Mutant HIF-1α Research Articles

Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation

Chondrosarcomas are the most common malignancy of cartilage and are associated with somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes. Somatic IDH mutations are also found in its benign precursor lesion, enchondromas, suggesting that IDH mutations are early events in malignant transformation. Human mutant IDH chondrosarcomas and mutant Idh mice that develop enchondromas investigated in our studies display glycogen deposition exclusively in mutant cells from IDH mutant chondrosarcomas and Idh1 mutant murine growth plates. Pharmacologic blockade of glycogen utilization induces changes in tumor cell behavior, downstream energetic pathways, and tumor burden invitro and invivo. Mutant IDH1 interacts with hypoxia-inducible factor 1α (HIF1α) to regulate expression of key enzymes in glycogen metabolism. Here, we show a critical role for glycogen in enchondromas and chondrosarcomas, which is likely mediated through an interaction with mutant IDH1 and HIF1α.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Alternative regulation of HIF-1α stability through Phosphorylation on Ser451

The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.

BMSC-Exosomes Carry Mutant HIF-1α for Improving Angiogenesis and Osteogenesis in Critical-Sized Calvarial Defects.

Repair and reconstruction of critical-sized bone defects has always been a difficult task in orthopedics. Hypoxia inducible factor-1α (HIF-1α) plays an important role in bone defect repair, it has the dual function of promoting osteogenesis and vascular regeneration, but it is quickly degraded by the body under normoxic conditions. Previously we prepared mutant HIF-1α, which has been shown to efficiently maintain cellular expression under normoxic conditions. In this study, we evaluated for the first time the role of exosomes of rat bone marrow mesenchymal stem cell carry mutant HIF-1α (BMSC-Exos-HIF1α) in repairing critical-sized bone defects. Evaluation of the effects of BMSC-Exos-HIF1α on bone marrow mesenchymal stem cells (BMSCs) proliferation and osteogenic differentiation by cell proliferation assay, alkaline phosphatase activity assay, alizarin red staining, real-time quantitative polymerase chain reaction. BMSC-Exos-HIF1α was loaded onto the β-TCP stent implanted in the bone defect area using a rat cranial critical-sized bone defect model, and new bone formation and neovascularization were detected in vivo by micro-CT, fluorescence labeling analysis, Microfil perfusion, histology and immunohistochemical analysis. In vitro results showed that BMSC-Exos-HIF1α stimulated the proliferation of BMSCs and up-regulated the expression level of bone-related genes, which was superior to bone marrow MSC exosomes (BMSC-Exos). In vivo results showed that BMSC-Exos-HIF1α combined with β-TCP scaffold promoted new bone regeneration and neovascularization in the bone defect area, and the effect was better than that of BMSC-Exos combined with β-TCP scaffold. In this study, the results showed that BMSC-Exos-HIF1α stimulated the proliferation and osteogenic differentiation of BMSCs and that BMSC-Exos-HIF1α combined with β-TCP scaffolds could repair critical-sized bone defects by promoting new bone regeneration and neovascularization.

Abstract 3561: HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition

Abstract Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels. We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system. To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG. Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy. Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.

Hypoxia inducible factor-1α/B-cell lymphoma 2 signaling impacts radiosensitivity of H1299 non-small cell lung cancer cells in a normoxic environment.

