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Tumor Biology |
, HIF-2, and Vascular Endothelial Growth Factor Expression and Their Regulation by the Phosphatidylinositol 3'-Kinase/Akt Signaling Pathway1
Imperial Cancer Research Fund, Molecular Oncology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and HIF-2 expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1 induction and phosphorylation under hypoxia. However, HIF-2 expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1 and HIF-2 expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1 expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1 expression also showed down-regulation of HIF-1 after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The major pathway regulating gene induction in response to hypoxia is under the control of the transcription factors HIF-1 and HIF-2 (15, 16, 17)
. HIF-1 and HIF-2 are regulated by ubiquitin-mediated proteolysis and are targeted for destruction by the pVHL in normoxia and stabilized under hypoxia (18, 19, 20, 21)
. Mutations in the VHL gene, as in renal cancer and VHL syndrome, result in constitutive expression of HIF-1 and HIF-2 in normoxia and a permanent transcriptional induction of hypoxia-responsive genes (20)
.
The mechanisms by which oncogenes interact with hypoxia signaling are poorly understood, but it has been shown that activation of the src oncogene increases HIF-1 mRNA and protein under hypoxia (22)
. In addition, exposure of cells to insulin, IGF-I, and IGF-II, also induces HIF-1 stabilization (23)
. Recently, the p42/p44 MAPK was found to phosphorylate HIF-1 and increase its ability to transactivate reporter genes (24)
.
Because many oncogenes signal partly through PI3K pathway (Ref. 25
; e.g., erbB2, mutant EGF receptor, ras, IGF-II, and PTEN), we investigated the role of this pathway in the stabilization of HIF-1 and HIF-2 under hypoxia. We studied the expression of both transcription factors in human breast cancer cell lines with different backgrounds of oncogene mutation or amplification and in breast cell lines permanently transfected with mutant ras or a key downstream kinase regulated by PI3K, Akt (25)
. The breast cell lines chosen present erbB2 amplification (SKBr3; Ref. 26
), ras mutation (MDA-MB-231; Ref. 27
), PTEN mutation and EGF receptor amplification (MDA-MB-468; Refs. 28
and 29
), or were EGF responsive (T47D). The mechanism of HIF-1 regulation was further analyzed in a renal cancer cell line defective in pVHL or in which interaction of HIF-1 with pVHL was abrogated by DFO, leading to HIF-1 stabilization.
Our results show that the PI3K inhibitor LY294002 significantly reduced HIF-1 expression and endogenous VEGF mRNA and protein in all cell lines tested, whereas HIF-2 was not affected. Down-regulation of HIF-1 protein expression by PI3K inhibitor appeared to be independent of the VHL pathway and did not involve an increased ubiquitination and proteasome degradation. Finally, mutant ras expression led to an increased expression of hypoxia-induced HIF-1 and HIF-2 via a PI3K-dependent pathway.
These results have therapeutic implications for the use of PI3K inhibitors to block the effect of oncogenes and growth factors related to the modulation of the HIF pathway and also for the use of PI3K inhibitors to block the effect of oncogenes and growth factors related to the modulation of the HIF pathway and also for the use of PI3K inhibitors in renal carcinoma, where constitutive HIF-1 activation occurs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and HIF-2 monoclonal antibodies (MoAb 28b and 190b, respectively) were produced in the laboratory and used as described previously (30)
.
Cell Culture.
MCF10A-V12-ras and MCF10A-EV, permanently transfected cell lines, were a gift from J. Downward (31
, 32)
(ICRF, London, United Kingdom). The MCF10A cell lines were cultured in 1:1 DMEM:Ham’s F-12 (both from Clare Hall Laboratories, ICRF) supplemented with 5% FCS, insulin (10 µg/ml), hydrocortisone (5 µg/ml), EGF (20 ng/ml), and cholera toxin (100 ng/ml; all chemicals were purchased from Sigma) and incubated under normoxic or hypoxic conditions for 16 h.
RCC4-VHL and RCC4-EV cell lines were a gift from P. J. Ratcliffe (Welcome Trust Centre for Human Genetics, Oxford, United Kingdom). The breast carcinoma cell lines T47D, SKBr3, MDA-MB-231 and MDA-MB-468 were obtained from American Type Culture Collection (Manassas, VA) and passaged using 0.25% trypsin-0.02% EDTA. All of these cell lines were cultured in DMEM (Clare Hall Laboratories, ICRF) supplemented with 10% FCS and 4 mM glutamine.
Subconfluent cells were used for all experiments. Culture medium was replaced, and protein kinase inhibitors were added 20 minutes before exposure to experimental conditions. Cells were routinely cultured in 20% O2 and 5% CO2 at 37°C and made hypoxic by placing them in a Heto-Holten Cell house 170 incubator (Heto-Holten, Camberley, United Kingdom) at 0.1% O2, 5% CO2 and balanced nitrogen. DFO was added to the culture medium at a final concentration of 100 µM.
RNA Analysis.
Total RNA was extracted using TRI Reagent (Sigma). Ribonuclease protection assays were performed essentially as described in Petersen et al. (33)
, using 10–30 µg of total RNA for VEGF121 detection. 32P-labeled riboprobes were generated using SP6, T3, or T7 RNA polymerase. U6 small nuclear RNA was used as a loading control (34)
. The templates used yielded protected fragments as follows: 517 bp for VEGF121 (accession nunber M95200); and 106 bp for U6 (nucleotides 1–107; GenBank accession no. X01366). After resolution on 8% polyacrylamide gels, quantification was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Protein Extraction and Immunoblot Analysis.
