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Clinical Cancer Research Vol. 9, 2416-2425, July 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Androgens Stimulate Hypoxia-inducible Factor 1 Activation via Autocrine Loop of Tyrosine Kinase Receptor/Phosphatidylinositol 3'-Kinase/Protein Kinase B in Prostate Cancer Cells1

Nicola J. Mabjeesh, Margaret T. Willard, Carrie E. Frederickson, Hua Zhong and Jonathan W. Simons2

Winship Cancer Institute, Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia 30322


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Purpose: Androgen deprivation is implicated in reducing neoangiogenesis in prostate cancer (PCA). Androgens regulate the expression of the vascular endothelial growth factor (VEGF); hypoxia stimulates VEGF expression through the activation of the transcriptional factor, hypoxia-inducible factor 1 (HIF-1). We tested the hypothesis that an effect of androgens on VEGF expression is regulated directly by HIF-1 and HIF-2, and antiandrogens block HIF function.

Experimental Design: Androgen and antiandrogen effects were evaluated on HIF-1{alpha} protein and HIF-1 transcriptional activation in human PCA cells.

Results: Dihydrotestosterone (DHT) activates HIF-1{alpha} nuclear protein expression in LNCaP cells but not in androgen receptor-negative PC-3 cells. HIF-1{alpha} expression is correlated with the transactivation of a hypoxia-responsive element-driven reporter gene and with the production of VEGF protein. The effect of DHT on HIF-1 was blocked by nonsteroidal antiandrogens, flutamide and bicalutamide. DHT does not affect HIF-1{alpha} mRNA levels but regulates HIF-1{alpha} protein expression through a translation-dependent pathway. PC-3 cells when incubated with increasing amounts of conditioned medium from LNCaP cells treated with DHT experienced a dose-dependent increase in HIF-1{alpha}. This induction was not seen either when LNCaP cells were treated with flutamide or conditioned medium were pretreated with antibody to the epidermal growth factor (EGF). HIF-1 activation by DHT was blocked by LY294002, a potent inhibitor of the phosphatidylinositol 3'-kinase signaling pathway, whereas HIF-1 activation by EGF, as ligand, was not inhibited by flutamide. In contrast, HIF-2{alpha} protein was not affected by androgens or antiandrogens.

Conclusion: Androgens activate HIF-1, driving VEGF expression in androgen-sensitive LNCaP cells. This regulation is mediated through an autocrine loop involving EGF/phosphatidylinositol 3'-kinase/protein kinase B, which in turn activate HIF-1{alpha} and HIF-1-regulated gene expression. Therapeutic actions of antiandrogens in PCA include inhibition of HIF-1 function.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
For >60 years androgen deprivation therapies have remained the mainstay of therapy for metastatic PCA.3 Antiandrogens have postulated recently to reduce angiogenesis as well as induce apoptosis in PCA (1) . More recently, p.o. available nonsteroidal antiandrogens like flutamide and bicalutamide block this axis clinically. The growth of PCA and other tumors is dependent on its blood supply and induction of new blood vessels from pre-existing ones through angiogenesis (2 , 3) . The discovery of angiogenesis-dependent tumor growth involves the release of soluble factors, including VEGF, transforming growth factor {alpha}, platelet-derived growth factor, acidic and basic fibroblast growth factors, insulin-like growth factor I, and matrix metalloproteinases (2 , 3) . VEGF is a critical one, which can be produced by many tumor cell types (4) . Elevated expression of VEGF has been shown in human and animal models of PCA (5 , 6) . VEGF expression is regulated by various growth factors, cytokines, estrogen, progesterone, and glucocorticoids (7, 8, 9, 10, 11, 12, 13, 14) . Hypoxia is thought to be the most potent stimulus for VEGF (15, 16, 17, 18) , and its expression is transcriptionally regulated by HIF-1 (19, 20, 21, 22) . Androgens regulate VEGF content in normal and malignant prostate cells (23, 24, 25, 26) . However, the mechanisms underlining the above observations are not clearly defined.

HIF-1 is a critical, genome-wide transcription regulator identified for O2 homeostasis responsive to hypoxic stress. HIF-1 controls the expression of >40 genes including VEGF, of which the protein products are involved in angiogenesis, erythropoiesis, glycolysis, and invasion (27) . HIF-1 is a heterodimer composed of HIF-1{alpha} and HIF-1ß subunits, which are basic helix-loop-helix-Per/Arnt/Sim domain proteins. HIF-1ß is constitutively expressed, whereas the expression of HIF-1{alpha} is maintained at low levels in most cells under normoxic conditions. Under hypoxic conditions, HIF-1{alpha} escapes proteasomal degradation and then translocates to the nucleus. The former process results from inhibiting the activity of oxygen-dependent prolyl hydroxylases that modify residues 564 and 402 (28) , and the latter process is mediated by nuclear localization signals. This enzymatic modification of HIF-1{alpha} is required for the binding of von Hippel-Lindau protein, which is the recognition component of an E3 ubiquitin-protein ligase that targets HIF-1{alpha} for proteasomal degradation. In contrast to the oxygen-dependent regulation of HIF-1{alpha} degradation, we and others reported that growth factor stimulation induces HIF-1{alpha} protein synthesis via a signal transduction pathway leading from receptor tyrosine kinases to PI3K to the serine/threonine kinases AKT and FRAP (mTOR; Refs. 29, 30, 31, 32, 33, 34 ).

