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1.  Myc post-transcriptionally induces HIF1 protein and target gene expression in normal and cancer cells 
Cancer Research  2011;72(4):949-957.
c-Myc is frequently overexpressed in tumors and plays an important role in the regulation of cancer metabolism. Hypoxia-inducible factor-1 (HIF1), the master regulator of the hypoxic response, enhances tumorigenesis and influences metabolism via upregulation of the glycolytic pathway and suppression of mitochondrial respiration. Together, deregulated Myc and HIF1 cooperate to lend metabolic advantages to proliferating cancer cells and contribute to the Warburg Effect. Here we show that overexpression of Myc significantly stabilizes the alpha subunit of HIF1 (HIF1alpha) under normoxic conditions and enhances HIF1alpha accumulation under hypoxic conditions in cells. Post-transcriptional regulation of HIF1α by Myc led to the induction of HIF1α gene targets. Normoxic HIF1α protein expression was also dependent on Myc. Functionally; HIF1α expression was required for Myc-induced anchorage-independent growth and cell proliferation. Myc-dependent stabilization of HIF1α involved either disruption of binding to the VHL complex or post-translational protein modifications. Taken together, our findings uncover a previously uncharacterized regulatory relationship between Myc and HIF1 that has important implications for cancer metabolism and development.
PMCID: PMC3288382  PMID: 22186139
2.  STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells 
Oncogene  2013;33(13):1670-1679.
Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.
PMCID: PMC3868635  PMID: 23604114
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
3.  Disulfiram deregulates HIF-α subunits and blunts tumor adaptation to hypoxia in hepatoma cells 
Acta Pharmacologica Sinica  2013;34(9):1208-1216.
Disulfiram is an aldehyde dehydrogenase inhibitor that was used to treat alcoholism and showed anticancer activity, but its anticancer mechanism remains unclear. The aim of this study was to investigate the effects of disulfiram on the hypoxia-inducible factor (HIF)-driven tumor adaptation to hypoxia in vitro.
Hep3B, Huh7 and HepG2 hepatoma cells were incubated under normoxic (20% O2) or hypoxic (1% O2) conditions for 16 h. The expression and activity of HIF-1α and HIF-2α proteins were evaluated using immunoblotting and luciferase reporter assay, respectively. Semi-quantitative RT-PCR was used to analyze HIF-mediated gene expression. Endothelial tubule formation assay was used to evaluate the anti-angiogenic effect.
Hypoxia caused marked expression of HIF-1α and HIF-1α in the 3 hepatoma cell lines, dramatically increased HIF activity and induced the expression of HIF downstream genes (EPO, CA9, VEGF-A and PDK1) in Hep3B cells. HIF-2α expression was positively correlated with the induction of hypoxic genes (CA9, VEGF-A and PDK1). Moreover, hypoxia markedly increased VEGF production and angiogenic potential of Hep3B cells. Disulfiram (0.3 to 2 μmol/L) inhibited hypoxia-induced gene expression and HIF activity in a dose-dependent manner. Disulfiram more effectively suppressed the viability of Hep3B cells under hypoxia, but it did not affect the cell cycle. Overexpression of HIF-2α in Hep3B cells reversed the inhibitory effects of disulfiram on hypoxia-induced gene expression and cell survival under hypoxia.
Disulfiram deregulates the HIF-mediated hypoxic signaling pathway in hepatoma cells, which may contribute to its anticancer effect. Thus, disulfiram could be used to treat solid tumors that grow in a HIF-dependent manner.
PMCID: PMC4003155  PMID: 23852087
disulfiram; hepatoma; hypoxia; HIF-2; VEGF; angiogenesis
4.  HIF-1α: a Valid Therapeutic Target for Tumor Therapy 
Hypoxia plays a major role in the induction of angiogenesis during tumor development. One mechanism by which tumor cells respond to a reduced oxygen level is via the activation of hypoxia-inducible factor-1 (HIF-1). HIF-1 is an oxygen-dependent transcriptional activator that plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1β subunit and the highly regulated HIF-1α subunits. The stability and activity of HIF-1α are regulated by various post-translational modifications, hydroxylation, acetylation, phosphorylation and sumoyaltion. Therefore, HIF-1α interacts with several protein factors including PHD, pVHL, ARD-1, SUMO and p300/CBP. Under normoxia, the HIF-1α subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubiquitin/proteasome pathway. The association of pVHL and HIF-1α under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, under the hypoxia condition, the HIF-1α subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Under hypoxic conditions, HIF-1 eventually acts as a master regulator of numerous hypoxia-inducible genes. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation and survival, and to glucose and iron metabolism. Moreover, it was reported that the activation of HIF-1α is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1α itself or the blocking of HIF-1α interacting proteins inhibits tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. Therefore, this review summarizes the molecular mechanism of HIF-1α stability, the biological functions of HIF-1 and its potential applications for cancer therapies.
