HIF-1α Degradation Research Articles

Human prolyl hydroxylase domain 2 reacts with O2 and 2-oxoglutarate to enable formation of inactive Fe(III).2OG.hypoxia-inducible-factor α complexes

Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O2 is present, Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases (human PHD1-3) promote HIFα degradation via prolyl-hydroxylation. We report crystallographic, spectroscopic, and biochemical characterization of stable and inactive PHD2.Fe(III).2OG complexes. Aerobic incubation of PHD2 with Fe(II) and 2OG enables formation of PHD2.Fe(III).2OG complexes which bind HIF1-2α to give inactive PHD2.Fe(III).2OG.HIF1-2α complexes. The Fe(III) oxidation state in the inactive complexes was shown by EPR spectroscopy. L-Ascorbate hinders formation of the PHD2.Fe(III).2OG.(+/-HIFα) complexes and slowly regenerates them to give the catalytically active PHD2.Fe(II).2OG complex. Crystallographic comparison of the PHD2.Fe(III).2OG.HIF2α complex with the analogous anaerobic Fe(II) complex reveals near identical structures. Exposure of the anaerobic PHD2.Fe(II).2OG.HIF2α crystals to O2 enables in crystallo hydroxylation. The resulting PHD2.product structure, manifests conformational changes compared to the substrate structures. The results have implications for the role of the PHDs in hypoxia sensing and open new opportunities for inhibition of the PHDs and other 2OG dependent oxygenases by promoting formation of stable Fe(III) complexes.

MiR-1307-5p enhances fibroblast transdifferentiation to exacerbate chronic obstructive pulmonary disease through regulating FBXL16/HIF1α axis

Chronic obstructive pulmonary disease (COPD) is an irreversible and progressive chronic inflammatory lung disease which affects millions of people worldwide. Activated fibroblasts are observed to accumulate in lung of COPD patients and promote COPD progression through aberrant extracellular matrix (ECM) deposition. In this study, we identified that miR-1307-5p expression was significantly increased in lung fibroblasts derived from COPD patients. Mechanistically, we found that upregulation of miR-1307-5p promoted TGF-β induced lung fibroblast activation and transdifferentiation. We also identified FBXL16 as a direct target for miR-1307-5p mediated myofibroblast activation in COPD. Knockdown of FBXL16 by siRNA prominently increased the expression of myofibroblast markers in MRC-5 fibroblasts after TGF-β administration. Ectopic expression of FBXL16 in MRC-5 counteracted miR-1307-5p agomir-induced fibroblast transdifferentiation. Furthermore, We found that miR-1307-5p promoted pulmonary fibroblast transdifferentiation through FBXL16 regulated HIF1α degradation. In general, our findings indicate that miR-1307-5p is important for COPD pathogenesis, and may serve as a potential target for COPD treatment.

USP7 depletion potentiates HIF2α degradation and inhibits clear cell renal cell carcinoma progression

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel Lindau (VHL) gene loss of function mutation, which leads to the accumulation of hypoxia-inducible factor 2α (HIF2α). HIF2α has been well-established as one of the major oncogenic drivers of ccRCC, however, its therapeutic targeting remains a challenge. Through an analysis of proteomic data from ccRCCs and adjacent non-tumor tissues, we herein revealed that Ubiquitin-Specific Peptidase 7 (USP7) was upregulated in tumor tissues, and its depletion by inhibitors or shRNAs caused significant suppression of tumor progression in vitro and in vivo. Mechanistically, USP7 expression is activated by the transcription factors FUBP1 and FUBP3, and it promotes tumor progression mainly by deubiquitinating and stabilizing HIF2α. Moreover, the combination of USP7 inhibitors and afatinib (an ERBB family inhibitor) coordinately induce cell death and tumor suppression. In mechanism, afatinib indirectly inhibits USP7 transcription and accelerates the degradation of HIF2α protein, and the combination of them caused a more profound suppression of HIF2α abundance. These findings reveal a FUBPs-USP7-HIF2α regulatory axis that underlies the progression of ccRCC and provides a rationale for therapeutic targeting of oncogenic HIF2α via combinational treatment of USP7 inhibitor and afatinib.

Crystallographic and Selectivity Studies on the Approved HIF Prolyl Hydroxylase Inhibitors Desidustat and Enarodustat.

Prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3) are 2-oxoglutarate (2OG)-dependent oxygenases catalysing C-4 hydroxylation of prolyl residues in α-subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), modifications that promote HIF-α degradation via the ubiquitin-proteasome pathway. Pharmacological inhibition of the PHDs induces HIF-α stabilisation, so promoting HIF target gene transcription. PHD inhibitors are used to treat anaemia caused by chronic kidney disease (CKD) due to their ability to stimulate erythropoietin (EPO) production. We report studies on the effects of the approved PHD inhibitors Desidustat and Enarodustat, and the clinical candidate TP0463518, on activities of a representative set of isolated recombinant human 2OG oxygenases. The three molecules manifest selectivity for PHD inhibition over that of the other 2OG oxygenases evaluated. We obtained crystal structures of Desidustat and Enarodustat in complex with the human 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), which, together with modelling studies, inform on the binding modes of Desidustat and Enarodustat to active site Fe(II) in 2OG oxygenases, including PHD1-3. The results will help in the design of selective inhibitors of both the PHDs and other 2OG oxygenases, which are of medicinal interest due to their involvement inter alia in metabolic regulation, epigenetic signalling, DNA-damage repair, and agrochemical resistance.

SOX9 promotes hypoxic pulmonary hypertension through stabilization of DPP4 in pulmonary artery smooth muscle cells

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH.

Hepatocyte aquaporin 8-mediated water transport facilitates bile dilution and prevents gallstone formation in mice

Although water channel aquaporin-8 (AQP8) has been implicated in hepatic bile formation and liver diseases associated with abnormal bile flow in human and animal studies, direct evidence of its involvement in bile secretion is still lacking. This study aimed to determine the role of AQP8 in bile secretion and gallstone formation. We generated various transgenic knock-in and knockout mouse models and assessed liver AQP8 expression by immunostaining and immunoblotting, hepatic bile secretion by cannulation of the common bile duct, cholesterol gallstone formation by feeding a high-fat lithogenic diet, and identified regulatory small molecules by screening the organic fractions of cholagogic Chinese herbs and biochemical characterization. We identified a novel expression pattern of AQP8 protein in the canalicular membrane of approximately 50% of the liver lobules. AQP8-deficient mice exhibited impaired hepatic bile formation, characterized by the secretion of concentrated bile with a lower flow rate and higher levels of bile lipids than that of wild-type littermates. AQP8-/- mice showed accelerated gallstone formation, which was rescued by AAV-mediated hepatic expression of AQP8 or AQP1. Moreover, we identified a small molecule, scutellarin, that upregulates hepatocyte AQP8 expression in vitro and in vivo. In AQP8+/+ mice, scutellarin significantly increased bile flow, decreased bile lipid concentrations, and prevented gallstone formation compared to AQP8-/- mice. Molecular studies revealed that scutellarin promoted the ubiquitination and degradation of HIF-1α, a transcriptional negative regulator of AQP8, by disrupting its interactions with HSP90. AQP8 plays a crucial role in facilitating water transport and bile dilution during hepatic bile formation, thereby mitigating gallstone formation in mice. Small-molecule intervention validated hepatocyte AQP8 as a promising drug target for gallstone therapy. The incidence of gallstone disease is high, and current drug treatments for gallstones are very limited, necessitating the identification of novel drug targets for developing new drugs with universal applicability. To our knowledge, this is the first study to provide direct evidence that hepatic water channel AQP8 plays a key role in bile dilution and gallstone formation. Modulation of hepatic water transport may provide a universal therapeutic strategy for all types of gallstone diseases.

DDR1 Drives Malignant Progression of Gastric Cancer by Suppressing HIF-1α Ubiquitination and Degradation.

The extracellular matrix (ECM) has been demonstrated to be dysregulated and crucial for malignant progression in gastric cancer (GC), but the mechanism is not well understood. Here, that discoidin domain receptor 1 (DDR1), a principal ECM receptor, is recognized as a key driver of GC progression is reported. Mechanistically, DDR1 directly interacts with the PAS domain of hypoxia-inducible factor-1α (HIF-1α), suppresses its ubiquitination and subsequently strengthens its transcriptional regulation of angiogenesis. Additionally, DDR1 upregulation in GC cells promotes actin cytoskeleton reorganization by activating HIF-1α/ Ras Homolog Family Member A (RhoA)/Rho-associated protein kinase 1 (ROCK1) signaling, which in turn enhances the metastatic capacity. Pharmacological inhibition of DDR1 suppresses GC progression and angiogenesis in patient-derived xenograft (PDX) and organoid models. Taken together, this work first indicates the effects of the DDR1-HIF-1α axis on GC progression and reveals the related mechanisms, providing experimental evidence for DDR1 as a therapeutic target for GC.

