Publications

Systemic Delivery of Microencapsulated 3-Bromopyruvate for the Therapy of Pancreatic Cancer

Abstract Purpose: This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with β-cyclodextrin (β-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: The presence of the β-CD–3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC50) of β-CD–3-BrPA, free 3-BrPA, β-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug in vitro. An orthotopic lucMiaPaCa-2 xenograft tumor model was used to investigate the in vivo efficacy. Results: β-CD–3-BrPA and free 3-BrPA demonstrated an almost identical IC50 profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. In vivo, animals treated with β-CD–3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the β-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for β-CD–3-BrPA. Conclusion: The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of β-CD–3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered. Clin Cancer Res; 20(24); 6406–17. ©2014 AACR.

HMG Domain Proteins

Abstract As described in Chapter 3, transcriptional regulators containing an HMG-type DNA-binding domain appear to function as architectural factors whose primary function is to bend DNA. The HMG domain is an 80-amino-acid motif that was first identified in the High Mobility group nuclear proteins HMGl and HMG2 and the RNA Pol I transcription factor UBF (Jantzen et al., 1990), which bind to DNA in a non-sequence specific manner. The HMG domain has subsequently been shown to de fine a large superfamily of DNA-binding proteins containing over 100 members from eukaryotic species that include yeast, plants, and animals spanning one billion years of evolutionary time (Grosschedl et al., 1994; Laudet et al., 1993). Phylogenetic analysis identified two large subfami lies consisting of sequence-specific DNA binding proteins containing a single HMG domain and non-sequence-specific DNA-binding proteins containing multiple HMG domains (Laudet et al., 1993). All of the se quence-specific HMG-domain proteins that have been analyzed bind to DNA in the minor groove and recognize the consensus sequence 5’-(A/ T)(A/T)CAA(A/T)G-3’ (reviewed by Pevny and Lovell-Badge, 1997). In this chapter, SRY and SOX9, two HMG domain-containing proteins that bind to DNA in a sequence-specific manner and are essential for normal testicular determination and differentiation, will be described.

RETRACTED: Anthracycline chemotherapy inhibits HIF-1 transcriptional activity and tumor-induced mobilization of circulating angiogenic cells

Using a cell-based reporter gene assay, we screened a library of drugs in clinical use and identified the anthracycline chemotherapeutic agents doxorubicin and daunorubicin as potent inhibitors of hypoxia-inducible factor 1 (HIF-1)-mediated gene transcription. These drugs inhibited HIF-1 by blocking its binding to DNA. Daily administration of doxorubicin or daunorubicin potently inhibited the transcription of a HIF-1-dependent reporter gene as well as endogenous HIF-1 target genes encoding vascular endothelial growth factor, stromal-derived factor 1, and stem cell factor in tumor xenografts. CXCR4 + /sca1 + , VEGFR2 + /CD34 + , and VEGFR2 + /CD117 + bone-marrow derived cells were increased in the peripheral blood of SCID mice bearing prostate cancer xenografts but not in tumor-bearing mice treated for 5 days with doxorubicin or daunorubicin, which dramatically reduced tumor vascularization. These results provide a molecular basis for the antiangiogenic effect of anthracycline therapy and have important implications for refining the use of these drugs to treat human cancer more effectively.

Supplementary Data from RETRACTED: Collagen Prolyl Hydroxylases Are Essential for Breast Cancer Metastasis

<p>Supplementary Data PDF file 764K, Supplementary Figure S1, HIF-1 dependent P4HA1 and P4HA2 induction Supplementary Figure S2, P4HA1 and P4HA2 knockdown inhibits breast cancer growth and metastasis Supplementary Figure S3, Collagen hydroxylase knockdown decreases collagen deposition in vivo Supplementary Figure S4, P4HA1 and P4HA2 knockdown inhibits growth and metastasis of MDA-MB-435 cells Supplementary Figure S5, Picrosirius red staining of mouse lungs Supplementary Figure S6, P4HA re-expression in HIF-deficient cells rescues collagen deposition and metastasis Supplementary Figure S7, DHB treatment of MDA-MB-231 Cells Supplementary Table S1, Real-time qPCR primers. Supplemental Table S2, Comparison of human P4HA1 mRNA versus mouse P4HA1 mRNA expression in tumors. Supplemental Table S3, Correlation of P4HA1 and P4HA2 mRNA expression with receptor status</p>