PMCID: PMC2843877  PMID: 20368827
ARD1; Angiogenesis; Anticancer therapy; Cell proliferation/survival; Glucose metabolism; HIF-1; Iron metabolism; PHD; SUMO; pVHL; p300/CBP; Transcription factor
5.  AB106. Differential regulation of LncRNA-SARCC suppresses VHL-mutant RCC cell proliferation yet promotes VHL-normal RCC cell proliferation via modulating androgen receptor/HIF-2α/C-MYC axis under hypoxia 
Translational Andrology and Urology  2016;5(Suppl 1):AB106.
It is well established that hypoxia contributes to tumor progression in a HIF-2α-dependent manner in renal cell carcinoma (RCC), yet the role of LncRNAs involved in hypoxia-mediated RCC progression remains unclear. Here we demonstrate that LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells. Mechanism dissection reveals that LncRNA-SARCC can post-transcriptionally regulate androgen receptor (AR) by physically binding and destabilizing AR protein to suppress AR/HIF-2α/C-MYC signals. In return, HIF-2α can transcriptionally regulate the LncRNA-SARCC expression via binding to hypoxia responsive elements (HREs) on the promoter of LncRNA-SARCC. The negative feedback modulation between LncRNA-SARCC/AR complex and HIF-2α signaling may then lead to differentially modulate RCC progression in a VHL-dependent manner. Together, these results may provide us a new therapeutic approach via targeting this newly identified signal from LncRNA-SARCC to AR-mediated HIF-2α/C-MYC signals against RCC progression.
Human samples—surgical specimens from human ccRCC tissues were obtained from 16 patients in the Department of Urology, Shanghai Tenth People’s Hospital, Tongji Medical School (Shanghai, China), freshly frozen in liquid nitrogen and stored at −80 °C until use. OCT-embedded blocks were sectioned until cut planes were >70% tumor. Sections were collected for DNA, RNA, and protein extraction. Samples were cataloged, clinical information on cases was obtained through chart review, and patient identifiers were removed before analysis. Informed consent was obtained from patients and the study was approved by the Institutional Review Board of Tongji Medical College. Immunohistochemistry—immunohistochemical staining was performed as previously described [40], with antibodies specific for HIF-1α, HIF-2α, C-MYC, Ki-67, AR (Abcam Inc., Cambridge, MA, USA; 1:200 dilution) and CAIX (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200 dilution). The reactivity degree was assessed by at least two pathologists without knowledge of the clinicopathological features of tumors. The degree of positivity was initially classified according to the percentage of positive tumor cells as the following: (−) >5% cells positive, (1+) 6–25% cells positive, (2+) 26–50% cells positive, and (3+) >50% cells positive. For AR, HIF-1α, HIF-2α, C-MYC, Ki-67 and CAIX, samples were scored as negative/weak, intermediate, or strong. Only cells with clear tumor cell morphology were scored. Cell culture and transfection—the human VHL-mut RCC cell lines SW839 (AR positive), OSRC-2 (AR positive), A498 (AR negative), 769-P (AR negative), 786-O (AR negative), and the human VHL-wt RCC cell lines Caki-1 (AR positive), Caki-2 (AR positive), and the immortalized proximal tubule epithelial cell line from normal adult human kidney HK-2 and 293T (AR positive) were originally purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and preserved in our lab. All RCC cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) in the humidified 5% CO2 environment at 37 °C. SW839-VHL cell lines were stably transfected with VHL-wt cDNA into VHL-mut SW839 cells (ATCC). To generate LncRNA-SARCC overexpressing or LncRNA-SARCC knocked-down stable clones of SW839, SW839-VHL and OSRC-2 cells were transfected with lentiviral vectors, pWPI-LncRNA-SARCC vs. pWPI-Vec or pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control, with the PAX2 packaging plasmid, and PMD2G envelope plasmid, then transfected into 293T cells for 48 hours to obtain the lentivirus soup followed by cryopreservation in −80 °C for later use. The cells were transfected using the lipofectamine 3000 (Invitrogen) reverse transfection protocol, according to the manufacturer’s instructions. Hypoxia—hypoxia (0.5% O2, 5% CO2, 94.5% N2) was achieved using an In Vivo2 hypoxic workstation (Ruskinn Technologies) or in a positive pressure chamber receiving gas from a custom-mixed tank (Airgas). Oxyrase® (Oxyrase) was used as a hypoxia mimetic at a final concentration of 100 mM while CoCl2 was used as a hypoxia mimetic at a final concentration of 200 µM. RNA immunoprecipitation (RIP)—native RIP was performed as described previously [50]. Briefly, SW839, SW839-VHL and OSRC-2 cells were lysed in RIPA lysis buffer (20 mM Tris-HCl/pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) supplemented with RNase