HIF-1α Inhibitor Research Articles

Abstract 4666: HC-7366, a potent GCN2 kinase activator, augments osimertinib therapy to delay resistance in EGFR mutant NSCLC models

Abstract Despite the efficacy seen with EGFR tyrosine kinase inhibitors (TKIs) in the treatment of EGFR mutant NSCLC, the long-term prognosis remains unfavorable due to resistance. Osimertinib is used as the first- and second-line treatment for EGFR exon 19 deletion, L858R mutation, and acquired T790M mutation. Resistance is driven by both EGFR-dependent and independent events, such as gene amplification and upregulation of HIF1α. Metabolic events also drive resistance by increasing oxidative phosphorylation (OXPHOS) caused by osimertinib-induced glycolysis inhibition. HiberCell has developed HC-7366, a GCN2 activator currently being investigated in a Ph1 clinical trial in patients with solid tumors (NCT05121948). GCN2 activation by HC-7366 drives the integrated stress response (ISR) by inducing the expression of ATF4. This results in translation inhibition, cell cycle arrest, HIF1α suppression, and apoptosis in cancer cells. Our preclinical data suggest HC-7366 can reduce glycolysis, TCA cycle metabolites, and OXPHOS, thereby causing mitochondrial dysregulation. We hypothesized that combination of HC-7366 with osimertinib could reduce resistance by evading the adaptive resistance mechanisms. We treated the NCI-H1975 xenograft model with HC-7366, a clinically relevant dose of osimertinib, and the combination for 21 days and then followed for tumor regrowth. At day 21, the HC-7366 group showed efficacy with tumor growth inhibition (TGI) of 55%, while osimertinib resulted in tumor regression of 97% with 8/10 CRs and 2/10 PRs. The combination had tumor regression of 99% with 10/10 CRs. After treatment cessation for 74 days, tumors in the osimertinib group quickly regrew with 6/10 tumors progressing and 4/10 mice tumor-free. In contrast, the combination group had only 2/10 tumors regrow with 8/10 mice tumor-free. In an EGFR-dependent resistance model NCI-H1975 (EGFR C797S), combination achieved 63% TGI versus 31% with osimertinib. In an EGFR-independent resistant PDX with EGFR exon 19 deletion, combination benefit was seen at 70% TGI compared to 40% for osimertinib, while a PDX with EGFR L858R showed a trend for combination benefit. To understand the mechanism of action, we examined osimertinib-resistance pathways to determine how GCN2 activation could affect them. Combination with HC-7366 reduced the expression of short-lived proteins involved in osimertinib resistance, including HIF1α, MET, cyclin D1, and CDK4. Combination decreased TIM17A and TIM23, proteins essential for mitochondrial protein translocation, potentially causing mitochondrial dysfunction. We found a proliferation reduction by Ki67 and an increase in apoptotic proteins CHOP, PUMA, and cleaved caspase 3. Taken together, these data reveal that combination of HC-7366 and osimertinib, and potentially other EGFR TKIs, may be an effective treatment strategy at targeting adaptive resistance mechanisms. Citation Format: Crissy Dudgeon, Sho Fujisawa, Olivia Funk, Feven Tameire, Takashi O. Kangas, Weiyu Zhang, Eric S. Lightcap, David Surguladze, Nandita Bose. HC-7366, a potent GCN2 kinase activator, augments osimertinib therapy to delay resistance in EGFR mutant NSCLC models [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 4666.