Negative Regulation of Hypoxic Responses via Induced Reptin Methylation

Data from Systemic Delivery of Microencapsulated 3-Bromopyruvate for the Therapy of Pancreatic Cancer

<div>Abstract<p><b>Purpose:</b> This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with β-cyclodextrin (β-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC).</p><p><b>Experimental Design:</b> The presence of the β-CD–3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC<sub>50</sub>) of β-CD–3-BrPA, free 3-BrPA, β-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug <i>in vitro</i>. An orthotopic <i>luc</i>MiaPaCa-2 xenograft tumor model was used to investigate the <i>in vivo</i> efficacy.</p><p><b>Results:</b> β-CD–3-BrPA and free 3-BrPA demonstrated an almost identical IC<sub>50</sub> profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. <i>In vivo</i>, animals treated with β-CD–3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the β-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for β-CD–3-BrPA.</p><p><b>Conclusion:</b> The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of β-CD–3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered. <i>Clin Cancer Res; 20(24); 6406–17. ©2014 AACR</i>.</p></div>

Hypoxia-inducible factors: roles in cardiovascular disease progression, prevention, and treatment

Abstract Hypoxia-inducible factors (HIF)-1 and HIF-2 are master regulators of oxygen homeostasis that regulate the expression of thousands of genes in order to match O2 supply and demand. A large body of experimental data links HIF activity to protection against multiple disorders affecting the cardiovascular system: ischemic cardiovascular disease (including coronary artery disease and peripheral artery disease), through collateral blood vessel formation and preconditioning phenomena; emphysema; lymphedema; and lung transplant rejection. In these disorders, strategies to increase the expression of one or both HIFs may be of therapeutic utility. Conversely, extensive data link HIFs to the pathogenesis of pulmonary arterial hypertension and drugs that inhibit one or both HIFs may be useful in treating this disease.

Burn wound homing of bone marrow-derived angiogenic cells is impaired by conditional knock out of the hypoxia-inducible factor 1beta gene

Sebastian, Raul MD; Rey, Sergio MD, PhD; Zhang, Xianjie MD, PhD; Sarkar, Kakali PhD; Liu, Lixin PhD; Marti, Guy P. MD; Amin, Firoozmand MD; Junkai, Du MD, PhD; Semenza, Gregg L. MD, PhD; Harmon, John W. MD, FACS Author Information

Levels of hypoxia‐inducible factor‐1α independently predict prognosis in patients with lymph node negative breast carcinoma

Abstract BACKGROUND Hypoxia‐inducible factor‐1 (HIF‐1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis to survive cellular hypoxia. Increased levels of HIF‐1α, the O 2 ‐regulated subunit of HIF‐1, were noted during breast carcinogenesis. In this study, the prognostic value of HIF‐1α expression and its correlation with various clinicopathologic variables in patients with invasive breast carcinoma were investigated. METHODS Expression levels of HIF‐1α, HER‐2/ neu , estrogen receptor, and progesterone receptor were analyzed in 150 patients with early‐stage breast carcinoma by immunohistochemistry. HER‐2/ neu gene amplification was investigated with automated fluorescent in situ hybridization. The mitotic activity index, histologic grade, and tumor type were assessed in hematoxylin and eosinstained specimens. Clinical data included disease‐free survival, overall survival, lymph node status, and tumor size. The data were analyzed with two‐sided univariate and multivariate tests, with P values < 0.05 considered significant. RESULTS High levels of HIF‐1α had an association of borderline significance with decreased overall survival ( P = 0.059) and disease‐free survival ( P = 0.110) that was ascribed completely to the subgroup of women with lymph node negative tumors ( n = 81 patients; P = 0.008 and P = 0.004, respectively). HER‐2/ neu immunoreactivity ( P < 0.001) and gene amplification ( P < 0.001), vascular endothelial growth factor expression ( P = 0.016), and Ki‐67 expression ( P < 0.001) were correlated strongly with HIF‐1α positivity, although none of those factors had an independent effect on survival. CONCLUSIONS Increased levels of HIF‐1α were associated independently with shortened survival in patients with lymph node negative breast carcinoma. Therefore, the use of immunohistochemical assessment of HIF‐1α as a new predictor of poor outcome may improve clinical decision‐making regarding adjuvant treatment of patients with lymph node negative breast carcinoma. Cancer 2003;97:1573–81. © 2003 American Cancer Society. DOI 10.1002/cncr.11246

Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

Chronic hypoxia induces polycythemia, pulmonary hypertension, right ventricular hypertrophy, and weight loss. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding proteins that mediate adaptive responses to hypoxia, including erythropoietin, vascular endothelial growth factor, and glycolytic enzymes. Expression of the HIF-1alpha subunit increases exponentially as O2 concentration is decreased. Hif1a-/- mouse embryos with complete deficiency of HIF-1alpha due to homozygosity for a null allele at the Hif1a locus die at midgestation, with multiple cardiovascular malformations and mesenchymal cell death. Hif1a+/- heterozygotes develop normally and are indistinguishable from Hif1a+/+ wild-type littermates when maintained under normoxic conditions. In this study, the physiological responses of Hif1a+/- and Hif1a+/+ mice exposed to 10% O2 for one to six weeks were analyzed. Hif1a+/- mice demonstrated significantly delayed development of polycythemia, right ventricular hypertrophy, pulmonary hypertension, and pulmonary vascular remodeling and significantly greater weight loss compared with wild-type littermates. These results indicate that partial HIF-1alpha deficiency has significant effects on multiple systemic responses to chronic hypoxia.

Reply to Lopez-Lazaro: Evidence that digoxin inhibits human cancer

We thank Dr. Lopez-Lazaro for his letter (1) raising the issue of whether our findings in mouse xenograft models (2) can be translated to cancer patients. Extensive differences between mice and humans with regard to the pharmacology of digoxin make extrapolation between species unreliable, and therefore it remains to be determined whether digoxin has anticancer effects at doses that are tolerated in humans. However, Dr. Elizabeth Platz (Department of Epidemiology, The Johns Hopkins Bloomberg School of Public Health) has kindly permitted us to cite her unpublished data: In a longitudinal study of 47,759 health professionals, a significantly decreased relative risk for prostate cancer (PCa) was observed among individuals taking digoxin after adjustment for age (P = 0.001), multiple variables (diabetes mellitus, physical activity, body mass index, height, family history, race, pack-years smoked, and dietary factors; P = 0.004), or multiple variables plus other medications (P = 0.008). Although HIF-1α levels in the PCa cases from this study were not determined, HIF-1α levels are increased in high-grade prostatic intraepithelial neoplasia, which is the precursor of invasive PCa (3) and, among PCa patients, increased HIF-1α levels in the diagnostic biopsy are associated with significantly decreased time to disease progression after radiotherapy or prostatectomy (4). Taken together with our preclinical studies (2), the epidemiological and clinical data cited above suggest the need for trials to determine whether digoxin has therapeutic effects in patients with PCa and possibly other cancers in which increased HIF-1α levels are associated with adverse outcome.

Figure S5 from Hypoxia Selectively Enhances Integrin α<sub>5</sub>β<sub>1</sub> Receptor Expression in Breast Cancer to Promote Metastasis

<p>Growth and metastasis characteristics of MDA-MB-231 subclones.</p>

Endothelial Hypoxia-Inducible Factor-2α Is Required for the Maintenance of Airway Microvasculature

Background: Hypoxia-inducible factors (HIFs), especially HIF-1α and HIF-2α, are key mediators of the adaptive response to hypoxic stress and play essential roles in maintaining lung homeostasis. Human and animal genetics studies confirm that abnormal HIF correlates with pulmonary vascular pathology and chronic lung diseases, but it remains unclear whether endothelial cell HIF production is essential for microvascular health. The large airway has an ideal circulatory bed for evaluating histological changes and physiology in genetically modified rodents. Methods: The tracheal microvasculature of mice, with conditionally deleted or overexpressed HIF-1α or HIF-2α, was evaluated for anatomy, perfusion, and permeability. Angiogenic signaling studies assessed vascular changes attributable to dysregulated HIF expression. An orthotopic tracheal transplantation model further evaluated the contribution of individual HIF isoforms in airway endothelial cells. Results: The genetic deletion of Hif-2α but not Hif-1α caused tracheal endothelial cell apoptosis, diminished pericyte coverage, reduced vascular perfusion, defective barrier function, overlying epithelial abnormalities, and subepithelial fibrotic remodeling. HIF-2α promoted microvascular integrity in airways through endothelial angiopoietin-1/TIE2 signaling and Notch activity. In functional tracheal transplants, HIF-2α deficiency in airway donors accelerated graft microvascular loss, whereas HIF-2α or angiopoietin-1 overexpression prolonged transplant microvascular perfusion. Augmented endothelial HIF-2α in transplant donors promoted airway microvascular integrity and diminished alloimmune inflammation. Conclusions: Our findings reveal that the constitutive expression of endothelial HIF-2α is required for airway microvascular health.