inhibitor, protease inhibitor cocktail. RNase-free DNase (NEB) (400 U) was then added to the lysates and incubated on-ice for 30 minutes. The cell lysates were diluted in the RIPA buffer and 50 µL of the supernatant saved as input for PCR analysis. A total of 500 µL of the supernatant was incubated with 4 µg of AR antibody overnight (normal rabbit IgG as control). Protein A/G beads were pre-blocked by 15 mg/mL BSA in PBS. Then pre-blocked beads were added to the antibody-lysate mixture and incubated for another 2 hours. The RNA/antibody complex was washed four times by RIPA buffer supplemented with RNase inhibitor, protease inhibitor cocktail. The RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol and subjected to RT-qPCR analysis. For UV cross-linking and RIP, cells were first subjected to formaldehyde cross-linking and then native RIP protocol was conducted. RNA-pull down assay—LncRNA-SARCC, LncRNA-SARCC (1.6 kb), LncRNA-SARCC (1.2 kb) or LncRNA-SARCC (0.8 kb) were in vitro transcribed respectively from the PCR generated T7 promoter driven DNA fragments and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One milligram of whole-cell lysates from SW839 cells were incubated with 3 µg of purified biotinylated transcripts for 1 hour at 25 °C and complexes were isolated with streptavidin agarose beads (Invitrogen). The AR protein present in the pull-down material was detected by standard immunoblot analysis. Chromatin immunoprecipitation assay (ChIP)—cells were cross-linked with 4% formaldehyde for 10 minutes followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300–1,000 bp long. Lysates were precleared sequentially with normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) and protein A-agarose. Anti-AR antibody (2.0 µg) was added to the cell lysates and incubated at 4 °C overnight. For the negative control, IgG was used in the reaction. Cell-cycle analysis—cells were plated at a density such that they would be 50% confluent on the day of analysis. Treatment (hypoxia) was then initiated over the next several days, so that all cells were in culture for the same amount of time and at similar confluency when harvested. BrdU analysis was performed following the standard protocol (Becton Dickinson) after a 20 min pulse with 10 mM BrdU. Cells were stained with Alexa 488 anti-BrdU (Invitrogen) and 0.1 M propidium iodide and analyzed in an LSR FACS machine (Becton Dickinson). For proliferation analysis with Hoechst staining, 104 cells were plated on 6-cm2 plates, with staining and counting done according to the manufacturer’s instructions (Invitrogen). Luciferase assay—cells were plated in 24-well plates and transfected with pGL3 reporter constructs using lipofectamine (Invitrogen) according to the manufacturer’s instruction. After transfection, DMEM media was added into the culture with dihydrotestosterone (DHT) with ethanol as vehicle control. pRL-TK was used as internal control. Luciferase activity was measured by Dual-Luciferase Assay (Promega) according to the manufacturer’s manual. Subcutaneous and renal capsule implantation—SW839 cells expressing pLKO1-sh-LncRNA-SARCC vs. pLKO1-shRNA-control (2×106) and SW839-VHL cells expressing pWPI-LncRNA-SARCC vs. pWPI-mock (2×106), were subcutaneously injected into one flank of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 8–9 weeks, mice were sacrificed, and tumors were excised and weighed. Similarly, cells were injected into left renal capsule of 6-week-old male athymic nude mice (NCI) (n=8 mice per group). After 7–8 weeks, mice were sacrificed, and tumors were excised and weighed. Studies on animals were conducted with approval from the Animal Research Ethics Committee of the University of Rochester Medical Center. Statistical analysis—unless otherwise stated, all data were shown as mean ± SD. The SPSS 12.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. The χ2 analysis and Fisher exact probability analysis were applied for comparison among the expression of AR, HIF-1α, HIF-2α, C-MYC and Ki-67 and individual clinicopathological features. Difference of tumor cells was determined by t test or analysis of variance.
(I) Hypoxia differentially regulates RCC cell proliferation in a VHL-dependent manner; (II) LncRNA-SARCC is differentially modulated by hypoxia in a VHL-dependent manner and is physically associated with AR; (III) LncRNA-SARCC interacts with AR and decreases AR protein stability; (IV) LncRNA-SARCC suppressed AR which alters HIF-2α/C-MYC signals under hypoxia; (V) mechanism dissection how AR modulates HIF-2α expression at the transcriptional level under hypoxia; (VI) mechanism dissection how HIF-2α modulates LncRNA-SARCC expression at the transcriptional level under hypoxia; (VII) LncRNA-SARCC differentially modulates RCC proliferation under hypoxia in the RCC in vivo mouse model.
