Publications

Purification and Characterization of Hypoxia-inducible Factor 1

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding protein that activates erythropoietin (Epo) gene transcription in Hep3B cells subjected to hypoxia or cobalt chloride treatment. HIF-1 DNA binding activity is also induced by hypoxia or cobalt in non-Epo-producing cells, suggesting a general role for HIF-1 in hypoxia signal transduction and transcriptional regulation. Here we report the biochemical purification of HIF-1 from Epo-producing Hep3B cells and non-Epo-producing HeLa S3 cells. HIF-1 protein was purified 11,250-fold by DEAE ion-exchange and DNA affinity chromatography. Analysis of HIF-1 isolated from a preparative gel shift assay revealed four polypeptides. Peptide mapping of these HIF-1 components demonstrated that 91-, 93-, and 94-kDa polypeptides had similar tryptic maps, whereas the 120-kDa polypeptide had a distinct profile. Glycerol gradient sedimentation analysis suggested that HIF-1 exists predominantly in a heterodimeric form and to a lesser extent as a heterotetramer. Partially purified HIF-1 bound specifically to the wild-type HIF-1 binding site from the EPO enhancer but not to a mutant sequence that lacks hypoxia-inducible enhancer activity. UV cross-linking analysis with purified HIF-1 indicated that both subunits of HIF-1 contact DNA directly. We conclude that in both cobalt chloride-treated HeLa cells and hypoxic Hep3B cells HIF-1 is composed of two different subunits: 120-kDa HIF-1 alpha and 91-94-kDa HIF-1 beta.

Targeting HIF-1 for cancer therapy

Erythropoietin ontogeny and organ distribution in mice.

Regulation of Osteogenesis-Angiogenesis Coupling by HIFs and VEGF

Abstract Bone is a highly vascularized tissue, but the function of angiogenesis in bone modeling and remodeling is still poorly defined, and the molecular mechanisms that regulate angiogenesis in bone are only partially elucidated. Genetic manipulations in mice have recently highlighted the critical role of the hypoxia-inducible-factor/vascular endothelial growth factor pathway in coupling angiogenesis and osteogenesis. In this brief perspective, we review the current understanding of the mechanisms responsible for this coupling. Elucidation of such mechanisms will expand our knowledge of bone development and homeostasis, and it may aid in the design of new therapies for accelerating bone regeneration and repair.

G-rich Oligonucleotides Inhibit HIF-1α and HIF-2α and Block Tumor Growth

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in tumor promotion by upregulating its target genes, which are involved in energy metabolism, angiogenesis, cell survival, invasion, metastasis, and drug resistance. The HIF-1alpha subunit, which is regulated by O2-dependent hydroxylation, ubiquitination, and degradation, has been identified as an important molecular target for cancer therapy. We have rationally designed G-rich oligodeoxynucleotides (ODNs) as inhibitors of HIF-1alpha for human cancer therapy. The lead compounds, JG243 and JG244, which form an intramolecular parallel G-quartet structure, selectively target HIF-1alpha and decreased levels of both HIF-1alpha and HIF-2alpha (IC50 < 2 micromol/l) and also inhibited the expression of HIF-1-regulated proteins [vascular endothelial growth factor (VEGF), Bcl-2, and Bcl-XL], but did not disrupt the expression of p300, Stat3, or p53. JG-ODNs induced proteasomal degradation of HIF-1alpha and HIF-2alpha that was dependent on the hydroxylase activity of prolyl-4-hydroxylase-2. JG243 and JG244 dramatically suppressed the growth of prostate, breast, and pancreatic tumor xenografts. Western blots from tumor tissues showed that JG-ODNs significantly decreased HIF-1alpha and HIF-2alpha levels and blocked the expression of VEGF. The JG-ODNs are novel anticancer agents that suppress tumor growth by inhibiting HIF-1.