Hypoxia inducible factor-1α (HIF-1α) is a critical transcriptional factor for the response of cells to hypoxic microenvironment and its expression induces resistance of hypoxic non-small-cell lung cancer (NSCLC) cells to radiotherapy. This study investigated how the activation of HIF-1α/B-cell lymphoma 2 (BCL-2) signaling under normoxic conditions impacted radiosensitivity of NSCLC cells. The recombinant pcDNA3.0-EGFP plasmids with wild-type or mutant HIF-1α complementary DNA (cDNA) were transfected into H1299 cells, an NSCLC cell line, establishing two H1299 sublines with high expression of HIF-1α. Compared with the levels of HIF-1α and BCL-2 proteins in non-transfected cells, increased levels of both proteins were found in transfected cells. Moreover, the expression of HIF-1α in non-transfected cells induced by chloride cobalt (CoCl2), a commonly used mimetic hypoxia reagent, was concomitant with the enhancement of BCL-2 expression. Conversely, reduction of HIF-1α expression by an inhibitor decreased the levels of BCL-2 proteins. The results revealed that the stabilization and expression of HIF-1α promoted the accumulation of BCL-2 proteins in H1299 cells. Subsequent experiments showed that intracellular HIF-1α/BCL-2 signaling was triggered in a normoxic environment after H1299 cells were exposed to irradiation, causing an elevated radioresistance. In contrast, blockage of HIF-1α/BCL-2 signaling leads to an elevated radiosensitivity. Proliferation of cells assay showed that, under normoxic conditions, population doubling times (PDTs) of irradiated cells were prolonged by suppression of HIF-1α/BCL-2 signaling. It is, therefore, indicated that HIF-1α/BCL-2 signaling activated by ionizing radiation reduces the radiosensitivity of H1299 cells independent of the hypoxic environment.

RGD Conjugated Hyperbranched Cationic Amylopectin Nanoparticle as Hypoxia Inducible Factor-1α Targeting Carrier Promotes Neovascularization in Cerebral Infarction

Promoting the regeneration of new blood vessels is a crucial treatment in cerebral infarction and hypoxia-inducible factor 1-α (HIF-1α) plays a pivotal role in the regulation of neovascularization. Nevertheless, the degradation of HIF-1α and poor homing into ischemic sites greatly limit its clinical application. In this study, we encapsulated mutant HIF-1α in RGD modified hyperbranched cationic amylopectin derivative conjugated with 3-(dimethylamino)-1-propylamine (RGD-DMAPA-Amyp) nanoparticles. RGD-DMAPA-Amyp nanoparticles exhibited good blood compatibility and low cytotoxicity in hemolysis and MTT assay, and in vivo study showed low concentrations of RGD-DMAPA-Amyp and DMAPA-Amyp had no significant toxicity to zebrafish embryos. Further to investigate the targeting ability of RGD-DMAPA-Amyp in human umbilical vein endothelial cells and rats model of transient middle cerebral artery occlusion, the in vitro and in vivo results showed RGD-DMAPA-Amyp could specifically bind to vascular endothelial cells. Moreover, the RGD-DMAPA-Amyp/HIF-1α administration significantly reduced the incidence of cerebral ischemia in zebrafish. The pro-angiogenic effect of RGD-DMAPA-Amyp/HIF-1α was evaluated on zebra fish model of vascular loss, RGD-DMAPA-Amyp/HIF-1α were found could improve the growth of subintestinal vessels (SIVs). Our results imply that RGD-DMAPA-Amyp/HIF-1α might alleviate cerebral infarction in vivo via promoting neovascularization and could potentially be a very promising approach for cerebral infarction or ischemic diseases.

Resveratrol integrates metabolic and growth effects in PC3 prostate cancer cells-involvement of prolyl hydroxylase and hypoxia inducible factor-1.

Resveratrol (RES) is a polyphenol produced by certain plant species that has been well studied due to its ability to slow the growth of cancer cells. In numerous cell types and tissues, RES has been demonstrated to promote mitochondrial biogenesis, fusion, and oxidative phosphorylation. The present study investigated the interaction between RES's effects on growth and metabolism in PC3 prostate cancer cells, and demonstrated that RES-mediated growth inhibition is only observed under conditions in which a metabolic shift from glucose fermentation to mitochondrial respiration can occur. When this shift was prevented by growing cells in galactose medium or by pharmacologically inhibiting prolyl hydroxylase (PHD) in order to stabilize hypoxia inducible factor-1α, RES did not effect mitochondrial fusion, biogenesis, respiration or cell growth. Similar results were observed in PC3 cells expressing a mutant HIF-1α lacking the prolines that are hydroxylated by PHD to promote its degradation. Thus, RES appears to slow PC3 cell growth by interfering with glucose fermentation and promoting respiration. Consistent with this, RES was observed to be particularly effective at inhibiting PC3 cell growth under hypoxic conditions that precluded increased reliance on oxidative phosphorylation. These observations are important in understanding how RES may affect cancer cell growth in vivo where hypoxia is common in growing tumours.

