Publications

Abstract HIF‐1 is a heterodimeric transcription factor, induced by hypoxia, that is composed of HIF‐1α and HIF‐1β protein subunits. It binds to promoter/enhancer elements and stimulates the transcription of hypoxia‐inducible target genes, including glucose transporter‐1 and the glycolytic enzymes. Because HIF‐1 activation might promote cell survival in hypoxic tissues, we studied the effect of permanent middle cerebral artery occlusion on the expression of HIF‐1α, HIF‐1β and several HIF‐1 target genes in adult rat brain. After focal ischaemia, mRNAs encoding HIF‐1α, glucose transporter‐1 and several glycolytic enzymes were up‐regulated in the peri‐infarct penumbra. This was observed by 7.5 h after the onset of ischaemia and increased further at 19 and 24 h. Regional cerebral blood flow was moderately decreased at 1 and 24 h after the ischaemia in areas of HIF‐1 and HIF‐1 target gene induction. Because hypoxia induces HIF‐1 in other tissues, systemic hypoxia (6% O 2 for 4.5 h) was also shown to increase HIF‐1α protein expression in the adult rat brain. It is proposed that decreased blood flow to the penumbra decreases the supply of oxygen and that this induces HIF‐1 and its target genes. This is the first study to show induction of HIF‐1 after focal ischaemia in brain. Increased expression of HIF‐1 target genes as a result of HIF‐1 activation by hypoxia may contribute to tissue viability in the hypoxic/ischaemic penumbra by increasing glucose transport and glycolysis.

The role of hypoxia-inducible factor (HIF)-1 in pancreatic β-cell response to intermittent hypoxia (IH) was examined. Studies were performed on adult wild-type (WT), HIF-1α heterozygous (HET), β-cell-specific HIF-1 −/− mice and mouse insulinoma (MIN6) cells exposed to IH patterned after blood O 2 profiles during obstructive sleep apnea. WT mice treated with IH showed insulin resistance, and pancreatic β-cell dysfunction manifested as augmented basal insulin secretion, and impaired glucose-stimulated insulin secretion and these effects were absent in HIF-1α HET mice. IH increased HIF-1α expression and elevated reactive oxygen species (ROS) levels in β-cells of WT mice. The elevated ROS levels were due to transcriptional upregulation of NADPH oxidase (NOX)-4 mRNA, protein and enzymatic activity, and these responses were absent in HIF-1α HET mice as well as in β-HIF-1 −/− mice. IH-evoked β-cell responses were absent in adult WT mice treated with digoxin, an inhibitor of HIF-1α. MIN6 cells treated with in vitro IH showed enhanced basal insulin release and elevated HIF-1α protein expression, and these effects were abolished with genetic silencing of HIF-1α. IH increased NOX4 mRNA, protein, and enzyme activity in MIN6 cells and disruption of NOX4 function by siRNA or scavenging H 2 O 2 with polyethylene glycol catalase blocked IH-evoked enhanced basal insulin secretion. These results demonstrate that HIF-1-mediated transcriptional activation of NOX4 and the ensuing increase in H 2 O 2 contribute to IH-induced pancreatic β-cell dysfunction.

In GBMs, the heterogeneous pattern of HIF-1alpha expression appears to be determined at least in part by tissue oxygenation, whereas in HBs the homogeneous expression of HIF-1alpha may be driven by an oncogenic rather than a physiologic stimulus.

Abstract Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor–positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. Cancer Res; 76(15); 4430–42. ©2016 AACR.

Abstract The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1α (HIF-1α). In hemangioblastomas and CC-RCCs, HIF-1α is constitutively overexpressed leading to increased transcription of HIF-1–regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1α, CXCR4, and its ligand stromal cell–derived factor-1α (SDF-1α) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1α within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1α was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1α protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1α mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1α mRNA was significantly decreased in HIF-1α-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1α and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.

HIF‐1 (hypoxia‐inducible factor‐1) is a key transcription factor that regulates several hypoxia‐inducible genes. Using a cell‐culture model, we recently reported that IH is a potent activator of HIF‐1‐mediated transcription ( Yuan et al. J. Biol. Chem. 2005 ). In the present study we examined the functional significance of HIF‐1 activation by CIH. Experiments were performed on mice that were either wild type (Wt) or heterozygousfor a loss‐of‐function (knockout) allele at the Hif1a locus, whichencodes the O 2 ‐regulated HIF‐1 subunit. Ten day exposure to CIH resulted in robust up‐regulation of HIF‐1 protein in the central nervous system of Wt but not in mutant mice. In Wt mice, CIH increased hypoxic sensitivity of the carotid bodies, and induced sensory long‐term facilitation (LTF). In sharp contrast, in Hif1a +/− mice, CIH neither augmented the hypoxic sensitivity nor induced sensory LTF. Wt mice exposed to CIH exhibited elevated blood pressures, increased plasma norepineprine, and enhanced ventilatory responses to acute hypoxia. In mutant mice, cardio‐respiratory responses to CIH were either absent or markedly attenuated. These results demonstrate that HIF‐1 plays a critical role in eliciting CIH‐induced cardio‐respiratory changes in part due to its effects on hypoxic sensing of the carotid body. Supported by NIH‐HL‐25830, and HL‐55338).

If the goal of LPC in PSR is to quench the expression of HIF-1-driven angiogenic mediators, our results support broad application of peripheral laser for its treatment.

Abstract Bone marrow–derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis. (Cancer Res 2006; 66(22): 10849-54)

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8 + T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8 + T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor–α). Costimulatory receptor gene expression was also induced when CD8 + T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.