of joint contracture was assessed by the measurement of knee joint range of motion, collagen deposition in joint capsule was examined with Masson staining, protein expression levels of HIF-1α, NLRP3, Caspase-1, GSDMD-N, TGF-β1, α-SMA and p-Smad3 in joint capsule were assessed using western blotting, and the morphological changes of fibroblasts were observed by transmission electron microscopy. The degree of total and arthrogenic contracture progressed from the first week and lasted until the first eight weeks after immobilization. The degree of total and arthrogenic contracture progressed rapidly in the first four weeks after immobilization and then progressed slowly. Masson staining indicated that collagen deposition in joint capsule gradually increased in the first 8 weeks following immobilization. Western blotting analysis showed that the protein levels of HIF-1α continued to increase during the first 8 weeks of immobilization, and the protein levels of pyroptosis-related proteins NLRP3, Caspase-1, GSDMD-N continued to increase in the first 4 weeks after immobilization and then decreased. The protein levels of fibrosis-related proteins TGF-β1, p-Smad3 and α-SMA continued to increase in the first 8 weeks after immobilization. Transmission electron microscopy showed that 4 weeks of immobilization induced cell membrane rupture and cell contents overflow, which further indicated the activation of pyroptosis. Knee extending joint contracture animal model can be established by external immobilization orthosis in rats, and the activation of hypoxia-mediated pyroptosis may play a stimulating role in the process of joint capsule fibrosis and joint contracture.

METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.

HIF-1α participates in the regulation of S100A16-HRD1-GSK3β/CK1α pathway in renal hypoxia injury

S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-β1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and β-catenin while decreasing GSK-3β. HIF-1α inhibition restored HRD1 and β-catenin upregulation and GSK-3β downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α‘s transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3β/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.

CD8 Cell PHD2 Deficiency Amplifies Pressure Overload-Induced Cardiac Inflammation through Metabolic Reprogramming of CD8 Cells

Prolyl hydroxylase domain (PHD) proteins function as oxygen sensors and regulate various cellular metabolism. PHD2 is well-known for its role in oxygen sensing and has been implicated in cardiac hypertrophy and fibrosis through HIF-2α. However, the specific impact and the mechanism of CD8 cell PHD2 deficiency on cardiac inflammation and heart failure (HF) development remains unclear. Here, we explored the role of CD8 cell-specific PHD2 deficiency (CD8-PHD2 KO) in HF development by using transverse aortic constriction (TAC) mouse model. Our findings reveal that CD8 PHD2 knockout exacerbated TAC-induced left ventricular (LV) hypertrophy, as evidenced by increased LV weight relative to bodyweight or tibial length. CD8-PHD2 KO also leads to a significant increase in lung and right ventricular weight relative to bodyweight or tibial length following TAC. Furthermore, CD8-PHD2 KO worsens TAC-induced reductions in LV ejection fraction, LV fractional shortening, and LV dilatation. Additionally, CD8-PHD2 KO exacerbates TAC-induced LV cardiomyocyte hypertrophy, fibrosis, and immune cell infiltration. Moreover, our study demonstrated that CD8-PHD2 KO amplified the infiltration and activation of antigen-presenting cells, CD4 T cells, and CD8 T cells in the heart and lungs after TAC. At the molecular level, we characterized the PHD2-deficient CD8 cells showed typical features of anaerobic glycolysis, which were paralleled by augmented hypoxia-inducible factor 1α (HIF-1α) protein levels. Collectively, these results highlight the critical role of PHD2 in CD8 cells in regulating cardiac inflammation and HF development in response to systolic overload. National Institutes of Health Project Number 5R01HL161085-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heat shock protein 90 and prolyl hydroxylase 2 co-regulate hypoxia-inducible factor-1α expression in porcine small intestinal epithelial cells under heat stress