RETRACTED: Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells

Overexpression of Rho kinase 1 (ROCK1) and the G protein RhoA is implicated in breast cancer progression, but oncogenic mutations are rare, and the molecular mechanisms that underlie increased ROCK1 and RhoA expression have not been determined. RhoA-bound ROCK1 phosphorylates myosin light chain (MLC), which is required for actin-myosin contractility. RhoA also activates focal adhesion kinase (FAK) signaling. Together, these pathways are critical determinants of the motile and invasive phenotype of cancer cells. We report that hypoxia-inducible factors coordinately activate RhoA and ROCK1 expression and signaling in breast cancer cells, leading to cell and matrix contraction, focal adhesion formation, and motility through phosphorylation of MLC and FAK. Thus, intratumoral hypoxia acts as an oncogenic stimulus by triggering hypoxia-inducible factor → RhoA → ROCK1 → MLC → FAK signaling in breast cancer cells.

Retraction: Induction of Hypoxia-inducible Factor 1 Activity by Muscarinic Acetylcholine Receptor Signaling

Abstract P3-07-62: Association of overexpression of hypoxia inducible factor 1α with response to neoadjuvant chemotherapy in early stage breast cancer

Abstract Background: Hypoxia inducible factor 1 alpha (HIF-1α) is a master transcription factor involved in multiple oncogenic processes. In breast cancer, HIF-1α overexpression is associated with increased resistance to radiation therapy, chemotherapy, and inferior disease-free and overall survival. Patients who achieve a pathologic complete response (pCR) following neoadjuvant therapy have improved survival outcomes. Limited data are available regarding the association between HIF-1α expression and rates of pCR. We investigated the relationship between HIF-1α overexpression and pCR rates following neoadjuvant chemotherapy for early breast cancer. Methods: Eligible women were those with HER2-negative, stage II-III breast cancer, who received anthracycline- and taxane-based neoadjuvant chemotherapy from 2002 to 2012, and were included in an institutional review board-approved Integrated Breast Cancer Research Database at Johns Hopkins. The database includes patient age, sex, menopausal status, breast cancer diagnosis, tumor histopathology, treatment history, laboratory data, and outcomes. Both diagnostic and surgical tissue blocks were retrieved from pathology archives. Whole section slides were prepared and analyzed by immunohistochemical staining with appropriate negative and positive controls. The intensity of cells positive for HIF-1α was estimated visually by a pathologist blinded to clinical data. A semi-quantitative scoring of nucleus expression was used to score HIF-1α expression: score 0 is defined as less than 1%, 1 is defined as 1-5%, 2 as 5-20% and 3 as &amp;gt;20% tumor cells positive for HIF-1α. Overall tumor HIF-1α negativity was defined as (0, 1) and positivity as (2, 3). We compared baseline HIF-1α status among responders (pCR defined as no invasive tumor in the breast or lymph nodes) and non-responders (no pCR) using Fisher's exact test, and evaluate the association between baseline and surgical specimens in those who did not achieve pCR using NcNemar's test. Results: A total of 122 women meeting the eligibility criteria underwent a definitive surgical procedure following neoadjuvant chemotherapy. Of those, 50 patients had no tissue blocks available at baseline and additional 16 patients' blocks did not contain sufficient tissue for analysis. Thus, tumors from 56 women were available for analysis. Median age was 50 (range 33-78), 54% were White and 35% Black; 41% and 59% of women had triple negative and hormone receptor-positive tumors, respectively; 71% women had Ki67&amp;gt;30%, and 80% were node-positive. Overall pCR was observed in 12 women (21%). We did not detect a significant association between HIF-1α score on the diagnostic specimen with pCR status (p=0.627). However, a positive HIF-1α score was significantly associated with positive lymph nodes (p=0.01). We observed a significant decrease in HIF-1α score following chemotherapy (p&amp;lt;0.001). Conclusions: We did not observe a clear association between HIF-1α expression and response to neoadjuvant chemotherapy. However, HIF-1α expression on a diagnostic specimen was statistically higher than following chemotherapy and was associated with lymph node positivity. Our study is limited by its retrospective nature and a small sample size. Citation Format: Zhi WI, Huang C-Y, Gabrielson E, Tully E, Cimino-Mathews A, Santa-Maria C, Jeter S, Kai C, Semenza G, Stearns V. Association of overexpression of hypoxia inducible factor 1α with response to neoadjuvant chemotherapy in early stage breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-62.

