Molecular Cancer Research Targeting the PI3-Kinase Pathway in Cancer Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Burroughs, K. D.
Right arrow Articles by DiAugustine, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burroughs, K. D.
Right arrow Articles by DiAugustine, R. P.
Molecular Cancer Research 1:312-322 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

Phosphatidylinositol 3-Kinase and Mek1/2 Are Necessary for Insulin-Like Growth Factor-I-Induced Vascular Endothelial Growth Factor Synthesis in Prostate Epithelial Cells: A Role for Hypoxia-Inducible Factor-1?

Kevin D. Burroughs1, Jennifer Oh1, J. Carl Barrett2 and Richard P. DiAugustine1

1 Hormones and Cancer Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC and
2 Center for Cancer Research, National Cancer Institute, Bethesda, MD

Requests for reprints: Richard P. DiAugustine, Hormones and Cancer Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Mail Drop D4-04, P. O. Box 12233, Research Triangle Park, NC 27709. Phone: (919) 541-3218; Fax: (919) 541-4704. E-mail: diaugus2{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Due to the importance of vascular endothelial growth factor (VEGF) in the neovascularization of solid tumors, a clear understanding of how VEGF is regulated in normal and tumor cells is warranted. We investigated insulin-like growth factor (IGF)-I-stimulated signaling pathways that increase the rate of VEGF synthesis in primary cultures of normal prostate epithelial cells (PrEC). IGF-I increased the secretion of VEGF165 into PrEC growth medium and stimulated transcription of a reporter gene driven by a 1.5-kb region of the VEGF promoter. Inhibition of either phosphatidylinositol 3-kinase (PI3-K) or Mek1/2 signaling pathways completely abrogated the IGF-I-induced increase in VEGF secretion and promoter activity, indicating a dependence on coordinate signaling from both pathways to produce this effect. Levels of the transcription factors hypoxia-inducible factor (HIF)-1 and Fos were elevated in response to IGF-I in a PI3-K-dependent and Mek1/2-dependent manner, respectively. The expression of an activator protein (AP)-1 dominant negative in an immortalized prostate epithelial cell line PZ-HPV-7 suppressed the IGF-I-induced increase in VEGF promoter activity. Mutation of the hypoxia response element (HRE), which mediates hypoxic stimulation of VEGF transcription, did not inhibit the effect of IGF-I on the VEGF promoter, despite the fact that this mutation inhibited PI3-K-stimulated VEGF promoter activity in prostate cancer cells. These data indicate that PI3-K signaling does not increase VEGF transcription through transactivation by HIF-1 at the HRE in normal PrEC. This work also suggests that an additional signal, not stimulated by IGF-I in PrEC, is needed for HIF-1 to stimulate transcription from the VEGF HRE.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Angiogenesis, or the formation of new blood vessels from preexisting endothelial cells, is an essential component of several normal and pathological biological events (1). Increasing attention has been given to the role of angiogenesis in solid tumors because it contributes to both the growth and metastatic spread of tumor cells (2). In this regard, intratumoral microvessel density has been demonstrated to be a strong prognostic indicator of metastatic potential for several tumors, including breast and prostatic carcinomas (PCa) (3, 4). Numerous studies have shown that the switch to an angiogenic phenotype occurs at an early stage during tumor progression, and preneoplastic lesions with increased angiogenic potential demonstrate an increased risk of malignant transformation (5, 6).

Over the last two decades, several growth factors along with their complementary receptors were discovered to regulate angiogenesis. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and a potent microvascular permeability factor. VEGF is a dimeric glycosylated Mr 34,000–46,000 protein that occurs as isoforms of 121, 145, 165, 189, and 206 amino acids as a consequence of alternative splicing of a single gene (7–9). This protein is one of the major factors promoting neovascularization of tumors and is commonly found in both premalignant and malignant lesions. Overexpression of VEGF is regularly observed in advanced PCa and also in a subset of microscopic, precancerous lesions in this organ (10–12). The use of a monoclonal antibody that neutralizes VEGF activity halted the growth of PCa tumor xenografts and prevented their metastasis (2). Taken together, these data implicate VEGF as an important factor in the progression of PCa.

Hypoxia stimulates transcription of VEGF and other genes by activating the hypoxia-inducible factor-1 (HIF-1) transcription factor (13). HIF-1 is composed of HIF-1{alpha} and the aryl hydrocarbon receptor nuclear translocator (ARNT) and regulates transcription through its association with DNA at specific sequences known as hypoxia response elements [HRE; Refs. (14, 15)]. The activity of HIF-1 is regulated in part by ubiquitin-mediated proteasomal degradation of HIF-1{alpha} under normoxic conditions (16). When cells are exposed to low ambient oxygen, HIF-1{alpha} is stabilized and translocates to the nucleus, where it binds ARNT and activates transcription (17). Recently, growth factors were shown to increase expression of HIF-1{alpha}- and HIF-1-mediated transcription via a phosphatidylinositol 3-kinase (PI3-K) mechanism. Whereas hypoxia prevents HIF-1{alpha} degradation, the PI3-K pathway stimulates the rate of HIF-1{alpha} translation (18–21). Therefore, growth factor signaling may induce a panel of HIF-dependent genes similar to hypoxia, albeit by a novel mechanism of HIF-1 activation.

A variety of growth factors, cytokines, and oncogenes stimulate VEGF mRNA expression (22–26). Insulin-like growth factor-I (IGF-I) was previously reported to increase VEGF expression in osteoblast-like cells, colon cancer cells, retinal pigment epithelial cells, bovine smooth muscle cells, and endometrial adenocarcinoma cells (27–30). Studies with the HT29 colon carcinoma cells indicate that IGF-I induces VEGF mRNA by transcriptional activation of the VEGF gene (28). Cells transiently transfected with a VEGF promoter-luciferase reporter construct revealed a marked increase in activity when treated with IGF-I. In addition, stimulation of VEGF transcript levels in the HT29 cells by IGF-I was blocked by actinomycin D and the half-life of VEGF mRNA was not prolonged by the growth factor.

The signaling pathways activated by IGF-I that increase VEGF synthesis have not been defined. IGF-I can activate both PI3-K and the Mek1/2 mitogen-activated protein kinase (MAPK) pathways in various cells (31). Substantial data indicate the involvement of these pathways in the regulation of VEGF production, with the responsible pathway dictated by the particular stimulus and cell type (32, 33). Because IGF-I is linked with both normal development (34) and tumor formation (35, 36) in the prostate gland, we investigated PI3-K and Mek1/2 pathways for their role in mediating the IGF-I-induced increase in VEGF synthesis in normal prostate epithelial cells (PrEC). We found that IGF-I increased VEGF secretion and also stimulated transcription of a reporter gene driven by a 1.5-kb region of the VEGF promoter. Both PI3-K and Mek1/2 pathways were required for IGF-I induction of VEGF transcription. Despite the induction of HIF-1{alpha} by IGF-I in PrEC, HIF-1 did not drive transcription through its binding site, the HRE, in the VEGF promoter. However, PCa cells secreted large amounts of VEGF, in part, due to constitutive activation of PI3-K signaling and HIF-1-mediated transcriptional activation through the HRE in the VEGF promoter.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
IGF-I Stimulates Synthesis of VEGF in PrEC
To determine whether signaling from the IGF-IR increases the rate of synthesis of VEGF in PrEC, confluent PrEC cultures were treated with LongR3-IGF-I, and the concentration of the VEGF165 isoform in culture medium was measured by ELISA. IGF-I dose-dependently increased the concentration of VEGF over a 24-h period with a maximum stimulation of approximately 2-fold occurring at 100 ng/ml (Fig. 1A). A time-course experiment showed that a single treatment of IGF-I at 100 ng/ml produced a sustained increase in the rate of VEGF secretion into medium over 72 h (Fig. 1B). In fact, Fig. 1C shows that the amount of VEGF produced in each 24-h period actually increased over the 72 h of treatment. Therefore, the availability of IGF-I to PrEC results in a sustained increase in the rate of VEGF production.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. IGF-I stimulates VEGF synthesis by PrEC. A. Confluent PrEC cultures were treated with the indicated concentrations of LongR3-IGF-I or vehicle for 24 h. The concentration of the VEGF165 isoform was measured in the medium by ELISA and normalized by the number of cells in each well. *, P <= 0.05 versus 0 ng/ml. B. The concentration of VEGF165 was determined after confluent PrEC cultures were treated for the indicated times with 100 ng/ml LongR3-IGF-I. *, P <= 0.05 versus vehicle-treated cultures at the same time point. C. Data are amounts of VEGF165 secreted at each 24-h period from the experiment in B.

