A strategy to prevent atherosclerosis via TNF receptor regulation.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. It has been known that development of atherosclerosis is closely related to activation of tumor necrosis factor α (TNF-α). The objective of this study was to elucidate the effects of TNF-α blockade with brusatol on endothelial activation under pro-atherosclerotic conditions. To this end, we examined the effects of brusatol on TNF-α-induced intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in human aortic endothelial cells (HAECs) using western blot and THP-1 adhesion assays. Brusatol induced a decrease in TNF-α-induced ICAM-1 and VCAM-1 expression through inhibiting TNFR1 expression, leading to suppression of endothelial inflammation independently of the NRF2 (nuclear factor erythroid 2-related factor 2) pathway. The mechanism underlying brusatol-induced TNF receptor 1 (TNFR1) inhibition was investigated with the aid of protein synthesis, co-immunoprecipitation, and cytokine arrays. Notably, brusatol inhibited TNFR1 protein synthesis and suppressed both the canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway and TNF-α-induced cytokine secretion. We further tested the functional effect of brusatol on atherosclerosis development in vivo using two different atherosclerosis mouse models, specifically, acute partial carotid ligation and conventional chronic high-fat diet-fed mouse models. Administration of brusatol led to significant suppression of atherosclerosis development in both mouse models. Our finding that brusatol prevents atherosclerosis via inhibition of TNFR1 protein synthesis supports the potential of downregulation of cell surface TNFR1 as an effective therapeutic approach to inhibit development of atherosclerosis.

Sulfated syndecan 1 is critical to preventing cellular senescence by modulating fibroblast growth factor receptor endocytosis.

Cellular senescence can be triggered by various intrinsic and extrinsic stimuli. We previously reported that silencing of 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) induces cellular senescence through augmented fibroblast growth factor receptor 1 (FGFR1) signaling. However, the exact molecular mechanism connecting heparan sulfation and cellular senescence remains unclear. Here, we investigated the potential involvement of heparan sulfate proteoglycans (HSPGs) in augmented FGFR1 signaling and cellular senescence. Depletion of several types of HSPGs revealed that cells depleted of syndecan 1 (SDC1) exhibited typical senescence phenotypes, and those depleted of PAPSS2-, SDC1-, or heparan sulfate 2-O sulfotransferase 1 (HS2ST1) showed decreased FGFR1 internalization along with hyperresponsiveness to and prolonged activation of fibroblast growth factor 2 (FGF2)-stimulated FGFR1- v-akt murine thymoma viral oncogene homolog (AKT) signaling. Clathrin- and caveolin-mediated FGFR1 endocytosis contributed to cellular senescence through the FGFR1-AKT-p53-p21 signaling pathway. Dynasore treatment triggered senescence phenotypes, augmented FGFR1-AKT-p53-p21 signaling, and decreased SDC1 expression. Finally, the replicatively and prematurely senescent cells were characterized by decreases of SDC1 expression and FGFR1 internalization, and an increase in FGFR1-AKT-p53-p21 signaling. Together, our results demonstrate that properly sulfated SDC1 plays a critical role in preventing cellular senescence through the regulation of FGFR1 endocytosis.

Fluorescent Detection of Methyl Mercury in Aqueous Solution and Live Cells Using Fluorescent Probe and Micelle Systems.

It is highly challenging to develop fast and sensitive fluorescent methods for monitoring organic mercury in purely aqueous solutions as well as live cells. Especially, selective fluorescent detection of methylmercury over inorganic mercury ions has not been reported. We developed a fast and sensitive fluorescent detection method for Hg2+ ions as well as methylmercury using an amino acid-based fluorescent probe (1) and SDS micelles. The fluorescent probe in SDS micelles detected sensitively and selectively Hg2+ ions and methylmercury among 16 metal ions in purely aqueous solution by the enhancement of the red emission at 575 nm, and the detection of methylmercury was completed within 1 min. The probe in SDS micelles with EDTA showed highly sensitive and selective turn on detection for methylmercury over Hg2+. The limit of detection was 9.1 nM for Hg2+ (1.8 ppb, R2 = 0.989) and 206 nM for CH3Hg+ (R2 = 0.997). 1 rapidly penetrated live cells and detected intracellular Hg2+ ions as well as CH3Hg+ by the enhancement of both red emissions and green emissions. Subsequent treatment of EDTA into the cell confirmed the selective detection of methylmercury in the cells. The present work indicated that the fluorescent probe with micelle systems provided a fast, sensitive, and selective detection method for monitoring inorganic mercury as well as methyl mercury.

Stimuli-Responsive Conformational Transformation of Peptides for Tunable Cytotoxicity.