Heat stress (HS) poses a substantial threat to animal growth and development, resulting in declining performance and economic losses. The intestinal system is susceptible to HS and undergoes intestinal hyperthermia and pathological hypoxia. Hypoxia-inducible factor-1α (HIF-1α), a key player in cellular hypoxic adaptation, is influenced by prolyl-4-hydroxylase 2 (PHD2) and heat shock protein 90 (HSP90). However, the comprehensive regulation of HIF-1α in the HS intestine remains unclear. This study aims to explore the impact of HS on pig intestinal mucosa and the regulatory mechanism of HIF-1α. Twenty-four Congjiang Xiang pigs were divided into the control and five HS-treated groups (6, 12, 24, 48, and 72 h). Ambient temperature and humidity were maintained in a thermally-neutral state (temperature–humidity index (THI) < 74) in the control group, whereas the HS group experienced moderate HS (78 < THI <84). Histological examination revealed villus exfoliation after 12 h of HS in the duodenum, jejunum, and ileum, with increasing damage as HS duration extended. The villus height to crypt depth ratio (V/C) decreased and goblet cell number increased with prolonged HS. Quantitative real-time PCR, Western blot, and immunohistochemistry analysis indicated increased expression of HIF-1α and HSP90 in the small intestine with prolonged HS, whereas PHD2 expression decreased. Further investigation in IPEC-J2 cells subjected to HS revealed that overexpressing PHD2 increased PHD2 mRNA and protein expression, while it decreases HIF-1α. Conversely, interfering with HSP90 expression substantially decreased both HSP90 and HIF-1α mRNA and protein levels. These results suggest that HS induces intestinal hypoxia with concomitant small intestinal mucosal damage. The expression of HIF-1α in HS-treated intestinal epithelial cells may be co-regulated by HSP90 and PHD2 and is possibly linked to intestinal hyperthermia and hypoxia.

The Essential Role of PGF2α/PTGFR in Molding Endometrial Breakdown and Vascular Dynamics, Regulated by HIF-1α in a Mouse Menstrual-like Model.

In women of childbearing age, extensive decidualization, shedding and remodeling of the endometrium during the menstrual cycle are fundamental for successful pregnancy. The role of prostaglandins (PGs) in menstruation has long been proposed in humans, and the rate-limiting enzyme cyclooxygenase was shown to play a key role in endometrial breakdown and shedding in a mouse menstrual-like model in our previous study. However, the specific types of PGs involved and their respective roles remain unclear. Therefore, our objective was to investigate the mechanism through which PGs regulate endometrial disintegration. In this study, the microscopy was observed by HE; the protein levels of prostaglandins E1 (PGE1), prostaglandins E2 (PGE2), prostaglandin F2α (PGF2α) and Prostaglandin I2 (PGI2) were detected by ELISA; the mRNA level of Pfgfr2, Vascular Endothelial Growth Factor(Vegf), Angiostatin and Hypoxia inducible factor-1α (Hif1α) were examined by real-time PCR; PTGFR Receptor (PTGFR), VEGF, Angiostatin and HIF-1α protein levels were investigated by western blotting; the locations of protein were observed by Immunohistochemistry; HIF-1α binding PTGFR promoter was detected by Chromatin Immunoprecipitation (ChIP) and real-time PCR. We found that the concentrations of PGE1, PGE2, and PGF2α all increased significantly during this process. Furthermore, Ptgfr mRNA increased soon after Progesterone (P4) withdrawal, and PTGFR protein levels increased significantly during abundant endometrial breakdown and shedding processes. PTGFR inhibitors AL8810 significantly suppressed endometrial breakdown and shedding, promoted Angiostatin expression, and reduced VEGF-A expressions and vascular permeability. And HIF-1α and PTGFR were mainly located in the luminal/gland epithelium, vascular endothelium, and pre-decidual zone. Interestingly, HIF-1α directly bound to Ptgfr promoter. Moreover, a HIF-1α inhibitor 2-methoxyestradiol (2ME) significantly reduced PTGFR expression and suppressed endometrial breakdown which was in accord with PTGFR inhibitor's effect. Similar changes occurred in human stromal cells relevant to menstruation in vitro. Our study provides evidence that PGF2α/PTGFR plays a vital role in endometrial breakdown via vascular changes that are regulated by HIF-1α during menstruation.

Salvianic acid A sodium facilitates cardiac microvascular endothelial cell proliferation by enhancing the hypoxia-inducible factor-1 alpha/vascular endothelial growth factor signalling pathway post-myocardial infarction.