 
IGF-I Stimulates VEGF Synthesis via PI3-K and Mek1/2 Signaling
Stimulation of the IGF-IR can activate multiple signaling pathways in target cells, including both PI3-K and Mek1/2 pathways (31). Immunoprecipitation of the IGF-IR in PrEC following exposure to IGF-I showed that the ß-subunit of the receptor was rapidly phosphorylated on tyrosine residues and that this phosphorylation was stable over 48 h (Fig. 2A). Likewise, p85, the regulatory subunit of the PI3-K enzyme, was present in IGF-IR immunoprecipitates only after stimulation with IGF-I. The association of p85 with IGF-IR was stable throughout the 48 h of treatment, indicating that the PI3-K enzyme was recruited to the activated IGF-IR. Additionally, Akt, which is a downstream target of PI3-K signaling, was rapidly and stably phosphorylated on serine 473 following IGF-I treatment (Fig. 2B). These data indicate that activation of the IGF-IR by IGF-I in PrEC initiates a prolonged PI3-K signaling response.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 2. IGF-I activates PI3-K and Erk1/2 signaling pathways in PrEC. Confluent PrEC cultures were treated with 100 ng/ml LongR3-IGF-I for the indicated times at which point cells were lysed. A. The ß-subunit of IGF-IR was immunoprecipitated and analyzed by Western blotting with anti-phosphotyrosine (PY), anti-p85 of PI3-K, and the ß-subunit of IGF-IR (IGF-IRß). B and C. Equal volumes of whole cell lysate were analyzed by Western blotting for Akt phosphorylated on serine 473 (P-Akt), total Akt, Erk1/2 phosphorylated at threonine 202 and tyrosine 204 (P-Erk1/2), and total Erk1/2.

 
The MAPKs Erk1/2 are activated via their phosphorylation by Mek1/2 kinases in response to numerous growth factors and cytokines (37). In PrEC, IGF-I treatment increased the level of phosphorylation of Erk1/2 in a biphasic manner (Fig. 2C). Phosphorylation occurred rapidly, then subsided by 2 h after IGF treatment; a second increase was observed at 24 h. At 48 h, levels of phosphorylated Erk1/2 had fallen below that of the untreated cells. The amount of phosphorylated Erk1/2 was not due to changes in the concentration of the two proteins in the cell, suggesting that changing dynamics between kinase and phosphatase activities occur in response to IGF-I. IGF-I is therefore capable of coordinately stimulating both PI3-K and Erk1/2 signaling pathways in PrEC.

To determine whether PI3-K or Mek1/2 pathway signaling mediated the stimulatory effect of IGF-I on VEGF synthesis, the pharmacological inhibitors LY294002 (PI3-K), PD98059 (selective Mek 1), or UO126 (Mek1/2) were used. We examined PrEC cultures from three separate individuals and observed in all cultures that IGF-I increased the concentration of VEGF in culture medium and that all three inhibitors completely inhibited this response, indicating the involvement of both pathways in the regulation of VEGF synthesis. Fig. 3A shows data from one of the three PrEC cultures tested. Additionally, Fig. 3B shows that the PI3-K and Mek1/2 pathways are each necessary, but not sufficient alone, to increase VEGF production. Either LY294002 or UO126 completely inhibited IGF-I stimulation of VEGF levels (Fig. 3B). Moreover, both compounds inhibited expression of VEGF in the absence of IGF-I stimulation, suggesting that constitutive activity in these pathways contributes to basal VEGF production. The use of LY294002 and UO126 together provided no additional inhibitory effect relative to their use alone. In PrEC, LY294002 had no effect on IGF-I-stimulated Erk1/2 phosphorylation, and neither PD98059 nor UO126 inhibited IGF-I-stimulated Akt phosphorylation (data not shown). Therefore, PI3-K and Mek1/2 function within independent signaling pathways in PrEC and each are necessary, but not sufficient, to confer an effect of IGF-I on VEGF synthesis.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. PI3-K and Mek1/2 mediate the effect of IGF-I on VEGF synthesis in PrEC. A. PrEC cultures were pretreated with DMSO vehicle, the PI3-K inhibitor LY294002 (LY), or Mek1/2 inhibitors PD98059 (PD) or UO126 (UO) at the indicated concentrations for 1 h before treatment with 100 ng/ml LongR3-IGF-I for 24 h in the continued presence of inhibitor. *, P <= 0.05 versus vehicle-treated cultures for each cell type. B. Similarly, PrEC cultures were pretreated with LY294002 and UO126 for 1 h then treated with 100 ng/ml LongR3-IGF-I or vehicle for 24 h in the presence of inhibitor. The concentration of VEGF165 was determined in the medium by ELISA and normalized by the number of cells in each well. *, P <= 0.05 versus matched vehicle-treated culture.

 
PI3-K and Mek1/2 Stimulate VEGF Gene Transcription in Response to IGF-I
To further address the mechanism whereby IGF-I increases VEGF synthesis, we created a luciferase reporter construct driven by 1.5 kb of the human VEGF gene promoter. After transient transfection of this plasmid into PrEC, treatment with IGF-I stimulated reporter activity approximately 2.5-fold. Either LY294002 or PD98059 inhibited the effect of IGF-I on promoter activity, indicating that both pathways contribute to the IGF-I-stimulated transcription of the VEGF gene (Fig. 4). Both inhibitors decreased the activity of the VEGF promoter fragment below that observed in the absence of IGF-I stimulation. This observation further supports that constitutive PI3-K and Mek1/2 signaling contribute to basal transcription of the VEGF gene. The potential for IGF-I to stimulate transcription from the VEGF promoter was also tested in an immortalized prostate epithelial cell line PZ-HPV-7. As was observed for PrEC cultures, IGF-I increased the concentration of VEGF165 protein in PZ-HPV-7 culture medium, an effect that was blocked by either PI3-K or Mek1/2 inhibition (data not shown). In transient transfection assays, IGF-I also increased activity of the hVEGF-Luc reporter in this cell line, and, as with PrEC, the effect of IGF-I on promoter activity was blocked by either LY294002 or PD98059 (Fig. 4).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. IGF-I stimulates transcription from the VEGF promoter through PI3-K and Mek1/2 signaling pathways. One microgram of the hVEGF-Luc plasmid and 20 ng of the normalizing vector pRL-CMV were transiently transfected into PrEC and the immortalized prostate epithelial cell line PZ-HPV-7. Cells were then treated as follows for 24 h: 0.05% DMSO (V), 100 ng/ml LongR3-IGF-I + 0.05% DMSO (I), 100 ng/ml LongR3-IGF-I + 25 µM LY294002 (I/LY), or 100 ng/ml LongR3-IGF-I + 20 µM PD98059 (I/PD). The firefly/Renilla luciferase values were expressed as "percent control" of the vehicle-treated cultures for PrEC and PZ-HPV-7. *, P <= 0.05 versus vehicle-treated cultures. **, P <= 0.05 versus IGF-I-treated cultures.

