HIF-1α Protein Translation Research Articles

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.

Enhanced lysosome escape mediated by 1,2-dicarboxylic-cyclohexene anhydride-modified poly-l-lysine dendrimer as a gene delivery system

Antisense oligodeoxynucleotide (ASODN) can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing, in turn, plays antitumor therapeutic roles. In the study, a novel HIF-1α ASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA. The physicochemical properties of nanocomposite were characterized using TEM, FTIR, DLS and zeta potentials. The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm, and according to the agarose gel retardation assay, the loading amount of ASODN accounted for 166.7 mg/g. The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells. The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL. The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite. In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit mRNA expression and HIF-1α protein translation at different levels. In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue. In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%. These results manifested that the GEL-DGL-FA-ASODN-DCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.

Montelukast inhibits hypoxia inducible factor-1α translation in prostate cancer cells

ABSTRACTThrough regulating the expression of hundreds of genes, hypoxia-inducible factor -1(HIF-1) plays a critical role in hypoxic adaption of cancer cells and is considered as a target for cancer therapy. Here we show that montelukast, a clinical leukotriene receptor antagonist for the treatment of asthma, inhibits hypoxia or CoCl2-induced HIF-1α activation and reduces its protein expression in prostate cancer cells. However, the other two leukotriene receptor antagonists, pranlukast and zafirlukast, cannot decrease HIF-1α protein, which indicates that montelukast-induced downregulation of HIF-1α is not mediated by leukotriene receptor. Neither proteasome inhibitor MG132 nor the lysosomal inhibitor chloroquine (CQ) can block montelukast-induced downregulation of HIF-1α protein. Interestingly, GSK2606414, a PKR-like endoplasmic reticulum kinase (PERK) inhibitor, abrogates montelukast-induced downregulation of HIF-1α under hypoxic conditions. However, montelukast increases phosphorylation of eIF-2α at Ser51. Moreover, montelukast inhibits the proliferation of prostate cancer cells, which can be reversed by overexpression of HIF-1α protein. In conclusion, we identify montelukast may be used as a novel agent for the treatment of prostate cancer by decreasing HIF-1α protein translation.

LncRNA MEG3 downregulation mediated by DNMT3b contributes to nickel malignant transformation of human bronchial epithelial cells via modulating PHLPP1 transcription and HIF-1α translation.

Long noncoding RNAs (lncRNAs) are emerging as key factors in various fundamental cellular biological processes, and many of them are likely to have functional roles in tumorigenesis. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA, and the decreased MEG3 expression has been reported in multiple cancer tissues. However, nothing is known about the alteration and role of MEG3 in environmental carcinogen-induced lung tumorigenesis. Our present study, for the first time to the best of our knowledge, discovered that environmental carcinogen nickel exposure led to MEG3 downregulation, consequently initiating c-Jun-mediated PHLPP1 transcriptional inhibition and hypoxia-inducible factor-1α (HIF-1α) protein translation upregulation, in turn resulting in malignant transformation of human bronchial epithelial cells. Mechanistically, MEG3 downregulation was attributed to nickel-induced promoter hypermethylation via elevating DNMT3b expression, whereas PHLPP1 transcriptional inhibition was due to the decreasing interaction of MEG3 with its inhibitory transcription factor c-Jun. Moreover, HIF-1α protein translation was upregulated via activating the Akt/p70S6K/S6 axis resultant from PHLPP1 inhibition in nickel responses. Collectively, we uncover that nickel exposure results in DNMT3b induction and MEG3 promoter hypermethylation and expression inhibition, further reduces its binding to c-Jun and in turn increasing c-Jun inhibition of PHLPP1 transcription, leading to the Akt/p70S6K/S6 axis activation, and HIF-1α protein translation, as well as malignant transformation of human bronchial epithelial cells. Our studies provide a significant insight into understanding the alteration and role of MEG3 in nickel-induced lung tumorigenesis.