For whole-cell extracts, cells were washed with ice-cold PBS and collected by scraping. Cell pellets were homogenized in extraction buffer [8 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 1% SDS, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM sodium orthovanadate] using an IKA Ultra-Turrax T8 homogenizer (Janke & Kunkel, Staufen, Germany) for 15 s at full speed. Protein levels in the extracts were quantified using the BioRad DC protein assay (BioRad, Hemel Hempstead, United Kingdom).
For immunoblotting, whole cell extracts (20 µg/lane) were resolved in 6% SDS polycrylamide gels and transferred onto Immobilon P membrane (Millipore, Bedford, MA) in 25 mM Tris-base, 190 mM glycine, and 15% methanol using a semidry blotter Imm-2 (W.E.P. Company, Concord, CA). Membranes were blocked with 5% fat-free milk and 0.05% Tween 20 in PBS. For HIF-1 detection, MoAb 28b (30)
was used at 4 µg/ml, and for HIF-2 detection, MoAb 190b (30)
was used at 2 µg/ml. PTEN monoclonal antibody and phospho-Akt (Ser-473) and Akt antibodies were used at the concentration recommended by the supplier. Detection of monoclonal and polyclonal antibodies was performed using horseradish peroxidase-conjugated goat antimouse immunoglobulins (Dako, Ely, United Kingdom) or horseradish peroxidase-conjugated swine antirabbit immunoglobulins (Dako), respectively, and enhanced chemiluminescence (Amersham Corp.). After analysis, membranes were stained with Ponceau S solution (Sigma) to verify equal protein loading and transfer. HIF-1 and HIF-2 protein expression was quantified by analyzing a representative autoradiograph with a densitometer, and normalized to a cross-reacting protein.
Determination of VEGF Protein Levels in Cell Culture Medium.
Subconfluent cells were grown in 6-well plates with 1.5 ml of medium for 16 h, either in air or under 0.1% oxygen. Cells supernatants were collected, clarified by centrifugation at 2000 rpm for 5 min, and stored at -20°C. Concomitantly, cells were harvested by trypsinization, and cell number was determined with an automated cell counter (Beckman Coulter, High Wycombe, United Kingdom). The amount of VEGF in the supernatant was determined with an ELISA kit (VEGF-ELISA; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. VEGF was expressed as picograms of VEGF protein per milliliter of medium and per 105 cells.
In Vitro Ubiquitination Assay.
[35S]Methionine-labeled HIF-1 protein was prepared by coupled transcription and translation reactions of human HIF-1 expression plasmid in rabbit reticulocyte lysate (TNT; Promega, Southhampton, United Kingdom). MDA-MB-468 and RCC4 cell lines were treated with 40 µM LY294002 or DMSO (carrier) for 16 h under normoxic conditions. To prepare cytoplasmic extracts, cells were washed twice with cold hypotonic extraction buffer [20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl2, and 1 mM DTT] and disrupted in a Dounce homogenizer. Crude extract was centrifuged at 10,000x g for 10 min at 4°C to remove cell debris and nuclei and stored in aliquots at -70°C (35)
.
Ubiquitination assays were carried out at 30°C in a total volume of 40 µl, containing 2 µl of in vitro translated [35S]methionine-labeled HIF-1, 27 µl of cytoplasmic cell extract, 4 µl of 10x ATP-regenerating system, 4 µl of 5 mg/ml ubiquitin (Sigma), and 0.83 µl of 150 µM ubiquitin aldehyde (Affiniti Research Products, Mamhead, United Kingdom). Aliquots were removed after 30, 90, and 270 min of incubation, mixed with SDS sample buffer, and analyzed by SDS-PAGE and autoradiography (35)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All of the cell lines studied showed a reduction of their basal VEGF protein level after 16 h of treatment with LY294002 (Fig. 1A)
. MDA-MB-231 cells expressing mutant ras and MDA-MB-468 cells lacking functional PTEN protein and overexpressing EGF receptor both showed the highest basal level of VEGF expression and the strongest inhibition (up to 40%) after treatment with LY294002 under normoxic conditions.
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To see whether LY294002 exerted its inhibitory effect on VEGF protein expression by interfering with the steady-state level of VEGF mRNA, ribonuclease protection assays were used. The results showed that treatment with LY294002 decreases the VEGF mRNA level in all cell lines tested (Fig. 1B)
.
To demonstrate the maximum effect ascribable to inhibition of the PI3K pathway, we carried out a dose-response analysis of the LY294002 inhibition of VEGF expression without cell death on MDA-MB-468 and MDA-MB-231 cell lines. We used a range of concentrations between 5 and 60 µM and exposed the cells to normoxia or hypoxia for 16 h (Fig. 1C)
. All concentrations of LY294002 tested resulted in a 30% inhibition of basal VEGF protein levels in both cell lines analyzed. The hypoxia-inducible level of VEGF shows 40% and 30% inhibition, respectively, for MDA-MB-468 and MDA-MB-231 cells at the lowest concentration of LY294002 tested and up to 70% inhibition with 60 µM LY294002 in both cell lines. These results suggest that both basal and hypoxia-induced VEGF protein expression are partially dependent on PI3K activity but that other signaling pathways are also stimulating the system. Because hypoxia inducibility of VEGF is HIF dependent, we analyzed the effects of LY294002 on HIF expression.
Effect of Kinase Inhibitors on HIF Expression in Vivo.