 
Activator Protein 1 Mediates an Effect of IGF-I on VEGF Gene Transcription
The 1.5-kb region of the human VEGF promoter contained in the hVEGF-Luc reporter plasmid has four putative activator protein (AP)-1 binding sites (7, 38, 39). Fos is induced as part of the immediate early response to mitogenic stimulation and dimerizes with Jun family members to form AP-1 transcription factors. Levels of Fos are induced in response to numerous, diverse stimuli, including growth factors, and can be regulated through the Mek/Erk signaling pathway (40). Western blotting of PrEC nuclear lysates demonstrated that Fos was induced by IGF-I within 2 h of treatment, with peak induction observed at 12 h (Fig. 5A). Fos remained elevated throughout the 48 h of IGF-I treatment. Mek1/2 pathway inhibitors completely abolished the effect of IGF-I on Fos in both PrEC and PZ-HPV-7 cells (Fig. 5B). The PI3-K inhibitor LY294002 had no effect, indicating that IGF-I induced Fos specifically through Mek1/2-dependent signaling. Jun was constitutively present and phosphorylated on serines 63 and 73 in PrEC (data not shown). Jun was only slightly induced by IGF-I without noticeably affecting the phosphorylation of this transcription factor.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 5. IGF-I increases levels of Fos in a Mek1/2-dependent manner. A. Equal volumes of nuclear protein lysates of confluent PrEC cultures treated with 100 ng/ml LongR3-IGF-I for the indicated times were analyzed by Western blotting with antisera to Fos. B. Nuclear lysates were made from PrEC and PZ-HPV-7 cultures pretreated with DMSO vehicle, 25 µM LY294002 (LY), 20 µM PD98059 (PD), or 10 µM UO126 (UO) for 1 h before stimulation with 100 ng/ml LongR3-IGF-I in the continued presence of inhibitor for 6 h. Equal volumes of lysates were probed with Fos antisera.

 
The AP-1 dominant negative A-Fos was used to determine the role of AP-1 transcription factors in IGF-I-stimulated VEGF expression. A-Fos binds to Jun with higher affinity than endogenous Fos, preventing dimerization of Jun with Fos and Jun family members and inhibiting the interaction of Jun with DNA (41). Cotransfection of an A-Fos expression vector along with hVEGF-Luc into PZ-HPV-7 cells had no effect on the basal level of transcription from the VEGF promoter (Fig. 6). Under these conditions, Fos levels were very low in the cell. However, the dominant negative inhibited the ability of IGF-I to stimulate reporter activity by more than 50%. This result implicates the participation of Fos and AP-1 proteins in IGF-I stimulation of VEGF gene transcription in PrEC, but not in the constitutive activity of the promoter.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 6. An AP-1 dominant negative blunts IGF-I-induced transcription from the VEGF promoter. PZ-HPV-7 cells were transiently transfected with 1 µg of the AP-1 dominant negative A-Fos or its empty control vector and allowed to recover for 24 h before transfection of 1 µg hVEGF-Luc and 20 ng pRL-CMV. Cells were then treated with vehicle or 100 ng/ml LongR3-IGF-I for 24 h and firefly/Renilla luciferase activities determined. *, P <= 0.05 versus IGF-I-treated control transfection. {blacksquare}, vehicle; {square}, IGF-I.

 
HIF-1 Does Not Mediate the Effect of IGF-I Through the VEGF HRE in PrEC
Because HIF-1 is a known inducer of VEGF gene transcription and others have recently shown that growth factor signaling can induce levels of HIF-1{alpha}, we investigated whether HIF-1 mediates an effect of IGF-I on VEGF promoter activity in PrEC. Stimulation of PrEC cultures with IGF-I increased levels of HIF-1{alpha} specifically in the nucleus (Fig. 7A). HIF-1{alpha} induction was observed by 4 h after IGF-I treatment and was maximal at 6 h. The concentration of HIF-1{alpha} in the nucleus remained higher than in untreated cells through 24 h, but after 48 h of treatment, HIF-1{alpha} fell to below pretreatment levels. Immunoprecipitation of HIF-1{alpha} from nuclear lysates of PrEC treated for 6 h with IGF-I coprecipitated ARNT; however, vehicle-treated cultures did not exhibit an interaction between HIF-1{alpha} and ARNT (Fig. 7B). Thus, IGF-I induces formation of the HIF-1 transcription factor in the nucleus of PrEC. Cotreatment of IGF-I-stimulated PrEC cultures with LY294002 prevented the increase in nuclear levels of HIF-1{alpha} (Fig. 7C). LY294002 did not cause an accumulation of HIF-1{alpha} in the cytoplasm, indicating that PI3-K signaling regulates the cellular concentration of HIF-1{alpha}, not simply its nuclear import (data not shown). Inhibition of Mek1/2 signaling had no effect on HIF-1{alpha} levels (Fig. 7C). HIF-1{alpha} was regulated by IGF-I in the same manner in PZ-HPV-7 cells (Fig. 7C). Therefore, IGF-I induces Fos and HIF-1 transcription factors through activation of two independent signaling pathways, Mek1/2 and PI3-K, respectively.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 7. IGF-I increases nuclear levels of the HIF-1 transcription factor in a PI3-K-dependent manner. A. Equal volumes of nuclear and cytoplasmic lysates of confluent PrEC cultures treated for the indicated times with 100 ng/ml LongR3-IGF-I were probed with antisera to HIF-1{alpha}. PrEC cultures were treated for 6 h with 130 µM desferrioxamine mesylate (Dfx) as a positive control to induce HIF-1{alpha}. B. Nuclear lysates of PrEC cultures treated for 6 h with 100 ng/ml LongR3-IGF-I or vehicle were immunoprecipitated (IP) with HIF-1{alpha} antisera. The precipitate was then probed with ARNT antisera. The nuclear lysate from IGF-I-treated PrEC served as a positive control for ARNT expression. C. Nuclear lysates were made from PrEC and PZ-HPV-7 cultures pretreated with DMSO vehicle, 25 µM LY294002 (LY), or 20 µM PD98059 (PD) for 1 h before stimulation with 100 ng/ml LongR3-IGF-I for 6 h. Equal volumes of lysates were probed with HIF-1{alpha} antisera.