Self-Produced O2 CNs-Based Nanocarriers of DNA Hydrophobization Strategy Triggers Photodynamic and Mitochondrial-Derived Ferroptosis for Hepatocellular Carcinoma Combined Treatment.

Hypoxia can aggravate tumor occurrence, development, invasion, and metastasis, and greatly inhibit the photodynamic therapy (PDT) effect. Herein, carbon nitride (CNs)-based DNA and photosensitizer co-delivery systems (BPSCNs) with oxygen-producing functions are developed to address this problem. Selenide glucose (Seglu) is used as the dopant to prepare red/NIR-active CNs (SegluCNs). The tumor-targeting unit Bio-PEG2000 is utilized to construct BPSCNs nanoparticles through esterification reactions. Furthermore, DNA hydrophobization is realized via mixing P53 gene with a positively charged mitochondrial-targeted near-infrared (NIR) emitting photosensitizer (MTTPY), which is encapsulated in non-cationic BPSCNs for synergistic delivery. Ester bonds in BPSCNs@MTTPY-P53 complexes can be disrupted by lipase in the liver to facilitate P53 release, upregulated P53 expression, and promoted HIF-1α degradation in mitochondria. In addition, the oxygen produced by the complexes improved the hypoxic microenvironment of hepatocellular carcinoma (HCC), synergistically downregulated HIF-1α expression in mitochondria, promoted mitochondrial-derived ferroptosis and enhanced the PDT effect of the MTTPY unit. Both in vivo and in vitro experiments indicated that the transfected P53-DNA, produced O2 and ROS by these complexes synergistically led to mitochondrial-derived ferroptosis in hepatoma cells through the HIF-1α/SLC7A11 pathway, and completely avoiding PDT resistance caused by hypoxia, exerting a significant therapeutic role in HCC treatment.

NXPH4 mediated by m5C contributes to the malignant characteristics of colorectal cancer via inhibiting HIF1A degradation

ObjectiveColorectal cancer (CRC) is a form of malignancy that exhibits a comparatively elevated occurrence and fatality rate. Given the relatively slower progress in diagnostic and therapeutic approaches for CRC, there is a need to investigate more accurate and efficient biomarkers.MethodsCore regulatory genes were screened using the TCGA database, and the expression of neurexophilin 4 (NXPH4) and its prognostic implications were validated using tissue microarray staining. The assessment of NXPH4 functions involved a range of experiments, including cellular, organoid, and murine models. Furthermore, a regulatory network between m5C, NXPH4, and HIF1A was established through several in vitro experiments.ResultsThe overexpression of NXPH4 is associated with unfavorable prognoses in patients with CRC and hepatocellular carcinoma. Additionally, it facilitates the progression of malignant tumors both in laboratory settings and in living organisms of colorectal carcinoma. Our research also reveals that NXPH4 mRNA can avoid degradation through RNautophagy, relying on an m5C-dependent mechanism. Moreover, NXPH4 amplifies the HIF signaling pathway and stabilizes HIF1A by competitively binding to PHD4.ConclusionsNXPH4, regulated by m5C, promotes malignant tumor progression and regulates the HIF pathway. Consequently, targeting NXPH4 through molecular therapies could potentially serve as an efficacious therapeutic strategy for the management of CRC exhibiting elevated NXPH4 expression.Graphical abstract

SENP1 reduces oxidative stress and apoptosis in renal ischaemia-reperfusion injury by deSUMOylation of HIF-1α.

Renal ischaemia-reperfusion injury (RIRI) is a primary cause of acute kidney damage, occurring frequently insituations like renal transplantation, yet the underlying mechanisms were not fully understood. Sentrin-specific protease 1 (SENP1) is an important member of the SENP family, which is widely involved in various diseases. However, the role of SENP1 in RIRI has been unclear. In our study, we discovered that SENP1 was involved in RIRI and reduced renal cell apoptosis and oxidative stress at elevated levels. Further mechanistic studies showed that hypoxia-inducible factor-1α (HIF-1α) was identified as a substrate of SENP1. Furthermore, SENP1 deSUMOylated HIF-1α, which reduced the degradation of HIF-1α, and exerted a renoprotective function. In addition, the protective function was lost after application of the HIF-1α specific inhibitor KC7F2. Briefly, our results fully demonstrated that SENP1 reduced the degradation of HIF-1α and attenuated oxidative stress and apoptosis in RIRI by regulating the deSUMOylation of HIF-1α, suggesting that SENP1 may serve as a potential therapeutic target for the treatment of RIRI.