Cardiac microvascular endothelial cells (CMECs) are important cells surrounding the cardiomyocytes in the heart that maintain microenvironment homeostasis. Salvianic acid A sodium (SAAS) has been reported to prevent myocardial infarction (MI) injury. However, the role of SAAS on CMEC proliferation remains unclear. CEMCs exposed to oxygen glucose deprivation (OGD) were used to explore the angiogenic abilities of SAAS. In vivo, C57BL/6 mice were divided into three groups: sham, MI and SAAS + MI groups. Compared to OGD group, SAAS led to a reduction in the apoptotic rate and an increase of the proliferation in vitro. Additionally, SAAS increased the protein levels of Bcl2, HIF-1α and vascular endothelial growth factor (VEGF) with the reduction of Bax. In terms of the specific mechanisms, SAAS might inhibit HIF-1α ubiquitination and enhance the HIF-1α/VEGF signalling pathway to increase CMEC proliferation. Furthermore, SAAS increased the density of vessels, inhibited myocardial fibrosis and improved cardiac dysfunction in vivo. The present study has revealed that SAAS could potentially be used as an active substance to facilitate CMEC proliferation post-MI.

High-fat diet consumption promotes adolescent neurobehavioral abnormalities and hippocampal structural alterations via microglial overactivation accompanied by an elevated serum free fatty acid concentration

Mounting evidence suggests that high-fat diet (HFD) consumption increases the risk for depression, but the neurophysiological mechanisms involved remain to be elucidated. Here, we demonstrated that HFD feeding of C57BL/6J mice during the adolescent period (from 4 to 8 weeks of age) resulted in increased depression- and anxiety-like behaviors concurrent with changes in neuronal and myelin structure in the hippocampus. Additionally, we showed that hippocampal microglia in HFD-fed mice assumed a hyperactive state concomitant with increased PSD95-positive and myelin basic protein (MBP)-positive inclusions, implicating microglia in hippocampal structural alterations induced by HFD consumption. Along with increased levels of serum free fatty acids (FFAs), abnormal deposition of lipid droplets and increased levels of HIF-1α protein (a transcription factor that has been reported to facilitate cellular lipid accumulation) within hippocampal microglia were observed in HFD-fed mice. The use of minocycline, a pharmacological suppressor of microglial overactivation, effectively attenuated neurobehavioral abnormalities and hippocampal structural alterations but barely altered lipid droplet accumulation in the hippocampal microglia of HFD-fed mice. Coadministration of triacsin C abolished the increases in lipid droplet formation, phagocytic activity, and ROS levels in primary microglia treated with serum from HFD-fed mice. In conclusion, our studies demonstrate that the adverse influence of early-life HFD consumption on behavior and hippocampal structure is attributed at least in part to microglial overactivation that is accompanied by an elevated serum FFA concentration and microglial aberrations represent a potential preventive and therapeutic target for HFD-related emotional disorders.

Abstract LB049: Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway

Abstract Lung cancer is the second most common cancer in both men and women in the United States. Even though treatments including chemotherapy, targeted therapy, and immunotherapy are available, lung cancer has been the leading cause of cancer deaths. The discovery and evaluation of new alternative medications targeting lung cancer are tremendously important for reducing lung cancer mortality. Phytochemicals are very desirable chemotherapeutic agents because of their greater safety profile. Isorhapontigenin (ISO), one of the oral bioavailable dietary stilbenes, can be found in various natural resources. Our result showed that ISO treatment suppressed the invasion and migration of human lung cancer cell lines A549, H23, and H1299 cells. Long non-coding RNA Maternally Expressed Gene 3 (MEG3), a tumor suppressor, was downregulated or lost in various primary human tumor tissues and cancer cell lines. The low expression of MEG3 is correlated with poor prognosis in non-small cell lung cancer (NSCLC). Our results showed that ISO treatment elevated MEG3 levels in these lung cancer cell lines. One of the major genetic causes of lung cancer is mutations in the epidermal growth factor receptor (EGFR) receptor tyrosine kinase, resulting in activation of EGFR which activates several signaling pathways involved in cell proliferation, drug sensitivity, and angiogenesis. Our results showed that ISO decreased the phosphorylation and protein levels of EGFR in A549 cells. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, regulates the activation of several genes involved in cell proliferation, angiogenesis, invasion, and metastasis. Our results showed that ISO decreased HIF-1α protein levels. Overexpression of MEG3 reduced p-EGFR and HIF-1α, indicating MEG3 negatively regulated EGFR and HIF-1α. Our data also showed that inhibition of EGFR by its tyrosine inhibitor decreased HIF-1α, suggesting that EGFR positively regulates HIF-1α. In cancer cells, Epithelial-Mesenchymal Transition(EMT) endows cells with increased invasiveness and tumor-initiating capacity, contributing to metastasis and resistance to therapeutics. Slug, one of the EMT transcription factors maintains cell plasticity during carcinogenesis. Our results showed that ISO treatment decreased Slugprotein levels in A549 and H23 cells. Transcription factor SOX2 regulates cancer cell proliferation, EMT, migration, invasion, metastasis, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy. Our results showed that ISO decreased SOX2 expression. Knockdown of MEG3 increased the expressions of Slug and SOX2, indicating that MEG3 negatively regulates Slug and SOX2. In conclusion, ISO elevated MEG3, inhibiting EGFR, HIF-1α, Slug, and SOX2, suppressing the migration and invasion of lung cancer cells. Citation Format: Sophia Shi, Zhuo Zhang, Jingxia Li, Huailu Tu, Max Costa. Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB049.