Exosomes secreted from mutant-HIF-1α-modified bone-marrow-derived mesenchymal stem cells attenuate early steroid-induced avascular necrosis of femoral head in rabbit.

Mesenchymal stem cells (MSCs)-derived exosomes exhibit protective effects on damaged or diseased tissues. Hypoxia-inducible factor 1α (HIF-1α) plays a critical role in bone development. However, HIF-1α is easily biodegradable under normoxic conditions. The bone-marrow-derived mesenchymal stem cells (BMSCs) were transfected with adenovirus carrying triple point-mutations (amino acids 402, 564, and 803) in the HIF-1α coding sequence (CDS). The mutant HIF-1α can efficiently express functional proteins under normoxic conditions. To date, no study has reported the role of exosomes secreted by mutant HIF-1α modified BMSCs in the recovery of the early steroid-induced avascular necrosis of femoral head (SANFH). In this study, we firstly analyzed exosomes derived from BMSCs modified by mutant (BMSC-ExosMU ) or wild-type HIF-1α (BMSC-ExosWT ). In vitro, we investigated the osteogenic differentiation capacity of BMSCs modified by BMSC-ExosMU or BMSC-ExosWT , and the angiogenesis effects of BMSC-ExosMU and BMSC-ExosWT on human umbilical vein endothelial cells (HUVECs). Besides, the healing of the femoral head was also assessed in vivo. We found that the potential of osteogenic differentiation of BMSCs treated with BMSC-ExosMU was higher than the wild-type group in vitro. In addition, BMSC-ExosMU stimulated the proliferation, migration, and tube formation of HUVECs in a dose-dependent manner. Compared with the BMSC-ExosWT or PBS control group, the injection of BMSC-ExosMU into the necrosis region markedly accelerated the bone regeneration and angiogenesis, which were indicated by the increased trabecular reconstruction and microvascular density. Taken together, our data suggest that BMSC-ExosMU facilitates the repair of SANFH by enhancing osteogenesis and angiogenesis.

Overexpression and Knockdown of Hypoxia-Inducible Factor 1 Disrupt the Expression of Steroidogenic Enzyme Genes and Early Embryonic Development in Zebrafish.

Hypoxia is an important environmental stressor leading to endocrine disruption and reproductive impairment in fish. Although the hypoxia-inducible factor 1 (HIF-1) is known to regulate the transcription of various genes mediating oxygen homeostasis, its role in modulating steroidogenesis-related gene expression remains poorly understood. In this study, the regulatory effect of HIF-1 on the expression of 9 steroidogenic enzyme genes was investigated in zebrafish embryos using a “gain-of-function and loss-of-function” approach. Eight of the genes, CYP11a, CYP11b2, 3β-HSD, HMGCR, CYP17a1, 17β-HSD2, CYP19a, and CYP19b, were found to be differentially upregulated at 24 and 48 hpf following zHIF-1α-ΔODD overexpression (a mutant zebrafish HIF-1α protein with proline-414 and proline-557 deleted). Knockdown of zHIF-1α also affected the expression pattern of the steroidogenic enzyme genes. Overexpression of zHIF-1α and hypoxia exposure resulted in downregulated StAR expression but upregulated CYP11a and 3β-HSD expression in zebrafish embryos. Conversely, the expression patterns of these 3 genes were reversed in embryos in which zHIF-1α was knocked down under normoxia, suggesting that these 3 genes are regulated by HIF-1. Overall, the findings from this study indicate that HIF-1–mediated mechanisms are likely involved in the regulation of specific steroidogenic genes.

Ursolic acid sensitizes radioresistant NSCLC cells expressing HIF-1α through reducing endogenous GSH and inhibiting HIF-1α.