G protein pathway suppressor 2 suppresses aerobic glycolysis through RACK1-mediated HIF-1α degradation in breast cancer

Aerobic glycolysis has been recognized as a hallmark of human cancer. G protein pathway suppressor 2 (GPS2) is a negative regulator of the G protein-MAPK pathway and a core subunit of the NCoR/SMRT transcriptional co-repressor complex. However, how its biological properties intersect with cellular metabolism in breast cancer (BC) development remains poorly elucidated. Here, we report that GPS2 is low expressed in BC tissues and negatively correlated with poor prognosis. Both in vitro and in vivo studies demonstrate that GPS2 suppresses malignant progression of BC. Moreover, GPS2 suppresses aerobic glycolysis in BC cells. Mechanistically, GPS2 destabilizes HIF-1α to reduce the transcription of its downstream glycolytic regulators (PGK1, PGAM1, ENO1, PKM2, LDHA, PDK1, PDK2, and PDK4), and then suppresses cellular aerobic glycolysis. Notably, receptor for activated C kinase 1 (RACK1) is identified as a key ubiquitin ligase for GPS2 to promote HIF-1α degradation. GPS2 stabilizes the binding of HIF-1α to RACK1 by directly binding to RACK1, resulting in polyubiquitination and instability of HIF-1α. Amino acid residues 70–92 aa of the GPS2 N-terminus bind RACK1. A 23-amino-acid-long GPS2-derived peptide was developed based on this N-terminal region, which promotes the interaction of RACK1 with HIF-1α, downregulates HIF-1α expression and significantly suppresses BC tumorigenesis in vitro and in vivo. In conclusion, our findings indicate that GPS2 decreases the stability of HIF-1α, which in turn suppresses aerobic glycolysis and tumorigenesis in BC, suggesting that targeting HIF-1α degradation and treating with peptides may be a promising approach to treat BC.

DAB2IP inhibits glucose uptake by modulating HIF-1α ubiquitination under hypoxia in breast cancer

Metabolic reprogramming has become increasingly important in tumor biology research. The glucose metabolic pathway is a major energy source and is often dysregulated in breast cancer. DAB2IP is widely reported to be a tumor suppressor that acts as a scaffold protein to suppress tumor malignancy in breast cancer. Interestingly, DAB2IP has also been found to be a potential regulator of glucose uptake; however, the exact mechanism remains unclear. In this study, we found that DAB2IP inhibited glucose uptake under hypoxia conditions in breast cancer cells by suppressing HIF-1α signals. Mechanically, DAB2IP interacted with the E3 ubiquitin ligase STUB1 via its PER domain, thus triggering STUB1 mediated HIF-1α ubiquitylation and degradation, and inhibit glucose metabolism and tumor progression. Deleting the PER domain abrogated the DAB2IP-related inhibitory effects on glucose uptake, intracellular ATP production, and lactic acid production in breast cancer cells. These findings elucidate the biological roles of DAB2IP in cancer-related glucose metabolism as well as a novel mechanism by which STUB1-driven HIF-1α ubiquitylated degradation is regulated in breast cancer.

CCT6A alleviates pulmonary fibrosis by inhibiting HIF-1α-mediated lactate production.

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive fibrotic lung disease. The development of IPF involves different molecular and cellular processes, and recent studies indicate that lactate plays a significant role in promoting the progression of the disease. Nevertheless, the mechanism by which lactate metabolism is regulated and the downstream effects remain unclear. The molecular chaperone CCT6A performs multiple functions in a variety of biological processes. Our research has identified a potential association between CCT6A and serum lactate levels in IPF patients. Herein, we found that CCT6A was highly expressed in type 2 alveolar epithelial cells (AEC2s) of fibrotic lung tissues and correlated with disease severity. Lactate increases the accumulation of lipid droplets in epithelial cells. CCT6A inhibits lipid synthesis by blocking the production of lactate in AEC2s and alleviates bleomycin-induced pulmonary fibrosis in mice. In addition, our results revealed that CCT6A blocks HIF-1α-mediated lactate production by driving the VHL-dependent ubiquitination and degradation of HIF-1α and further inhibits lipid accumulation in fibrotic lungs. In conclusion, we propose that there is a pivotal regulatory role of CCT6A in lactate metabolism in pulmonary fibrosis, and strategies aimed at targeting these key molecules could represent potential therapeutic approaches for pulmonary fibrosis.