Signaling Pathways That Protect the Heart Against Apoptosis Induced by Ischemia and Reperfusion

Ischemia and reperfusion injury commonly occurs in ischemic heart disease, resulting in apoptotic or necrotic cell death. Apoptotic cell death is highly regulated. Two mechanisms of apoptosis involve the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Both pathways lead to the activation of effector caspases, resulting in cell death. The mitochondrial pathway plays a key role in initiating apoptosis after ischemia and reperfusion. The phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) signaling pathways protect the heart against ischemia and reperfusion injury. They inhibit mitochondrial cytochrome c release into the cytosol by regulating the Bcl-2 family proteins and activating the mitoKATP channel, thereby blocking the process of apoptosis.

VEGF Secreted by Hypoxic Müller Cells Induces MMP-2 Expression and Activity in Endothelial Cells to Promote Retinal Neovascularization in Proliferative Diabetic Retinopathy

In proliferative diabetic retinopathy (PDR), retinal ischemia promotes neovascularization (NV), which can lead to profound vision loss in diabetic patients. Treatment for PDR, panretinal photocoagulation, is inherently destructive and has significant visual consequences. Therapies targeting vascular endothelial growth factor (VEGF) have transformed the treatment of diabetic eye disease but have proven inadequate for treating NV, prompting exploration for additional therapeutic options for PDR patients. In this regard, extracellular proteolysis is an early and sustained activity strictly required for NV. Extracellular proteolysis in NV is facilitated by the dysregulated activity of matrix metalloproteinases (MMPs). Here, we set out to better understand the regulation of MMPs by ischemia in PDR. We demonstrate that accumulation of hypoxia-inducible factor-1α in Müller cells induces the expression of VEGF, which, in turn, promotes increased MMP-2 expression and activity in neighboring endothelial cells (ECs). MMP-2 expression was detected in ECs in retinal NV tissue from PDR patients, whereas MMP-2 protein levels were elevated in the aqueous of PDR patients compared with controls. Our findings demonstrate a complex interplay among hypoxic Müller cells, secreted angiogenic factors, and neighboring ECs in the regulation of MMP-2 in retinal NV and identify MMP-2 as a target for the treatment of PDR.

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

A Novel EGLN1/PHD2 High-Frequency Variant in Tibetans Protects Against Hypoxia-Induced Polycythemia.

Abstract Abstract 2079 The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. As a high altitude population, Tibetans are genetically adapted to the environmental stress of high altitude, having normal hemoglobin concentration both at sea level and at high altitude. Several haplotypes have undergone positive selection in Tibetans, including variations at or near the EGLN locus, which encodes prolyl 4-hydroxylase (PHD2). PHD2 triggers the degradation of hypoxia-inducible factors (HIFs) that mediate many physiological responses to hypoxia, including erythropoiesis. We now describe two high-frequency Tibetan variants, PHD2D4E and PHD2C127S, that originated on the same haplotype about ∼6,000 years ago and contribute functionally to the Tibetan high-altitude phenotype. The PHD2D4E, C127S variant exhibits a lower Km value for O2, suggesting that it promotes HIF degradation under hypoxia. Because HIF directly stimulates erythropoiesis, the measured kinetic PHD2D4E, C127S properties were experimentally reproduced in native PHD2D4E, C127S erythroid progenitors that had decreased proliferation under hypoxic conditions, whereas wild-type progenitors have increased proliferation. Our results demonstrate that the Tibetan selected PHD2D4E, C127S variant contributes to protection from polycythemia at high altitude. We describe the first high-altitude adaptive mutations in Tibetans and demonstrate that they abrogate hypoxia-induced HIF-mediated augmentation of erythropoiesis, explaining Tibetan protection from polycythemia at high altitude. Disclosures: No relevant conflicts of interest to declare.

Study of hypoxia in human head and neck tumors by pimonidazole binding and HIF-1 alpha expression