Abstract 4221: Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models

Abstract The monocarboxylic acid transporter 4 (Mct-4), a downstream biomarker of hypoxia inducing factor (HIF)-1α, is involved in the cellular response to hypoxia, as indicated by the hypoxic response element in its promoter region. Using a tumorsphere assay as an in vitro 3-dimensional (3D) model generated using 384-well ultra-low attachment (ULA) plates for cell proliferation analysis using a plate-based image cytometer, we identify a hypoxic response in the tumorsphere model that is distinct from that of cells grown under 2-dimensional (2D) normoxic conditions and demonstrate a key role for Mct-4 in enabling 3D growth. The tumorsphere model yields evidence of an essential role for Mct-4 in multiple cell lines which were genetically modified to underexpress and overexpress Mct-4, evidence not apparent in a standard 2D model of growth in the same cell lines. In addition, we identify the effects of overexpressing Mct-4 in cancer cell migration using a transwell chamber assay. We also show that the response to hypoxia may be circumvented by transfection with a CMV promoter driven Mct-4, which confers constitutive 3D growth, wherein tumorsphere growth inhibition by small molecule HIF-1α inhibitors is mitigated. Finally, we demonstrate quantifiable gene/protein expression differences between 2D and 3D cancer models based on the normoxic and hypoxic conditions. Therefore, the tumorsphere 3D model generated using 384-well ULA plates in combination with high-throughput image cytometer is demonstrated to provide a convenient, robust, and reproducible tool and method for the elucidation of mechanisms of action underlying tumor growth and migration in the hypoxic tumor microenvironment. Citation Format: James McDonald, Leo Li-Ying Chan, Sarah Kessel, Bo Lin, Paul Weingarten. Characterization and comparison of hypoxia inducing factors on tumor growth and metastasis in 2D and 3D tumor models [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 4221.

Abstract 2918: Potential for a novel therapeutic target: Effects of lactate & lactylation in expression of key regulatory cancer proteins

Abstract The purpose of our study is to investigate the immunologic, metastatic, and gene regulation effects of lactate in the context of cancer in normoxic and hypoxic conditions to identify novel biomarkers and potential therapeutic targets. We aim to modify the in-vitro cancer microenvironment to show how lactate and lactylation can be exploited using existing cancer treatments. While hyperlactatemia is defined as lactate levels between 2 mmol/L and 4 mmol/L (Foucher et al 2023), tumor biopsies have demonstrated extracellular concentrations as high as 40 mM (Pérez-Tomás et al 2020). Through CTG viability assays we identified lactate concentrations that PANC1 cells can survive, which are in agreement with the in-vivo findings of Brizel et al (2001). We observed pancreatic cancer cells achieving over 50% viability for 5 days at 50 mM lactate concentrations while at 30 mM on day 5 they proliferated past their initial plating numbers. Furthermore, Brizel et al (2001) found that patients with high tumor lactate concentrations had a significantly higher incidence of metastatic relapse. Through our scratch assay, we show qualitative evidence of increased mobility and inferred migration potential of PANC1 at 7.5mM [lactate] compared to the absence of lactate. We have also observed increased migration at higher lactate concentrations for which we are pursuing quantitative measurements to evaluate significance. Migration experiments will be followed by the investigation of gene regulation (eg. mRNA seq, lactylation profile) involved in the observed increased mobility. Lactate has been shown to affect immune regulation, namely suppression of iNKT (Fu et al, 2020), macrophages (Yang et al, 2020), dendritic cell differentiation and antigen recognition/presentation (Wang et al, 2022), however not much is known about its effect on NK cells and their cytokine expression. Through NK92MI/PANC1 cocultures we show the effects of lactate concentration on the killing ability of NK cells (in the works). And plan on investigating the cytokine profile leading to the phenotypic killing differences. Lastly, via lactate-titrated incubation of PANC1 cells in normoxia we have demonstrated a positive correlation of HIF1a expression, pan-lactyllysine lactylation, and microenvironment lactate concentration. We speculate HIF1a inhibition post-lactate-sensitization may induce cancer killing and we will investigate this theory in normoxia and hypoxia using existing treatment options. Citation Format: Christos P. Costeas, Connor Purcell, Wafik S. El-Deiry. Potential for a novel therapeutic target: Effects of lactate & lactylation in expression of key regulatory cancer proteins [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 2918.