In previous studies, the present authors demonstrated that effective sensitization of ionizing radiation-induced death of tumor cells, including non-small cell lung cancer (NSCLC) cells, could be produced by oleanolic acid (OA), a pentacyclic triterpenoid present in plants. In the present study, it was investigated whether ursolic acid (UA), an isomer of OA, had also the capacity of sensitizing radioresistant NSCLC cells. The radioresistant cell line H1299/M-hypoxia inducible factor-1α (HIF-1α) was established by transfection with a recombinant plasmid expressing mutant HIF-1α (M-HIF-1α). Compared with parental H1299 cells and H1299 cells transfected with empty plasmid, H1299/M-HIF-1α cells had lower radiosensitivity. Following the use of UA to treat NSCLC cells, elevation of the radiosensitivity of cells was observed by MTT assay. The irradiated H1299/M-HIF-1α cells were more sensitive to UA pretreatment than the irradiated cells with empty plasmid and control. The alteration of DNA damage in the irradiated cells was further measured using micronucleus (MN) assay. The combination of UA treatment with radiation could induce the increase of cellular MN frequencies, in agreement with the change in the tendency observed in the cell viability assay. It was further shown that the endogenous glutathione (GSH) contents were markedly attenuated in the differently irradiated NSCLC cells with UA (80 µmol/l) pretreatment through glutathione reductase/5,5′-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. The results revealed that UA treatment alone could effectively decrease the GSH content in H1299/M-HIF-1α cells. In addition, the inhibition of HIF-1α expression in radioresistant cells was confirmed by western blotting. It was then concluded that UA could upregulate the radiosensitivity of NSCLC cells, and in particular reduce the refractory response of cells expressing HIF-1α to ionizing radiation. The primary mechanism is associated with reduction of endogenous GSH and inhibition of high expression of intracellular HIF-1α. UA should therefore be deeply studied as a potential radiosensitizing reagent for NSCLC radiotherapy.

Mutant hypoxia-inducible factor 1α modified bone marrow mesenchymal stem cells ameliorate cerebral ischemia.

Hypoxia-inducible factor 1α (HIF1α) plays stimulatory roles in revascularization in the ischemic area of cerebral ischemia. However, the hydroxylation of proline at 564 and asparagine at 803 in the HIF1α coding sequence facilitated the degradation of HIF1α and inhibited the transcription activity of the HIF1α promoter under normoxic conditions and confined the pro-angiogenic efficacy of HIF1α. In the present study, the HIF1α mutant containing P564A and N803A was constructed by site‑directed mutagenesis. Rat bone marrow mesenchymal stem cells (BMSCs) were infected with adenoviral particles containing HIF1α mutant at multiplicity of infection of 150. The HIF1α mRNA and protein levels under hypoxia and normoxic conditions were compared using reverse transcription-polymerase chain reaction and western blot analysis. To explore the therapeutic effect of mutant HIF1α on the cerebral ischemia, BMSCs overexpressing mutant HIF1α were transplanted in the rat middle cerebral artery occlusion model (MCAO). The motor function and cerebral infarct size were evaluated using modified neurological severity score and triphenyltetrazolium chloride (TTC) staining within four weeks after MCAO. Vascular endothelial growth factor (VEGF) protein expression was detected by western blot analysis. Microvessel density and angiogenesis were detected by immunohistochemistry to evaluate the recovery of the brain ischemia. The HIF1α mutant containing P564A and N803A could be expressed under normoxic conditions. Transplantation of BMSCs stably expressing mutant HIF1α significantly improved motor function, reduced cerebral infarction and increased VEGF protein expression revascularization at days 7, 14 and 28 (p<0.05). Therefore, the HIF1α mutant containing P564A and N803A may be a potential target for the treatment of the cerebral ischemia.

Visualization of Hypoxia‐Inducible Factor 1α‐p300 Interactions in Live Cells by Fluorescence Resonance Energy Transfer

Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications.

