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1.  Synergistic Suppression of Early Phase of Adipogenesis by Microsomal PGE Synthase-1 (PTGES1)-Produced PGE2 and Aldo-Keto Reductase 1B3-Produced PGF2α 
PLoS ONE  2012;7(9):e44698.
We recently reported that aldo-keto reductase 1B3-produced prostaglandin (PG) F2α suppressed the early phase of adipogenesis. PGE2 is also known to suppress adipogenesis. In this study, we found that microsomal PGE2 synthase (PGES)-1 (mPGES-1; PTGES1) acted as the PGES in adipocytes and that PGE2 and PGF2α synergistically suppressed the early phase of adipogenesis. PGE2 production was detected in preadipocytes and transiently enhanced at 3 h after the initiation of adipogenesis of mouse adipocytic 3T3-L1 cells, followed by a quick decrease; and its production profile was similar to the expression of the cyclooxygenase-2 (PTGS2) gene. When 3T3-L1 cells were transfected with siRNAs for any one of the three major PTGESs, i.e., PTGES1, PTGES2 (mPGES-2), and PTGES3 (cytosolic PGES), only PTGES1 siRNA suppressed PGE2 production and enhanced the expression of adipogenic genes. AE1-329, a PTGER4 (EP4) receptor agonist, increased the expression of the Ptgs2 gene with a peak at 1 h after the initiation of adipogenesis. PGE2-mediated enhancement of the PTGS2 expression was suppressed by the co-treatment with L-161982, a PTGER4 receptor antagonist. Moreover, AE1-329 enhanced the expression of the Ptgs2 gene by binding of the cyclic AMP response element (CRE)-binding protein to the CRE of the Ptgs2 promoter; and its binding was suppressed by co-treatment with L-161982, which was demonstrated by promoter luciferase and chromatin immunoprecipitation assays. Furthermore, when 3T3-L1 cells were caused to differentiate into adipocytes in medium containing both PGE2 and PGF2α, the expression of the adipogenic genes and the intracellular triglyceride level were decreased to a greater extent than in medium containing either of them, revealing that PGE2 and PGF2α independently suppressed adipogenesis. These results indicate that PGE2 was synthesized by PTGES1 in adipocytes and synergistically suppressed the early phase of adipogenesis of 3T3-L1 cells in cooperation with PGF2α through receptor-mediated activation of PTGS2 expression.
PMCID: PMC3436788  PMID: 22970288
2.  Variants of gene for microsomal prostaglandin E2 synthase show association with disease and severe inflammation in rheumatoid arthritis 
Microsomal PGE synthase 1 (mPGES-1) is the terminal enzyme in the induced state of prostaglandin E2 (PGE2) synthesis and constitutes a therapeutic target for rheumatoid arthritis (RA) treatment. We examined the role of the prostaglandin E synthase (PTGES) gene polymorphism in susceptibility to and severity of RA and related variations in the gene to its function. The PTGES gene polymorphism was analyzed in 3081 RA patients and 1900 controls from two study populations: Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) and the Leiden Early Arthritis Clinic (Leiden EAC). Baseline disease activity score (DAS28) was employed as a disease severity measure. mPGES-1 expression was analyzed in synovial tissue from RA patients with known genotypes using immunohistochemistry. In the Swedish study population, among women a significant association with risk for RA was observed for PTGES single-nucleotide polymorphisms (SNPs) in univariate analysis and for the distinct haplotype. These results were substantiated by meta-analysis of data from EIRA and Leiden EAC studies with overall OR 1.31 (95% confidence interval 1.11–1.56). Several PTGES SNPs were associated with earlier onset of disease or with higher DAS28 in women with RA. Patients with the genotype associated with higher DAS28 exhibited significantly higher mPGES-1 expression in synovial tissue. Our data reveal a possible influence of PTGES polymorphism on the pathogenesis of RA and on disease severity through upregulation of mPGES-1 at the sites of inflammation. Genetically predisposed individuals may develop earlier and more active disease owing to this mechanism.