Targeting HIF-1α with Specific DNA Yokes for Effective Anticancer Therapy.

Hypoxia, a ubiquitous hallmark in cancer, underscores the significance of targeting HIF-1α, the principal transcriptional factor of hypoxic responses, for effective cancer therapy. Herein, DNA yokes, a novel class of DNA nanomaterials harboring specific HIF-1α binding sequences (hypoxia response elements, HREs), are introduced as nanopharmaceuticals for cancer treatment. Comprising a basal tetrahedral DNA nanostructure and four HRE-bearing overhanging chains, DNA yokes exhibit exceptional stability and prolonged intracellular retention. The investigation reveals their capacity to bind HIF-1α, thereby disrupting its interaction with the downstream genomic DNAs and impeding transcriptional activity. Moreover, DNA yokes facilitate HIF-1α degradation via the ubiquitination pathway, thereby sequestering it from downstream targets and ultimately promoting its degradation. In addition, DNA yokes attenuate cancer cell proliferation, migration, and invasion under hypoxic conditions, while also displaying preferential accumulation within tumors, thereby inhibiting tumor growth and metastasis in vivo. This study pioneers a novel approach to cancer therapy through the development of DNA-based drugs characterized by high stability and low toxicity to normal cells, positioning DNA yokes as promising candidates for cancer treatment.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

Polyphyllin I ameliorates gefitinib resistance and inhibits the VEGF/VEGFR2/p38 pathway by targeting HIF-1a in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been administered as the first-line therapy for patients with EGFR mutations in LUAD, but it is almost inevitable that resistance to EGFR-TKIs therapy eventually arises. Polyphyllin I (PPI), derived from Paris polyphylla rhizomes, has been shown to have potent anti-cancer properties in a range of human cancer types including LUAD. However, the role of PPI in gefitinib resistance and the underlying mechanism remain elusive. PurposeTo evaluate the antitumor impacts of PPI on gefitinib resistance cells and investigate its molecular mechanism. MethodsCCK-8, wound healing, transwell assay, and xenograft model were performed to determine the anti-cancer effects of PPI as well as its ability to overcome gefitinib resistance. Immunoblotting, co-immunoprecipitation, phospho-RTK antibody array, qRT-PCR, and immunofluorescence were utilized to explore the mechanism by which PPI overrides gefitinib resistance. ResultsPPI inhibited cell survival, growth, and migration/invasion in both gefitinib-sensitive (PC9) and -resistant (PC9/GR) LUAD cells (IC50 at 2.0 μM). Significantly, treatment with PPI at 1.0 μM resensitized the resistant cells to gefitinib. Moreover, cell-derived xenograft experiments revealed that the combination of PPI and gefitinib overcame gefitinib resistance. The phospho-RTK array and immunoblotting analyses showed PPI significant inhibition of the VEGFR2/p38 pathway. In addition, molecular docking suggested the interaction between PPI and HIF-1α. Mechanistically, PPI reduced the protein expression of HIF-1α in both normoxia and hypoxia conditions by triggering HIF-1α degradation. Moreover, HIF-1α protein but not mRNA level was elevated in gefitinib-resistant LUAD. We further demonstrated that PPI considerably facilitated the binding of HIF-1α to VHL. ConclusionsWe present a novel discovery demonstrating that PPI effectively counteracts gefitinib resistance in LUAD by modulating the VEGF/VEGFR2/p38 pathway. Mechanistic investigations unveil that PPI facilitates the formation of the HIF-1α /VHL complex, leading to the degradation of HIF-1α and subsequent inhibition of angiogenesis. These findings uncover a previously unidentified mechanism governing HIF-1α expression in reaction to PPI, providing a promising method for therapeutic interventions targeting EGFR-TKI resistance in LUAD.