Recent publications have emphasized the role of the MAPK and PI3K pathways on HIF expression and HIF-1 phosphorylation (24
, 36, 37, 38)
. For the following experiments, we selected the cell lines MDA-MB-468 and T47D because they gave a strong hypoxic induction of HIF-1 and HIF-2 protein expression (39)
and because both cell lines showed a higher molecular weight species of HIF-1 on Western blots.4
This high molecular weight species corresponds to a phosphorylated form of HIF-1 because it was prevented by the addition of alkaline phosphatase to the extract (24)
.
To determine whether PI3K activity was required for HIF-1 expression and phosphorylation, we treated MDA-MB-468 and T47D cells with different kinase inhibitors before incubating them for 16 h in normoxia, hypoxia, or with DFO, an iron chelator known to mimic hypoxia (Fig. 2A)
. The results showed a stronger effect of the kinase inhibitors on HIF-1 expression in MDA-MB-468 cell lines than in T47D cells. Indeed, in the former cell line, HIF-1 expression under hypoxia was totally inhibited by LY294002; in comparison, HIF-1 expression in the same cell line was reduced by PD98059, an ERK kinase (MEK-1) inhibitor, but was not affected by SB203580, a p38 MAPK inhibitor. In both cell lines, the addition of LY294002 before the incubation with DFO resulted in a complete loss of inducible HIF-1 expression. However, expression remained largely unaffected in cells treated with PD98059 or SB203580. There was no effect of the kinase inhibitors on the lower molecular weight form of HIF-1 expressed under normoxia (data not shown). Analysis of HIF-2 expression in these cell lines showed no significant effect with any of the kinase inhibitors tested under hypoxia (Fig. 2B)
. However, HIF-2-induced expression with DFO was slightly reduced in the presence of LY294002. Similar effects on HIF-1 expression were found for MDA-MB-231 and SKBr3 cell lines (Fig. 2C)
. In addition, HIF-2 expression was not modulated by kinase inhibitor treatment in the latter cell lines (data not shown).
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regulation, we studied the expression of the tumor suppressor gene PTEN and the phosphorylated form of Akt, the key downstream kinase regulated by PI3K, in the panel of breast cell lines. PTEN protein was expressed at about the same level in three of the cell lines tested (MDA-MB-231, SKBr3, and T47D), but it was not detectable in MDA-MB-468 cells (Fig. 3)
. After 16 h of exposure to hypoxia, the PTEN protein level did not change in any of the breast cell lines. Akt was expressed at a similar level in all cell lines and was not affected by hypoxic exposure. Phospho-Akt was highly expressed in MDA-MB-468 cells featuring the PTEN mutation and was detectable at a low level in T47D (these two cell lines presented a strong HIF-1 expression). However, in both cell lines, the level of phospho-Akt was not induced after 16 h of exposure to hypoxia. In addition, shorter hypoxic exposures of these cell lines (30 min and 1 h) did not show any up-regulation of phospho-Akt (data not shown). Phospho-Akt expression was not detectable in SKBr3 and MDA-MB-231 cell lines.
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The exact mechanism by which mutant ras exerts its impact on VEGF expression is not well understood. One of the best-characterized signaling pathways activated by ras involves a cascade of interactions comprising Raf-1, MAPK kinase, or MEK, and its substrates, MAPKs, also known as ERK1 and ERK2 (40
, 41)
. Further emphasis on the existence of other pathways became apparent with the observation that PI3K activity is required for ras-dependent transformation and that the catalytic subunit of this enzyme (p110) can synergize with activated Raf-1 (42
, 43)
. Using MCF10A cells stably transfected with V12-ras (MCF10A-V12ras), we showed an increased expression of VEGF mRNA and protein under normoxia and hypoxia in comparison to cells containing the empty vector control (MCF10A-EV; Fig. 4, A and B
). In both cell lines, the expression of VEGF mRNA and protein was partially inhibited by the addition of the PI3K inhibitor LY294002 and the MEK-1 inhibitor PD98059.
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and HIF-2 in these cell lines and the effects of kinase inhibitors on the hypoxia-induced level of these transcription factors’ subunits (Fig. 5, A and B)
. The cells expressing V12-ras showed an increased expression of both HIF-1 and HIF-2 under hypoxia compared with empty vector control cells. However, ras overexpression did not result in an up-regulation of HIF-2 expression under normoxia. Two independent clones expressing V12-ras were tested and showed similar results (data not shown). The quantification of two independent experiments showed a 2-fold further increase in HIF-1 expression and a 1.5-fold further increase in HIF-2 expression under hypoxia of MCF10A-V12ras-expressing cell lines. This effect could be prevented for both HIF-1 and HIF-2 by the addition of either PI3K and MEK-1 inhibitors, but not p38 MAPK inhibitor (Fig. 5B)
. Hypoxia-induced HIF-1 expression was totally inhibited by LY294002 in MCF10A-EV but was not affected by either MEK-1 or p38 MAPK inhibitors, and HIF-2 expression was only slightly reduced by LY294002.
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Expression in VHL Mutant Renal Carcinoma Cell Line. protein under normoxic conditions, which leads to a constitutive overexpression of HIF-1 (20)
. It has been shown recently that pVHL binds directly to HIF-1, targeting HIF for ubiquitination and proteasome degradation (21
, 35
, 44)
. To determine whether PI3K-induced HIF-1 expression was dependent on VHL status, we performed a similar analysis of endogenous VEGF and HIF-1 expression using a VHL-deficient renal carcinoma cell line (RCC4). The RCC4 cell line was permanently transfected with the wild-type human VHL cDNA (RCC4-VHL) or with an empty vector control (RCC4-EV; Ref. 20
).