PMCID: PMC3172931  PMID: 21448233
mPGES-1; gene polymorphism; rheumatoid arthritis; gender
3.  Hypoxia and Prostaglandin E Receptor 4 Signalling Pathways Synergise to Promote Endometrial Adenocarcinoma Cell Proliferation and Tumour Growth 
PLoS ONE  2011;6(5):e19209.
The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1–4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.
PMCID: PMC3093383  PMID: 21589857
4.  HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells 
World Journal of Gastroenterology : WJG  2014;20(47):17894-17904.
AIM: To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC).
METHODS: Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction.
RESULTS: Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo.
CONCLUSION: HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2.
PMCID: PMC4273139  PMID: 25548487
Esophageal squamous cell carcinoma; Hypoxia-inducible factor-1α; VE-cadherin; RNA interference; Vasculogenic mimicry
5.  Hypoxia-inducible factor-2α is essential in activating the COX2/mPGES-1/PGE2 signaling axis in colon cancer 
Carcinogenesis  2012;34(1):163-169.
Cyclooxygenase 2 (COX2) is overexpressed in 80% of colon adenocarcinomas. However, the mechanism leading to aberrant COX2 expression in tumors is unclear. Intestinal epithelium-specific disruption of the von Hippel–Lindau tumor suppressor protein (VHL) in adenomatous polyposis coli (Apc)min/+ mice (VhlΔIE/Apcmin/+) resulted in constitutive activation of hypoxia-inducible factor (HIF), robustly enhanced colon carcinogenesis and potentiated COX2 expression in normal colon epithelium and tumors. In this study, we hypothesize that HIF regulates COX2 expression in colon tumors, and this regulation is critical for HIF-mediated colon carcinogenesis. COX2 was demonstrated to be a direct target gene of HIF-2α, and genetic disruption of HIF-2α abolished the induction of COX2 in tumors. Furthermore, inhibition of COX2 by nimesulide reduced HIF-2α-induced colon tumor formation. Interestingly, the levels of prostaglandin E2 (PGE2), the downstream effector of COX2, remained elevated in normal and tumor tissues of the nimesulide-treated VhlΔIE/Apcmin/+ mice. Further examination revealed that the terminal PGE2 synthesis enzyme microsomal prostaglandin E synthase 1 (mPGES-1) was overexpressed in the colon of VhlΔIE/Apcmin/+ mice. mPGES-1 was demonstrated to be a direct target gene of HIF-2α, and genetic disruption of HIF-2α abolished the induction of mPGES-1 in colon tumors. Together, our findings demonstrate that HIF-2α is a major regulator of COX2/mPGES-1/PGE2 pathway in colon tumors.
PMCID: PMC3534191  PMID: 23042097
6.  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
7.  Increased HIF-1alpha expression in tumor cells and lymphocytes of tumor microenvironments predicts unfavorable survival in esophageal squamous cell carcinoma patients 
The expression of hypoxia-induced factor (HIF)-1α is up-regulated in tumor microenvironments under hypoxia condition. However, the prognostic significance of HIF-1α in esophageal squamous cell carcinoma (ESCC) is still elusive. We measured the HIF-1α expression by immunochemistry in tumor specimens from 136 resected ESCC; in the current study, the HIF-1α expression in tumor cells was significantly associated with tumor stage (P = 0.003) and lymph node metastasis (P = 0.006); whereas the HIF-1α expression in tumor-infiltrating lymphocytes (TILs) had no relationship with patients’ clinicopathological parameters. Patients with high HIF-1α expression in tumor cells or in TILs showed worse survival related to those with low HIF-1α expression. Multivariate analysis demonstrated that expression of HIF-1α in TILs was an independent factor for DFS (P = 0.007) and OS (P = 0.013). Additionally, the expression of HIF-1α in tumor cells was an independent factor for DFS (P = 0.037) and OS (P = 0.033) in locoregional ESCC patients, whereas the expression of HIF-1α in TILs was an independent factor for DFS (P = 0.048) and OS (P = 0.039) in metastatic ESCC patients. Correlation analysis revealed that expressions of HIF-1α in tumor cells and in TILs were positively correlated, and patients with combined high HIF-1α in both tumor cells and TILs had the worst survivals (P < 0.05). These findings suggest that the HIF-1α expressions in different cell populations of ESCC microenvironments have different clinical relevance and prognostic impact on patients.