Hypoxia inducible factor-1α facilitates transmissible gastroenteritis virus replication by inhibiting type I and type III interferon production

Transmissible gastroenteritis virus (TGEV) is characterized by watery diarrhea, vomiting, and dehydration and is associated with high mortality especially in newborn piglets, causing significant economic losses to the global pig industry. Hypoxia inducible factor-1α (HIF-1α) has been identified as a key regulator of TGEV-induced inflammation, but understanding of the effect of HIF-1α on TGEV infection remains limited. This study found that TGEV infection was associated with a marked increase in HIF-1α expression in ST cells and an intestinal organoid epithelial monolayer. Furthermore, HIF-1α was shown to facilitate TGEV infection by targeting viral replication, which was achieved by restraining type I and type III interferon (IFN) production. In vivo experiments in piglets demonstrated that the HIF-1α inhibitor BAY87–2243 significantly reduced HIF-1α expression and inhibited TGEV replication and pathogenesis by activating IFN production. In summary, we unveiled that HIF-1α facilitates TGEV replication by restraining type I and type III IFN production in vitro, ex vivo, and in vivo. The findings from this study suggest that HIF-1α could be a novel antiviral target and candidate drug against TGEV infection.

HIF-1α in cartilage homeostasis, apoptosis, and glycolysis in mice with steroid-induced osteonecrosis of the femoral head.

With the prevalence of coronavirus disease 2019, the administration of glucocorticoids (GCs) has become more widespread. Treatment with high-dose GCs leads to a variety of problems, of which steroid-induced osteonecrosis of the femoral head (SONFH) is the most concerning. Since hypoxia-inducible factor 1α (HIF-1α) is a key factor in cartilage development and homeostasis, it may play an important role in the development of SONFH. In this study, SONFH models were established using methylprednisolone (MPS) in mouse and its proliferating chondrocytes to investigate the role of HIF-1α in cartilage differentiation, extracellular matrix (ECM) homeostasis, apoptosis and glycolysis in SONFH mice. The results showed that MPS successfully induced SONFH in vivo and vitro, and MPS-treated cartilage and chondrocytes demonstrated disturbed ECM homeostasis, significantly increased chondrocyte apoptosis rate and glycolysis level. However, compared with normal mice, not only the expression of genes related to collagens and glycolysis, but also chondrocyte apoptosis did not demonstrate significant differences in mice co-treated with MPS and HIF-1α inhibitor. And the effects observed in HIF-1α activator-treated chondrocytes were similar to those induced by MPS. And HIF-1α degraded collagens in cartilage by upregulating its downstream target genes matrix metalloproteinases. The results of activator/inhibitor of endoplasmic reticulum stress (ERS) pathway revealed that the high apoptosis rate induced by MPS was related to the ERS pathway, which was also affected by HIF-1α. Furthermore, HIF-1α affected glucose metabolism in cartilage by increasing the expression of glycolysis-related genes. In conclusion, HIF-1α plays a vital role in the pathogenesis of SONFH by regulating ECM homeostasis, chondrocyte apoptosis, and glycolysis.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, −10 and −11

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, −10, and −11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, −10, and −11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, −10, and −11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, −10 and −11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

Potential Effects of NO-Induced Hypoxia-Inducible Factor-1α on Yak Meat Tenderness during Post-Mortem Aging.

The objective of this study was to investigate the mechanism underlying nitric oxide (NO)-induced hypoxia-inducible factor-1α (HIF-1α) and its impact on yak muscle tenderness during post-mortem aging. The Longissimus thoracis et lumborum (LTL) muscle of yak were incubated at 4 °C for 0, 3, 6, 9, 12, 24, and 72 h after treatment with 0.9% saline, NO activator, or a combination of the NO activator and an HIF-1α inhibitor. Results indicated that elevated NO levels could increase HIF-1α transcription to achieve stable expression of HIF-1α protein (P < 0.05). Additionally, elevated NO triggered HIF-1α S-nitrosylation, which further upregulated the activity of key glycolytic enzymes, increased glycogen consumption, accelerated lactic acid accumulation, and decreased pH (P < 0.05). These processes eventually improved the tenderness of yak muscle during post-mortem aging (P < 0.05). The results demonstrated that NO-induced activation of HIF-1α S-nitrosylation enhanced glycolysis during post-mortem aging and provided a possible pathway for improving meat tenderness.