Leucine improves thiram-induced tibial dyschondroplasia and gut microbiota dysbiosis in broilers

Thiram, a commonly used agricultural insecticide and fungicide, has been found to cause tibial dyschondroplasia (TD) in broilers, leading to substantial economic losses in the poultry industry. In this study, we aimed to investigate the mechanism of action of leucine in mitigating thiram-induced TD and leucine effects on gut microbial diversity. Broiler chickens were randomly divided into five equal groups: control group (standard diet), thiram-induced group (thiram 80 mg/kg from day 3 to day 7), and different concentrations of leucine groups (0.3%, 0.6%, 0.9% leucine from day 8 to day 18). Performance indicator analysis and tibial parameter analysis showed that leucine positively affected thiram-induced TD broilers. Additionally, mRNA expressions and protein levels of HIF-1α/VEGFA and Ihh/PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. The results showed that leucine recovered lameness disorder by downregulating the expression of HIF-1α, VEGFA, and PTHrP while upregulating the expression of Ihh. Moreover, the 16 S rRNA sequencing revealed that the leucine group demonstrated a decrease in the abundance of harmful bacteria compared to the TD group, with an enrichment of beneficial bacteria responsible for producing short-chain fatty acids, including Alistipes, Paludicola, CHKCI002, Lactobacillus, and Erysipelatoclostridium. In summary, the current study suggests that leucine could improve the symptoms of thiram-induced TD and maintain gut microbiota homeostasis.

Abstract 1971: Olverembatinib, a novel multikinase inhibitor, demonstrates superior antitumor activity in succinate dehydrogenase (SDH)-deficient neoplasms