Cetuximab Reverses the Warburg Effect by Inhibiting HIF-1–Regulated LDH-A

Hypoxia-inducible factor-1 (HIF-1) plays a critical role in reprogramming cancer metabolism toward aerobic glycolysis (i.e., the Warburg effect), which is critical to supplying cancer cells with the biomass needed for proliferation. Previous studies have shown that cetuximab, an EGF receptor-blocking monoclonal antibody, downregulates the alpha subunit of HIF-1 (HIF-1α) through the inhibition of EGF receptor downstream cell signaling and that downregulation of HIF-1α is required for cetuximab-induced antiproliferative effects. However, the mechanism underlying these actions has yet to be identified. In this study, we used the Seahorse XF96 extracellular flux analyzer to assess the effect of cetuximab treatment on changes in glycolysis and mitochondrial respiration, the two major energy-producing pathways, in live cells. We found that cetuximab downregulated lactate dehydrogenase A (LDH-A) and inhibited glycolysis in cetuximab-sensitive head and neck squamous cell carcinoma (HNSCC) cells in an HIF-1α downregulation-dependent manner. HNSCC cells with acquired cetuximab resistance expressed a high level of HIF-1α and were highly glycolytic. Overexpression of a HIF-1α mutant (HIF-1α/ΔODD) conferred resistance to cetuximab-induced G1 phase cell-cycle arrest, which could be overcome by knockdown of LDH-A expression. Inhibition of LDH-A activity with oxamate enhanced the response of cetuximab-resistant cells to cetuximab. Cetuximab had no noticeable inhibitory effect on glycolysis in nontransformed cells. These findings provide novel mechanistic insights into cetuximab-induced cell-cycle arrest from the perspective of cancer metabolism and suggest novel strategies for enhancing cetuximab response.

Triple Point-Mutants of Hypoxia-Inducible Factor-1α Accelerate In Vivo Angiogenesis in Bone Defect Regions

To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α (HIF1α) in angiogenesis in bone defect regions under normoxic conditions.1. Triple point-mutations (in amino acids 402, 564, and 803) in the HIF1α coding sequence (CDS) were induced by polymerase chain reaction. The triple mutant HIF1α (402/564/803) was inserted into the adenovirus pAdEasy-1 system for complete viral packaging and titer measurements. 2. For the in vitro experiment, rabbit bone marrow mesenchymal stem cells (MSCs) were divided into four experimental groups. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP). The HIF1α mRNA, protein and VEGF protein expression levels in infected cells in each experimental group were measured. 3. As in the in vivo experiment, the MSCs were divided into four groups and infected with the viral solutions from each complementary in vitro group and cultured under normoxic conditions. The MSCs were used as seed cells and transplanted into an apatite-wollastonite magnetic bioactive glass-ceramic (AW MGC) vector to construct artificial tissue-engineering scaffolds that were then implanted into the in vivo rabbit radial bone defect model. The animals from each group were killed 8weeks after the surgery, and the tissues from the implantation region were harvested for the evaluation of the angiogenesis. 1. The 402,564, and 803 amino acids in CDS area were point mutated into alanine; three types of recombinant adenovirus were successfully constructed, packaged, and characterized. 2. The expression levels of HIF1α mRNA in A and B groups were significantly higher than those in the C and D groups (P<0.05). The HIF1α and VEGF protein expression levels in A group were significantly higher than those in the other three groups (P<0.05). 3. There was prominent angiogenesis in bone defect regions in group A animals. 1. Triple point-mutants of HIF1α efficiently expressed functional proteins under normoxic conditions.2. Triple point-mutants HIF1α effectively promoted in vivo angiogenesis in bone defect regions.

Abstract LB-486: Overexpression of a mutated, constitutively active HIF2α promotes the carcinogenesis of TRACK mouse

Abstract Background: VHL disease patients have an increased risk of developing clear cell renal cell carcinomas (ccRCC). Increased expression of HIF1α and/or HIF2α has been proposed as a key step in ccRCC carcinogenesis. We recently reported a mouse model of VHL renal cancer, the TRACK model (Fu et al, Cancer research. 2011;71(21):6848-56.). In the TRACK model, proximal tubule (PT) cell overexpression of a triple mutant (P402A, P564A and N803A) HIF1α in the mouse mimics human VHL kidney disease. Methods: Here we report a mouse model expressing a triple mutant (P405A, P531A and N847A) HIF2α in PT cells. Mutant HIF2α was expressed in both WT and HIF1α-expressing C57BL/6 mice. Results: Transgenic (TG) mice expressing this HIF2α construct specifically in PT cells exhibit vacuolization consistent with glycogen accumulation and hydropic degeneration. The activation of HIF2α was confirmed by increased expression of HIF2α target genes, e.g. CA9. Neoplastic transformation was not observed in the kidneys of HIF2α TG+ mice. In HIF1α/HIF2α-expressing mice, disorganized PT cells mimicking in situ carcinoma was observed in 5 mo old mouse kidneys, which was seen only at 14 mo in mice expressing only mutant HIF1α. Conclusions: These data suggest HIF2α accelerates carcinogenesis in the mutant HIF1α TRACK model. These mouse models provide excellent models to study VHL renal disease and carcinogenesis, and provide an opportunity for chemoprevention studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-486. doi:1538-7445.AM2012-LB-486