The role of Sirtuin 1 in regulation of fibrotic genes expression in pre-adipocytes.

Considering inhibition of pre-adipocyte cells differentiation in adipose tissue fibrosis, we aimed to explore whether Sirt1 and Hif-1α in pre-adipocytes have a significant effect on fibrotic gene expression. 3T3-L1 pre-adipocytes were transfected with SIRT1-specific siRNA, confirmed by real-time polymerase chain reaction (RT-PCR) and western blotting. Additionally, cells were treated with varying concentrations of resveratrol and sirtinol as the activator and inhibitor of Sirt1, respectively. Involvement of Hif-1α was evaluated by treatment with echinomycin. Subsequently, we assessed the gene and protein expressions related to fibrosis in the extracellular matrix of adipose tissue, including collagen VI (Col VI), lysyl oxidase (Lox), matrix metalloproteinase-2 (Mmp-2), Mmp-9, and osteopontin (Opn) in pre-adipocytes through RT-PCR and western blot. The current study demonstrated that Sirt1 knockdown and reduced enzyme activity significantly increased the expression of Col VI, Lox, Mmp-2, Mmp-9, and Opn genes in the treated 3T3-L1 cells compared to the control group. Interestingly, resveratrol significantly decreased the gene expression related to the fibrosis pathway. Inhibition of Hif-1α by echinomycin led to a significant reduction in Col VI, Mmp-2, and Mmp-9 gene expression in the treated group compared to the control. This study highlights that down-regulation of Sirt1 might be a predisposing factor in the emergence of adipose tissue fibrosis by enhancing the expression of extracellular matrix (ECM) components. Activation of Sirt1, similar to suppressing of Hif-1α in pre-adipocytes may be a beneficial approach for attenuating fibrotic gene expression. The online version contains supplementary material available at 10.1007/s40200-024-01389-4.

Gentiopicroside improves non-alcoholic steatohepatitis by activating PPARα and suppressing HIF1.

Gentiopicroside (GPS) is a highly water-soluble small-molecule drug and the main bioactive secoiridoid glycoside of Gentiana scabra that has been shown to have hepatoprotective effects against non-alcoholic steatohepatitis (NASH), a form of non-alcoholic fatty liver disease (NAFLD) that can progress to cirrhosis and hepatocellular carcinoma. However, the effects of GPS on NASH and the underlying mechanisms remain obscure. Firstly, a high-fat, high-cholesterol (HFHC) diet and a high-sugar solution containing d-fructose and d-glucose were used to establish a non-alcoholic steatohepatitis (NASH) mice model. Secondly, we confirmed GPS supplementation improve metabolic abnormalities and reduce inflammation in NASH mice induced by HFHC and high-sugar solution. Then we used metabolomics to investigate the mechanisms of GPS in NASH mice. Metabolomics analysis showed GPS may work through the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway and glycine, serine, and threonine metabolism. Functional metabolites restored by GPS included serine, glycine, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Western blot and qRT-PCR analysis confirmed GPS improve NASH by regulating PPARα and Hypoxia-Inducible Factor-1α (HIF-1α) signaling pathways. In vitro, studies further demonstrated EPA and DHA enhance fatty acid oxidation through the PPARα pathway, while serine and glycine inhibit oxidative stress through the HIF-1α pathway in palmitic acid-stimulated HepG2 cells. Our results suggest GPS's anti-inflammatory and anti-steatosis effects in NASH progression are related to the suppression of HIF-1α through the restoration of L-serine and glycine and the activation of PPARα through increased EPA and DHA.

CoCl2-Induced hypoxia promotes hPDLSCs osteogenic differentiation through AKT/mTOR/4EBP-1/HIF-1α signaling and facilitates the repair of alveolar bone defects.