PMCID: PMC4129000  PMID: 25120765
Esophageal squamous cell carcinoma; HIF-1α; tumor microenvironments; clinical prognosis; tumor-infiltrating lymphocytes
8.  Crucial Role of IL1beta and C3a in the In Vitro-Response of Multipotent Mesenchymal Stromal Cells to Inflammatory Mediators of Polytrauma 
PLoS ONE  2015;10(1):e0116772.
Multipotent mesenchymal stromal cells (MSC) exert immune-modulatory effects and support tissue regeneration in various local trauma models. In case of a polytrauma, high amounts of danger-associated molecular patterns are released, leading to a systemic increase of inflammatory mediators. The influence of such a complex inflammatory microenvironment on human MSC is mainly unknown so far. Therefore, we investigated the effects of a defined serum-free polytrauma “cocktail” containing ILͳbeta, IL6, IL8 and the anaphylatoxins C3a and C5a, in concentrations corresponding to those measured in the blood of polytrauma patients, on human MSC in vitro. The polytrauma cocktail induced directed migration of MSC with C3a representing its major soluble chemoattractive agent. Furthermore, the polytrauma cocktail and IL1beta upregulated the expression of MMP1 indicating a potential role of IL1beta to enhance MSC migration in the tissue context. COX2, PTGES and TSG6 were also found to be upregulated upon stimulation with the polytrauma cocktail or IL1beta, but not through other single factors of the polytrauma cocktail in pathophysiologically relevant concentrations. An RNA expression array of 84 inflammation-related genes revealed that both the polytrauma cocktail and IL1beta induced C3, CSF1, TLR3 and various chemokines without major qualitative or quantitative differences. These results indicate that IL1beta is a crucial mediator of the polytrauma cocktail in terms of immune-modulation and MMP1 expression. Thus, upon encountering the primary sterile, inflammatory milieu of a polytrauma, endogenous or systemically transfused MSC might be able to migrate to sites of injury, secrete TSG6 and PGE2 and to influence macrophage biology as observed in local trauma models.
PMCID: PMC4285554  PMID: 25562599
9.  MicroRNA response to hypoxic stress in soft tissue sarcoma cells: microRNA mediated regulation of HIF3α 
BMC Cancer  2014;14:429.
Hypoxia is often encountered in solid tumors and known to contribute to aggressive tumor behavior, radiation- and chemotherapy resistance resulting in a poor prognosis for the cancer patient. MicroRNAs (miRNAs) play a role in the regulation of the tumor cell response to hypoxia, however, not much is known about the involvement of miRNAs in hypoxic signalling pathways in soft tissue sarcomas (STS).
A panel of twelve STS cell lines was exposed to atmospheric oxygen concentrations (normoxia) or 1% oxygen (hypoxia) for up to 48 h. Hypoxic conditions were verified and miRNA expression profiles were assessed by LNA™ oligonucleotide microarrays and RT-PCR after 24 h. The expression of target genes regulated by hypoxia responsive miRNAs is examined by end-point PCR and validated by luciferase reporter constructs.
Exposure of STS cell lines to hypoxic conditions gave rise to upregulation of Hypoxia Inducible Factor (HIF) 1α protein levels and increased mRNA expression of HIF1 target genes CA9 and VEGFA. Deregulation of miRNA expression after 24 h of hypoxia was observed. The most differentially expressed miRNAs (p < 0.001) in response to hypoxia were miR-185-3p, miR-485-5p, miR-216a-5p (upregulated) and miR-625-5p (downregulated). The well-known hypoxia responsive miR-210-3p could not be reliably detected by the microarray platform most likely for technical reasons, however, its upregulation upon hypoxic stress was apparent by qPCR. Target prediction algorithms identified 11 potential binding sites for miR-485-5p and a single putative miR-210-3p binding site in the 3’UTR of HIF3α, the least studied member of the HIF family. We showed that HIF3α transcripts, expressing a 3’UTR containing the miR-485-5p and miR-210-3p target sites, are expressed in all sarcoma cell lines and upregulated upon hypoxia. Additionally, luciferase reporter constructs containing the 3’UTR of HIF3α were used to demonstrate regulation of HIF3α by miR-210-3p and miR-485-5p.