Pomolic acid suppresses HIF1α/VEGF-mediated angiogenesis by targeting p38-MAPK and mTOR signaling cascades

BackgroundPomolic acid (PA), an active triterpenoid from Euscaphis japonica, inhibits the proliferation of a variety of cancer cells, but the molecular mechanisms of the anti-angiogenic potential of PA have not been fully elucidated in breast cancer cells. Hypothesis/PurposeWe investigated the molecular mechanisms underlying the anti-angiogenic effect of PA in epidermal growth factor (EGF)-responsive human breast cancer cells, MCF-7 and MDA-MB-231, and human umbilical vascular endothelial cells (HUVEC). Study design/MethodsEffects of PA on EGF-induced HIF1α/VEGF expression in MCF-7, MDA-MB-231 and HUVEC were assayed. As to the mechanisms, EGF-mediated MAPKs, PI3K/Akt, and mTOR signaling pathway were performed. Wound healing and invasion assay, tube formation assay, immunoblot assay, real-time PCR, luciferase gene assay, electrophoretic mobility shift assay and immunofluorescence staining were used for assessment. ResultsPA significantly and selectively suppressed EGF-induced HIF1α/VEGF expression, whereas it did not affect the expression of HIF1β in MCF-7 and MDA-MB-231. Furthermore, PA inhibited EGF-induced angiogenesis in vitro and downregulated HIF1α/VEGF expression in HUVEC. Mechanistically, we found that the inhibitory effects of PA on HIF1α/VEGF expression are associated with inhibition of HIF1α/VEGF expression through an EGF-dependent mechanism. In addition, PA suppressed the EGF-induced phosphorylation of p38-MAPK and mTOR. ConclusionPA suppresses EGF-induced HIF1α protein translation by inhibiting the p38-MAPK and mTOR kinase signaling pathways and plays a novel anti-angiogenic role.

The N-terminal region of p27 inhibits HIF-1α protein translation in ribosomal protein S6-dependent manner by regulating PHLPP-Ras-ERK-p90RSK axis.

P27 was identified as a tumor suppressor nearly two decades, being implicated in cell-cycle control, differentiation, senescence, apoptosis and motility. Our present study, for the first time to the best of our knowledge, revealed a potential role of p27 in inhibiting S6-mediated hypoxia-inducible factor-1α (HIF-1α) protein translation, which contributed to the protection from environmental carcinogen (sodium arsenite)-induced cell transformation. Our findings showed that depletion of p27 expression by knockout and knockdown approaches efficiently enhanced S6 phosphorylation in arsenite response via overactivating Ras/Raf/MEK/ERK pathway, which consequently resulted in the stimulation of p90RSK (90 kDa ribosomal S6 kinase), a direct kinase for S6 phosphorylation. Although PI3K/AKT pathway was also involved in S6 activation, blocking AKT and p70S6K activation did not attenuate arsenite-induced S6 activation in p27−/− cells, suggesting p27 specifically targeted Ras/ERK pathway rather than PI3K/AKT pathway for inhibition of S6 activation in response to arsenite exposure. Further functional studies found that p27 had a negative role in cell transformation induced by chronic low-dose arsentie exposure. Mechanistic investigations showed that HIF-1α translation was upregulated in p27-deficient cells in an S6 phosphorylation-dependent manner and functioned as a driving force in arsenite-induced cell transformation. Knockdown of HIF-1α efficiently reversed arsenite-induced cell transformation in p27-depleted cells. Taken together, our findings provided strong evidence showing that by targeting Ras/ERK pathway, p27 provided a negative control over HIF-1α protein synthesis in an S6-dependent manner, and abrogated arsenite-induced cell transformation via downregulation of HIF-1α translation.

Translational suppression of HIF-1α by miconazole through the mTOR signaling pathway.

Miconazole is an imidazole antifungal agent that has amply been used in the treatment of superficial mycosis. Preliminary data indicate that miconazole may also induce anticancer effects. As yet, however, little is known about the therapeutic efficacy of miconazole on cancer and the putative mechanism(s) involved. Here, we show that miconazole suppresses hypoxia inducible factor-1α (HIF-1α) protein translation in different cancer-derived cells. The effect of miconazole on HIF-1α expression was examined by Western blotting and reverse transcriptase polymerase chain reaction assays in human U87MG and MCF-7 glioma and breast cancer-derived cell lines, respectively. The transcriptional activity of the HIF-1 complex was confirmed using a luciferase assay. To assess whether angiogenic factors are increased under hypoxic conditions in these cells, vascular endothelial growth factor (VEGF) levels were measured by ELISA. Metabolic labeling was performed to examine HIF-1α protein translation and global protein synthesis. The role of the mammalian target of rapamycin (mTOR) signaling pathway was examined to determine translation regulation of HIF-1α after miconazole treatment. Miconazole was found to suppress HIF-1α protein expression through post-transcriptional regulation in U87MG and MCF-7 cells. The suppressive effect of HIF-1α protein synthesis was found to be due to inhibition of mTOR. Miconazole significantly inhibited the transcriptional activity of the HIF-1 complex and the expression of its target VEGF. Moreover, miconazole was found to suppress global protein synthesis by inducing phosphorylation of the translation initiation factor 2α (eIF2α). Our data indicate that miconazole plays a role in translational suppression of HIF-1α. We suggest that miconazole may represent a novel therapeutic option for the treatment of cancer.