LncRNA-SARCC is differentially regulated by hypoxia in a VHL-dependent manner both in tissue culture and in human RCC clinical samples. LncRNA-SARCC can suppress hypoxic cell cycle progression in the VHL-mutant RCC cells while derepress it in the VHL-restored RCC cells.
PMCID: PMC4842606
Renal cell carcinoma; LncRNA; hypoxia; androgen receptor
6.  HIF-α effects on c-Myc distinguish two subtypes of sporadic VHL-deficient clear cell renal carcinoma 
Cancer cell  2008;14(6):435-446.
VHL tumor suppressor loss results in hypoxia inducible factor-alpha (HIF-α) stabilization, and occurs in 70% of sporadic clear cell renal carcinomas (ccRCCs). To determine whether opposing influences of HIF-1α and HIF-2α on c-Myc activity regulate human ccRCC progression, we analyzed VHL genotype and HIF-α expression in 160 primary tumors, which segregated into three groups with distinct molecular characteristics. Interestingly, ccRCCs with intact VHL, as well as pVHL-deficient, HIF-1α/HIF-2α expressing ccRCCs, exhibited enhanced Akt/mTOR and ERK/MAPK signaling. In contrast, pVHL-deficient ccRCCs expressing only HIF-2α displayed elevated c-Myc activity, resulting in enhanced proliferation and resistance to replication stress. These reproducible distinctions in ccRCC behavior delineate HIF-α effects on c-Myc in vivo and suggest molecular criteria for selecting targeted therapies.
Constitutive HIF activity is clearly associated with ccRCC tumorigenesis; however, the influence of individual HIF-α subunits on cell growth mechanisms in vivo is unknown. Few dominant oncogenic pathways have been identified within ccRCC, making it difficult to select optimal targeted therapies for patients, or to predict disease outcome, except by grade and stage. Cell culture experiments indicate that HIF-1α inhibits the c-Myc oncoprotein, whereas HIF-2α potentiates c-Myc transcriptional activity and cellular proliferation. The findings reported here indicate that HIF-1α and HIF-2α promote distinct oncogene activation in human ccRCCs, and reveal a critical role for HIF-2α and c-Myc in promoting genomic integrity. These results suggest that evaluating pVHL status and HIF-α expression may aid targeted therapy selection for human ccRCCs.
PMCID: PMC2621440  PMID: 19061835
7.  HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs) 
Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs.
We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs.
We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF).
The present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.
PMCID: PMC3978922  PMID: 23947803
8.  Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response 
Cellular signalling  2013;25(9):1895-1903.
Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactoral enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 require distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity.
PMCID: PMC3700616  PMID: 23707522
hypoxia; HIF; enhanceosome; transcription factors; tumor microenvironment; transcription
9.  Analysis of hypoxia-inducible factor alpha polyploidization reveals adaptation to Tibetan plateau in the evolution of schizothoracine fish 
Hypoxia-inducible factor (HIF) is a master regulator that mediates major changes in gene expression under hypoxic conditions. Though HIF family has been identified in many organisms, little is known about this family in schizothoracine fish.
Duplicated hif-α (hif-1αA, hif-1αB, hif-2αA, and hif-2αB) genes were identified in schizothoracine fish. All the deduced HIF-α proteins contain the main domains (bHLH-PAS, ODDD, and TAD), also found in humans. Evidence suggests a Cyprinidae-specific deletion, specifically, a conserved proline hydroxylation motif LxxLAP, in the NODD domain of schizothoracine fish HIF-1αA. In addition, a schizothoracine-specific mutation was observed in the CODD domain of the specialized and highly specialized schizothoracine fish HIF-1αB, which is the proline hydroxylation motif mutated into PxxLAP. Standard and stochastic branch-site codon model analysis indicated that only HIF-1αB has undergone positive selection, which may have led to changes in function. To confirm this hypothesis, HIF-αs tagged with Myc were transfected into HEK 293 T cells. Each HIF-1αB was found to significantly upregulate luciferase activity under normoxic and hypoxic conditions, which indicated that the HIF-1αB protein was more stable than other HIF-αs.
All deduced HIF-α proteins of schizothoracine fish contain important domains, like their mammalian counterparts, and each HIF-α is shorter than that of human. Our experiments reveal that teleost-specific duplicated hif-α genes played different roles under hypoxic conditions, and HIF-1αB may be the most important regulator in the adaptation of schizothoracine fish to the environment of the Tibetan Plateau.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0192-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4162920  PMID: 25205386
HIF-α; Schizothoracine fish; Hypoxia; Positive selection; Tibetan Plateau adaptation
10.  Signal Transducer and Activator of Transcription 3 is required for hypoxia-inducible factor-1α RNA expression in both tumor cells and tumor-associated myeloid cells 
Molecular cancer research : MCR  2008;6(7):1099-1105.