Up-regulated HIF-1{alpha} expression has been observed in >70% of cancers including PCA as compared with adjacent normal tissues (35 , 36) , and is likely achieved through both epigenetic mechanisms (intratumoral hypoxia) and genetic alterations (mutations in tumor suppressor genes and oncogene activation; Ref. 29 ). Overexpression of HIF-1{alpha} or HIF-1-dependent genes is associated with aggressive behavior in human cancers in vitro as well as in clinical specimens (35 , 37, 38, 39, 40, 41, 42, 43, 44, 45) . Hypoxic regions exist in human prostate carcinoma and increasing levels of hypoxia are associated with higher clinical stages (46) . In mouse xenograft models, tumor growth and angiogenesis are inhibited by small molecules and genetic strategies that disrupt HIF-1 activity but are stimulated by HIF-1{alpha} overexpression (27 , 47) .

We tested the hypothesis that an antiangiogenic effect of antiandrogens in androgen-responsive PCA cells can be regulated by blocking HIF-1 transcriptional pathway. We found that DHT stimulates HIF-1{alpha} protein expression, HIF-1 transcriptional activity, and VEGF production in LNCaP cells, whereas flutamide reduced these effects. Our experiments indicate that androgenic induction of HIF-1{alpha} protein expression and function are regulated in part through an autocrine loop mechanism involving the PI3K/AKT pathway in PCA cells.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Culture Conditions.
The human PCA cell lines LNCaP and PC-3 were purchased from American Type Culture Collection (Manassas, VA), and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere and 5% CO2 in air. The cells were subjected to hypoxia in a sealed modular incubator chamber (Billups-Rothenberg, Del Mar, CA) flushed with 1% O2, 5% CO2, and 94% N2 (1% O2), or to normoxia the cells were placed directly in a 5% CO2 and 95% air incubator (20% O2), and cultured at 37°C.

Reagents and Antibodies.
DHT and flutamide were obtained from Sigma-Aldrich (St. Louis, MO). CHX was from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). LY294002 was purchased from Alexis Biochemicals (San Diego, CA). Human recombinant EGF was purchased from Life Technologies, Inc. (Rockville, MD). R1881 was from Perkin-Elmer, Inc. (Boston, MA). Bicalutamide (Casodex) was a generous gift from Dr. Leland W. Chung (Emory University, Atlanta, GA). Purified mouse monoclonal anti-HIF-1{alpha} antibody was obtained from BD Transduction Laboratories (Lexington, KY). Polyclonal human antibody against HIF-2{alpha} was purchased from Novus Biologicals (Littleton, CO). Antibodies against human AR, actin, and VEGF (A-20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal human antibody to human TOPO-I was purchased from TopoGEN (Columbus, OH). The monoclonal antibody to rhEGF was purchased from R & D Systems, Inc. (Minneapolis, MN). The PhosphoPlus AKT (Ser473) Antibody kit for analysis of the phopholylation status of AKT and antibody to phospho-p44/42 MAPK (Thr202/Tyr204) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Secondary antibodies were horseradish peroxidase-conjugated and purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Hormonal Treatment of LNCaP Cells.
Only a low passage number (up to 10) of LNCaP cells were used. Cells were seeded in either six-well or 100-mm cell culture dishes and grown in RPMI 1640 containing 10% fetal bovine serum until 50% confluence. The medium was then replaced with phenol-red free RPMI 1640 containing 10% charcoal stripped serum (androgen-free medium). After 24 h, the androgen-free medium was refreshed and 1 nM DHT (dissolved in 100% ethanol), 1 µM flutamide (dissolved in DMSO), or vehicle (0.1% ethanol and/or 0.1% DMSO) was added. The medium was changed at 2-day intervals.