Minocycline inhibits angiogenesis in vitro through the translational suppression of HIF-1α

Minocycline was recently found to be effective against cancer. However, the precise molecular mechanisms of minocycline in cancer are poorly understood. Hypoxia-inducible factor-1 (HIF-1, a heterodimeric transcription factor composed of HIF-1α and β) activates the transcription of genes that are involved in angiogenesis in cancer. In this study, we found that minocycline significantly inhibits HIF-1α protein expression and suppresses HIF-1 transcriptional activity. The tube formation assay showed that minocycline has anti-angiogenic activity and suppresses hypoxia-induced vascular endothelial growth factor (VEGF) expression. The metabolic labeling assay showed that minocycline reduces HIF-1α protein translation and global protein synthesis. In addition, minocycline suppresses mTOR signaling and increases the phosphorylation of eIF2α, which is known to be related to the translational regulation of HIF-1α expression. These findings collectively indicate that minocycline is a potential inhibitor of HIF-1α and provide new insight into the discovery of drugs for cancer treatment.

Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content.

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1α protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1α was increased by 1.69±0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1α expression by bilirubin. HIF-1α expression decreased by 10 µM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1α concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1α mRNA expression. In coonclusion, bilirubin enhances HIF-1α transcription as well as the up-regulation of HIF-1α protein translation through the attenuation of ROS and subunits of NADPH oxidase.

Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action.

HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway

Hypoxia induces the expansion of glioblastoma stem cells (GSCs), but the mechanism underlying it is still unclear. Here, we supply evidence that hypoxia-inducible factor-1α (HIF-1α) induced activation of Notch pathway is essential for hypoxia-mediated maintenance of GSC. Either depletion of HIF-1α or inactivation of Notch signaling partly inhibits the hypoxia-mediated maintenance of GSC. Further data suggest a role for HIF-1α in the interaction and stabilization of intracellular domain of Notch (NICD), and activation of Notch signaling. The mRNA level of HIF-1α and Notch target gene FABP7 was elevated in GSC. And the STAT3 pathway responsible for the HIF-1α gene transcription, the phosphatidylinositol 3-kinase-Akt and ERK1/2, both of which are contributed to HIF-1α protein translation, are also preferentially activated in GSC. Inhibition of these pathways partly reduces the hypoxia-induced activation of the Notch pathway and subsequent GSC maintenance. Taken together, our findings suggest that HIF-1α requires Notch pathway to drive the maintenance of GSC. The activated regulation of HIF-1α makes GSC more sensitive to hypoxia-mediated maintenance. These findings enhance our understanding of mechanism of hypoxia-mediated GSC expansion and provide HIF-1α as an attractive target for glioblastoma therapy.

Tanshinone IIA–Induced Attenuation of Lung Injury in Endotoxemic Mice Is Associated with Reduction of Hypoxia-Inducible Factor 1α Expression

Inhibiting hypoxia-inducible factor (HIF)-1α activity has been proposed as a novel therapeutic target in LPS-induced sepsis syndrome. We have reported that tanshinone IIA (TIIA) can reduce LPS-induced lethality and lung injury in mice, but the precise mechanisms have not been fully described. Therefore, the present study investigated whether the protective effect of TIIA was related to the inhibition of LPS-induced HIF-1α expression and what mechanisms accounted for it. This study showed that TIIA pretreatment improved LPS-induced biochemical and cellular changes and reduced the production of inflammatory cytokines. Pretreatment with TIIA decreased LPS-induced HIF-1α expression in vivo and in vitro. TIIA did not affect the LPS-induced HIF-1α mRNA level but inhibited HIF-1α protein translation by the inhibition of the PI3K/AKT and MAPK pathways and related protein translational regulators, such as p70S6K1, S6 ribosomal protein, 4E-BP1, and eIF4E, and promoted HIF-1α protein degradation via the proteasomal pathway in LPS-stimulated macrophages. These observations partially explain the antiinflammatory effects of TIIA, which provides scientific basis for its application for the treatment of acute lung injury/acute respiratory distress syndrome or sepsis.