In VHL-competent cells, VEGF basal expression was up-regulated under hypoxia or DFO, and treatment with LY294002 inhibited up to 50% of VEGF expression under all conditions (Fig. 6A)
. However, the basal VEGF expression was elevated in RCC4 defective cell lines and slightly up-regulated under hypoxia or DFO, but there was no significant effect of LY294002 on VEGF expression in these cell lines. Analysis of VEGF mRNA expression followed a similar pattern (Fig. 6B)
, with a strong inhibition of basal and hypoxia-induced mRNA expression in the RCC4-VHL cell line treated with PI3K inhibitor and a slight down-regulation of basal VEGF mRNA expression in the RCC4 mutant cell line.
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was induced under hypoxia and induced to a higher level with DFO in the RCC4-VHL cell line (Fig. 6C)
. Treatment with LY294002 completely inhibited hypoxic induction of HIF-1 and reduced the protein expression in RCC4-VHL cells incubated with DFO. However, ERK and p38 MAPK inhibitors did not show any effect on HIF-1 expression under hypoxia. In RCC4 mutant cell lines, HIF-1 was constitutively expressed at a high level, and treatment with the PI3K inhibitor reduced HIF-1 expression under normoxia and DFO and reduced it to a greater extent under hypoxia, but levels were still nearly as high as those induced by hypoxia in cells with wild-type VHL.
Although HIF-1 inhibition by the PI3K inhibitor was clearly more efficient in VHL-competent cells, a diminution of HIF-1 expression was apparent in the VHL-defective cells. These results show that PI3K-regulated HIF-1 expression is not dependent on pVHL expression.
Effect of PI3K Inhibitor on HIF-1 Ubiquitination in Vitro.
To test whether HIF-1 down-regulation by inhibition of the PI3K pathway was via increased proteolysis through the ubiquitin-proteasome pathway, we used an in vitro ubiquitination assay for HIF-1 in cell extracts, as described in Cockman et al. (35)
. In this assay, cytoplasmic cell extracts from MDA-MB-468 and RCC4 cell lines were incubated with [35S]methionine-labeled HIF-1 prepared in vitro from reticulocyte lysates, and ubiquitination was maintained by an ATP-regenerating system. Addition of ubiquitin resulted in the conversion of HIF-1 species to a high molecular weight 35S-labeled protein ladder of polyubiquitinated HIF-1 (Fig. 7)
. Comparison of cytoplasmic cell extracts from MDA-MB-468 and RCC4-VHL cell lines preincubated with LY294002 for 16 h showed no difference in the rate of HIF-1 ubiquitination as compared with those not preincubated with LY294002. Control experiments done in the absence of an ATP-regenerating system showed for both cell lines a reduced in vitro ubiquitination of HIF-1 and no effect of LY294002 (data not shown). Finally, although HIF-1 ubiquitination was clearly more efficient in VHL-competent cells, a low level of ubiquitination could be demonstrated in the defective cell lines (data not shown). These results demonstrate that the PI3K pathway does not regulate VHL-dependent ubiquitination and proteasome degradation of HIF-1.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, HIF-2, and endogenous VEGF production. We used four breast cancer cell lines with different activated oncogenes representing tumor heterogeneity to assess the generality of the regulation, and we also used stably transfected breast and renal cancer cell lines to study specific pathways.
In all cases we found that a PI3K-selective inhibitor, LY294002 (45)
, at nontoxic doses down-regulated VEGF production up to 50% at the mRNA and protein level in response to hypoxia. However, similar effects were also found in normoxia, although the absolute effect was much greater in hypoxia. Thus, a substantial component of VEGF induced by hypoxia is dependent on a PI3K pathway in cells with ras mutations, PTEN mutation, or erbB2 amplification. Because HIF-1 and HIF-2 are key regulators of VEGF expression in response to hypoxia, we analyzed the effects of LY294002 on HIF induction. In all cell lines tested, the PI3K inhibitor substantially reduced the expression of the total amount and phosphorylated form of HIF-1 protein under hypoxia but did not affect HIF-2 expression. The reduction of HIF-1 expression was similar in magnitude to the VEGF inhibition by LY294002, but there was only a partial effect of PD98059, the ERK (MEK-1) inhibitor, and no effect of SB203580, the p38 MAPK inhibitor. The absence of effect on HIF-2 expression may highlight the difference between these two transcription factors in their mechanism of regulation because it was recently found that HIF-2 is much more susceptible to redox regulation because of a cysteine residue in a critical region that differs from HIF-1 (46)
. This difference may explain the reason for cells to express both factors, i.e., the factors respond to different environmental stresses.
Because Akt is one of the key downstream mediators of PI3K activation, we analyzed the effects of hypoxia acutely and chronically on phospho-Akt expression and found no regulation of the kinase by hypoxia in the breast cancer cell lines. These results should be contrasted with two reports analyzing the interactions of PTEN and PI3K on HIF-1 and VEGF expression (37
, 38)
. In the report by Zhong et al. (37)
, a panel of prostate cancer cell lines was used, and LY294002 was found to have similar effects on HIF-1 expression, but HIF-2 was not analyzed. The regulation of the hypoxia signaling pathway was analyzed on hypoxia response element (HRE)-reporter constructs and showed much more marked effects than did our study on endogenous VEGF. They also showed that hypoxia did not activate phospho-Akt expression. Finally, their study was carried out at 1% oxygen, and our experiments were realized under 0.1% oxygen.