Hypoxia-inducible factor 1 (HIF-1) is a potent tumorigenic factor. Its alpha subunit (HIF-1α), which is tightly regulated in normal tissues, is elevated in tumors due to hypoxia and overactive growth signaling pathways. Although much is known about HIF-1α regulation in cancer cells, crucial molecular targets that affect HIF-1α levels modulated by both hypoxia and oncogenic signaling pathways remain to be identified. Additionally, whether and how the tumor microenvironment contributes to HIF-1α accumulation is unclear. This study demonstrates a novel mechanism by which HIF-1α availability is regulated in both cancer cells and in myeloid cells in the tumor microenvironment. We show a requirement of Stat3 for HIF-1α RNA expression under both hypoxia and growth signaling conditions. Furthermore, tumor-derived myeloid cells express elevated levels of HIF-1α mRNA relative to their counterparts from normal tissues in a Stat3-dependent manner. Additionally, Stat3 activity in the non-transformed cells in the tumor milieu impacts HIF-1α RNA expression of the entire growing tumor. Consistent with a role of Stat3 in regulating HIF-1α RNA transcription, elevated Stat3 activity increases HIF-1α promoter activity, and Stat3 protein binds to the HIF-1α promoter in both transformed cells and in growing tumors. Taken together, these findings demonstrate a novel mode by which HIF-1α is regulated not only in cancer cells but also in the tumor associated inflammatory cells, suggesting Stat3 as an important molecular target for inhibiting the oncogenic potential of HIF-1 induced by both hypoxia and overactive growth signaling pathways prevalent in cancer.
PMCID: PMC2775817  PMID: 18644974
Neuro-Oncology  2014;16(Suppl 2):ii27-ii28.
BACKGROUND: Von Hippel-Lindau disease (VHL)is a genetic condition predisposing to the development of multiple specific tumors, mainly CNS and retinal hemangioblastomas and clear cell renal cancer (CCRC). Protein pVHL, codified by VHL gene, is an important component of the functional complex responsible for degrading hypoxia inducible factors (HIF1alpha and HIF2alpha). In hypoxic condition, or in absence of functional protein VHL, an accumulation of HIF results, and thus an activation of gene transcription related to angiogenesis, and cell proliferation and survival. A number of works supports a role of unregulated amplification of HIF activity and of proteins codified by HIF-inducible genes as oncogenic, promoting the development of some tumors as CCRC, in VHL patients. Their role is still unknown in the appearance of hemangioblastomas, as there is no information about the level of synthesis and transcription of HIF proteins in these tumors. The main objective of our study is to determine the levels of proteins HIF-1alpha and HIF-2alpha and the eventual associated VEGF variation. MATERIAL AND METHODS: Tissue from nine hemangioblastomas (cerebellar, spinal cord, brainstem, brain) resected from VHL patients was obtained immediately after each tumor resection, and cultured for growing. Six primary cultures were obtained, and analyzed by Flow Cytometry for cell population component evaluation and by Western-Blot in order to measure the levels of proteins HIF-1alpha, HIF-2alpha and of VEGF. RESULTS: Cellular characterization of the 6 primary cultures obtained from 9 hemangioblastomas has shown they are composed of stromal cells (more than 50% CD99 + cells), endothelial cells (15% CD34+ cells), and pericytes (30% NG2+ cells). In the study, 25-40% of stromal cells, 3-15% of endothelial cells, and 5-30% of pericytes had increased levels of HIF-1alpha and HIF-2alpha. Furthermore, Western-Blot assay showed a very high concentration of VEGF in hemangioblastoma cells, 2 to 4 times more than in cell lines characterized by over-expression of VEGF (lines derived from colon cancer and from retinal pigmentary epithelium). No correlation of HIF expression in culture and cell proliferation in the original tumor (Mib-1) has been found. CONCLUSIONS: Our results show that proteins HIF-1alpha and HIF-2alpha are over-expressed in hemangioblastomas from VHL patients, suggesting a role of these proteins in the development and growth of these tumors. The results support the exploration of possible treatments directed to inactivate HIF1alpha and HIF-2alpha proteins as new targets, in order to control de proliferation of hemangioblastomas in these patients.
PMCID: PMC4185871
12.  Selection, Analysis and Improvement of Anti-Angiogenesis Compounds Identified by an Anti-HIF-1α Screening and Validation System 
Journal of Cancer  2016;7(14):1926-1938.