 
To determine whether HIF-1 increases the rate of VEGF gene transcription in response to IGF-I by binding to the HRE at -975 of the human VEGF promoter, we mutated the hVEGF-Luc construct at the HIF-1 binding site to make the HREm1 reporter construct. A three-nucleotide substitution was made that was previously shown (13, 42) to prevent HIF-1 binding and transcriptional activation (Fig. 8). When transfected into PZ-HPV-7 cells, the mutant promoter was activated by IGF-I to the same extent as that of the wild-type promoter. Additionally, HREm1 retained sensitivity to LY294002, which we previously showed prevents IGF-I-stimulated HIF-1{alpha} up-regulation. Therefore, induction of the HIF-1 transcription factor by IGF-I does not lead to increased transcription of the VEGF gene through the HRE. This observation was surprising given previous observations by others that PI3-K signaling via HIF-1 stimulates transcription from the VEGF promoter and other heterologous promoters containing a HRE in PCa cell lines (19, 20). We, therefore, asked whether the HRE was important for VEGF promoter activity in two PCa cell lines, PC-3 and TSU-Pr1, which contain constitutively active PI3-K signaling and constitutively express HIF-1{alpha}. In both of these cell lines, Akt was phosphorylated on serine 473 in the absence of growth factor stimulation. In addition, LY294002 or UO126 inhibited the phosphorylation of Akt and decreased the constitutive production of VEGF (data not shown). Mutation of the HRE in the VEGF promoter decreased constitutive VEGF promoter activity by more than 50% in PC-3 and TSU-Pr1 cells (Fig. 9), Furthermore, mutation of the HRE completely abrogated sensitivity of the promoter to PI3-K inhibition by LY294002 in PC-3 cells. The effect of LY294002 was also greatly diminished in TSU-Pr1 cells. These results suggest that, while PI3-K is sufficient to increase nuclear levels of HIF-1, a separate signal apart from PI3-K is necessary to produce a HIF-1 transcription factor capable of stimulating transcription from the VEGF promoter. This separate signal is apparently activated in PCa cells, but not in PrEC.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 8. The VEGF HRE does not mediate the PI3-K-dependent effect of IGF-I on the VEGF promoter in PrEC. PZ-HPV-7 cells were transiently transfected with 1 µg hVEGF-Luc or HREm1 in which the HRE was mutated as indicated along with 20 ng pRL-CMV. Cells were then treated as follows for 24 h: 0.05% DMSO (V), 100 ng/ml LongR3-IGF-I + 0.05% DMSO (I), 100 ng/ml LongR3-IGF-I + 25 µM LY294002 (I/LY), or 100 ng/ml LongR3-IGF-I + 20 µM PD98059 (I/PD). The firefly/Renilla luciferase values were expressed as percent control of the vehicle-treated cultures. The sequence of the wild-type and mutated HRE in hVEGF-Luc and HREm1-Luc, respectively, are shown. The three base pair substitution made in HREm1-Luc is underlined. *, P <= 0.05 versus matched vehicle-treated cultures. **, P <= 0.05 versus matched IGF-I-treated cultures.

 


View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 9. The VEGF HRE mediates the effect of constitutive PI3-K signaling on the VEGF promoter in PCa cells. PC-3 and TSU-Pr1 cells were transiently transfected with 1 µg of either hVEGF-Luc or HREm1 along with 20 ng of pRL-CMV. Cells were then treated with either DMSO vehicle (V), 50 µM LY294002 (LY), or 10 µM UO126 (UO) for 24 h. Firefly/Renilla luciferase values are expressed as percent control of vehicle-treated cells transfected with hVEGF-Luc. *, P <= 0.05 versus matched vehicle-treated hVEGF-Luc transfections. **, P <= 0.05 versus matched vehicle-treated HREm1 transfections. {blacksquare}, hVEGF; {square}, HREm1.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The present data have shown that IGF-I increases the rate of synthesis of VEGF in cultured, normal PrEC and does so, at least in part, through the stimulation of VEGF gene transcription. IGF-I increased the activity of the PI3-K and Mek1/2 signaling pathways; each pathway was necessary, but not sufficient, to mediate an effect of IGF-I on VEGF production. In addition, both pathways functioned to regulate the rate of transcription from the VEGF promoter. The level of Fos was stimulated by IGF-I in a Mek-dependent manner, and inhibition of AP-1 with the A-Fos dominant negative indicated that AP-1 proteins are involved in regulating VEGF transcription. Although HIF-1{alpha} protein levels were stimulated by IGF-I in PrEC, mutation of the HRE in the VEGF promoter had no effect on IGF-I-stimulated VEGF transcription, indicating that the effect of PI3-K signaling on VEGF transcription occurred either through a novel mechanism involving HIF-1 or through some other unknown factor. Additionally, in contrast to PrEC, mutation of the VEGF HRE decreased promoter activity in PCa cells and almost completely abolished sensitivity of the promoter to PI3-K inhibition. This observation suggests that PI3-K stimulation of nuclear HIF-1 levels is not sufficient to stimulate HIF-1's ability to activate the VEGF promoter through the HRE, but that an additional cue, not active in normal PrEC, is necessary.

The proangiogenic potential of IGF-I has now been recognized in numerous cell types and tissues, indicating that IGF-I may contribute to the regulation of tissue vascularization in a widespread manner. This effect of IGF-I may stem from two separate mechanisms of action. First, IGF-I exerts direct effects on endothelial cells where IGF-IR signaling cooperates with VEGF receptors in stimulating signal transduction pathways involved in an angiogenic response (43, 44). Second, IGF-I can work indirectly, as observed in the current work, by stimulating the synthesis of angiogenic factors, such as VEGF (27–30). IGF-I, in mediating the effects of growth hormone, stimulates the physiological growth of lean body mass during development, and exogenous administration of recombinant IGF-I or overexpression of endogenous IGF-I in transgenic animals produces an increase in whole body weight and organomegaly of certain visceral organs (45). It is reasonable that such increases in tissue mass must be accompanied by new vascularization. Angiogenesis may be one of multiple responses to IGF-I that are required for this growth factor to stimulate tissue growth in vivo. These observations have meaningful implications for cancer prevention and treatment. High serum IGF-I is now linked to an increased risk of common human cancers (35, 36, 46–48), and autocrine stimulation of the IGF-IR is a regularly observed event in the progression of solid tumors (49). Because angiogenesis is a rate-limiting step for solid tumor growth beyond 1–2 mm3 and facilitates entry of malignant cells into the circulation (50), the ability to attenuate the biological effects of IGF-IR signaling and subsequent angiogenic effects may, therefore, slow tumor growth and progression to metastatic disease. This idea is supported by xenograft models in which inhibition of the IGF-IR slowed tumor growth, progression, and metastasis (51–55).

In PrEC, the stimulation of VEGF synthesis by IGF-I required signaling from both PI3-K and Mek1/2 pathways. A recent report has indicated a similar mechanism for IGF-I activation of eukaryotic initiation factor eIF-2B in neuronal cells (56). In both studies, primary cultures of normal cells were used which should allow analysis of signaling transduction pathways that are not altered by malignant progression or immortalization. It is likely that other cases will be found where activation of multiple independent signaling pathways is required to regulate a single downstream target. With regard to VEGF transcription, a mechanism of activation that requires signaling from more than one pathway provides more points of regulation and perhaps finer control of activity by integrating numerous cellular cues. Given the now evident role of VEGF in malignant growth, such a mechanism could also provide additional protection against VEGF overexpression from aberrant activity in a single pathway. Interestingly, the amounts of phosphorylated Akt (57) and Erk1/2 (58, 59) are elevated with increasing grade in PCa specimens with respect to adjacent, normal epithelium, indicating an increase in the activity of both PI3-K and Mek1/2 signaling pathways, respectively.

AP-1 transcription factors were previously identified as inducers of VEGF synthesis. Dominant negative Jun inhibits stimulation of VEGF transcription in response to ionizing radiation and lead (60, 61). Forced overexpression of Fos and Jun together increased VEGF promoter activity by 4-fold in ovarian cancer cells (62). Additionally, more direct evidence of a role for Fos in VEGF expression was reported by Saez et al. (63). These authors examined the effect of homozygous deletion of the c-fos gene on skin cancer development in mice. Papillomas that developed in c-fos-/- mice lacked external vascularity, and VEGF mRNA expression was reduced 5- to 10-fold in mutant mice. In the current study, we find that IGF-I activates Fos through Mek-dependent signaling and that inhibition of AP-1 activity in normal PrEC decreases the activity of the VEGF promoter specifically when stimulated by IGF-I.