Here we provide evidence for the miRNA mediated regulation of HIF3α by hypoxia responsive miRNAs in STS, which may help to tightly regulate and fine-tune the hypoxic response. This provides a better insight into the mechanisms underlying the hypoxic response in STS and may ultimately yield information on novel prognostic and predictive markers or targets for treatment.
PMCID: PMC4065608  PMID: 24927770
miRNA; Hypoxia; HIF3α; Soft tissue sarcomas; miR-210-3p; miR-485-5p
10.  Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation 
PLoS ONE  2012;7(11):e48342.
Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE2). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE2 production after HIPK2 depletion and how to modulate it.
Methodology/Principal findings
We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE2 generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE2/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation.
These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE2/VEGF production; and 3) zinc treatment of HIPK2 depleted cells restored DCs maturation.
PMCID: PMC3492329  PMID: 23144866
11.  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
12.  Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis1 
Neuro-Oncology  2005;7(2):134-153.
Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors α and β, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.
PMCID: PMC1871894  PMID: 15831232
13.  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
14.  HIF-1α and HIF-2α have divergent roles in colon cancer 
Hypoxia-inducible factor (HIF)-1 and HIF-2 are heterodimeric transcription factors that mediate the cellular response to hypoxia. Their key regulatory subunits, HIF-1α and HIF-2α, are induced similarly by hypoxia, but their functional roles in cancer may be distinct and isoform-specific. SW480 colon cancer cells with stable expression of siRNA to HIF-1α or HIF-2α or both were established. HIF-1α-deficient cells displayed lower rates of proliferation and migration, but HIF-2α-deficient cells exhibited enhanced anchorage independent growth in a soft agar assay. Xenograft studies revealed that HIF-1α deficiency inhibited overall tumor growth, whereas deficiency of HIF-2α stimulated tumor growth. In human colon cancer tissues, expression of HIF-1α and to a lesser extent, HIF-2α, was linked to upregulation of VEGF and tumor angiogenesis. However, loss of expression of HIF-2α but not HIF-1α was strongly correlated with advanced tumor stage. DNA microarray analysis identified distinct sets of HIF-1α and HIF-2α target genes that may explain these phenotypic differences. Collectively, these findings suggest that HIF isoforms may have differing cellular functions in colon cancer. In particular, HIF-1α promoted the growth of SW480 colon cancer cells but HIF-2α appeared to restrain growth. Consequently, therapeutic approaches that target HIF may need to consider these isoform-specific properties.
PMCID: PMC2682346  PMID: 19030186
colon cancer; HIF; angiogenesis
15.  Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1 
PLoS ONE  2012;7(11):e49744.
Sulforaphane (SFN) is a dietary cancer preventive with incompletely characterized mechanism(s) of cancer prevention. Since prostaglandin E2 (PGE2) promotes cancer progression, we hypothesized that SFN may block PGE2 synthesis in cancer cells. We found that SFN indeed blocked PGE2 production in human A549 cancer cells not by inhibiting COX-2, but rather by suppressing the expression of microsomal prostaglandin E synthase (mPGES-1), the enzyme that directly synthesizes PGE2. We identified the Hypoxia Inducible Factor 1 alpha (HIF-1α) as the target of SFN-mediated mPGES-1 suppression. SFN suppressed HIF-1α protein expression and the presence of HIF-1α at the mPGES-1 promoter, resulting in reduced transcription of mPGES-1. Finally, SFN also reduced expression of mPGES-1 and PGE2 production in A549 xenograft tumors in mice. Together, these results point to the HIF-1α, mPGES-1 and PGE2 axis as a potential mediator of the anti-cancer effects of SFN, and illustrate the potential of SFN for therapeutic control of cancer and inflammation. Harmful side effects in patients taking agents that target the more upstream COX-2 enzyme render the downstream target mPGES-1 a significant target for anti-inflammatory therapy. Thus, SFN could prove to be an important therapeutic approach to both cancer and inflammation.