‘The Metabolism of Tumours’: 70 Years Later

Otto Warburg's classic treatise on the reprogramming of tumour metabolism from oxidative to glycolytic metabolism was published in London in 1930. Although the Warburg effect is one of the most universal characteristics of solid tumours, the molecular basis for this phenomenon has only recently been elucidated by studies indicating that increased expression of genes encoding glucose transporters and glycolytic enzymes in tumour cells is mediated by the transcription factors c-MYC and HIF-1. Whereas c-myc is a direct target for oncogenic mutations, expression of hypoxia-inducible factor 1 (HIF-1) is indirectly up-regulated via gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes that result increased HIF-1alpha protein expression and/or increased HIF-1 transcriptional activity in a cell-type-specific manner. As a result of genetic alterations and intratumoral hypoxia, HIF-1alpha is overexpressed in the majority of common human cancers relative to the surrounding normal tissue. In human breast cancer and brain tumours, HIF-1alpha overexpression is strongly correlated with tumour grade and vascularity.

Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2.

Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other proteins involved in O2 homeostasis and tumor progression. The expression and transcriptional activity of the HIF-1alpha subunit is regulated by the cellular O2 concentration. We demonstrate that insulin, insulin-like growth factor (IGF)-1, and IGF-2 induce expression of HIF-1alpha, which is required for expression of genes encoding IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3. These data provide a novel mechanism by which HIF-1alpha overexpression may occur in tumor cells and contribute to an autocrine growth factor loop.

Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways

Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.

The intravenous anesthetic propofol inhibits hypoxia‐inducible factor 1 activity in an oxygen tension‐dependent manner

Hypoxia elicits a wide range of responses that occur at different organizational levels in the body. Hypoxia is not only a signal for energy conservation and metabolic change, but triggers expression of a select set of genes. The transcription factor hypoxia‐inducible factor 1 (HIF‐1) is now appreciated to be a master factor of the gene induction. Although knowledge on molecular mechanisms of HIF‐1 activation in response to hypoxia is accumulating, the molecular mechanism of maintenance of HIF‐1 activity under normoxic conditions remains to be elucidated. We demonstrate that the intravenous anesthetic propofol reversibly inhibits HIF‐1 activity and the gene expression mediated by HIF‐1 by blocking the synthesis of the HIF‐1α subunit under 20% or 5% O 2 conditions, but not under 1% O 2 conditions.

Reciprocal Regulation of DUSP9 and DUSP16 Expression by HIF1 Controls ERK and p38 MAP Kinase Activity and Mediates Chemotherapy-Induced Breast Cancer Stem Cell Enrichment