The role of HIF-1 in mediating the stimulatory effects of hypoxia on VEGF transcription is well established. Additionally, growth factors such as insulin, epidermal growth factor (EGF), and heregulin or loss of PTEN tumor suppressor gene function also stimulate HIF-1{alpha} levels through increased PI3-K activity (18–21, 64). The mechanism whereby heregulin increased HIF-1{alpha} levels was shown to involve a PI3-K/Akt/FRAP (FK506 binding protein-Rapamycin-associated protein) pathway. Activation of this pathway increased the rate of HIF-1{alpha} protein synthesis, rather than inhibiting its degradation as what occurs during hypoxia. This same pathway was responsible for increasing HIF-1{alpha} in response to insulin as well (65). We also found that nuclear levels of HIF-1{alpha} in PrEC were indeed regulated specifically by a PI3-K/Akt pathway without contribution from Mek1/2 signaling. Recently, however, IGF-I induction of HIF-1{alpha} in HCT116 colon cancer cells was shown to involve both PI3-K and Mek1/2 signaling (66). In that study, inhibition of PI3-K or Mek1/2 signaling blocked IGF-I-induced phosphorylation of the translational regulatory proteins p70 S6 kinase and eukaryotic initiation factor-4E binding protein 1 (4E-BP1). Therefore, it appears that growth factor regulation of HIF-1{alpha} functions by a mechanism distinct from hypoxia and that the signaling pathways regulating HIF-1 in response to IGF-I may differ depending on cell type.

Although we find that IGF-I increased nuclear HIF-1{alpha} levels in PrEC cultures through the PI3-K pathway, mutation of the HRE at -975 in the VEGF promoter had no effect on the IGF-I-stimulated increase in reporter activity. Likewise, EGF was found to increase VEGF transcription in a glioblastoma cell line in a PI3-K-dependent manner that did not involve the HRE (67). Whether HIF-1 enhances VEGF transcription in PrEC via a novel mechanism not involving its binding to the HRE is unknown, but this possibility is supported by observations from other investigators. Forsythe et al. (13) reported that removal of the HRE decreased hypoxia-induced VEGF promoter activity approximately 50% in Hep3B cells; however, the activity of the VEGF promoter lacking the HRE was still modulated by HIF-1{alpha} overexpression or a HIF-1{alpha} dominant negative, indicating that HIF-1 may play additional roles in VEGF gene expression.

It was recently reported that under hypoxic conditions, Jun can potentiate HIF-1 activation of a heterologous promoter containing an HRE (68). This effect of Jun was dependent on the presence of an intact HRE. In the current study, a Fos dominant negative that blocks Jun function inhibited IGF-I-induced VEGF promoter activity. However, mutation of the HRE had no effect on VEGF promoter activity in response to IGF-I. Additionally, the authors of the aforementioned study did not report whether the interaction between Jun and HIF-1{alpha} had an effect on transcription from an AP-1 sensitive promoter. In an effort to understand the sensitivity of the VEGF promoter to Mek1/2 signaling and the Fos dominant negative, we mutated the AP-1 site that lies downstream of the VEGF HRE at position 937. This site was previously shown to contribute to hypoxic stimulation of the VEGF promoter (39). Mutation of this AP-1 site did not inhibit transcription of the VEGF promoter reporter construct in response to IGF-I (data not shown). Other putative AP-1 binding sites exist within the 1.5-kb fragment of the VEGF promoter used in this study. Therefore, it is not clear whether an interaction between Jun and HIF-1 could account for data observed in our study.

The induction of VEGF transcription in various cell lines by growth factors and cytokines, such as transforming growth factor {alpha}, platelet-derived growth factor-BB, tumor necrosis factor {alpha}, basic fibroblast growth factor, and interleukin-1ß, is mediated by a region of the promoter proximal to the transcription start site (22–25). These studies implicated Sp1 and AP-2 transcription factors in these effects. With the exception of interleukin-1ß effects, which were mediated by p38 MAPK, the signaling pathways responsible for transcriptional activation in response to these growth factors were not examined. A deletion analysis of the VEGF promoter in PrEC has not yet been performed, so it is uncertain whether PI3-K promotes VEGF transcription through this site. Neither IGF-treated PrEC nor PCa cell lines in which PI3-K was constitutively activated revealed phosphorylation of p38 MAPK.

Our findings in PCa cells suggest that a separate signaling pathway not transduced by the IGF-IR or PI3-K is necessary to produce a HIF-1 transcription factor capable of acting through the VEGF HRE. Unlike normal PrEC, mutation of the HRE decreased basal promoter activity in PCa cells by more than 50% and abrogated sensitivity of the promoter to PI3-K inhibition. Therefore, HIF-1 is capable of driving VEGF transcription from the HRE in PCa cells by a mechanism not stimulated by IGF-I in normal cells. Evidence that other growth factor pathways can contribute to HIF-1 activity was provided by a study where MCF-7 cells were exposed to heregulin (21). The concentration of HIF-1{alpha} and activity of the VEGF promoter increased in cells treated with this ligand. Mutation of the HRE sequence blocked heregulin-induced activity of a HIF-1-responsive promoter containing a HRE upstream of a basal SV40 promoter, but the effects of mutating the HRE within the context of the VEGF promoter were not examined. When an expression construct containing the HIF-1{alpha} transactivation domain fused to the GAL4 DNA binding domain was transfected into MCF-7 cells, heregulin had no effect on a promoter containing five GAL4 binding sites, despite the fact that hypoxia stimulated the promoter approximately 5-fold. The authors concluded that heregulin, while increasing protein levels of HIF-1{alpha} and HIF-1 DNA binding activity, did not stimulate the transactivational capabilities of this transcription factor. Thus, the regulation of HIF-1 activity in heregulin-stimulated MCF-7 cells may be similar to what was observed in the present study. Because HIF-1 is a potent transactivator of the VEGF gene and HIF-1{alpha} is overexpressed in many human cancers (69), this transcription factor is implicated in the neovascularization of solid tumors. Therefore, it will be of interest to determine what other factors contribute to HIF-1's transactivational functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture
Primary cultures of normal, human PrEC, and prostate epithelial cell growth medium (PrEGM) were purchased from Clonetics (San Diego, CA). The immortalized PZ-HPV-7 cell line was a generous gift of Dr. Donna M. Peehl (Stanford University) and was generated from transfection of human papilloma virus 18 DNA into a primary culture of normal human PrEC (70). Both PrEC and PZ-HPV-7 were maintained in PrEGM. Cell treatments were performed on confluent cultures in PrEGM lacking EGF, insulin, and bovine pituitary extract (PrEGM-GF) following two rinses with sterile HEPES-buffered saline and overnight growth factor starvation in PrEGM-GF. Treatment of cells with IGF-I was done using LongR3-IGF-I (Diagnostic Systems Laboratories, Webster, TX) which exhibits relatively low affinity for the family of IGF binding proteins. Experiments with primary cell cultures were performed before passage 6. PC-3 and TSU-Pr1 PCa cell lines were maintained in DMEM/F12 + 10% fetal bovine serum and antibiotics. Treatment of cancer cell lines was done in DMEM/F12 without serum. The kinase inhibitors LY294002, PD98059, and UO126 were purchased from Calbiochem (La Jolla, CA); the levels of kinase inhibitors used did not affect prostate cell viability over a period of 24 h. Desferrioxamine mesylate was purchased from Sigma Chemical Co. (St. Louis, MO).