PMCID: PMC3500324  PMID: 23166763
16.  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
17.  Several fusion genes identified by whole transcriptome sequencing in a spindle cell sarcoma with rearrangements of chromosome arm 12q and MDM2 amplification 
International Journal of Oncology  2014;45(5):1829-1836.
Spindle cell tumors are clinically heterogeneous but morphologically similar neoplasms that can occur anywhere, mostly in adult patients. They are treated primarily with surgery to which is often added adjuvant or neoadjuvant radiation. Sub-classification of spindle cell sarcomas requires integration of histology, clinicopathological parameters, immunohistochemistry, cytogenetics (including fluorescence in situ hybridization) and/or molecular genetics. Some of the tumor subtypes are characterized by the presence of distinct chromosomal translocations and fusion genes. When no signs of differentiation are seen, the diagnosis by exclusion becomes undifferentiated spindle cell sarcoma. Cytogenetic, RNA sequencing and RT-PCR analyses were performed on a case of spindle cell sarcoma. The karyotype of the primary tumor was 46,X,del(X)(p?11p?22), der(12)(12pter→12q?22::12q?15→ q?22::16p11→16pter),-16,+r(12). MDM2 was found amplified in both the primary tumor and a metastasis. RNA-Seq of the primary tumor identified four fusion genes, PTGES3-PTPRB, HMGA2-DYRK2, TMBIM4-MSRB3 and USP15-CNTN1, in which all the partner genes map to the q arm of chromosome 12. In material from the metastasis, RT-PCR detected the PTGES3-PTPRB, HMGA2-DYRK2 and TMBIM4-MSRB3 whereas no USP15-CNTN1 fusion transcript was found. Because MDM2 amplification and the fusion transcripts PTGES3-PTPRB, HMGA2-DYRK2 and TMBIM4-MSRB3 were found both in the primary tumor and in the metastasis, they are components of the same clone and may be involved both in initiation and progression of the tumor. Which of them is pathogenetically primary remains unknown.
PMCID: PMC4203330  PMID: 25176350
fusion genes; whole transcriptome sequencing; spindle cell sarcoma; MDM2 amplification; rearrangements of chromosome Arm 12q; PTGES3-PTPRB; HMGA2-DYRK2; TMBIM4-MSRB3; USP15-CNTN1
18.  Characterization of the Activation of Protein Tyrosine Phosphatase, Receptor-Type, Z Polypeptide 1 (PTPRZ1) by Hypoxia Inducible Factor-2 Alpha 
PLoS ONE  2010;5(3):e9641.
Hypoxia inducible factors (HIFs) are the principal means by which cells upregulate genes in response to hypoxia and certain other stresses. There are two major HIFs, HIF-1 and HIF-2. We previously found that certain genes are preferentially activated by HIF-2. One was protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1). PTPRZ1 is overexpressed in a number of tumors and has been implicated in glioblastoma pathogenesis.
Methodology/Principal Findings
To understand the preferential activation of PTPRZ1 by HIF-2, we studied the PTPRZ1 promoter in HEK293T cells and Hep3B cells. Through deletion and mutational analysis, we identified the principal hypoxia response element. This element bound to both HIF-1 and HIF-2. We further identified a role for ELK1, an E26 transformation-specific (Ets) factor that can bind to HIF-2α but not HIF-1α, in the HIF-2 responsiveness. Knock-down experiments using siRNA to ELK1 decreased HIF-2 activation by over 50%. Also, a deletion mutation of one of the two Ets binding motifs located near the principal hypoxia response element similarly decreased activation of the PTPRZ1 promoter by HIF-2. Finally, chromatin immunoprecipitation assays showed binding of HIF and ELK1 to the PTPRZ1 promoter region.