Bone defects are characterized by a hypoxic environment, which affects bone tissue repair. However, the role of hypoxia in the repair of alveolar bone defects remains unclear. Human periodontal ligament stem cells (hPDLSCs) are high-quality seed cells for repairing alveolar bone defects, whose behavior changes under hypoxia. However, their mechanism of action is not known and needs to be elucidated. We hypothesized that hypoxia might be beneficial to alveolar bone defect repair and the osteogenic differentiation of hPDLSCs. To test this hypothesis, cobalt chloride (CoCl2) was used to create a hypoxic environment, both in vitro and in vivo. In vitro study, the best osteogenic effect was observed after 48 h of hypoxia in hPDLSCs, and the AKT/mammalian target of rapamycin/eukaryotic translation initiation factor 4e-binding protein 1 (AKT/mTOR/4EBP-1) signaling pathway was significantly upregulated. Inhibition of the AKT/mTOR/4EBP-1 signaling pathway decreased the osteogenic ability of hPDLSCs under hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) expression. The inhibition of HIF-1α also decreased the osteogenic capacity of hPDLSCs under hypoxia without significantly affecting the level of phosphorylation of AKT/mTOR/4EBP-1. In vitro study, Micro-CT and tissue staining results show better bone regeneration in hypoxic group than control group. These results suggested that hypoxia promoted alveolar bone defect repair and osteogenic differentiation of hPDLSCs, probably through AKT/mTOR/4EBP-1/HIF-1α signaling. These findings provided important insights into the regulatory mechanism of hypoxia in hPDLSCs and elucidated the effect of hypoxia on the healing of alveolar bone defects. This study highlighted the importance of physiological oxygen conditions for tissue engineering.

Changes of bone remodeling, cartilage damage and apoptosis-related pathways in broilers with femoral head necrosis 1

Femoral head necrosis (FHN) is a common leg disorder in the poultry industry often leads to significant cartilage damage. The mechanism behind abnormal apoptosis in FHN broilers, leading to cartilage damage, remains unclear; although endoplasmic reticulum stress (ERS) has been found to play a role in glucocorticoid-induced FHN broilers. In this study, we collected samples from broilers with femoral head separation (FHS) and femoral head separation accompanied with growth plate lacerations (FHSL) in a broiler farm. The aim was to investigate the potential association between the severity of FHN, bone remodeling and cartilage damage. Additionally, primary chondrocytes were treated with methylprednisolone (MP) to construct an in vitro FHN model, followed by inhibition or activation of ERS or hypoxia inducible factor-1α (HIF-1α) to further investigate the mechanism of apoptosis in cartilage. The results suggested that cartilage appeared to be the appropriate tissue to investigate the potential mechanisms of FHN, as the degree of cartilage damage was found to be closely related to the severity of the disease. Bone quality was only affected in FHSL broilers, although factors related to bone metabolism were significantly altered among FHN-affected broilers. In addition, cartilage in FHN-affected broilers exhibited high levels of apoptosis and upregulated expression of ERS-related and HIF-1α, which was consistent with both in vivo and in vitro findings after MP treatment. The results were further supported by treatment with HIF-1α or ERS inhibition or activation. In conclusion, bone remodeling and cartilage homeostasis were affected in FHN broilers, but only cartilage damage was significantly exacerbated with FHN development. Moreover, activation of ERS or HIF-1α resulted in apoptosis in cartilage, thus exhibiting a significant correlation with FHN severity.

SKP alleviates the ferroptosis in diabetic kidney disease through suppression of HIF-1α/HO-1 pathway based on network pharmacology analysis and experimental validation.