Abstract SDH deficiency, characterized by loss of SDHB, occurs in many tumors including paraganglioma, pheochromocytoma, renal, and gastrointestinal stromal tumor (GIST). Olverembatinib, a novel multikinase inhibitor, targets a broad spectrum of kinases and has promising efficacy in SDH-deficient GIST (Qiu H et al. J Clin Oncol. 2023). Here, we assessed antitumor effects of olverembatinib in preclinical models of SDH-deficient cancers and explored potential mechanisms of action (MOA). Antiproliferation assays were conducted using CellTiter-Glo®. MOA analyses were performed using western blotting. In vivo activity was evaluated using an SDHB knock-out [KO])-derived mouse xenograft model. Olverembatinib had superior antiproliferative activity (vs other approved TKIs) in SDH-deficient cell lines (IC50, 0.129-5.132 μM), including Jurkat clone E6-1 (SDHB mutation [mut]), OS-RC-2 (SDHA and SDHB mut), RKO (SDHA mut), and rat pheochromocytoma PC12 cells with SDHB KO (PC12#5F7) (Table 1). In PC12#5F7 cells, olverembatinib decreased HIF-1α, HIF-2α, VEGFA, and FGFR1 protein levels and inhibited phosphorylation of FGFR1, IGF-1R, SRC, AKT, and ERK1/2. Increased cleavage of caspase-3 and PARP-1 was observed, suggesting induction of apoptosis. In PC12#5F7 (SDHB KO)-derived xenograft models, olverembatinib demonstrated dose-dependent antitumor activity at 10 and 20 mg/kg (QOD), with tumor growth inhibition rates of 27.4% and 52.2%, respectively. At 20 mg/kg, olverembatinib showed superior efficacy vs ponatinib (10 mg/kg) and rogaratinib (50 mg/kg). Results from western blot analyses were similar in both tumor tissues and cell lines. Olverembatinib exerts promising antitumor effects in SDH-deficient neoplasms and can modulate multiple signal pathways involved in angiogenesis, apoptosis, proliferation, and survival. The results provide a preclinical rationale for future development of olverembatinib in SDH-deficient cancers. Table 1. Antiproliferation activity of TKIs in SDH-deficient cell lines IC50 μM, mean ± SD Cell lines Acute lymphoblastic leukemia Renal cell carcinoma Colon cancer Rat pheochromocytoma carcinoma Jurkat, clone E6-1 OS-RC-2 RKO PC12 PC12#5F7 SDH status* SDHB: missense_variant, p.A15T SDHA: missense_variant, p.G184R; SDHB: stop gained, p.W218Ter SDHA: missense_variant, p.P279S SDHB competent SDHB exon 2+3 KO (heterozygote) by CRISPR-Cas9 Compound Olverembatinib (KIT, PDGFR, SRC, VEGFR, FGFR, FLT-3, RET) 0.149 ± 0.04 0.135 ± 0.083 0.129 ± 0.136 9.600 ± 1.407(n = 4) 5.132 ± 1.950 Imatinib (KIT, PDGFR) &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 Ripretinib (KIT, PDGFR) 1.559 ± 0.294 8.781 ± 1.725 1.247 ± 0.933 &amp;gt; 10 &amp;gt; 10 Avapritinib (PDGFRA exon 18 mut) &amp;gt; 8.071 ± 2.728 &amp;gt; 10 ± 0 &amp;gt; 10 ± 0 &amp;gt; 10 &amp;gt; 10 Ponatinib (KIT, PDGFR, VEGFR, FGFR) 2.136 ± 2.107 0.809 ± 0.443 0.274 ± 0.241 &amp;gt; 10 8.537 ± 3.553 Dasatinib (KIT, SRC, PDGFR, FGFR) 2.025 ± 0.644 0.029 ± 0.003 0.382 ± 0.320 &amp;gt; 10 &amp;gt; 10 Sunitinib (VEGFR) 4.289 ± 0.280 &amp;gt; 5.099 ± 0.207 3.789 ± 0.385 &amp;gt; 10 &amp;gt; 10 Regorafenib (VEGFR) &amp;gt; 10 5.110 ± 0.588 2.145 ± 0.812 &amp;gt; 10 &amp;gt; 10 Sorafenib (VEGFR) &amp;gt; 10 3.581 ± 0.255 3.875 ± 0.170 &amp;gt; 10 &amp;gt; 10 Pazopanib (VEGFR, FGFR) &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 Rogaratinib (FGFR) &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 Infigratinib (FGFR) 4.581 ± 0.342 3.166 ± 0.791 2.374 ± 2.311 &amp;gt; 10 &amp;gt; 10 Pemigatinib (FGFR) 4.642 ± 1.842 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 &amp;gt; 10 *From CrownBio database. Citation Format: Yan Xiong, Eric Liang, Ping Min, Huidan Yu, Bingxing Wu, Guoqin Zhai, Dajun Yang, Yifan Zhai. Olverembatinib, a novel multikinase inhibitor, demonstrates superior antitumor activity in succinate dehydrogenase (SDH)-deficient neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1971.