These results identify HIF-binding and Ets-binding motifs on the PTPRZ1 promoter and provide evidence that preferential activation of PTPRZ1 by HIF-2 results at least in part from cooperative binding of HIF-2 and ELK1 to nearby sites on the PTPRZ1 promoter region. These results may have implications in tumor pathogenesis and in understanding neurobiology, and may help inform the development of novel tumor therapy.
PMCID: PMC2835759  PMID: 20224786
19.  Prostaglandin E2 Exerts Homeostatic Regulation of Pulmonary Vascular Remodeling in Allergic Airway Inflammation 
Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE2 in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE2 synthase (mPGES)-1. Mice lacking mPGES-1 (ptges−/− mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE2 in the bronchoalveolar lavage fluids of Der f-treated ptges−/− mice were ~80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges−/− mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1-derived PGE2 was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges−/− mice. These vascular changes were suppressed by the administration of the stable PGE2 analog 16, 16-dimethyl PGE2, or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE2. Thus, mPGES-1 and its product, PGE2, protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.
PMCID: PMC3721536  PMID: 20028661
20.  The NADPH Oxidase Subunit NOX4 Is a New Target Gene of the Hypoxia-inducible Factor-1 
Molecular Biology of the Cell  2010;21(12):2087-2096.
NADPH oxidases generate reactive oxygen species (ROS). We studied the role of NOX4 under hypoxia. Hypoxia enhanced NOX4 expression in lung smooth-muscle cells and lung tissue due to HIF-1α binding and activation of the NOX4 promoter. HIF-1α–dependent NOX4 induction restored ROS levels after hypoxia and induced proliferation by hypoxia. The following citations were not referenced in the reference list or the reference/citation is not styled correctly: Kietzmann et al., 1999.
NADPH oxidases are important sources of reactive oxygen species (ROS), possibly contributing to various disorders associated with enhanced proliferation. NOX4 appears to be involved in vascular signaling and may contribute to the response to hypoxia. However, the exact mechanisms controlling NOX4 levels under hypoxia are not resolved. We found that hypoxia rapidly enhanced NOX4 mRNA and protein levels in pulmonary artery smooth-muscle cells (PASMCs) as well as in pulmonary vessels from mice exposed to hypoxia. This response was dependent on the hypoxia-inducible transcription factor HIF-1α because overexpression of HIF-1α increased NOX4 expression, whereas HIF-1α depletion prevented this response. Mutation of a putative hypoxia-responsive element in the NOX4 promoter abolished hypoxic and HIF-1α–induced activation of the NOX4 promoter. Chromatin immunoprecipitation confirmed HIF-1α binding to the NOX4 gene. Induction of NOX4 by HIF-1α contributed to maintain ROS levels after hypoxia and hypoxia-induced proliferation of PASMCs. These findings show that NOX4 is a new target gene of HIF-1α involved in the response to hypoxia. Together with our previous findings that NOX4 mediates HIF-1α induction under normoxia, these data suggest an important role of the signaling axis between NOX4 and HIF-1α in various cardiovascular disorders under hypoxic and also nonhypoxic conditions.
PMCID: PMC2883952  PMID: 20427574
21.  Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α 
Increased levels of hypoxia and hypoxia-inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category “Response to hypoxia” was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia-responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF-1α shRNA or Dox at low concentrations blocked HIF-1α induction of VEGF-A by 84–97% and carbonic anhydrase 9 by 83–93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF-1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α target genes that may promote resistance to antiangiogenic and other therapies. HIF-1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.
What’s new?
Despite their initial promise, anti-angiogenic therapies have been a disappointment in the clinic. One reason is that solid tumors often become resistant to these drugs. Tumors that respond poorly to this type of therapy have increased activation of the hypoxia-induced transcription factor HIF-1α which can enhance tumor survival and progression. In this study, the authors report that this evasive resistance can be overcome by adding low-dose doxorubicin or shRNA to inhibit HIF-1α activity. They are thus developing a clinical trial combining the angiogenesis inhibitor bevacizumab with metronomic doxorubicin in sarcoma patients.