Diabetic kidney disease (DKD) represents a microvascular complication of diabetes mellitus. Shenkang Pills (SKP), a traditional Chinese medicine formula, has been widely used in the treatment of DKD and has obvious antioxidant effect. Ferroptosis, a novel mode of cell death due to iron overload, has been shown to be associated with DKD. Nevertheless, the precise effects and underlying mechanisms of SKP on ferroptosis in diabetic kidney disease remain unclear. The active components of SKP were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Protein-protein interaction (PPI) network and Herb-ingredient-targets gene network were constructed using Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted utilizing the Metascape system database. Additionally, an in vivo model of DKD induced by Streptozotocin (STZ) was established to further investigate and validate the possible mechanisms underlying the effectiveness of SKP. We retrieved 56 compounds and identified 223 targets of SKP through the TCMSP database. Key targets were ascertained using PPI network analysis. By constructing a Herb-Ingredient-Targets gene network, we isolated the primary active components in SKP that potentially counteract ferroptosis in diabetic kidney disease. KEGG pathway enrichment analysis suggested that SKP has the potential to alleviate ferroptosis through HIF signaling pathway, thereby mitigating renal injury in DKD. In animal experiments, fasting blood glucose, 24h urine protein, urea nitrogen and serum creatine were measured. The results showed that SKP could improve DKD. Results from animal experiments were also confirmed the efficacy of SKP in alleviating renal fibrosis, oxidative stress and ferroptosis in DKD mice. These effects were accompanied by the significant reductions in renal tissue expression of HIF-1α and HO-1 proteins. The mRNA and immunohistochemistry results were the same as above. SKP potentially mitigating renal injury in DKD by subduing ferroptosis through the intricacies of the HIF-1α/HO-1 signaling pathway.

PX-478, an HIF-1α inhibitor, impairs mesoCAR T cell antitumor function in cervical cancer.

Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable success in treating hematological malignancies. However, its efficacy against solid tumors, including cervical cancer, remains a challenge. Hypoxia, a common feature of the tumor microenvironment, profoundly impacts CAR T cell function, emphasizing the need to explore strategies targeting hypoxia-inducible factor-1α (HIF-1α). In this study, we evaluated the effects of the HIF-1α inhibitor PX-478 on mesoCAR T cell function through in-silico and in vitro experiments. We conducted comprehensive analyses of HIF-1α expression in cervical cancer patients and examined the impact of PX-478 on T cell proliferation, cytokine production, cytotoxicity, and exhaustion markers. Our in-silico analyses revealed high expression of HIF-1α in cervical cancer patients, correlating with poor prognosis. PX-478 effectively reduced HIF-1α levels in T and HeLa cells. While PX-478 exhibited dose-dependent inhibition of antigen-nonspecific T and mesoCAR T cell proliferation, it had minimal impact on antigen-specific mesoCAR T cell proliferation. Notably, PX-478 significantly impaired the cytotoxic function of mesoCAR T cells and induced terminally exhausted T cells. Our results underscore the significant potential and physiological relevance of the HIF-1α pathway in determining the fate and function of both T and CAR T cells. However, we recognize the imperative for further molecular investigations aimed at unraveling the intricate downstream targets associated with HIF-1α and its influence on antitumor immunity, particularly within the context of hypoxic tumors. These insights serve as a foundation for the careful development of combination therapies tailored to counter immunosuppressive pathways within hypoxic environments and fine-tune CAR T cell performance in the intricate tumor microenvironment.

VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation

The Von Hippel–Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Retraction: Multimodal targeting of tumor vasculature and cancer stem-like cells in sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy.

Retraction: Multimodal targeting of tumor vasculature and cancer stem-like cells in sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy.

HIV coinfection exacerbates HBV-induced liver fibrogenesis through a HIF-1α- and TGF-β1-dependent pathway

Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-β1 signaling. The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-β1 (TGF-β1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-β1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-β1-induced fibrogenesis. Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-β1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-β1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.

