Bing-Hua Jiang

Hypoxia Response Elements in the Aldolase A, Enolase 1, and Lactate Dehydrogenase A Gene Promoters Contain Essential Binding Sites for Hypoxia-inducible Factor 1

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5′-RCGTG-3′ and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.

Dimerization, DNA Binding, and Transactivation Properties of Hypoxia-inducible Factor 1

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1α subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1α and HIF-1β (ARNT; aryl hydrocarbon receptor nuclear translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA. In vitro-translated HIF-1α and HIF-1β generated a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 present in nuclear extracts from hypoxic cells. Compared to 826-amino acid, full-length HIF-1α, amino acids 1-166 mediated heterodimerization with HIF-1β (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1α eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1α and HIF-1β (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1α (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1α construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.

Nickel Compounds Act through Phosphatidylinositol-3-kinase/Akt-Dependent, p70S6k-Independent Pathway to Induce Hypoxia Inducible Factor Transactivation and Cap43 Expression in Mouse Epidermal Cl41 Cells

Nickel compounds are a somewhat unique class of carcinogens. Previous studies have demonstrated that NiCl2 exposure leads to marked induction of hypoxia inducible factor 1 (HIF-1) in human osteosarcoma and BALB/c 3T3 cells, a transcription factor that has been considered to play an important role in tumor promotion and progression. However, the signal transduction pathways leading to HIF-1 induction are not well understood. The present study indicated that exposure of mouse epidermal Cl41 cells to either Ni3S2 or NiCl2 resulted in activation of phosphatidylinositol 3-kinase (PI-3K), Akt, and p70 S6 kinase (p70S6k). Inhibition of PI-3K, Akt, and p70S6k by overexpression of a dominant-negative mutant of PI-3K (Δp85) impaired nickel-induced HIF-1 transactivation. Furthermore, an overexpression of the dominant-negative Akt mutant (Akt-T308A/S473A) blocked nickel-induced Akt phosphorylation and HIF-1 transactivation, whereas inhibition of p70S6k activation by pretreatment of cells with rapamycin did not show significant inhibitory effects on HIF-1 transactivation induced by nickel compounds. Consistent with HIF-1 transactivation, inhibition of the PI-3K/Akt pathway by either overexpression of Δp85 or Akt-T308A/S473A caused dramatic inhibition of Cap43 protein expression induced by nickel compounds, whereas pretreatment of cells with rapamycin did not exhibit inhibition of Cap43 induction. These results demonstrated that nickel compounds induce HIF-1 transactivation and Cap43 protein expression through a PI-3K/Akt-dependent and p70S6k-independent pathway. This study should help us understand the signal transduction pathways involved in the carcinogenic effects of nickel compounds.

Hypoxia-inducible Factor-1 Mediates Transcriptional Activation of the Heme Oxygenase-1 Gene in Response to Hypoxia

Exposure of rats to hypoxia (7% O2) markedly increased the level of heme oxygenase-1 (HO-1) mRNA in several tissues. Accumulation of HO-1 transcripts was also observed after exposure of rat aortic vascular smooth muscle (VSM) cells to 1% O2, and this induction was dependent on gene transcription. Activation of the mouse HO-1 gene by all agents thus far tested is mediated by two 5′-enhancer sequences, SX2 and AB1, but neither fragment was responsive to hypoxia in VSM cells. Hypoxia-dependent induction of the chloramphenicol acetyltransferase (CAT) reporter gene was mediated by a 163-bp fragment located approximately 9.5 kilobases upstream of the transcription start site. This fragment contains two potential binding sites for hypoxia-inducible factor 1 (HIF-1). A role for HIF-1 in HO-1 gene regulation was established by the following observations: 1) HIF-1 specifically bound to an oligonucleotide spanning these sequences, 2) mutation of these sequences abolished HIF-1 binding and hypoxia-dependent gene activation in VSM cells, 3) hypoxia increased HIF-1α and HIF-1β protein levels in VSM cells, and 4) hypoxia-dependent HO-1 mRNA accumulation was not observed in mutant hepatoma cells lacking HIF-1 DNA-binding activity. Taken together, these data demonstrate that hypoxia induces HO-1 expression in animal tissues and cell cultures and implicate HIF-1 in this response.

Transactivation and Inhibitory Domains of Hypoxia-inducible Factor 1α MODULATION OF TRANSCRIPTIONAL ACTIVITY BY OXYGEN TENSION

Hypoxia-inducible factor 1 (HIF-1) binds to cis-acting hypoxia-response elements within the erythropoietin, vascular endothelial growth factor, and other genes to activate transcription in hypoxic cells. HIF-1 is a basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β subunits. Here, we demonstrate that HIF-1α contains two transactivation domains located between amino acids 531 and 826. When expressed as GAL4 fusion proteins, the transcriptional activity of these domains increased in response to hypoxia. Fusion protein levels were unaffected by changes in cellular O2 tension. Two minimal transactivation domains were localized to amino acid residues 531–575 and 786–826. The transcriptional activation domains were separated by amino acid sequences that inhibited transactivation. Deletion analysis demonstrated that the gradual removal of inhibitory domain sequences (amino acids 576–785) was associated with progressively increased transcriptional activity of the fusion proteins, especially in cells cultured at 20% O2. Transcriptional activity of GAL4/HIF-1α fusion proteins was increased in cells exposed to 1% O2, cobalt chloride, or desferrioxamine, each of which also increased levels of endogenous HIF-1α protein but did not affect fusion protein levels. These results indicate that increased transcriptional activity mediated by HIF-1 in hypoxic cells results from both increased HIF-1α protein levels and increased activity of HIF-1α transactivation domains.