In the study by Zundel et al. (38)
, Akt was found to be stimulated after 30 min of hypoxia in a glioblastoma cell line (oxygen tension was not stated). Thus, the two recent studies conflict, and our data support the former. This may reflect tissue specificity in the regulation of the PI3K signaling pathway. The authors showed that HIF-1 induction was dependent on PI3K expression and that decreased PI3K activity with PTEN expression totally inhibited HIF-1 production. Surprisingly, transient PTEN restoration blocked hypoxic induction of HIF-responsive genes. However, in our study, cell lines with wild-type PTEN and low phospho-Akt levels were able to respond readily to hypoxia. These contrasting results show the potential tissue or tumor type specificity of HIF signaling modulation.
The selective effect of PD98059 is compatible with evidence of a direct phosphorylation of HIF-1 by MAPK (24)
, but there is no direct effect of the other kinases. Indeed, there was no evidence of interaction between PI3K and HIF-1, and HIF-1 did not present consensus PI3K phosphorylation sites (38)
.
Because ras activation has been well recognized to be synergistic with hypoxia in up-regulating VEGF (13)
and to act via PI3K among other pathways (3)
, we analyzed the effects of constitutive overexpression of mutant ras in MCF10A cells. Basal and hypoxia-inducible HIF-1 and, to a small extent, hypoxia-inducible HIF-2 were up-regulated in V12-ras clones. This effect could also be blocked by LY294002, which substantially reduced VEGF mRNA and protein expression. This result provides a mechanism for the ras effects on VEGF expression and synergy with hypoxia. The breast cell lines studied are of epithelial origin, and Rak et al. (4)
have recently demonstrated that regulation of VEGF expression was mediated via the PI3K pathway in ras-transformed epithelial cell lines, as opposed to ras-transformed fibroblasts that involved the MAPK pathway. However, in that study, the effects of hypoxia were not examined. Chen et al. (47)
recently reported a similar effect of ras on the glut1 reporter via up-regulation of HIF 1 in rat cell lines.
Mazure et al. (3)
, using transient transfection of HREs into Ha-ras-transformed NIH3T3 and Rat-1 cells, demonstrated a significant reduction of VEGF promotor activity under hypoxia after treatment with wortmannin, another inhibitor of PI3K. In contrast to our study, the authors found no effect of the inhibitor on basal levels of VEGF promoter activity. In addition, overexpression of a dominant negative mutant of the p85 regulatory subunit of PI3K inhibited VEGF promoter induction under hypoxia. They concluded that PI3K mediated VEGF induction through HREs. Our results support these findings but suggest that the mechanism will be through ras increasing HIF-1 protein expression via a PI3K-mediated step.
To evaluate the mechanism further, we analyzed a pathway known to stabilize HIF-1 expression to see whether the effect of PI3K depended on HIF interaction with pVHL. In a renal cancer cell line mutant for VHL, HIF-1 was constitutively stabilized, and the PI3K inhibitor still down-regulated HIF-1 expression. In cell lines treated with DFO, the VHL-HIF-1 complex is abrogated, and HIF-1 is stabilized; however, addition of PI3K inhibitor led to a decrease in HIF-1 to a similar basal level in both cell lines. In addition, there was no significant effect of PI3K inhibitor on in vitro ubiquitination of HIF-1 in MDA-MB-468 and RCC4-VHL cell lines.
These results suggest a novel pathway for HIF-1 protein regulation independent of VHL and therefore of potential importance not only to breast, prostate, and gliomas but also to renal cancer with constitutive HIF-1 activation. The effect of inhibiting PI3K does not involve enhanced activity of ubiquitination but could be due to enhanced sensitivity of the underphosphorylated HIF-1 to degradation. The mechanism is unlikely to involve the conventional proteasome pathway with VHL because the effect occurs in cell with VHL mutations. Our results are in accordance with the existence of multiple pathways regulating HIF-1 expression, but the effect of each pathway appears to be partly cell type specific.
The high basal level of VEGF protein and mRNA in RCC4-VHL mutant cells is related to stable HIF-1 protein expression and could explain the lack of effect of the PI3K inhibitor on VEGF level in these cell lines. Indeed, HIF-1 is highly expressed in these cells at the beginning of the experiments and is decreased after 16 h of treatment with LY294002 to a basal level but is still able to induce VEGF expression. This high level of HIF-1 expression may be responsible for the final amount of VEGF protein and mRNA in the cultured cells.
Overall this study shows that HIF-1, but not HIF-2, is regulated by a PI3K-dependent pathway, which contributes about 50% of the endogenous hypoxia-inducible VEGF, in a range of breast cancer cell lines. Our study extends previous work by showing that HIF activation occurs as a result of ras activation via a PI3K pathway VHL. These results have major implications for the broad spectrum use of PI3K pathway inhibitors against tumor angiogenesis mediated by a wide range of oncogene pathways upstream and downstream from PI3K.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Imperial Cancer Research Fund. 
2 To whom requests for reprints should be addressed. Phone: 44-1865-222457; Fax: 44-1865-222431; E-mail: aharris.lab{at}icrf.icnet.uk
3 The abbreviations used are: VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; PI3K, phosphatidylinositol 3'-kinase; MAP, mitogen-activated protein; VHL, von Hippel-Lindau; pVHL, VHL protein; IGF, insulin-like growth factor; EGF, epidermal growth factor; ICRF, Imperial Cancer Research Fund; DFO, desferrioxamine; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; HRE, hypoxia response element.