VEGF ELISA
Confluent PrEC cultures growing in 24-well dishes were treated as indicated in duplicate. Culture medium was harvested and cell counts were performed on trypsinized cells. The concentration of the VEGF165 isoform in culture medium was determined using a commercially available ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

Immunoprecipitation and Western Blotting
Whole cell lysates were made by rinsing cells twice with cold PBS then scraping cells in lysis-immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4)] containing protease inhibitor cocktail (4 µg/ml 4-amidinophenyl-methanesulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin) and phosphatase inhibitor cocktail (1 mM sodium vanadate, 1 mM NaF, 50 µM Na2MoO4). Insoluble material was spun down at full speed in a microcentrifuge and the supernatant stored at -80°C. Nuclear and cytoplasmic lysates were prepared using the Pierce (Rockford, IL) NE-PER kit according to the manufacturer's protocol. Protease and phosphatase inhibitor cocktails as described above were added to the nuclear and cytoplasmic lysis buffers before use. Immunoprecipitation was performed at 4°C in lysis-immunoprecipitation buffer by incubating antibody directed to the protein of interest with protein A-Sepharose (Amersham, Arlington Heights, IL) or protein G-Sepharose (Zymed, San Francisco, CA) before incubation with protein lysates. Before PAGE, cell lysates and immunoprecipitated antigen-antibody complexes were boiled 5 min in 4x NuPAGE sample buffer (Invitrogen, San Diego, CA) containing 1 mM DTT. Proteins were separated on NuPAGE gels and transferred to PVDF membrane (Millipore, Marlborough, MA) with a semi-dry electrotransfer apparatus (Bio-Rad, Hercules, CA). Blots were blocked and probed in TBST [150 mM NaCl, 0.1% Tween 20, and 10 mM Tris-HCl (pH 7.5)] containing 5% milk for all antibodies except the anti-phosphotyrosine PY20-HRP (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) which was blocked in 5% BSA. Western blots were developed with a chemiluminescent system. Additional antibodies used were directed against IGF-IRß, ARNT (Novus Biologicals, Littleton, CO); Fos, p85 (Upstate Biotechnology, Lake Placid, NY); phospho-Ser473-Akt, Akt, phospho-Erk1/2, Erk1/2 (Cell Signaling Technology, Beverly, MA); HIF-1{alpha} (Clones OZ12 and OZ15; NeoMarkers, Fremont, CA).

Transient Transfections
hVEGF-Luc was constructed with a 1.5-kb PCR fragment of the human VEGF gene promoter region cloned into the XhoI and HindIII sites of the pGL3-Control firefly luciferase reporter plasmid (Promega, Madison, WI). The PCR fragment containing XhoI and HindIII sites was generated from the following primers: 5'-CTCGAGCCAGGTCAGAAACCAGCCA-3' (upstream) and 5'-AAGCTTCTCCCCGCTACCAGCCGAC-3' (downstream). The product was subsequently subcloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen) before sequence verification by direct sequencing and cloning into pGL3. hVEGF-Luc HREm1 was generated using the GeneEditor in vitro Site-Directed Mutagenesis System (Promega) and the primer 5'-GCCAGACTCCACAGTGCATAAAAGGGCTCCAACAGGTCCTCTTCCC-3' and its complement. The A-Fos dominant negative expression vector and the empty vector control CMV-500 were a generous gift from Dr. Charles Vinson (National Cancer Institute). pRL-CMV (Promega), which contains the Renilla luciferase gene driven by the cytomegalovirus immediate early enhancer/promoter region, was used as a control for transfection efficiency.

Cells were plated in six-well dishes and grown to 50–60% confluence in maintenance medium. For reporter assays not involving dominant negative expression vectors, cells were rinsed twice with buffered saline and transfected with 1 µg hVEGF-Luc and 20 ng pRL-CMV in 2 ml PrEGM-GF or DMEM/F12 without serum. FuGENE 6 was the transfection reagent (Roche, Palo Alto, CA). After 3 h exposure to transfection medium, cultures were refed with the appropriate medium containing the indicated treatments. Reporter activity was determined 24 h after treatment using the Dual Luciferase Reporter Assay System (Promega) on a DYNEX luminometer. In studies utilizing the A-Fos dominant negative, cells were transfected with the appropriate expression vector in 2 ml PrEGM. After 6 h exposure to the plasmid, cells were refed with PrEGM and incubated overnight before transfection with the reporter plasmid in PrEGM-GF as described above. Reporter gene data are the result of triplicate trials and were calculated as the ratio of firefly luciferase to Renilla luciferase luminescence.

Statistical Analysis
Presented data represent the mean±SD. Statistical significance between values was determined by analysis of variance and the Tukey-Kramer honestly significant difference test using JMP software from the SAS Institute, Inc., Cary, NC.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We are grateful to Drs. Cynthia A. Afshari and John P. O'Bryan for their critique of the manuscript and Michael P. Walker for his assistance in preparation of the manuscript.

Received September 9, 2002; revised December 30, 2002; accepted January 16, 2003.