Cancer cells resort to activating hypoxia-inducible factor-1 (HIF-1) as one of several responses to hypoxic conditions. Overexpression of HIF-1, the transcriptional regulator for a group of malignant-pathway related genes including vascular endothelial growth factor (VEGF), is associated with increased tumor growth, vascularization, and metastasis. HIF-1 is composed of an inducible subunit, HIF-1α and a constitutively expressed subunit, HIF-1ß. HIF-1 activity is mainly dependent on the level of HIF-1α protein, the inducible and regulatory subunit of the HIF-1 heterodimer complex; thus, identification of novel anti-HIF-1α agents will lead to effective blockage of the HIF-1 (HIF-1α)-mediated “switch-on” function for those malignant-pathway related genes and suppression of the HIF-1α/VEGF-mediated signaling pathway that promotes cancer progression and metastasis.
While there is an extremely large number of small molecule compounds in the database (compound libraries), the currently existing screening system is inefficient and time-consuming; or, at best, the application of the existing screening system is very limited as it is usually not coupled with biological validation processes. The further development of potential drugs is partly hindered due to the cumbersome steps in between the primary screen and consequent validation: the slow, exhausted and sometimes lack of a linked biological validation process contributes to the dismal fate of scant compounds uncovered in the primary screen.
To improve upon the status quo, we developed a prototype screening system that is coupled anti-HIF-1α primary screen with secondary anti-VEGF/anti-angiogenesis validation screens. We used breast cancer cells as the model to select potent anti-HIF-1α small-molecule compounds by their abilities to inhibit transactivation of a VEGF promoter fused to a luciferase reporter gene under hypoxia. Positive compounds were then validated by a series of assays that confirm compounds' anti-HIF-1α activities including measurement of their effects on HIF-1α downstream VEGF gene expression and angiogenic ability of breast cancer cells. Moreover, we demonstrated that we could further improve the compound's potency of anti-HIF-1α and anti-angiogenesis by modifying the identified lead to synthesize a superior (novel) drug.
PMCID: PMC5118656  PMID: 27877208
hypoxia-inducible factor-1; anti-angiogenesis
13.  Inhibiting αv Integrins Decreases the Malignant Characteristics of Medulloblastoma 
Hypoxia induces an aggressive phenotype in some brain tumors in part due to hypoxia inducible factor 1α (HIF-1α) and integrin expression. The importance of hypoxia in medulloblastoma is unclear and the interaction of HIF-1α and c-Myc in medulloblastoma has not been explored. The objective of this project was to determine if hypoxia induces an aggressive phenotype in human medulloblastoma cells that constitutively express high (D283 Med) or low (DAOY) levels of c-Myc and to determine if blocking αv integrins with the monoclonal antibody, intetumumab, inhibits hypoxia-induced cellular stress responses.
Cells were grown at 21% and 1% O2 and in the presence or absence of intetumumab. Measures of malignancy evaluated included cell proliferation, cell migration, and expression of vascular endothelial growth factor (VEGF), αv integrins, HIF-1α, and c-Myc.
Both cell lines robustly expressed αv integrins. Hypoxic DAOY cells had significantly increased proliferation compared to normoxic controls (P < 0.05) whereas D283 Med cells did not. Both cell lines exhibited a dose-dependent decrease in proliferation when treated with intetumumab, (P < 0.05). Hypoxia did not increase DAOY migration. Regardless, intetumumab significantly inhibited migration at both oxygen conditions (P < 0.05). Intetumumab significantly decreased VEGF levels in DAOY cells at both oxygen conditions (P < 0.05) and in normoxic D283 cells (P < 0.01). Neither cell line had increased HIF-1α expression in response to hypoxia. However, hypoxic D283 Med cells grown in the presence of intetumumab demonstrated significantly decreased c-Myc expression (P < 0.05).
Hypoxia did not clearly induce a more aggressive phenotype in medulloblastoma cells. Despite this, intetumumab decreased medulloblastoma cell proliferation and migration and variably decreased VEGF and c-Myc expression in hypoxic conditions. Targeting αv integrins represents a promising potential adjuvant modality in the treatment of medulloblastoma, particularly subtypes that metastasize and overexpress VEGF and c-Myc.
PMCID: PMC3709582  PMID: 23082872
medulloblastoma; intetumumab; αv integrin; hypoxia; c-Myc; HIF-1α
14.  HIF1α deficiency reduces inflammation in a mouse model of proximal colon cancer 
Disease Models & Mechanisms  2015;8(9):1093-1103.
Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates the adaptation of cells to hypoxic microenvironments, for example inside solid tumours. Stabilisation of HIF1α can also occur in normoxic conditions in inflamed tissue or as a result of inactivating mutations in negative regulators of HIF1α. Aberrant overexpression of HIF1α in many different cancers has led to intensive efforts to develop HIF1α-targeted therapies. However, the role of HIF1α is still poorly understood in chronic inflammation that predisposes the colon to carcinogenesis. We have previously reported that the transcription of HIF1α is upregulated and that the protein is stabilised in inflammatory lesions that are caused by the non-steroidal anti-inflammatory drug (NSAID) sulindac in the mouse proximal colon. Here, we exploited this side effect of long-term sulindac administration to analyse the role of HIF1α in colon inflammation using mice with a Villin-Cre-induced deletion of Hif1α exon 2 in the intestinal epithelium (Hif1αΔIEC). We also analysed the effect of sulindac sulfide on the aryl hydrocarbon receptor (AHR) pathway in vitro in colon cancer cells. Most sulindac-treated mice developed visible lesions, resembling the appearance of flat adenomas in the human colon, surrounded by macroscopically normal mucosa. Hif1αΔIEC mice still developed lesions but they were smaller than in the Hif1α-floxed siblings (Hif1αF/F). Microscopically, Hif1αΔIEC mice had significantly less severe colon inflammation than Hif1αF/F mice. Molecular analysis showed reduced MIF expression and increased E-cadherin mRNA expression in the colon of sulindac-treated Hif1αΔIEC mice. However, immunohistochemistry analysis revealed a defect of E-cadherin protein expression in sulindac-treated Hif1αΔIEC mice. Sulindac sulfide treatment in vitro upregulated Hif1α, c-JUN and IL8 expression through the AHR pathway. Taken together, HIF1α expression augments inflammation in the proximal colon of sulindac-treated mice, and AHR activation by sulindac might lead to the reduction of E-cadherin protein levels through the mitogen-activated protein kinase (MAPK) pathway.
Summary: HIF1α deficiency reduces inflammation in the mouse proximal colon but is associated with defective E-cadherin expression in colon epithelial cells when mice lacking intestinal epithelium expression of Hif1α are challenged with sulindac.
PMCID: PMC4582097  PMID: 26183215
HIF1α; MIF; AHR; E-cadherin; Sulindac; Colon inflammation
15.  Combinatorial regulation of neuroblastoma tumor progression by N-Myc and hypoxia inducible factor HIF-1α 
Cancer research  2010;70(24):10351-10361.
In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. Because hypoxia contributes to aggressive tumor phenotypes, predominantly via two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, we examined hypoxia responses in MYCN amplified neuroblastoma cells. We demonstrate here that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN amplified neuroblastoma cells and primary tumors in comparison to samples without MYCN amplification. Our results showed that interplay between N-Myc and HIF-1α plays critical roles in neuroblastoma. For example, high levels of N-Myc override HIF-1α inhibition of cell cycle progression, enabling continued proliferation under hypoxia. Furthermore, both HIF-1α and N-Myc are essential for the Warburg effect (aerobic glycolysis) in neuroblastomas by activating the transcription of multiple glycolytic genes. Of note, expression of Phosphoglycerate Kinase 1 (PGK1), Hexokinase 2 (HK2) and Lactate Dehydrogenase A (LDHA), were each significantly higher in MYCN amplified neuroblastomas compared to tumors without MYCN amplification. Interestingly, MYCN amplified neuroblastoma cells are “addicted” to LDHA enzymatic activity, as its depletion completely inhibits tumorigenesis in vivo. Thus, our results provide mechanistic insights explaining how MYCN amplified neuroblastoma cells contend with hypoxic stress and paradoxically how hypoxia contributes to neuroblastoma aggressiveness through combinatorial effects of N-Myc and HIF-1α. These results also suggest LDHA represents a novel, pharmacologically tractable target for neuroblastoma therapeutics.
PMCID: PMC3005134  PMID: 20961996
N-Myc; hypoxia inducible factor; neuroblastoma; tumorigenesis; Warburg effect
16.  HIF-1α Promotes A Hypoxia-Independent Cell Migration 
Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.
PMCID: PMC2946250  PMID: 20882121
VEGF; cell migration; hypoxia; HIF-1α
17.  Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions 
Molecular Cancer  2009;8:104.
Hypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.
HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1α and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1α expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 μM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1α expression. However, a 24 h treatment with 10 μM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels.
We speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1α expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1α in melanoma biology increases its importance as a therapeutic target.
PMCID: PMC2781803  PMID: 19919690
18.  Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus 
Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.
We have cloned and characterized two distinct HIF-alpha cDNAs – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57–68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41–47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp.
There are at least two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp.
PMCID: PMC1473195  PMID: 16623959
19.  Hypoxia Promotes Migration and Induces CXCR4 Expression via HIF-1α Activation in Human Osteosarcoma 
PLoS ONE  2014;9(3):e90518.