Received 4/ 5/01. Accepted 7/26/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(hif-1-) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem., 272: 22642-22647, 1997. is mediated by an O-2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA, 95: 7987-7992, 1998. protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. Proc. Natl. Acad. Sci. USA, 97: 4748-4753, 2000. and insulin-like growth factor 2. Cancer Res., 59: 3915-3918, 1999. (HIF-1 ) and enhance the transcriptional activity of HIF-1. J. Biol Chem., 274: 32631-32637, 1999.. Blood, 92: 2260-2268, 1998. binding and ubiquitylation by the Von Hippel-Lindau tumor suppressor protein. J. Biol. Chem., 275: 25733-25741, 2000. expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res., 60: 1541-1545, 2000. and the HIF-like factor. J. Biol. Chem., 275: 4618-4627, 2000.This article has been cited by other articles:
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 |
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  J. S.P. Yuen, E. Akkaya, Y. Wang, M. Takiguchi, S. Peak, M. Sullivan, A. S. Protheroe, and V. M. Macaulay Validation of the type 1 insulin-like growth factor receptor as a therapeutic target in renal cancer Mol. Cancer Ther., June 1, 2009; 8(6): 1448 - 1459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Burkitt, S. Y. Chun, D. T. Dang, and L. H. Dang Targeting both HIF-1 and HIF-2 in human colon cancer cells improves tumor response to sunitinib treatment Mol. Cancer Ther., May 1, 2009; 8(5): 1148 - 1156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Ma, T. Lim, J. Xu, H. Tang, Y. Wan, H. Zhao, M. Hossain, P. H. Maxwell, and M. Maze Xenon Preconditioning Protects against Renal Ischemic-Reperfusion Injury via HIF-1{alpha} Activation J. Am. Soc. Nephrol., April 1, 2009; 20(4): 713 - 720. [Abstract] [Full Text] [PDF]
|
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|
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 |
|
 G. Yang, Q. Sun, Y. Teng, F. Li, T. Weng, and X. Yang PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1{alpha} signaling and endoplasmic reticulum stress Development, November 1, 2008; 135(21): 3587 - 3597. [Abstract] [Full Text] [PDF]
|
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 I. Ader, L. Brizuela, P. Bouquerel, B. Malavaud, and O. Cuvillier Sphingosine Kinase 1: A New Modulator of Hypoxia Inducible Factor 1{alpha} during Hypoxia in Human Cancer Cells Cancer Res., October 15, 2008; 68(20): 8635 - 8642. [Abstract] [Full Text] [PDF]
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 G. G. Van den Eynden, M. Smid, S. J. Van Laere, C. G. Colpaert, I. Van der Auwera, T. X. Bich, P. van Dam, M. A. den Bakker, L. Y. Dirix, E. A. Van Marck, et al. Gene Expression Profiles Associated with the Presence of a Fibrotic Focus and the Growth Pattern in Lymph Node-Negative Breast Cancer Clin. Cancer Res., May 15, 2008; 14(10): 2944 - 2952. [Abstract] [Full Text] [PDF]
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X. Li, Y. Lu, K. Liang, T. Pan, J. Mendelsohn, and Z. Fan Requirement of hypoxia-inducible factor-1{alpha} down-regulation in mediating the antitumor activity of the anti-epidermal growth factor receptor monoclonal antibody cetuximab Mol. Cancer Ther., May 1, 2008; 7(5): 1207 - 1217. [Abstract] [Full Text] [PDF]
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E. Furuta, S. K. Pai, R. Zhan, S. Bandyopadhyay, M. Watabe, Y.-Y. Mo, S. Hirota, S. Hosobe, T. Tsukada, K. Miura, et al. Fatty Acid Synthase Gene Is Up-regulated by Hypoxia via Activation of Akt and Sterol Regulatory Element Binding Protein-1 Cancer Res., February 15, 2008; 68(4): 1003 - 1011. [Abstract] [Full Text] [PDF]
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J. R. Garlich, P. De, N. Dey, J. D. Su, X. Peng, A. Miller, R. Murali, Y. Lu, G. B. Mills, V. Kundra, et al. A Vascular Targeted Pan Phosphoinositide 3-Kinase Inhibitor Prodrug, SF1126, with Antitumor and Antiangiogenic Activity Cancer Res., January 1, 2008; 68(1): 206 - 215. [Abstract] [Full Text] [PDF]
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B. Fu, J. Xue, Z. Li, X. Shi, B.-H. Jiang, and J. Fang Chrysin inhibits expression of hypoxia-inducible factor-1{alpha} through reducing hypoxia-inducible factor-1{alpha} stability and inhibiting its protein synthesis Mol. Cancer Ther., January 1, 2007; 6(1): 220 - 226. [Abstract] [Full Text] [PDF]
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 P. Schoffski, H. Dumez, P. Clement, A. Hoeben, H. Prenen, P. Wolter, S. Joniau, T. Roskams, and H. Van Poppel Emerging role of tyrosine kinase inhibitors in the treatment of advanced renal cell cancer: a review Ann. Onc., August 1, 2006; 17(8): 1185 - 1196. [Abstract] [Full Text] [PDF]
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D. T. Dang, F. Chen, L. B. Gardner, J. M. Cummins, C. Rago, F. Bunz, S. V. Kantsevoy, and L. H. Dang Hypoxia-Inducible Factor-1{alpha} Promotes Nonhypoxia-Mediated Proliferation in Colon Cancer Cells and Xenografts Cancer Res., February 1, 2006; 66(3): 1684 - 1693. [Abstract] [Full Text] [PDF]