Abstract TP26: Hypothermic and Non-Hypothermic Neuroprotection by Phenothiazine in Acute Ischemic Stroke: Modulation of NLRP3 Inflammasome-Related-Inflammation via Different Pathways of RIPK1/RIPK3-DRP1 or HIF-1α

Background: Inflammation after successful revascularization worsened outcomes in ischemic stroke. NLRPyrin domain containing 3 (NLRP3) inflammasome mediated inflammatory responses, in which receptor-interacting protein kinase (RIPK1) activated dynamin-related protein 1 (DRP1) in a RIPK3-dependent fashion. Hypoxia-inducible factor 1α (HIF-1α) regulated ischemic inflammation through NLRP3 inflammasome complex, thus apoptosis after stroke. Phenothiazine, including chlorpromazine and promethazine (C+P), induced hypothermic and non-hypothermic neuroprotection. We determined effects of RIPK1/RIPK3-DRP1 or HIF-1α pathways underlying the dual neuroprotection by C+P. Methods: 144 adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO), followed by 24 h reperfusion. 8mg/kg of C+P was injected at the onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation (NLRP3, ASC, Caspase-1, IL-1β and IL-18) and cytochrome C-apoptosis (cytochrome C, APAF-1, Caspase-9 and Caspase-3) were assessed. Apoptotic cell death, and the infiltration of neutrophils and macrophages were evaluated. Co-localization of RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. Results: C+P significantly reduced brain inflammation, neuronal apoptosis and immune cell infiltration, with or without hypothermia. C+P with induced-hypothermia and RIPK1 inhibitor significantly reduced infarct volumes, the expression of RIPK1, RIPK3, DRP-1, NLRP3-inflammation process and cytochrome C-apoptosis, as well as the co-localization of RIPK1/RIPK3, suggesting the hypothermic effect underlying the RIPK1/RIPK3-DRP1 pathway. In non-hypothermia effect, HIF-1α expression was reduced by C+P or HIF-1α inhibitor, in concomitant with reduced NLRP3 inflammasome, cytochrome C-apoptosis, and decreased interaction of HIF-1α and NLRP3. Conclusion: Hypothermic and non-hypothermic neuroprotection of C+P involve different pathways. The hypothermic effect was via RIPK1/RIPK3-DRP1 signaling, while non-hypothermic effect was mediated by HIF-1α. This provides a theoretical basis for future precise exploration of hypothermic and non-hypothermic neuroprotection.

Abstract WMP1: Neuroprotection by Normobaric Oxygen (NBO): Modulating Inflammasome (NLRP3) Activation and Stress Granule Formation in Ischemic Stroke

Background: NBO has emerged as a promising neuroprotective strategy for ischemic stroke. Key to comprehending these effects is understanding its mechanisms. This study examines the pivotal roles of HIF-1α as an oxygen sensor, the protective emergence of stress granules (SGs) in response to various stimuli, and the inflammatory cell death process known as pyroptosis, triggered by inflammasomes. We elucidated the interplay between HIF-1α, SGs, inflammasomes in ischemia/reperfusion injury and in NBO-induced neuroprotection. Methods: A total of 132 adult male SD rats were subjected to 2 h middle cerebral artery occlusion (MCAO), followed by 2, 6, 24 or 48 h of reperfusion. At the onset of reperfusion, oxygen was inhaled for 2 h at concentration of 95% and flow rate of 2 L/min. YC-1 (HIF-1α inhibitor) was administered 2 h before MCAO. Brain damage was evaluated by infarct volumes (TTC), LDH and ROS levels (ELISA), apoptotic cell death (flow cytometry and TUNEL). Pyroptosis was evaluated by flow cytometry. mRNA and protein levels of HIF-1α and inflammasome related factors, including IL-18, IL-1β, ASC, NLRP3, TXNIP, N-GSDMD, cleaved-Caspase-1, as well as SGs proteins including DDX3X, G3BP1, TIA-1 were examined. Co-IP was used to detect the interaction between DDX3X and G3BP1, as well as DDX3X and NLRP3. Results: Infarct volumes, LDH and ROS levels, apoptotic and pyroptotic cell death, as well as HIF-1α expression were all increased after ischemia/reperfusion. All these increases were reversed by either NBO or YC-1. NBO or YC-1 further reversed the increase in expression of NLRP3 inflammasome related proteins and suppressed the interaction between DDX3X and NLRP3, thereby suppressing inflammasome activation. NBO or YC-1 stimulated the expressions of SGs proteins and facilitated the interaction between DDX3X and G3BP1, thereby promoting the formation of SGs, in association with cell protection. Conclusion: Our findings highlight the neuroprotective effects of NBO in ischemic stroke. This protection is attributed to its dual capacity to inhibit NLRP3 inflammasome activation while simultaneously promoting formation of SGs. Central to these processes, HIF-1α, as an instrumental modulator, sheds light on potential therapeutic targets for stroke intervention.