PMCID: PMC3677782  PMID: 22684860
sarcomas; hypoxia; HIF-1α; VEGF-A
22.  Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells 
Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.
PMCID: PMC4317221
Cell density; COX-2; EP4 prostanoid receptor; GPCRs; HCA-7 cells; HIF-1α; human colon cancer; PGE 2
23.  Correlation among 16 biological factors [p53, p21waf1, MIB-1 (Ki-67), p16INK4A, cyclin D1, E-cadherin, Bcl-2, TNF-α, NF-κB, TGF-β, MMP-7, COX-2, EGFR, HER2/neu, ER, and HIF-1α] and clinical outcomes following curative chemoradiation therapy in 10 patients with esophageal squamous cell carcinoma 
Oncology Letters  2013;5(3):903-910.
The expression levels of 16 proteins were analyzed to identify prognostic correlations in esophageal squamous cell carcinoma (ESCC) treated with concurrent chemoradiation therapy (CCRT). The immunohistochemical expression levels of p53, p21waf1, molecular immunology borstel-1 (MIB-1, Ki-67), p16INK4A, cyclin D1, E-cadherin, Bcl-2, tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, transforming growth factor (TGF)-β, matrix metalloproteinase (MMP)-7, cyclooxygenase (COX)-2, epidermal growth factor receptor (EGFR), human EGFR type 2 (HER2/neu), estrogen receptor (ER) and hypoxia-inducible factor (HIF)-1α were studied in 10 cases of ESCC treated with CCRT. The patients underwent CCRT between 2000 and 2010. The mean patient age was 68.1 years (range, 46-80 years). The numbers of patients in stages I, II, III and IV of the disease were 2, 2, 3 and 3, respectively. Of the tumors, 8 were positive for p53, 6 for p21waf1, 7 for MIB-1 (Ki-67), 7 for p16INK4A, 7 for cyclin D1, 8 for E-cadherin, 3 for Bcl-2, 0 for TNF-α, 5 for NF-κB, 7 for TGF-β, 9 for MMP-7, 7 for COX-2, 5 for EGFR, 1 for HER2/neu, 1 for ER and 7 for HIF-1α. The 2-year overall survival rate of patients expressing high levels of MIB-1 was 71% (±17%) compared with 0% (P=0.019) for those expressing low levels. For NF-κB, the rate was 0% for patients with high levels compared with 100% (P<0.018) for those with low levels. The 2-year local control rates of HER2/neu were 0% in patients expressing high levels and 88% (±12%) in patients expressing low levels (P=0.027). The 2-year disease-free survival rates of HER2/neu and ER were 0% for patients expressing high levels compared with 56% (±17%) for those with low levels (P=0.027). There were no significant correlations between the expression levels of the other proteins and clinical outcomes. In the present study, high levels of MIB-1 and low levels of NF-κB, HER2 and ER were shown to be good prognostic factors following definitive CCRT for ESCC.
PMCID: PMC3576225  PMID: 23426606
esophageal cancer; imunohistochemistry; squamous cell carcinoma; prognostic factors; biological markers
24.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
25.  Targeting Hypoxia in Cancer Cells by Restoring Homeodomain Interacting Protein-Kinase 2 and p53 Activity and Suppressing HIF-1α 
PLoS ONE  2009;4(8):e6819.
The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) is a crucial regulator of p53 apoptotic function. HIPK2 is also a transcriptional co-repressor of hypoxia-inducible factor-1α (HIF-1α) restraining tumor angiogenesis and chemoresistance. HIPK2 can be deregulated in tumors by several mechanisms including hypoxia. Here, we sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1α, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance.
Methodology/Principal Findings
Upon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1α expression and inhibiting the p53-apoptotic response to drug. Cobalt suppressed HIPK2 recruitment onto HIF-1α promoter. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 response to drug. Zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1α promoter, repression of MDR1, Bcl2, and VEGF genes, and activation of the p53 apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes.
We show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment.
PMCID: PMC2729407  PMID: 19714248

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