Reactive Oxygen Species Regulate Angiogenesis and Tumor Growth through Vascular Endothelial Growth Factor

Reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. However, the direct roles of endogenous ROS production still remain to be elucidated. In this study, we found that high levels of ROS were spontaneously produced by ovarian and prostate cancer cells. This elevated ROS production was inhibited by NADPH oxidase inhibitor diphenylene iodonium (DPI) and mitochondria electron chain inhibitor rotenone in the cells. To further analyze the source of ROS production, we found that ovarian cancer cells have much higher expression of NOX4 NADPH oxidase, and that specific inhibition of NADPH oxidase subunit p47(phox) diminished ROS production. To analyze the functional relevance of ROS production, we showed that ROS regulated hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in ovarian cancer cells. Elevated levels of endogenous ROS were required for inducing angiogenesis and tumor growth. NOX4 knockdown in ovarian cancer cells decreased the levels of VEGF and HIF-1 alpha and tumor angiogenesis. This study suggests a new mechanism of higher ROS production in ovarian cancer cells and provides strong evidence that endogenous ROS play an important role for cancer cells to induce angiogenesis and tumor growth. This information may be useful to understand the new mechanism of cancer cells in inducing tumorigenesis and to develop new therapeutic strategy by targeting ROS signaling in human cancer in the future.

PI3K/PTEN signaling in angiogenesis and tumorigenesis.

Phosphatidylinositol 3-kinase (PI3K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway play an important role in multiple cellular functions such as cell metabolism, proliferation, cell-cycle progression, and survival. PI3K is activated by growth factors and angiogenesis inducers such as vascular endothelial growth factor (VEGF) and angiopoietins. The amplification and mutations of PI3K and the loss of the tumor suppressor PTEN are common in various kinds of human solid tumors. The genetic alterations of upstream and downstream of PI3K signaling molecules such as receptor tyrosine kinases and AKT, respectively, are also frequently altered in human cancer. PI3K signaling regulates tumor growth and angiogenesis by activating AKT and other targets, and by inducing HIF-1 and VEGF expression. Angiogenesis is required for tumor growth and metastasis. In this review, we highlight the recent studies on the roles and mechanisms of PI3K and PTEN in regulating tumorigenesis and angiogenesis, and the roles of the downstream targets of PI3K for transmitting the signals. We also discuss the crosstalk of these signaling molecules and cellular events during tumor growth, metastasis, and tumor angiogenesis. Finally, we summarize the potential applications of PI3K, AKT, and mTOR inhibitors and their outcome in clinical trials for cancer treatment.

MiR-21 Induced Angiogenesis through AKT and ERK Activation and HIF-1α Expression

MicroRNAs (miRNAs) are endogenous, small noncoding RNAs that play important roles in various cellular functions and tumor development. Recent studies have indicated that miR-21 is one of the important miRNAs associated with tumor growth and metastasis, but the role and molecular mechanism of miR-21 in regulating tumor angiogenesis remain to be elucidated. In this study, miR-21 was overexpressed by transfecting pre-miR-21 into human prostate cancer cells and tumor angiogenesis was assayed using chicken chorioallantoic membrane (CAM). We found that overexpression of miR-21 in DU145 cells increased the expression of HIF-1α and VEGF, and induced tumor angiogenesis. AKT and extracellular regulated kinases (ERK) 1/2 are activated by miR-21. Inhibition of miR-21 by the antigomir blocked this process. Overexpression of the miR-21 target, PTEN, also inhibited tumor angiogenesis by partially inactivating AKT and ERK and decreasing the expression of HIF-1 and VEGF. The AKT and ERK inhibitors, LY294002 and U0126, suppressed HIF-1α and VEGF expression and angiogenesis. Moreover, inhibition of HIF-1α expression alone abolished miR-21-inducing tumor angiogenesis, indicating that HIF-1α is required for miR-21-upregulated angiogenesis. Therefore, we demonstrate that miR-21 induces tumor angiogenesis through targeting PTEN, leading to activate AKT and ERK1/2 signaling pathways, and thereby enhancing HIF-1α and VEGF expression; HIF-1α is a key downstream target of miR-21 in regulating tumor angiogenesis.

Cross-talk between Epidermal Growth Factor Receptor and Hypoxia-inducible Factor-1α Signal Pathways Increases Resistance to Apoptosis by Up-regulating Survivin Gene Expression

Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.

Cyanidin-3-glucoside, a Natural Product Derived from Blackberry, Exhibits Chemopreventive and Chemotherapeutic Activity

Epidemiological data suggest that consumption of fruits and vegetables has been associated with a lower incidence of cancer. Cyanidin-3-glucoside (C3G), a compound found in blackberry and other food products, was shown to possess chemopreventive and chemotherapeutic activity in the present study. In cultured JB6 cells, C3G was able to scavenge ultraviolet B-induced ·OH and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} radicals. In vivo studies indicated that C3G treatment decreased the number of non-malignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene-initiated mouse skin. Pretreatment of JB6 cells with C3G inhibited UVB- and TPA-induced transactivation of NF-κB and AP-1 and expression of cyclooxygenase-2 and tumor necrosis factor-α. These inhibitory effects appear to be mediated through the inhibition of MAPK activity. C3G also blocked TPA-induced neoplastic transformation in JB6 cells. In addition, C3G inhibited proliferation of a human lung carcinoma cell line, A549. Animal studies showed that C3G reduced the size of A549 tumor xenograft growth and significantly inhibited metastasis in nude mice. Mechanistic studies indicated that C3G inhibited migration and invasion of A549 tumor cells. These finding demonstrate for the first time that a purified compound of anthocyanin inhibits tumor promoter-induced carcinogenesis and tumor metastasis in vivo.