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N. Skuli, S. Monferran, C. Delmas, I. Lajoie-Mazenc, G. Favre, C. Toulas, and E. Cohen-Jonathan-Moyal Activation of RhoB by Hypoxia Controls Hypoxia-Inducible Factor-1{alpha} Stabilization through Glycogen Synthase Kinase-3 in U87 Glioblastoma Cells Cancer Res., January 1, 2006; 66(1): 482 - 489. [Abstract] [Full Text] [PDF]
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I. Roodink, J. Raats, B. van der Zwaag, K. Verrijp, B. Kusters, H. van Bokhoven, M. Linkels, R. M.W. de Waal, and W. P.J. Leenders Plexin D1 Expression Is Induced on Tumor Vasculature and Tumor Cells: A Novel Target for Diagnosis and Therapy? Cancer Res., September 15, 2005; 65(18): 8317 - 8323. [Abstract] [Full Text] [PDF]
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 T. Funasaka, T. Yanagawa, V. Hogan, and A. Raz Regulation of phosphoglucose isomerase/autocrine motility factor expression by hypoxia FASEB J, September 1, 2005; 19(11): 1422 - 1430. [Abstract] [Full Text] [PDF]
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 K. M. Connor, S. Subbaram, K. J. Regan, K. K. Nelson, J. E. Mazurkiewicz, P. J. Bartholomew, A. E. Aplin, Y.-T. Tai, J. Aguirre-Ghiso, S. C. Flores, et al. Mitochondrial H2O2 Regulates the Angiogenic Phenotype via PTEN Oxidation J. Biol. Chem., April 29, 2005; 280(17): 16916 - 16924. [Abstract] [Full Text] [PDF]
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C. P. Bracken, M. L. Whitelaw, and D. J. Peet Activity of Hypoxia-inducible Factor 2{alpha} Is Regulated by Association with the NF-{kappa}B Essential Modulator J. Biol. Chem., April 8, 2005; 280(14): 14240 - 14251. [Abstract] [Full Text] [PDF]
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 B. Kaur, F. W. Khwaja, E. A. Severson, S. L. Matheny, D. J. Brat, and E. G. Van Meir Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis Neuro-oncol, April 1, 2005; 7(2): 134 - 153. [Abstract] [PDF]
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J. Fang, C. Xia, Z. Cao, J. Z. Zheng, E. Reed, and B.-H. Jiang Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways FASEB J, March 1, 2005; 19(3): 342 - 353. [Abstract] [Full Text] [PDF]
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R. Blum, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, and Y. Kloog Ras Inhibition in Glioblastoma Down-regulates Hypoxia-Inducible Factor-1{alpha}, Causing Glycolysis Shutdown and Cell Death Cancer Res., February 1, 2005; 65(3): 999 - 1006. [Abstract] [Full Text] [PDF]
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 I. Vucenik, A. Passaniti, M. I. Vitolo, K. Tantivejkul, P. Eggleton, and A. M. Shamsuddin Anti-angiogenic activity of inositol hexaphosphate (IP6) Carcinogenesis, November 1, 2004; 25(11): 2115 - 2123. [Abstract] [Full Text] [PDF]
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 K. K. K. Sheares, T. K. Jeffery, L. Long, X. Yang, and N. W. Morrell Differential effects of TGF-{beta}1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L919 - L927. [Abstract] [Full Text] [PDF]
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U. E. Knies-Bamforth, S. B. Fox, R. Poulsom, G. I. Evan, and A. L. Harris c-Myc Interacts with Hypoxia to Induce Angiogenesis In vivo by a Vascular Endothelial Growth Factor-Dependent Mechanism Cancer Res., September 15, 2004; 64(18): 6563 - 6570. [Abstract] [Full Text] [PDF]
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Z. Cao, J. Fang, C. Xia, X. Shi, and B.-H. Jiang trans-3,4,5'-Trihydroxystibene Inhibits Hypoxia-Inducible Factor 1{alpha} and Vascular Endothelial Growth Factor Expression in Human Ovarian Cancer Cells Clin. Cancer Res., August 1, 2004; 10(15): 5253 - 5263. [Abstract] [Full Text] [PDF]
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C. Segrelles, S. Ruiz, M. Santos, J. Martinez-Palacio, M. F. Lara, and J. M. Paramio Akt mediates an angiogenic switch in transformed keratinocytes Carcinogenesis, July 1, 2004; 25(7): 1137 - 1147. [Abstract] [Full Text] [PDF]
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R. L. Elstrom, D. E. Bauer, M. Buzzai, R. Karnauskas, M. H. Harris, D. R. Plas, H. Zhuang, R. M. Cinalli, A. Alavi, C. M. Rudin, et al. Akt Stimulates Aerobic Glycolysis in Cancer Cells Cancer Res., June 1, 2004; 64(11): 3892 - 3899. [Abstract] [Full Text] [PDF]
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B. Kaufman, O. Scharf, J. Arbeit, M. Ashcroft, J. M. Brown, R. K. Bruick, J. D. Chapman, S. M. Evans, A. J. Giaccia, A. L. Harris, et al. Proceedings of the Oxygen Homeostasis/Hypoxia Meeting Cancer Res., May 1, 2004; 64(9): 3350 - 3356. [Abstract] [Full Text] [PDF]
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G. Powis and L. Kirkpatrick Hypoxia inducible factor-1{alpha} as a cancer drug target Mol. Cancer Ther., May 1, 2004; 3(5): 647 - 654. [Abstract] [Full Text] [PDF]
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J. Zhou, T. Schmid, R. Frank, and B. Brune PI3K/Akt Is Required for Heat Shock Proteins to Protect Hypoxia-inducible Factor 1{alpha} from pVHL-independent Degradation J. Biol. Chem., April 2, 2004; 279(14): 13506 - 13513. [Abstract] [Full Text] [PDF]
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 J. I. Bardos, N.-M. Chau, and M. Ashcroft Growth Factor-Mediated Induction of HDM2 Positively Regulates Hypoxia-Inducible Factor 1{alpha} Expression Mol. Cell. Biol., April 1, 2004; 24(7): 2905 - 2914. [Abstract] [Full Text] [PDF]