The stimuli-responsive conformational transformation of peptides possessing a constrained form triggered by specific biological microenvironment would provide an effective strategy for the development of highly specific peptide therapeutics. Here, we developed a peptide containing a cytotoxic helical KLA sequence with therapeutic specificity through the use of stimuli-responsive conformational transformation. The KLA peptide is modified to form a cyclic structure to allow for constrained helicity that confers low cytotoxicity. The modified KLA peptide is electrostatically complexed to hyaluronic acid to facilitate enhanced endocytosis into the cancer cells. After endocytosis, the peptide is released from the complex into the cellular cytoplasm by hyaluronidases on the surface of the cellular membrane. Specific intracellular stimuli then trigger the release of the strain that suppresses peptide helicity, and the inherent helical conformation of the KLA peptide is restored. Therefore, the stimuli-responsive conformational transformation of a peptide from low to high helicity selectively induces cell death by disruption of the plasma and mitochondrial membrane.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.

Quinacrine-Mediated Inhibition of Nrf2 Reverses Hypoxia-Induced 5-Fluorouracil Resistance in Colorectal Cancer.

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for the systemic treatment of colorectal cancer (CRC), but its effectiveness against CRC is limited by increased 5-FU resistance caused by the hypoxic tumor microenvironment. The purpose of our study was to assess the feasibility of using quinacrine (QC) to increase the efficacy of 5-FU against CRC cells under hypoxic conditions. QC reversed the resistance to 5-FU induced by hypoxia in CRC cell lines, as determined using ATP-Glo cell viability assays and clonogenic survival assays. Treatment of cells with 5-FU under hypoxic conditions had no effect on the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a regulator of cellular resistance to oxidative stress, whereas treatment with QC alone or in combination with 5-FU reduced Nrf2 expression in all CRC cell lines tested. Overexpression of Nrf2 effectively prevented the increase in the number of DNA double-strand breaks induced by QC alone or in combination with 5-FU. siRNA-mediated c-Jun N-terminal kinase-1 (JNK1) knockdown inhibited the QC-mediated Nrf2 degradation in CRC cells under hypoxic conditions. The treatment of CRC xenografts in mice with the combination of QC and 5-FU was more effective in suppressing tumor growth than QC or 5-FU alone. QC increases the susceptibility of CRC cells to 5-FU under hypoxic conditions by enhancing JNK1-dependent Nrf2 degradation.

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells.

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial-mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.

Mesoporous Silica Nanocarriers with Cyclic Peptide Gatekeeper: Specific Targeting of Aminopeptidase†N and Triggered Drug Release by Stimuli-Responsive Conformational Transformation.

Utilizing stimuli-responsive conformational transformation of a cyclic peptide as a gatekeeper for mesoporous nanocarriers has several advantages such as facile introduction of targeting capabilities, low enzymatic degradation during blood circulation and enhanced specific binding to selected cells. In this report, a Asn-Gly-Arg (NGR)-containing dual-functional cyclic peptide gatekeeper on the surface of mesoporous nanocarrier is prepared not only for active targeting of the aminopeptidase N (APN) expressed on cancer cells but also stimuli-responsive intracellular drug release triggered by a glutathione (GSH)-induced conformational transformation of the peptide gatekeeper. The peptide gatekeeper on the surface of nanocarriers exhibits on-off gatekeeping by conformational transformation triggered by intracellular glutathione of the cancer cells. H1299 cells (high APN expression) showed greater uptake of the nanocarrier by endocytosis and higher apoptosis than A549 cells (low APN expression).

Selective and Sensitive Detection of Heavy Metal Ions in 100% Aqueous Solution and Cells with a Fluorescence Chemosensor Based on Peptide Using Aggregation-Induced Emission.

A fluorescent peptidyl chemosensor for the detection of heavy metal ions in aqueous solution as well as in cells was synthesized on the basis of the peptide receptor for the metal ions using an aggregation-induced emission fluorophore. The peptidyl chemosensor (1) bearing tetraphenylethylene fluorophore showed an exclusively selective turn-on response to Hg(2+) among 16 metal ions in aqueous buffered solution containing NaCl. The peptidyl chemosensor complexed Hg(2+) ions and then aggregated in aqueous buffered solution, resulting in the significant enhancement (OFF-On) of emissions at around 470 nm. The fluorescent sensor showed a highly sensitive response to Hg(2+), and about 1.0 equiv of Hg(2+) was enough for the saturation of the emission intensity change. The detection limit (5.3 nM, R(2) = 0.99) of 1 for Hg(2+) ions was lower than the maximum allowable level of Hg(2+) in drinking water by EPA. Moreover, the peptidyl chemosensor penetrated live cells and detected intracellular Hg(2+) ions by the turn-on response.