Abstract 2096: A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy

Abstract Background: Ferroptosis is a novel form of cell death driven by iron-dependent lipid peroxidation, which disrupts cellular membranes resulting in immune-activating death. A variety of tumor types, particularly those associated with enhanced lipid and/or iron content, or which experience increased oxidative stress (including drug tolerant persister cells), are sensitive to ferroptosis, rendering ferroptosis a promising therapeutic strategy for cancer. However, currently available ferroptosis inducers show limited activity in vivo, suggesting a need for novel molecular targets to validate the clinical utility of ferroptosis induction. Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of VHL resulting in constitutive activation of HIF-1α and HIF-2α, which contribute to disease progression, including by promoting lipid accumulation that sensitizes ccRCC cells to ferroptosis. Methods: We performed a high-throughput screen to identify small molecule inhibitors of HIF-2α followed by structure-activity-relationship studies for validation. The molecular target of these inhibitors was identified using the Drug Affinity Responsive Target Stability assay, and target validation was confirmed using siRNA and NMR. In vitro efficacy was determined in a panel of cancer cell lines and in vivo anti-tumor efficacy was verified using mouse models of cancer. Results: We identified a novel molecule, KD061, which decreases HIF-1/2α and induces ferroptosis in vivo through oral administration by targeting Iron Sulfur Cluster Assembly 2 (ISCA2). ISCA2 is a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex and ISCA2 inhibition either pharmacologically or using siRNA triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 inhibition also decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation. These studies reveal a novel function of ISCA2 in extra-mitochondrial Fe-S assembly that may be limiting in cancer due to the increased need for these proteins in DNA replication and maintenance. Outside of ccRCC, a 92-cell line screen reveals a variety of tumor types sensitive to ISCA2 inhibition including melanoma and clear cell ovarian cancer. Treatment of tumor-bearing mice with KD061 resulted in &amp;gt; 65% tumor growth inhibition, associated with significantly increased lipid peroxidation, thus confirming ferroptosis in vivo, and with decreased HIF-1/2α. The anti-tumor efficacy of KD061 exceeded that of α-PD1 and was well tolerated at the therapeutic dose. Conclusions: We describe the first orally available inducer of ferroptosis with significant anti-tumor efficacy in ccRCC and other tumor types. Citation Format: Yangsook Song Green, Maria Ferreira dos Santos, Simone Ciofi-Baffoni, Xiaohui Liu, Mei Yee Koh. A novel, orally available ferroptosis inducer targeting Fe-S assembly with significant in vivo anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2096.

Abstract 2991: Global analysis of miR-210 and miR-21 dysregulation and direct targeting in thyroid cancer during hypoxia

Abstract Introduction: Hypoxia is frequent in solid tumors and stabilizes Hypoxia Inducible Factor-1 (HIF-1). HIF-1, in turn, activates several cellular pathways that promote tumor growth and resistance to chemotherapy. MicroRNA (miRNA) miR-210 is a direct HIF-1 target. HIF-1 binds to a Hypoxia-Response Element (HRE) in the miR-210 promoter, up-regulating its expression. miR-21 is the most widely reported oncogenic miRNA in cancer. miRNAs complexed with Argonaute-2 (AGO2) bind target mRNAs to inhibit translation. Given that a single miRNA can have hundreds of predicted mRNA targets, we sought to identify mRNA targets of miR-210 and miR-21. Identifying dysregulated target genes of miR-210 and miR-21 may highlight new therapeutic targets. Methods: The SW1736 (anaplastic thyroid cancer) cell line was cultured at 2% (hypoxic) or atmospheric (normoxic) O2 for 24 or 48hrs. HIF-1α accumulation relative to normoxia was analyzed by Western blot. miR-210-3p expression was measured relative to normoxia by RT-qPCR. Small and total RNA-sequencing was performed to identify additional differentially expressed miRNAs and mRNAs in hypoxia after 48hrs. To identify direct miRNA targets, SW1736 cells were cross-linked by UV irradiation after 48hrs of normoxic or hypoxic conditions. AGO2 covalently cross-linked to miRNA and target mRNAs were purified by immunoprecipitation after DNA and RNA digestion. miRNA and target mRNAs protected by AGO2 were ligated to generate chimeric miRNA:mRNA molecules followed by sequencing. miRNA:mRNA chimeras were analyzed using the SCRAP bioinformatic pipeline. Results: miR-210-3p expression was up-regulated by ~11-fold at 24hrs and ~19-fold at 48hrs, which was consistent with increased HIF-1α protein levels assessed by Western blot. miR-210-3p and HIF-1α levels were highest after 48hr of hypoxia relative to normoxia. Small RNA-seq showed that miR-210-3p was the only miRNA significantly up-regulated (&amp;gt;2-fold) in hypoxia (48hr) in SW1736. Further, miRNA:mRNA chimeric sequencing results confirmed that miRNAs primarily interacted with the 3’UTRs and coding sequences of mRNA. miR-210-3p was most abundant in hypoxia and found in chimeras with HIF-1α mRNA as well as some oncogenic and tumor suppressor transcripts involved in metabolism and mRNA processing. miR-21-5p was the most abundant chimera miRNA in both normoxia and hypoxia and was primarily bound to tumor suppressor mRNAs associated with cell cycle arrest, TGFB signaling, and inhibition of tumor invasion and migration. Conclusions: miR-210-3p and miR-21-5p elevated levels and interactions indicate hypoxia and malignancy in solid tumors including breast, thyroid, prostate, and lung. Further analysis of other miRNA:mRNA interactions and functional validation may aid in identifying novel therapies for solid tumors. Citation Format: Bonita H. Powell, William T. Mills, Andrey Turchinovich, Yongchun Wang, Martha A. Zeiger, Christopher B. Umbricht, Mollie K. Meffert, Kenneth W. Witwer. Global analysis of miR-210 and miR-21 dysregulation and direct targeting in thyroid cancer during hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2991.