Wild-type IDH2 is a therapeutic target for triple-negative breast cancer

Mutations in isocitrate dehydrogenases (IDH) are oncogenic events due to the generation of oncogenic metabolite 2-hydroxyglutarate. However, the role of wild-type IDH in cancer development remains elusive. Here we show that wild-type IDH2 is highly expressed in triple negative breast cancer (TNBC) cells and promotes their proliferation in vitro and tumor growth in vivo. Genetic silencing or pharmacological inhibition of wt-IDH2 causes a significant increase in α-ketoglutarate (α-KG), indicating a suppression of reductive tricarboxylic acid (TCA) cycle. The aberrant accumulation of α-KG due to IDH2 abrogation inhibits mitochondrial ATP synthesis and promotes HIF-1α degradation, leading to suppression of glycolysis. Such metabolic double-hit results in ATP depletion and suppression of tumor growth, and renders TNBC cells more sensitive to doxorubicin treatment. Our study reveals a metabolic property of TNBC cells with active utilization of glutamine via reductive TCA metabolism, and suggests that wild-type IDH2 plays an important role in this metabolic process and could be a potential therapeutic target for TNBC.

Ganoderic acid D attenuates gemcitabine resistance of triple-negative breast cancer cells by inhibiting glycolysis via HIF-1α destabilization

BackgroundGemcitabine (GEM) resistance is the primary reason why combination chemotherapy is limited in triple-negative breast cancer (TNBC). Ganoderic acid D (GAD), a natural triterpenoid compound obtained from Ganoderma lucidum, has been shown to have antitumor activities. However, whether GAD can reverse GEM resistance in TNBC requires further investigation. PurposeThis study investigated whether and how GAD could reverse GEM resistance in TNBC as an antitumor adjuvant. MethodsThe effects of GAD on cell proliferation, cell cycle, and glycolysis were studied in vitro using a GEM-resistant (GEM-R) TNBC cell model. We enriched key pathways affected by GAD using proteomics techniques. Western blotting and qPCR were used to detect the expression of glycolysis-related genes after GAD treatment. A mouse resistance model was established using GEM-R TNBC cells, and hematoxylin-eosin staining and immunohistochemistry were used to assess the role of GAD in reversing resistance in vivo. ResultsCellular functional assays showed that GAD significantly inhibited proliferation and glucose uptake in GEM-R TNBC cells. GAD reduces HIF-1α accumulation in TNBC cells under hypoxic conditions through the ubiquitinated protease degradation pathway. Mechanistically, GAD activates the p53/MDM2 pathway, promoting HIF-1α ubiquitination and proteasomal degradation and downregulating HIF-1α-dependent glycolysis genes like GLUT1, HK2, and PKM2. Notably, GAD combined with gemcitabine significantly reduced the growth of GEM-R TNBC cells in a subcutaneous tumor model. ConclusionsThis study reveals a novel antitumor function of GAD, which inhibits glycolysis by promoting HIF-1α degradation in GEM-R TNBC cells, offering a promising therapeutic strategy for TNBC patients with GEM resistance.

Tumor-associated macrophage-induced circMRCKα encodes a peptide to promote glycolysis and progression in hepatocellular carcinoma

The tumor-associated macrophages (TAMs) play multifaceted roles in the progression of hepatocellular carcinoma (HCC). However, the involvement of circular RNAs in the interplay between TAMs and HCC remains unclear. Based on Transwell co-culturing and circular RNA sequencing, this study revealed that TAMs enhanced tumor glycolysis and progression by upregulating circMRCKα in HCC cells. Patients with HCC who exhibited elevated circMRCKα levels presented significantly reduced overall survival and greater cumulative recurrence. Notably, we identified a novel functional peptide of 227 amino acids named circMRCKα-227aa, encoded by circMRCKα. Mechanistically, circMRCKα-227aa bound to USP22 and enhanced its protein level to obstruct HIF-1α degradation via the ubiquitin-proteasome pathway, thereby augmenting HCC glycolysis and progression. In clinical HCC samples, a positive correlation was observed between the expression of circMRCKα and the number of infiltrating CD68+ TAMs and expression of USP22. Furthermore, circMRCKα emerged as an independent prognostic risk factor both individually and in conjunction with CD68+ TAMs and USP22. This study illustrated that circMRCKα-227aa, a novel TAM-induced peptide, promotes tumor glycolysis and progression via USP22 binding and HIF-1α upregulation, suggesting that circMRCKα and TAMs could be combined as therapeutic targets in HCC.

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.