Robust neuroprotective effects of intranasally delivered iNOS siRNA encapsulated in gelatin nanoparticles in the postischemic brain.

The therapeutic efficacy of intranasal iNOS siRNA delivery was investigated in the postischemic rat brain after encapsulating on in gelatin nanoparticles (GNPs; diameter 188.0 ± 60.9 nm) cross-linked with 0.0667% glutaraldehyde (GA). Intranasally delivered GNPs were found in extracellular and intracellular compartments of many brain regions, including the olfactory bulb, cerebral cortex, and striatum at 1 hour after infusion and continued to be detected for days. Infarct volumes were markedly suppressed (maximal reduction to 42.1 ± 2.6%) at 2 days after 60 minutes of middle cerebral artery occlusion (MCAO) when iNOS siRNA/GNPs were delivered at 6 hours post-MCAO. In addition, this protective effect was manifested by reductions in neurological and behavioral deficits that were sustained for 2 weeks. Therapeutic potency of iNOS siRNA/GNPs was significantly greater and sustained longer than that of bare siRNA and prolonged and efficient iNOS by iNOS siRNA/GNP is responsible for the robust neuroprotective effect.

TRIM22 induces cellular senescence by targeting PHLPP2 in hepatocellular carcinoma.

The ubiquitin-proteasome system is a vital protein degradation system that is involved in various cellular processes, such as cell cycle progression, apoptosis, and differentiation. Dysregulation of this system has been implicated in numerous diseases, including cancer, vascular disease, and neurodegenerative disorders. Induction of cellular senescence in hepatocellular carcinoma (HCC) is a potential anticancer strategy, but the precise role of the ubiquitin-proteasome system in cellular senescence remains unclear. In this study, we show that the E3 ubiquitin ligase, TRIM22, plays a critical role in the cellular senescence of HCC cells. TRIM22 expression is transcriptionally upregulated by p53 in HCC cells experiencing ionizing radiation (IR)-induced senescence. Overexpression of TRIM22 triggers cellular senescence by targeting the AKT phosphatase, PHLPP2. Mechanistically, the SPRY domain of TRIM22 directly associates with the C-terminal domain of PHLPP2, which contains phosphorylation sites that are subject to IKKß-mediated phosphorylation. The TRIM22-mediated PHLPP2 degradation leads to activation of AKT-p53-p21 signaling, ultimately resulting in cellular senescence. In both human HCC databases and patient specimens, the levels of TRIM22 and PHLPP2 show inverse correlations at the mRNA and protein levels. Collectively, our findings reveal that TRIM22 regulates cancer cell senescence by modulating the proteasomal degradation of PHLPP2 in HCC cells, suggesting that TRIM22 could potentially serve as a therapeutic target for treating cancer.

FLRT2 prevents endothelial cell senescence and vascular aging by regulating the ITGB4/mTORC2/p53 signaling pathway.

The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.

NQO1 prevents radiation-induced aneuploidy by interacting with Aurora-A.

Aneuploidy is the most common characteristic of human cancer cells. It also causes genomic instability, which is involved in the initiation of cancer development. Various lines of evidence indicate that nicotinamide adenine dinucleotide(P)H quinone oxidoreductase 1 (NQO1) plays an important role in cancer prevention, but the molecular mechanisms underlying this effect have not yet been fully elucidated. Here, we report that ionizing radiation (IR) induces substantial aneuploidy and centrosome amplification in NQO1-deficient cancer cells, suggesting that NQO1 plays a crucial role in preventing aneuploidy. NQO1 deficiency markedly increased the protein stability of Aurora-A in irradiated cancer cells. Small interfering RNA targeting Aurora-A effectively attenuated IR-induced centrosome amplification concerned with aneuploidy in NQO1-deficient cancer cells. Furthermore, we found that NQO1 specifically binds to Aurora-A via competing with the microtubule-binding protein, TPX2 (targeting protein for Xklp2), and contributes to the degradation of Aurora-A. Our results collectively demonstrate that NQO1 plays a key role in suppressing IR-induced centrosome amplification and aneuploidy through a direct interaction with Aurora-A.

Ratiometric Fluorescence Sensing System for Lead Ions Based on Self-Assembly of Bioprobes Triggered by Specific Pb2+-Peptide Interactions.