Elevated aerobic glycolysis driven by p62-mTOR axis promotes arsenic-induced oncogenic phenotypes in human mammary epithelial cells.

Chronic arsenic exposure is considered to increase the risk of breast cancer. p62 is a multifunctional adaptor protein that controls myriad cellular processes and is overexpressed in breast cancer tissues. Although previous studies have indicated the involvement of p62 accumulation in arsenic tumorigenesis, the underlying mechanism remains obscure. Here, we found that 0.1 µM or 0.5 µM arsenite exposure for 24 weeks induced oncogenic phenotypes in human mammary epithelial cells. Elevated aerobic glycolysis, cell proliferation capacity, and activation of p62-mTOR pathway, as indicated by increased protein levels of p62, phosphorylated-mTOR (p-mTOR) and hypoxia-inducible factor 1α (HIF1α), were observed in chronically arsenite-exposed cells, and of note in advance of the onset of oncogenic phenotypes. Moreover, p62 silencing inhibited acquisition of oncogenic phenotypes in arsenite-exposed cells. The protein levels of p-mTOR and HIF1α, as well as aerobic glycolysis and cell proliferation, were suppressed by p62 knockdown. In addition, re-activation of p‑mTOR reversed the inhibitory effects of p62 knockdown. Collectively, our data suggest that p62 exerts an oncogenic role via mTORC1 activation and acts as a key player in glucose metabolism during arsenite-induced malignant transformation, which provides a new mechanistic clue for the arsenite carcinogenesis.

GSH-related enzyme activity and tumor relation: glutathione peroxidase and glutathione reductase status under hypoxia in HepG2 cells

Abstract Objectives In hepatocellular carcinoma (HCC), tumorigenesis, hypoxia and reactive oxygen species (ROS) play a crucial role in altering the tumor microenvironment (TME). Until now, the time-dependent alteration of hypoxia-inducible factor-1α (HIF-1α), the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) under hypoxic conditions in HCC were not clear. Consequently, our main target was to investigate the role of GPx and GR status in HCC cell line (HepG2) under hypoxic conditions. Methods HIF-1α protein levels in cell lysates were determined by ELISA assay and protein expressions were identified using western blot. GPx and GR activity levels of the cell lysates were measured spectrophotometrically. Results HIF-1α protein levels were determined under normoxic and hypoxic conditions (p&lt;0.001). Also, HIF-1α protein levels and expressions were observed under time-dependent hypoxic conditions, the HIF-1α protein level is found to be reached its peak point at 4 h in the HepG2 cell line. We also have detected decreased activity levels of GPx and increased GR activity levels under hypoxia for 4 h (p&lt;0.001). Conclusions More than 4 h of exposure to hypoxic environment reducted the HIF-1α levels in HCC cells. According to the results, we suggest the ideal exposure time to hypoxic conditions as 4 h for the HepG2 cell line. In addition, hypoxia also stimulated the activity levels of GPx and GR. Our results suggest that the activity levels of GPx and/or GR enzymes may be therapeutic targets in the hypoxia-dependent HCC tumorigenesis process.