Cellular adaptation to a hypoxic microenvironment is essential for tumor progression and is largely mediated by HIF-1α through coordinated regulation of hypoxia-responsive genes. The chemokine SDF-1α and its unique receptor CXCR4 have been implicated in organ-specific metastases of many cancers. In this study, we investigated the response of osteosarcoma cells to hypoxia and the expression of CXCR4 and HIF-1α in human osteosarcoma specimens and explored the roles of CXCR4 and HIF-1α in the cell migration process.
Methodology/Principal Findings
We performed immunohistochemistry, immunocytochemistry, quantitative real-time PCR, Western blots and fluorescent reporter assays to evaluate the correlation between CXCR4 and HIF-1α expression in human osteosarcoma specimens or SOSP-9607 cells under normoxic and hypoxic conditions. Transwell assays were used to assess cell migration under different conditions. Exposure of SOSP-9607 cells to hypoxic conditions resulted in significantly increased migration. When SOSP-9607 cells were subjected to hypoxic conditions, the mRNA and protein levels of CXCR4 were significantly increased in a time-dependent manner. Moreover, siHIF-1α significantly decreased the mRNA and protein levels of CXCR4 under hypoxia, whereas pcDNA-HIF-1α significantly increased the mRNA and protein levels of CXCR4 under normoxia. A luciferase reporter gene study showed that siHIF-1α reduced pGL3-CXCR4 luciferase activity. Furthermore, coexpression of HIF-1α and CXCR4 was significantly higher in patients with distant metastasis compared with those without metastasis.
The hypoxia-HIF-1α-CXCR4 pathway plays a crucial role during the migration of human osteosarcoma cells, and targeting this pathway might represent a novel therapeutic strategy for patients suffering from osteosarcoma.
PMCID: PMC3949690  PMID: 24618817
20.  CDK1 stabilizes HIF-1α via direct phosphorylation of Ser668 to promote tumor growth 
Cell Cycle  2013;12(23):3689-3701.
Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intratumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to anticancer therapies. Here, we show that cyclin-dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators. Overexpression of CDK1 and/or cyclin B1 is sufficient to stabilize HIF-1α under normoxic conditions, whereas inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady-state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668. HIF-1α is stabilized under normoxic conditions during G2/M phase via CDK1-mediated phosphorylation of Ser668. A phospho-mimetic construct of HIF-1α at Ser668 (S668E) is significantly more stable under both normoxic and hypoxic conditions, resulting in enhanced transcription of HIF-1 target genes and increased tumor cell invasion and migration. Importantly, HIF-1α (S668E) displays increased tumor angiogenesis, proliferation, and tumor growth in vivo compared with wild-type HIF-1α. Thus, we have identified a novel link between CDK1 and HIF-1α that provides a potential molecular explanation for the elevated HIF-1 activity observed in primary and metastatic tumors, independent of hypoxia, and offers a molecular rationale for the clinical translation of CDK inhibitors for use in tumors with constitutively active HIF-1.
PMCID: PMC3903720  PMID: 24189531
CDK1; HIF-1α; angiogenesis; cell cycle; hypoxia
21.  Notch1 is required for hypoxia-induced proliferation, invasion and chemoresistance of T-cell acute lymphoblastic leukemia cells 
Notch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL). Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α) activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL.
T-ALL cell lines (Jurkat, Sup-T1) transfected with HIF-1α or Notch1 small interference RNA (siRNA) were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot.
Hypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2) and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression.
Notch1 signalling is required for hypoxia/HIF-1α-induced proliferation, invasion and chemoresistance in T-ALL. Pharmacological inhibitors of HIF-1α or Notch1 signalling may be attractive interventions for T-ALL treatment.
PMCID: PMC3544631  PMID: 23289374
T-cell acute lymphoblastic leukemia; Hypoxia; HIF-1α; Notch1; Proliferation; Invasion; Chemoresistance
22.  The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme 
PLoS ONE  2014;9(1):e86102.
Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.
PMCID: PMC3900478  PMID: 24465898
23.  Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions 
Oncology Letters  2016;12(2):1034-1040.
Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1α/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.
PMCID: PMC4950743  PMID: 27446390
hypoxia inducible factor-1α; toll-like receptor 4 signaling pathway; HepG2 cell line; anti-tumor effects
24.  A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1α (HIF-1α) does not impair Pro-564 hydroxylation 
Molecular Cancer  2003;2:31.
The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).
Patients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.
The Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.
PMCID: PMC212228  PMID: 14521712
25.  STAT3 or USF2 Contributes to HIF Target Gene Specificity 
PLoS ONE  2013;8(8):e72358.
The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.
PMCID: PMC3749168  PMID: 23991099

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