Abstract 3094: Microtubule-dependent regulation of HIF-1α

Abstract Hypoxia inducible factor-1α (HIF-1α) is a pro-angiogenic transcription factor over-expressed in over 70% of human cancers. We have shown that microtubule-targeting drugs (MTDs) such as the taxanes, inhibit HIF-1α protein translation and transcriptional activity. The underlying mechanism involves trafficking of HIF-1α mRNA on dynamic microtubules as a requirement for its active translation. MTD-induced microtubule disruption resulted in polysome release of HIF-1α mRNA, followed by its enrichment to Argonaute-2 containing P-bodies, where HIF-specific miRNAs were also accumulated and repressed its translation. This process was reversible following microtubule repolymerization, and both Argonaute-2 knock-down and removal of HIF's UTR regions abrogated Taxol's inhibitory activity. Together these results suggest that microtubule dynamics tightly regulate HIF-1α's translation by altering its mRNA localization. To identify additional, similarly regulated mRNAs, we are currently isolating Argonaute-2 bound mRNAs from untreated versus MTD-treated cells and analyzing them by RNA Seq. We are also trying to identify the “zip-code” sequence within the UTR regions of HIF-1α that confers microtubule sensitivity. Interestingly, preliminary data from our lab have also shown that microtubule disruption inhibits the hypoxia-induced nuclear accumulation of HIF-1α protein and the subsequent activation of HIF response element (HRE)-containing genes, such as VEGF. Immunofluorescence followed by confocal microscopy revealed that HIF-1α protein colocalized with the microtubule cytoskeleton. This result is further supported by transmission electron microscopy (TEM), co-immunoprecipitation, and microtubule co-sedimentation assays. Inhibition of the minus-end directed motor protein, dynein, prevented HIF-1α nuclear accumulation, further implicating microtubule dynamics in HIF-1α protein trafficking and activity. Thus far, we have shown that HIF-1α is regulated by microtubules at both the level of protein synthesis and protein trafficking. To date, the only example where HIF-1α activity is not microtubule-dependent is in Renal Cell Carcinoma (RCC), where HIF-1α is constitutively active due to VHL mutations. MTD treatment failed to inhibit HIF-1α in VHL-null RCCs at concentrations that disrupted microtubules, while reintroduction of wild-type VHL did not restore the ability of MTDs to inhibit HIF-1α. Furthermore, MTD treatment of RCC cells did not release HIF-1α mRNA from polysomes, and HIF-1α protein did not bind microtubules. These results suggest that the regulation of HIF-1α has become independent of the microtubule cytoskeleton, which may contribute to the chemoresistant nature of RCCs. Further understanding of the microtubule dependent regulation of HIF-1α, and lack thereof in RCCs, will help us better elucidate the biology of HIF-1α translation and will have important therapeutic implications given the wide use of MTDs in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3094. doi:10.1158/1538-7445.AM2011-3094

Pentamidine reduces expression of hypoxia-inducible factor-1α in DU145 and MDA-MB-231 cancer cells

Pentamidine is an aromatic diamine used for the treatment of human protozoa infections. Recently, pentamidine has been reported to exhibit anticancer properties. In this study, we report that pentamidine inhibits expression of hypoxia-inducible factor (HIF)-1α in cancer cells. Pentamidine decreased HIF-1α protein translation and enhanced its protein degradation in DU145 prostate cancer and MDA-MB-231 breast cancer cells. In parallel with reduction of de novo synthesis of HIF-1α, pentamidine was able to suppress global protein translation, an effect accompanied by the reduction of eIF4F complex formation and also the induction of eIF2α phosphorylation. These results show that pentamidine is a potential inhibitor of HIF-1α and its potential as a cancer therapeutic reagent warrants further study.

Abstract 306: KRas mediated induction of HIF-1α in normoxia promotes tumorigenesis and identifies HIF-1α as a therapeutic target