Abstract 4641: Hypoxia activated pro-drug TH-302 induces hypoxia-dependent anti-leukemia activity in vitro and in vivo

Abstract Hypoxia in tumors is generally associated with chemoresistance and radioresistance. Hypoxia-inducible factor-1α (HIF-1α) was expressed in the bone marrow in 58% of primary acute myelogenous (AML) cases by immunohistochemistry (n=156). We have recently demonstrated a marked expansion of hypoxia in leukemia bone marrow (Benito PLoSOne 2011) and demonstrated hypoxia promotes chemoresistance in acute leukemias. Normoxic stabilization of HIF-1α using inhibitor of prolyl hydroxylases DMOG, or overexpression through infection with lentiviral non-degradable mutant HIF-1α, significantly diminished apoptosis induced by vincristine and etoposide in REH and Nalm-6 cells. These findings suggest that hypoxia mediates chemoresistance at least in part through HIF-1a. TH-302 is a 2-nitroimidazole of the cytotoxin bromo-isophosphoramide mustard that upon hypoxia-dependent activation induces DNA alkylation, a novel approach to target leukemia. We report the in vitro and in vivo anti leukemia activity of TH-302. In vitro, TH-302 exhibited potent anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04µM to 2.3µM. The hypoxic cytotoxicity ratios (aerobic IC50/hypoxic IC50) were 11 and 92 in ALL (REH, NALM6) and 14, 80 and 116 in AML (OCI-AML3, KG-1, KBM5). The drug at 5-7.5µM also exhibited specific hypoxia dependent anti-leukemia activity in primary ALL and AML samples (normoxic vs hypoxic specific apoptosis defined as %AnnV positive cells +/− SEM normalized to untreated control cells): ALL-1: 5.1+/−0.5% vs 65.3+/−4.2%; ALL-2: 8.3+/−1.9% vs 28.4+/−6.3%; AML 1: 2.6+/−0.2% vs 40.3+/−1.5%). In in vivo leukemia models TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 x106/ml; TH-302, 2.5+/−2.1 x106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003). Finally, anti-leukemia efficacy of TH-302 was tested in vitro in combination with chemotherapy commonly used for treatment of AML (AraC, doxorubicin) and with demethylating agents azacitidine (5-AZA) and decitabine (DAC). Combination TH-302 with AraC or with DAC were synergistic in killing OCI-AML3 and MOLM-13 AML cells under hypoxia (combination index values of 0.4 and 0.67 for AraC plus TH-302 in MOLM13 and OCI-AML3, respectively; 0.3 for DAC plus TH-302 in OCI-AML3). In summary, our findings suggest that hypoxia and HIF-1α constitute important factors in the survival of leukemic blasts within the BM microenvironment. The results support targeting hypoxia with a hypoxia-activated drug such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 clinical study of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4641. doi:1538-7445.AM2012-4641

HDAC4 Protein Regulates HIF1α Protein Lysine Acetylation and Cancer Cell Response to Hypoxia

Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.