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 G. Hopfl, O. Ogunshola, and M. Gassmann HIFs and tumors--causes and consequences Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2004; 286(4): R608 - R623. [Abstract] [Full Text] [PDF]
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Y. Mizukami, J. Li, X. Zhang, M. A. Zimmer, O. Iliopoulos, and D. C. Chung Hypoxia-Inducible Factor-1-Independent Regulation of Vascular Endothelial Growth Factor by Hypoxia in Colon Cancer Cancer Res., March 1, 2004; 64(5): 1765 - 1772. [Abstract] [Full Text] [PDF]
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 H.-T. Yuan, X.-Z. Li, J. E. Pitera, D. A. Long, and A. S. Woolf Peritubular Capillary Loss after Mouse Acute Nephrotoxicity Correlates with Down-Regulation of Vascular Endothelial Growth Factor-A and Hypoxia-Inducible Factor-1{alpha} Am. J. Pathol., December 1, 2003; 163(6): 2289 - 2301. [Abstract] [Full Text]
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D. Mottet, V. Dumont, Y. Deccache, C. Demazy, N. Ninane, M. Raes, and C. Michiels Regulation of Hypoxia-inducible Factor-1{alpha} Protein Level during Hypoxic Conditions by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3{beta} Pathway in HepG2 Cells J. Biol. Chem., August 15, 2003; 278(33): 31277 - 31285. [Abstract] [Full Text] [PDF]
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T. T.-L. Tang and L. A. Lasky The Forkhead Transcription Factor FOXO4 Induces the Down-regulation of Hypoxia-inducible Factor 1{alpha} by a von Hippel-Lindau Protein-independent Mechanism J. Biol. Chem., August 8, 2003; 278(32): 30125 - 30135. [Abstract] [Full Text] [PDF]
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L. Zhang, N. Yang, D. Katsaros, W. Huang, J.-W. Park, S. Fracchioli, C. Vezzani, I. A. Rigault de la Longrais, W. Yao, S. C. Rubin, et al. The Oncogene Phosphatidylinositol 3'-Kinase Catalytic Subunit {alpha} Promotes Angiogenesis via Vascular Endothelial Growth Factor in Ovarian Carcinoma Cancer Res., July 15, 2003; 63(14): 4225 - 4231. [Abstract] [Full Text] [PDF]
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N. J. Mabjeesh, M. T. Willard, C. E. Frederickson, H. Zhong, and J. W. Simons Androgens Stimulate Hypoxia-inducible Factor 1 Activation via Autocrine Loop of Tyrosine Kinase Receptor/Phosphatidylinositol 3'-Kinase/Protein Kinase B in Prostate Cancer Cells Clin. Cancer Res., July 1, 2003; 9(7): 2416 - 2425. [Abstract] [Full Text] [PDF]
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M. C. A. Duyndam, S. T. M. Hulscher, E. van der Wall, H. M. Pinedo, and E. Boven Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite J. Biol. Chem., February 21, 2003; 278(9): 6885 - 6895. [Abstract] [Full Text] [PDF]
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 K. Reisinger, R. Kaufmann, and J. Gille Increased Sp1 phosphorylation as a mechanism of hepatocyte growth factor (HGF/SF)-induced vascular endothelial growth factor (VEGF/VPF) transcription J. Cell Sci., January 15, 2003; 116(2): 225 - 238. [Abstract] [Full Text] [PDF]
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 M. Mayerhofer, P. Valent, W. R. Sperr, J. D. Griffin, and C. Sillaber BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1alpha , through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin Blood, November 15, 2002; 100(10): 3767 - 3775. [Abstract] [Full Text] [PDF]
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 K. A. Seta, Z. Spicer, Y. Yuan, G. Lu, and D. E. Millhorn Responding to Hypoxia: Lessons From a Model Cell Line Sci. Signal., August 20, 2002; 2002(146): re11 - re11. [Abstract] [Full Text] [PDF]
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C. Treins, S. Giorgetti-Peraldi, J. Murdaca, G. L. Semenza, and E. Van Obberghen Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway J. Biol. Chem., July 26, 2002; 277(31): 27975 - 27981. [Abstract] [Full Text] [PDF]
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C. B. Knobbe, A. Merlo, and G. Reifenberger Pten signaling in gliomas Neuro-oncol, July 1, 2002; 4(3): 196 - 211. [Abstract] [PDF]
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A. M. Arsham, D. R. Plas, C. B. Thompson, and M. C. Simon Phosphatidylinositol 3-Kinase/Akt Signaling Is Neither Required for Hypoxic Stabilization of HIF-1alpha nor Sufficient for HIF-1-dependent Target Gene Transcription J. Biol. Chem., April 19, 2002; 277(17): 15162 - 15170. [Abstract] [Full Text] [PDF]
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M. Alvarez-Tejado, A. Alfranca, J. Aragones, A. Vara, M. O. Landazuri, and L. del Peso Lack of Evidence for the Involvement of the Phosphoinositide 3-Kinase/Akt Pathway in the Activation of Hypoxia-inducible Factors by Low Oxygen Tension J. Biol. Chem., April 12, 2002; 277(16): 13508 - 13517. [Abstract] [Full Text] [PDF]
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 C. K. Sen, S. Khanna, M. Venojarvi, P. Trikha, E. C. Ellison, T. K. Hunt, and S. Roy Copper-induced vascular endothelial growth factor expression and wound healing Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1821 - H1827. [Abstract] [Full Text] [PDF]
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