ALKBH1 promotes HIF-1α-mediated glycolysis by inhibiting N-glycosylation of LAMP2A

ALKBH1 is a typical demethylase of nucleic acids, which is correlated with multiple types of biological processes and human diseases. Recent studies are focused on the demethylation of ALKBH1, but little is known about its non-demethylase function. Here, we demonstrate that ALKBH1 regulates the glycolysis process through HIF-1α signaling in a demethylase-independent manner. We observed that depletion of ALKBH1 inhibits glycolysis flux and extracellular acidification, which is attributable to reduced HIF-1α protein levels, and it can be rescued by reintroducing HIF-1α. Mechanistically, ALKBH1 knockdown enhances chaperone-mediated autophagy (CMA)-mediated HIF-1α degradation by facilitating the interaction between HIF-1α and LAMP2A. Furthermore, we identify that ALKBH1 competitively binds to the OST48, resulting in compromised structural integrity of oligosaccharyltransferase (OST) complex and subsequent defective N-glycosylation of LAMPs, particularly LAMP2A. Abnormal glycosylation of LAMP2A disrupts lysosomal homeostasis and hinders the efficient degradation of HIF-1α through CMA. Moreover, NGI-1, a small-molecule inhibitor that selectively targets the OST complex, could inhibit the glycosylation of LAMPs caused by ALKBH1 silencing, leading to impaired CMA activity and disruption of lysosomal homeostasis. In conclusion, we have revealed a non-demethylation role of ALKBH1 in regulating N-glycosylation of LAMPs by interacting with OST subunits and CMA-mediated degradation of HIF-1α.

MITF regulates the subcellular location of HIF1α through SUMOylation to promote the invasion and metastasis of daughter cells derived from polyploid giant cancer cells.

High concentrations of cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs) in various tumors, which can produce daughter cells with strong proliferative, migratory and invasive abilities via asymmetric division. To study the role of hypoxia‑inducible factor (HIF) 1α in the formation of PGCCs, colon cancer cell lines Hct116 and LoVo were used as experimental subjects. Western blotting, nuclear and cytoplasmic protein extraction and immunocytochemical experiments were used to compare the changes in the expression and subcellular localization of HIF1α, microphthalmia‑associated transcription factor (MITF), protein inhibitor of activated STAT protein4 (PIAS4) and von Hippel‑Lindau disease tumor suppressor (VHL) after treatment with CoCl2. The SUMOylation of HIFα was verified by co‑immunoprecipitation assay. After inhibiting HIF1α SUMOylation, the changes in proliferation, migration and invasion abilities of Hct116 and LoVo were compared by plate colony formation, wound healing and Transwell migration and invasion. In addition, lysine sites that led to SUMOylation of HIF1α were identified through site mutation experiments. The results showed that CoCl2 can induce the formation of PGCCs with the expression level of HIF1α higher in treated cells than in control cells. HIF1α was primarily located in the cytoplasm of control cell. Following CoCl2 treatment, the subcellular localization of HIF1α was primarily in the nuclei of PGCCs with daughter cells (PDCs). After treatment with SUMOylation inhibitors, the nuclear HIF1α expression in PDCs decreased. Furthermore, their proliferation, migration and invasion abilities also decreased. After inhibiting the expression of MITF, the expression of HIF1α decreased. MITF can regulate HIF1α SUMOylation. Expression and subcellular localization of VHL and HIF1α did not change following PIAS4 knockdown. SUMOylation of HIF1α occurs at the amino acid sites K391 and K477 in PDCs. After mutation of the two sites, nuclear expression of HIF1α in PDCs was reduced, along with a significant reduction in the proliferation, migration and invasion abilities. In conclusion, the post‑translation modification regulated the subcellular location of HIF1α and the nuclear expression of HIF1α promoted the proliferation, migration and invasion abilities of PDCs. MITF could regulate the transcription and protein levels of HIF1α and participate in the regulation of HIF1α SUMOylation.