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. (Lond.), 1: 27–31, 1995.[Medline]
  2. Melnyk, O., Zimmerman, M., Kim, K. J., and Shuman, M. Neutralizing anti-vascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model. J. Urol., 161: 960–963, 1999.[Medline]
  3. Heimann, R., Ferguson, D., Gray, S., and Hellman, S. Assessment of intratumoral vascularization (angiogenesis) in breast cancer prognosis. Breast Cancer Res. Treat., 52: 147–158, 1998.[Medline]
  4. Brawer, M. K. Quantitative microvessel density. A staging and prognostic marker for human prostatic carcinoma. Cancer, 78: 345–349, 1996.[Medline]
  5. Brawer, M. K., Deering, R. E., Brown, M., Preston, S. D., and Bigler, S. A. Predictors of pathologic stage in prostatic carcinoma. The role of neovascularity. Cancer, 73: 678–687, 1994.[Medline]
  6. Engels, K., Fox, S. B., Whitehouse, R. M., Gatter, K. C., and Harris, A. L. Distinct angiogenic patterns are associated with high-grade in situ ductal carcinomas of the breast. J. Pathol., 181: 207–212, 1997.[Medline]
  7. Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J. C., and Abraham, J. A. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J. Biol. Chem., 266: 11947–11954, 1991.[Abstract/Free Full Text]
  8. Houck, K. A., Ferrara, N., Winer, J., Cachianes, G., Li, B., and Leung, D. W. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol. Endocrinol., 5: 1806–1814, 1991.[Abstract]
  9. Poltorak, Z., Cohen, T., Sivan, R., Kandelis, Y., Spira, G., Vlodavsky, I., Keshet, E., and Neufeld, G. VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J. Biol. Chem., 272: 7151–7158, 1997.[Abstract/Free Full Text]
  10. Ferrer, F. A., Miller, L. J., Andrawis, R. I., Kurtzman, S. H., Albertsen, P. C., Laudone, V. P., and Kreutzer, D. L. Vascular endothelial growth factor (VEGF) expression in human prostate cancer: in situ and in vitro expression of VEGF by human prostate cancer cells. J. Urol., 157: 2329–2333, 1997.[Medline]
  11. Harper, M. E., Glynne-Jones, E., Goddard, L., Thurston, V. J., and Griffiths, K. Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells. Br. J. Cancer, 74: 910–916, 1996.[Medline]
  12. Mazzucchelli, R., Montironi, R., Santinelli, A., Lucarini, G., Pugnaloni, A., and Biagini, G. Vascular endothelial growth factor expression and capillary architecture in high-grade PIN and prostate cancer in untreated and androgen-ablated patients. Prostate, 45: 72–79, 2000.[Medline]
  13. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol., 16: 4604–4613, 1996.[Abstract]
  14. Wang, G. L. and Semenza, G. L. Purification and characterization of hypoxia-inducible factor 1. J. Biol. Chem., 270: 1230–1237, 1995.[Abstract/Free Full Text]
  15. Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA, 92: 5510–5514, 1995.[Abstract/Free Full Text]
  16. Salceda, S. and Caro, J. Hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) 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.[Abstract/Free Full Text]
  17. Jiang, B. H., Rue, E., Wang, G. L., Roe, R., and Semenza, G. L. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem., 271: 17771–17778, 1996.[Abstract/Free Full Text]
  18. Zelzer, E., Levy, Y., Kahana, C., Shilo, B. Z., Rubinstein, M., and Cohen, B. Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1{alpha}/ARNT. EMBO J., 17: 5085–5094, 1998.[Medline]
  19. Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, M. M., Simons, J. W., and Semenza, G. L. Modulation of hypoxia-inducible factor 1{alpha} 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.[Abstract/Free Full Text]
  20. Jiang, B. H., Jiang, G., Zheng, J. Z., Lu, Z., Hunter, T., and Vogt, P. K. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth & Differ., 12: 363–369, 2001.[Abstract/Free Full Text]
  21. Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., and Semenza, G. L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol. Cell. Biol., 21: 3995–4004, 2001.[Abstract/Free Full Text]
  22. Gille, J., Swerlick, R. A., and Caughman, S. W. Transforming growth factor-{alpha}-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO J., 16: 750–759, 1997.[Medline]
  23. Finkenzeller, G., Sparacio, A., Technau, A., Marme, D., and Siemeister, G. Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression. Oncogene, 15: 669–676, 1997.[Medline]
  24. Ryuto, M., Ono, M., Izumi, H., Yoshida, S., Weich, H. A., Kohno, K., and Kuwano, M. Induction of vascular endothelial growth factor by tumor necrosis factor {alpha} in human glioma cells. Possible roles of SP-1. J. Biol. Chem., 271: 28220–28228, 1996.[Abstract/Free Full Text]
  25. Tanaka, T., Kanai, H., Sekiguchi, K., Aihara, Y., Yokoyama, T., Arai, M., Kanda, T., Nagai, R., and Kurabayashi, M. Induction of VEGF gene transcription by IL-1ß is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J. Mol. Cell. Cardiol., 32: 1955–1967, 2000.[Medline]
  26. Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirasawa, S., Sasazuki, T., and Kerbel, R. S. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res., 55: 4575–4580, 1995.[Abstract/Free Full Text]
  27. Goad, D. L., Rubin, J., Wang, H., Tashjian, A. H., Jr., and Patterson, C. Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I. Endocrinology, 137: 2262–2268, 1996.[Abstract]
  28. Akagi, Y., Liu, W., Zebrowski, B., Xie, K., and Ellis, L. M. Regulation of vascular endothelial growth factor expression in human colon cancer by insulin-like growth factor-I. Cancer Res., 58: 4008–4014, 1998.[Abstract/Free Full Text]
  29. Punglia, R. S., Lu, M., Hsu, J., Kuroki, M., Tolentino, M. J., Keough, K., Levy, A. P., Levy, N. S., Goldberg, M. A., D'Amato, R. J., and Adamis, A. P. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes, 46: 1619–1626, 1997.[Abstract]
  30. Bermont, L., Lamielle, F., Fauconnet, S., Esumi, H., Weisz, A., and Adessi, G. L. Regulation of vascular endothelial growth factor expression by insulin-like growth factor-I in endometrial adenocarcinoma cells. Int. J. Cancer, 85: 117–123, 2000.[Medline]
  31. Blakesley, V. A., Scrimgeour, A., Esposito, D., and Le Roith, D. Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev., 7: 153–159, 1996.[Medline]
  32. Milanini, J., Vinals, F., Pouyssegur, J., and Pages, G. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts. J. Biol. Chem., 273: 18165–18172, 1998.[Abstract/Free Full Text]
  33. Jung, Y. D., Nakano, K., Liu, W., Gallick, G. E., and Ellis, L. M. Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cells. Cancer Res., 59: 4804–4807, 1999.[Abstract/Free Full Text]
  34. Ruan, W., Powell-Braxton, L., Kopchick, J. J., and Kleinberg, D. L. Evidence that insulin-like growth factor I and growth hormone are required for prostate gland development. Endocrinology, 140: 1984–1989, 1999.[Abstract/Free Full Text]
  35. Chan, J. M., Stampfer, M. J., Giovannucci, E., Gann, P. H., Ma, J., Wilkinson, P., Hennekens, C. H., and Pollak, M. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study. Science (Washington, DC), 279: 563–566, 1998.[Abstract/Free Full Text]
  36. Stattin, P., Bylund, A., Rinaldi, S., Biessy, C., Dechaud, H., Stenman, U. H., Egevad, L., Riboli, E., Hallmans, G., and Kaaks, R. Plasma insulin-like growth factor-I, insulin-like growth factor-binding proteins, and prostate cancer risk: a prospective study. J. Natl. Cancer Inst., 92: 1910–1917, 2000.[Abstract/Free Full Text]
  37. Seger, R. and Krebs, E. G. The MAPK signaling cascade. FASEB J., 9: 726–735, 1995.[Abstract]
  38. Damert, A., Ikeda, E., and Risau, W. Activator-protein-1 binding potentiates the hypoxia-inducible factor-1-mediated hypoxia-induced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells. Biochem. J., 327: 419–423, 1997.