Abstract Oncogenic mutation of the KRas enzyme occurs frequently in human tumors and results in the constitutive activation of multiple signaling pathways. KRas mutation occurs in three specific codons, 12, 13, or 61, and involves the substitution of multiple amino acids. We observed in cancer cell lines containing activating mutations in KRas but not wild type KRas, that the Hypoxia Inducible Factor 1 α (HIF-1α) is expressed and functional under normoxic conditions. HIF-1α itself has been implicated in multiple aspects of tumorigenesis, including survival and invasion. We have found that exogenous expression of wild type KRas or the introduction of oncogenic KRas into transformed cells, causes the expression of HIF-1α in normoxia, but has little or no effect on HIF-1α induction by hypoxia. Additionally, in cancer cells, the deletion of the oncogenic KRas allele, while preserving the wild type allele, silenced the normoxic expression of HIF-1α. To determine the mechanisms by which KRas elicits the expression of HIF-1α, we quantitated the expression of 176 phospho or total proteins utilized a Reverse Phase Protein Array (RPPA) of 74 highly characterized non small cell lung cancer lines (NSCLC's) including 22 NSCLS with oncogenic KRas under three different culture conditions (with serum, without serum and starved then stimulated with serum for 30 minutes). From this RPPA, we found that NSCLC lines containing oncogenic KRas expressed active serine 473 Akt at differing levels, dependent upon which amino acid substitution was present in KRas. Additionally, we found that the KRas activation of Akt signaling, along with other KRas dependent signals, was responsible for HIF-1 stabilization in a hypoxia independent manner. As previously suggested we find that through KRas and Akt driven signaling, KRas is able to overcome the oxygen driven mechanisms responsible for HIF-1α degradation by increased HIF-1α protein translation However, we additionally found that aerobic glycolysis driven by KRas and Akt, specifically the accumulation of the products of glycolysis, also contribute to HIF-1α expression in normoxia. Finally, genome wide mRNA profiling of a KRas transformed line with a stable shRNA mediated knockdown of HIF-1α compared it a vector control showed that under normoxic conditions, knockdown of HIF-1α down regulates several genes important to transformation, including growth factors previously associated with KRas induced transformation. This data suggests inhibitors of HIF-1α may be effective in tumors harboring the KRas oncogenes. Additionally, Akt and HIF-1α inhibitors may be more active when combined with other agents targeted to other signaling pathways activated by KRas in order to bypass resistance mediated by redundant KRas driven signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 306.

Kinome Sirna Screen Identifies SMG-1 as a Negative Regulator of Hypoxia-inducible Factor-1α in Hypoxia

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1α activity in hypoxia, in part through blocking MAPK activation.

Differential Regulation of Hypoxia-Inducible Factor-1 through Receptor Tyrosine Kinase Transactivation in Vascular Smooth Muscle Cells

Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes expressed in hypoxic conditions. In vascular smooth muscle cells, the vasoactive hormone angiotensin II (Ang II) is a very potent inducer and activator of HIF-1. As opposed to hypoxia, which induces HIF-1alpha by protein stabilization, Ang II induced HIF-1alpha through transcriptional and translational mechanisms. Interestingly, a number of intracellular signaling events triggered by Ang II are mediated by the transactivation of receptor tyrosine kinases. The major receptor tyrosine kinases shown to be transactivated by Ang II in vascular smooth muscle cells are the epidermal growth factor receptor and the IGF-I receptor. In this study, we demonstrate that the transactivation of both these receptor tyrosine kinases is involved in HIF-1 complex activation by Ang II. More interestingly, this modulation of HIF-1 is at different degrees and through different pathways. Our results show that transactivation of IGF-I receptor is essential for HIF-1alpha protein translation through phosphatidylinositol 3-kinase/p70S6 kinase pathway activation, and epidermal growth factor receptor transactivation is implicated in HIF-1 complex activation through the stimulation of the p42/p44 MAPK pathway. Our results therefore show that Ang II-induced receptor tyrosine kinase transactivation is essential in both the induction and activation of HIF-1. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation.

UVC inhibits HIF-1α protein translation by a DNA damage- and topoisomerase I-independent pathway

Hypoxia inducible factor 1 (HIF-1) is a key player in cancer progression and an attractive target for cancer therapy. Several small molecule inhibitors of HIF-1alpha also induce a DNA damage response. However, whether or not DNA damage is required for or associated with the inhibition of HIF-1alpha protein accumulation is poorly understood. In this report we investigated the effects of distinct DNA damaging conditions on the hypoxic induction of HIF-1alpha protein in cancer cell lines. We demonstrate that in addition to topotecan (TPT), a known inhibitor of HIF-1alpha, UVC, but not other DNA damaging agents (cisplatin, ionizing radiation and doxorubicin), inhibited HIF-1alpha protein accumulation in a dose-dependent, p53-independent fashion. Low doses UVC decreased HIF-1alpha translation without affecting global protein synthesis. Inhibition of HIF-1alpha by UVC required ongoing RNA transcription, but not DNA replication. Moreover, a functional ATR was required for the activation of DNA damage-dependent responses by both UVC and TPT, but was dispensable for the inhibition of HIF-1alpha protein. Notably, unlike TPT, inhibition of HIF-1alpha protein by UVC did not require topoisomerase I, suggesting a similar yet distinct mode of action. Our data reveal that UVC is a novel signal associated with inhibition of HIF-1alpha protein accumulation, and they uncouple the DNA damage-dependent signaling pathway exerted by UVC and TPT from HIF-1alpha inhibition.