Ultrasound molecular imaging of angiogenesis induced by mutant forms of hypoxia-inducible factor-1α

Targeted point mutants of hypoxia-inducible factor-1α (HIF-1α) are potential optimal agents for angiogenesis therapy. Data are limited regarding the angiogenic response of HIF-1α mutants. We aimed to compare the angiogenic effect of wild-type and mutant HIF-1α by contrast ultrasound molecular imaging (UMI) of α(v)-integrin expression. The wild-type gene of human HIF-1α, a gene with double mutations (HIF-1α(564/803)), a gene with triple mutations (HIF-1α(564/803/402)), or the LacZ gene (control) was transfected into the ischaemic hind limbs of C57BL/6 mice using an adenovirus vector. The video intensity of microbubbles targeted to α(v)-integrins in the ischaemic limbs increased along with the number of point mutations of HIF-1α. Immunohistochemical expression of endothelial α(v)-integrins was higher in the mutant HIF-1α(564/803/402) group than the other groups as was the density of both capillaries and arterioles in ischaemic muscle. Expression of both the mRNA and protein for HIF-1α and VEGF was significantly higher in the mutant HIF-1α(564/803/402) group than in the other groups. The half-life of HIF-1α and VEGF mRNA was longer in HIF-1α mutant-transfected cells than in wild-type HIF-1α or LacZ-transfected cells. HIF-1α mutants were more effective for enhancing angiogenesis in ischaemic muscle tissue than wild-type HIF-1α, and the response could be qualitatively evaluated by UMI of α(v)-integrins expression.

Abstract 2857: Expression of mutated, constitutively active HIF1α in the mouse kidney results in early stage clear cell renal cell carcinoma

Abstract Introduction: Renal cell carcinoma (RCC) is the most common primary kidney cancer, with clear cell carcinoma (ccRCC) representing ∼75% of all RCCs. In patients with Von Hippel-Lindau (VHL) disease, inactivation of the VHL tumor suppressor gene results in increased expression of HIF1α, the development of numerous premalignant lesions as well as multifocal ccRCC. Previously, no mouse model that simulates human ccRCC has been established. We report the first transgenic mouse model that closely resembles the development of ccRCC. Methods: Three mutations (P402A, P564A and N803A) were introduced into human HIF1α cDNA by site-directed mutagenesis. Gamma-glutamyl transpeptidase (GGT or γ-GT) is specifically expressed in the proximal tubules of the kidney starting 3 weeks after birth. GGT promoter, triple mutant HIF1α ORF and a beta globin poly A were cloned to create the γ-HIF1α-M3 plasmid. A linearized fragment with the vector sequence removed was injected into the pronuclei of one-cell mouse embryos to create γ-HIF1α-M3 transgenic mice. Results: Five of 51 founder mice harbored the integrated target gene by Southern Blot analysis (1 lacked Germline transmission). Expression of the HIF1α transgene mRNA was confirmed by RT-PCR. Triple mutant HIF1α was expressed in the kidneys of transgenic positive (TG+) mice (lines 8, 25, 32 and 43) and not other organs tested (spleen, liver, heart, lung, intestine, skeletal muscle). TG+ mice and transgenic negative (TG-) littermates were sacrificed at 6-7 months. Histological review revealed clusters of “clear” cells with distorted, abnormal shapes in the outer cortex of TG+ kidneys. These “clear” cells contained large amounts of glycogen and lipid in their cytoplasm, as shown by Periodic Acid/Schiff stain and Oil Red O stain, respectively. Immunohistochemical analysis of HIF1α regulated genes revealed greatly increased expression of CA-IX, Glut-1, and VEGF in these “clear” cells, as well as an increased number of blood vessels (angiogenesis) as determined by Cd-31b. Although there was no clear evidence of neoplasia in 6-7 month old mice, pre-malignant “clear” cells exhibited increased proliferation (PCNA expression) and increased DNA double strand breaks (γH2AX expression). Analysis of the kidneys of 1.5 year-old TG+ mice revealed multiple renal cysts and disorganized proximal tubules that display an intratubular proliferation of atypical clear cells, suggesting a pre-invasive or in-situ tumor. Conclusions: Expression of mutated, constitutively active HIF1α protein in mouse kidney proximal tubule cells results in a transgenic phenotype similar to that observed in patients with VHL disease, including the development of premalignant lesions, multiple renal cysts and early ccRCC. This Transgenic Model of Cancer of the Kidney (TRACK) provides a model to study the genetic and molecular events that result in the development of ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2857. doi:10.1158/1538-7445.AM2011-2857