  39. Kimura, H., Weisz, A., Kurashima, Y., Hashimoto, K., Ogura, T., D'Acquisto, F., Addeo, R., Makuuchi, M., and Esumi, H. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood, 95: 189–197, 2000.[Abstract/Free Full Text]
  40. Janknecht, R., Cahill, M. A., and Nordheim, A. Signal integration at the c-fos promoter. Carcinogenesis (Lond.), 16: 443–450, 1995.[Free Full Text]
  41. Olive, M., Krylov, D., Echlin, D. R., Gardner, K., Taparowsky, E., and Vinson, C. A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis. J. Biol. Chem., 272: 18586–18594, 1997.[Abstract/Free Full Text]
  42. Semenza, G. L. and Wang, G. L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol., 12: 5447–5454, 1992.[Abstract/Free Full Text]
  43. Smith, L. E., Shen, W., Perruzzi, C., Soker, S., Kinose, F., Xu, X., Robinson, G., Driver, S., Bischoff, J., Zhang, B., Schaeffer, J. M., and Senger, D. R. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat. Med., 5: 1390–1395, 1999.[Medline]
  44. Hellstrom, A., Perruzzi, C., Ju, M., Engstrom, E., Hard, A. L., Liu, J. L., Albertsson-Wikland, K., Carlsson, B., Niklasson, A., Sjodell, L., LeRoith, D., Senger, D. R., and Smith, L. E. Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity. Proc. Natl. Acad. Sci. USA, 98: 5804–5808, 2001.[Abstract/Free Full Text]
  45. Jones, J. I. and Clemmons, D. R. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev., 16: 3–34, 1995.[Medline]
  46. Hankinson, S. E., Willett, W. C., Colditz, G. A., Hunter, D. J., Michaud, D. S., Deroo, B., Rosner, B., Speizer, F. E., and Pollak, M. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet, 351: 1393–1396, 1998.[Medline]
  47. Ma, J., Pollak, M. N., Giovannucci, E., Chan, J. M., Tao, Y., Hennekens, C. H., and Stampfer, M. J. Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J. Natl. Cancer Inst., 91: 620–625, 1999.[Abstract/Free Full Text]
  48. Yu, H., Spitz, M. R., Mistry, J., Gu, J., Hong, W. K., and Wu, X. Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis. J. Natl. Cancer Inst., 91: 151–156, 1999.[Abstract/Free Full Text]
  49. Khandwala, H. M., McCutcheon, I. E., Flyvbjerg, A., and Friend, K. E. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr. Rev., 21: 215–244, 2000.[Abstract/Free Full Text]
  50. Zetter, B. R. Angiogenesis and tumor metastasis. Annu. Rev. Med., 49: 407–424, 1998.[Medline]
  51. Reiss, K., D'Ambrosio, C., Tu, X., Tu, C., and Baserga, R. Inhibition of tumor growth by a dominant negative mutant of the insulin-like growth factor I receptor with a bystander effect. Clin. Cancer Res., 4: 2647–2655, 1998.[Abstract]
  52. Dunn, S. E., Ehrlich, M., Sharp, N. J., Reiss, K., Solomon, G., Hawkins, R., Baserga, R., and Barrett, J. C. A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer. Cancer Res., 58: 3353–3361, 1998.[Abstract/Free Full Text]
  53. Burfeind, P., Chernicky, C. L., Rininsland, F., and Ilan, J. Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo. Proc. Natl. Acad. Sci. USA, 93: 7263–7268, 1996.[Abstract/Free Full Text]
  54. Long, L., Rubin, R., Baserga, R., and Brodt, P. Loss of the metastatic phenotype in murine carcinoma cells expressing an antisense RNA to the insulin-like growth factor receptor. Cancer Res., 55: 1006–1009, 1995.[Abstract/Free Full Text]
  55. Chernicky, C. L., Yi, L., Tan, H., Gan, S. U., and Ilan, J. Treatment of human breast cancer cells with antisense RNA to the type I insulin-like growth factor receptor inhibits cell growth, suppresses tumorigenesis, alters the metastatic potential, and prolongs survival in vivo. Cancer Gene Ther., 7: 384–395, 2000.[Medline]
  56. Quevedo, C., Alcazar, A., and Salinas, M. Two different signal transduction pathways are implicated in the regulation of initiation factor 2B activity in insulin-like growth factor-1-stimulated neuronal cells. J. Biol. Chem., 275: 19192–19197, 2000.[Abstract/Free Full Text]
  57. Paweletz, C. P., Charboneau, L., Bichsel, V. E., Simone, N. L., Chen, T., Gillespie, J. W., Emmert-Buck, M. R., Roth, M. J., Petricoin, I. E., and Liotta, L. A. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene, 20: 1981–1989, 2001.[Medline]
  58. Gioeli, D., Mandell, J. W., Petroni, G. R., Frierson, H. F., Jr., and Weber, M. J. Activation of mitogen-activated protein kinase associated with prostate cancer progression. Cancer Res., 59: 279–284, 1999.[Abstract/Free Full Text]
  59. Price, D. T., Rocca, G. D., Guo, C., Ballo, M. S., Schwinn, D. A., and Luttrell, L. M. Activation of extracellular signal-regulated kinase in human prostate cancer. J. Urol., 162: 1537–1542, 1999.[Medline]
  60. Park, J. S., Qiao, L., Su, Z. Z., Hinman, D., Willoughby, K., McKinstry, R., Yacoub, A., Duigou, G. J., Young, C. S., Grant, S., Hagan, M. P., Ellis, E., Fisher, P. B., and Dent, P. Ionizing radiation modulates vascular endothelial growth factor (VEGF) expression through multiple mitogen activated protein kinase dependent pathways. Oncogene, 20: 3266–3280, 2001.[Medline]
  61. Hossain, M. A., Bouton, C. M., Pevsner, J., and Laterra, J. Induction of vascular endothelial growth factor in human astrocytes by lead. Involvement of a protein kinase C/activator protein-1 complex-dependent and hypoxia-inducible factor 1-independent signaling pathway. J. Biol. Chem., 275: 27874–27882, 2000.[Abstract/Free Full Text]
  62. Hu, Y. L., Tee, M. K., Goetzl, E. J., Auersperg, N., Mills, G. B., Ferrara, N., and Jaffe, R. B. Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells. J. Natl. Cancer Inst., 93: 762–768, 2001.[Abstract/Free Full Text]
  63. Saez, E., Rutberg, S. E., Mueller, E., Oppenheim, H., Smoluk, J., Yuspa, S. H., and Spiegelman, B. M. c-fos is required for malignant progression of skin tumors. Cell, 82: 721–732, 1995.[Medline]
  64. Zundel, W., Schindler, C., Haas-Kogan, D., Koong, A., Kaper, F., Chen, E., Gottschalk, A. R., Ryan, H. E., Johnson, R. S., Jefferson, A. B., Stokoe, D., and Giaccia, A. J. Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev., 14: 391–396, 2000.[Abstract/Free Full Text]
  65. Treins, C., Giorgetti-Peraldi, S., Murdaca, J., Semenza, G. L., and Van Obberghen, E. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J. Biol. Chem., 277: 27975–27981, 2002.[Abstract/Free Full Text]
  66. Fukuda, R., Hirota, K., Fan, F., Jung, Y. D., Ellis, L. M., and Semenza, G. L. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J. Biol. Chem., 277: 38205–38211, 2002.[Abstract/Free Full Text]
  67. Maity, A., Pore, N., Lee, J., Solomon, D., and O'Rourke, D. M. Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3'-kinase and distinct from that induced by hypoxia. Cancer Res., 60: 5879–5886, 2000.[Abstract/Free Full Text]
  68. Alfranca, A., Gutierrez, M. D., Vara, A., Aragones, J., Vidal, F., and Landazuri, M. O. c-Jun and hypoxia-inducible factor 1 functionally cooperate in hypoxia-induced gene transcription. Mol. Cell. Biol., 22: 12–22, 2002.[Abstract/Free Full Text]
  69. Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D., Buechler, P., Isaacs, W. B., Semenza, G. L., and Simons, J. W. Overexpression of hypoxia-inducible factor 1{alpha} in common human cancers and their metastases. Cancer Res., 59: 5830–5835, 1999.[Abstract/Free Full Text]
  70. Weijerman, P. C., Konig, J. J., Wong, S. T., Niesters, H. G., and Peehl, D. M. Lipofection-mediated immortalization of human prostatic epithelial cells of normal and malignant origin using human papillomavirus type 18 DNA. Cancer Res., 54: 5579–5583, 1994.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
T. R. Graham, H. E. Zhau, V. A. Odero-Marah, A. O. Osunkoya, K. S. Kimbro, M. Tighiouart, T. Liu, J. W. Simons, and R. M. O'Regan
Insulin-like Growth Factor-I-Dependent Up-regulation of ZEB1 Drives Epithelial-to-Mesenchymal Transition in Human Prostate Cancer Cells
Cancer Res., April 1, 2008; 68(7): 2479 - 2488. <