Siah2 inhibitor and the metabolic antagonist Oxamate retard colon cancer progression and downregulate PD1 expression.

Solid tumors such as colon cancer are characterized by rapid and sustained cell proliferation, which ultimately results in hypoxia, induction of hypoxia-inducible factor-1α (HIF-1α), and activation of glycolysis to promote tumor survival and immune evasion. We hypothesized that a combinatorial approach of menadione (MEN) as an indirect HIF-1α inhibitor and sodium oxamate (OX) as a glycolysis inhibitor may be a promising treatment strategy for colon cancer. We investigated the potential efficacy of this combination for promoting an antitumor immune response and suppressing tumor growth in a rat model of colon cancer. Colon cancer was induced by once-weekly subcutaneous injection of 20 mg/kg dimethylhydrazine (DMH) for 16 weeks. Control rats received the vehicle and then no further treatment (negative control) or MEN plus OX for 4 weeks (drug control). Dimethylhydrazine-treated rats were then randomly allocated to four groups: DMH alone group and other groups treated with MEN, OX, and a combination of (MEN and OX) for 4 weeks. Serum samples were assayed for the tumor marker carbohydrate antigen (CA19.9), while expression levels of HIF-1α, caspase-3, PHD3, LDH, and PD1 were evaluated in colon tissue samples by immunoassay and qRT-PCR. Additionally, Ki-67 and Siah2 expression levels were examined by immunohistochemistry. The combination of MEN plus OX demonstrated a greater inhibitory effect on the expression levels of HIF-1α, Siah2, LDH, Ki-67, and PD1, and greater enhancement of caspase-3 and PHD3 expression in colon cancer tissues than either drug alone. Simultaneous targeting of hypoxia and glycolysis pathways by a combination of MEN and OX could be a promising therapy for inhibiting colon cancer cell growth and promoting antitumor immunity [1].

LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α

Hypoxia-inducible factor 1 (HIF1) is critically important for driving angiogenesis and tumorigenesis. Linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin ligase capable of catalyzing protein linear ubiquitination to date, is implicated in cell signaling and associated with cancers. However, the role and mechanism of LUBAC in regulating the expression and function of HIF1α, the labile subunit of HIF1, remain to be elucidated. Herein we showed that LUBAC increases HIF1α protein expression in cultured cells and tissues of human lung cancer and enhances HIF1α DNA-binding and transcriptional activities, which are dependent upon LUBAC enzymatic activity. Mechanistically, LUBAC increases HIF1α stability through antagonizing HIF1α decay by the chaperone-mediated autophagy (CMA)-lysosome pathway, thereby potentiating HIF1α activity. We further demonstrated that HIF1α selectively interacts with HOIP (the catalytic subunit of LUBAC) primarily in the cytoplasm. LUBAC catalyzes linear ubiquitination of HIF1α at lysine 362. Linear ubiquitination shields HIF1α from interacting with heat-shock cognate protein of 70 kDa and lysosome-associated membrane protein type 2 A, two components of CMA. Consequently, linear ubiquitination confers protection against CMA-mediated destruction of HIF1α, increasing HIF1α stability and activity. We found that prolyl hydroxylation is not a perquisite for LUBAC’s effects on HIF1α. Functionally, LUBAC facilitates proliferation, clonogenic formation, invasion and migration of lung cancer cells. LUBAC also boosts angiogenesis and exacerbates lung cancer growth in mice, which are greatly compromised by inhibition of HIF1α. This work provides novel mechanistic insights into the role of LUBAC in regulating HIF1α homeostasis, tumor angiogenesis and tumorigenesis of lung cancer, making LUBAC an attractive therapeutic target for cancers.