Lead is one of the most toxic substances. However, there are few ratiometric fluorescent probes for sensing Pb2+ in aqueous solution as well as living cells because specific ligands for Pb2+ ions have not been well characterized. Considering the interactions between Pb2+ and peptides, we developed ratiometric fluorescent probes for Pb2+ based on the peptide receptor in two steps. First, we synthesized fluorescent probes (1-3) based on the tetrapeptide receptor (ECEE-NH2) containing hard and soft ligands by conjugation with diverse fluorophores that showed excimer emission when they aggregated. After investigation of fluorescent responses to metal ions, benzothiazolyl-cyanovinylene was evaluated as an appropriate fluorophore for ratiometric detection of Pb2+. Next, we modified the peptide receptor to decrease the number of hard ligands and/or to replace Cys with disulfide bond and methylated Cys for improving selectivity and cell permeability. From this process, we developed two fluorescent probes (3 and 8) among the probes (1-8) that exhibited remarkable ratiometric sensing properties for Pb2+ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb2+, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb2+-peptide interactions of the probes caused nanosized aggregates in which the fluorophores of the probes came close each other, exhibiting excimer emission. In particular, 8 based on tetrapeptide bearing a disulfide bond and two carboxyl groups with a good permeability successfully quantified intracellular uptake of Pb2+ in live cells through ratiometric fluorescent signals. The ratiometric sensing system based on specific metal-peptide interactions and excimer emission process could provide a valuable tool to quantify Pb2+ in live cells and pure aqueous solutions.

NQO1 regulates cell cycle progression at the G2/M phase.

Rationale: Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of the cell cycle regulator, cyclin-dependent kinase subunit-1 (CKS1), at the G2/M phase through effects on the stability of c­Fos. Methods: The roles of the NQO1/c-Fos/CKS1 signaling pathway in cell cycle progression were analyzed in cancer cells using synchronization of the cell cycle and flow cytometry. The mechanisms underlying NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied using siRNA approaches, overexpression systems, reporter assays, co-immunoprecipitation, pull-down assays, microarray analysis, and CDK1 kinase assays. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Results: Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos, which has been implicated in cancer proliferation, differentiation, and development as well as patient survival, and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulation of cell cycle progression at the G2/M phase. Notably, a NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Conclusions: Collectively, our results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase in cancer through effects on c­Fos/CKS1 signaling.

Novel histone deacetylase inhibitor CG200745 induces clonogenic cell death by modulating acetylation of p53 in cancer cells.

Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

The radiosensitivity of endothelial cells isolated from human breast cancer and normal tissue in vitro.

We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.

Tuning of Peptide Cytotoxicity with Cell Penetrating Motif Activatable by Matrix Metalloproteinase-2.

Although diverse cell penetrating motifs not only from naturally occurring proteins but also from synthetic peptides have been discovered and developed, the selectivity of cargo delivery connected to these motifs into the desired target cells is generally low. Here, we demonstrate the selective cytotoxicity tuning of an anticancer KLA peptide with a cell penetrating motif activatable by matrix metalloproteinase-2 (MMP2). The anionic masking sequence introduced at the end of the KLA peptide through an MMP2-cleavable linker is selectively cleaved by MMP2 and the cationic cell penetrating motif is activated. Upon treatment of the peptide to H1299 cells (high MMP2 level), it is selectively internalized into the cells by MMP2, which consequently induces membrane disruption and cell death. In contrast, the peptide shows negligible cytotoxicity toward A549 cancer cells with low MMP2 levels. Furthermore, the selective therapeutic efficacy of the peptide induced by MMP2 is also corroborated using in vivo study.

HIF-1α Inhibition Improves Anti-Tumor Immunity and Promotes the Efficacy of Stereotactic Ablative Radiotherapy (SABR).

High-dose hypofractionated radiation such as SABR (stereotactic ablative radiotherapy) evokes an anti-tumor immune response by promoting a series of immune-stimulating processes, including the release of tumor-specific antigens from damaged tumor cells and the final effector phase of immune-mediated lysis of target tumor cells. High-dose hypofractionated radiation also causes vascular damage in tumors, thereby increasing tumor hypoxia and upregulation of hypoxia-inducible factors HIF-1α and HIF-2α, the master transcription factors for the cellular response to hypoxia. HIF-1α and HIF-2α are critical factors in the upregulation of immune suppression and are the master regulators of immune evasion of tumors. Consequently, SABR-induced increase in anti-tumor immunity is counterbalanced by the increase in immune suppression mediated by HIFα. Inhibition of HIF-1α with small molecules such as metformin downregulates immunosuppressive pathways, including the expression of immune checkpoints, and it improves or restores the anti-tumor immunity stimulated by irradiation. Combinations of HIFα inhibitors, particularly HIF-1α inhibitors, with immune checkpoint blocking antibodies may represent a novel approach to boost the overall anti-tumor immune profile in patients and thus enhance outcomes after SABR.