Abstract Triple-negative breast cancer (TNBC) has a poor prognosis due to its aggressive characteristics and lack of targeted therapies. Cytotoxic chemotherapy may reduce tumor bulk, but leaves residual disease due to the persistence of chemotherapy-resistant breast cancer stem cells (BCSC), which are critical for tumor recurrence and metastasis. Here, we demonstrate that hypoxia-inducible factor (HIF)-1–dependent regulation of mitogen-activated protein kinase (MAPK) signaling pathways contributes to chemotherapy-induced BCSC enrichment. Chemotherapy increased DUSP9 expression and decreased DUSP16 expression in a HIF1–dependent manner, leading to inhibition of ERK and activation of p38 signaling pathways, respectively. Inhibition of ERK caused transcriptional induction of the pluripotency factor Nanog through decreased inactivating phosphorylation of FoxO3, while activation of p38 stabilized Nanog and Klf4 mRNA through increased inactivating phosphorylation of RNA-binding protein ZFP36L1, both of which promoted specification of the BCSC phenotype. Inhibition of HIF1 or p38 signaling blocked chemotherapy-induced pluripotency factor expression and BCSC enrichment. These surprising results delineate a mechanism by which a transcription factor switches cells from ERK to p38 signaling in response to chemotherapy and suggest that therapeutic targeting of HIF1 or the p38 pathway in combination with chemotherapy will block BCSC enrichment and improve outcome in TNBC. Significance: These findings provide a molecular mechanism that may account for the increased relapse rate of women with TNBC who are treated with cytotoxic chemotherapy and suggest that combining chemotherapy with an inhibitor of HIF1 or p38 activity may increase patient survival. Cancer Res; 78(15); 4191–202. ©2018 AACR.

Enhanced Interferon-γ Gene Expression in T Cells and Reduced Ovalbumin-Dependent Lung Eosinophilia in Hypoxia-Inducible Factor-1-α-Deficient Mice

&lt;i&gt;Background:&lt;/i&gt; There is growing evidence that hypoxia-inducible transcription factors are involved in the pathophysiology of asthma. Hypoxia-inducible factor-1α (HIF-1α) in particular controls the expression of many hypoxia regulated genes, but whether HIF-1α directly contributes to allergen-driven immune responses is not known. &lt;i&gt;Methods:&lt;/i&gt; Partially HIF-1α-deficient mice (HIF-1α&lt;sup&gt;+/–&lt;/sup&gt;) or wild-type littermate controls were used in all experiments. Spleen CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and cytokine secretion was measured in vitro. Mice were sensitized by intraperitoneal injection of ovalbumin (Ova) plus alum, and then challenged by intranasal Ova followed by bronchoalveolar lavage (BAL) and isolation of spleen cells. BAL cells were counted and the differential determined using cytospin, and splenocytes were incubated with Ova to measure recall cytokine production. &lt;i&gt;Results:&lt;/i&gt; Interferon-γ secretion was significantly higher in anti-CD3 plus anti-CD28 stimulated CD4+ T cells obtained from HIF-1α&lt;sup&gt;+/–&lt;/sup&gt; mice compared to wild-type controls. HIF-1α&lt;sup&gt;+/–&lt;/sup&gt; mice were protected from lung eosinophilia 72 h after allergen challenge, in association with enhanced secretion of interferon-γ in recall responses of splenocytes. &lt;i&gt;Conclusions:&lt;/i&gt; HIF-1α contributes to allergic immune responses and lung eosinophilia in a mouse model of asthma.

Fig. S5 from Reciprocal Regulation of DUSP9 and DUSP16 Expression by HIF1 Controls ERK and p38 MAP Kinase Activity and Mediates Chemotherapy-Induced Breast Cancer Stem Cell Enrichment

&lt;p&gt;p38 MAPK regulates stability of pluripotency factor mRNAs.&lt;/p&gt;

Functional Analysis of the Role of Hypoxia-Inducible Factor 1 in the Pathogenesis of Hypoxic Pulmonary Hypertension

Proceedings of the Oxygen Homeostasis/Hypoxia Meeting

Abstract The first Oxygen Homeostasis/Hypoxia Meeting was held on February 12, 2003, at the Sheraton National Hotel, Washington, D.C. The meeting was hosted by Drs. S. Percy Ivy and Giovanni Melillo of the National Cancer Institute, NIH. The purpose of the meeting was to stimulate collaborations among the participants who are engaged in different areas of hypoxia research and application, including basic research on hypoxia, and its induction and consequences; the development of drugs targeting hypoxia and factors involved in pathways leading to (or controlled by) hypoxia; and the development and application of hypoxia imaging techniques and reagents.