Protein Isolation and Western Blot Analysis.
Cells were washed twice with ice-cold PBS and then harvested, scraped into ice-cold PBS, and pelleted by centrifugation at 500 x g for 5 min at 4°C. NE and CE were prepared as described previously (48) . Briefly, the packed cells were resuspended in 10 mM Tris HCl (pH 7.5), 1.5 mM MgCl2, and 10 mM KCl freshly supplemented with 2 mM DTT, 0.4 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml apportioning, 2 µg/ml pepstatin, and 1 mM Na3VO4. Cells were kept on ice for 10 min. Nuclei were pelleted by centrifugation at 17,000 x g for 10 min at 4°C. The CE was kept, and the pellet was resuspended in 0.5 M NaCl, 20 mM Tris HCl (pH 7.5), 20% glycerol, and 1.5 mM MgCl2 freshly supplemented with the protease and phosphatase inhibitors listed above, and then rotated for 30 min in the cold room. The NE was cleared by centrifugation at 20,000 x g for 30 min at 4°C.

Proteins (30–60 µg/lane) from CEs or NEs were resolved by 7.5% SDS-PAGE, electrotransferred to nitrocellulose membrane, and incubated with the primary antibody. Immunoreactivity was visualized by incubating the membrane with horseradish peroxidase-conjugated secondary antiserum, followed by the treatment with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). For detection with another antibody, the membranes were stripped using a restore Western blot stripping buffer (Pierce, Rockford, IL) and reprobed with the desired antibody. TOPO-I and actin antibodies were used as a loading control for NE and CE, respectively.

VEGF and PSA Measurement.
LNCaP culture media were collected, centrifuged to remove cellular debris, and stored at -70°C until assayed for VEGF or PSA. VEGF assay was performed using a commercially available ELISA kit (R & D Systems, Inc.). PSA protein levels were determined using Microparticle Enzyme Immunoassay (Abbott IMx PSA assay; Abbott Laboratories, Abbott Park, IL). Results between wells were standardized according to the amount of VEGF or PSA per total protein per well as measured in cell lysates and expressed as pg of VEGF protein per ml supernatant and ng of PSA protein per ml supernatant.

Transient Transfections and Reporter Gene Assay.
LNCaP cells growing in six-well culture plates were transfected in triplicate with 1 µg/well of reporter plasmid (pBI-GL V6L) containing HREs from the VEGF gene using GenePorter transfection reagent (Gene Therapy Sys, Inc., San Diego, CA) as described (49 , 50) . After 5 h of transfection, the cells were allowed to recover overnight in androgen-free medium. The cells were then washed twice with PBS, and replenished with androgen-free medium and vehicle or reagent as indicated in the figure legends. Duplicate sets of transfected cell culture dishes were then separated and incubated either under normoxic or hypoxic conditions for 16 h. Luciferase activity was measured with commercial kit TROPIX (Bedford, MA) using a BMG Labtechnologies LUMIstar Galaxy luminometer and following the manufacturer’s instructions. Arbitrary Luciferase activity units were normalized to the amount of protein in each assay point. Protein concentration was determined using a BCA protein assay kit (Pierce).

Isolation and Analysis of RNA.
Total RNA was isolated using TRIzol Reagent (Life Technologies, Inc.) and was subjected (15 µg/sample) to Northern blotting using human HIF-1{alpha} cDNA probe (593-bp HindIII/MspI fragment) as described (51) or GAPDH and ß-actin probes (Ambion, Inc., Austin, TX).

Data Analysis.
Experiments presented in the figures are representative of three or more different repetitions. Quantification of band densities was performed using the public domain NIH Image (version 1.61). Statistical analysis was performed using a one-way ANOVA test (P < 0.05 was considered statistically significant).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Androgens Induce Expression of HIF-1{alpha} Protein and HIF-1 Activity.
We first examined the effect of DHT and flutamide on HIF-1{alpha} and HIF-2{alpha} protein expression in LNCaP cells starting at 24 h after the culture medium was changed to androgen-free medium under normoxic conditions (Fig. 1A)Citation . Treatment with DHT showed an ~4-fold increase in HIF-1{alpha} after 24 h, which then increased to a maximum ~10-fold increase after 48 h. Additional incubation resulted in a gradual decrease in the levels of the protein. The time-dependent changes in expression of HIF-1{alpha} in LNCaP cells were similar to those of PSA (Fig. 1B)Citation and VEGF (Fig. 1C)Citation in the culture medium, but all were still higher in the presence of DHT than control and flutamide at each time point. VEGF protein expression in cytoplasmic fraction was induced by DHT as well. Changes in nuclear HIF-2{alpha} protein levels were minimal compared with HIF-1{alpha} (Fig. 1A)Citation . Clinically relevant in vitro concentrations of flutamide were selected for testing. Fultamide (1 µM) inhibited the induction of HIF-1{alpha} protein by DHT (Fig. 2A)Citation . To measure the transcriptional activity of HIF-1, we used a reporter gene assay (Fig. 2B)Citation . LNCaP cells were transiently transfected with a construct containing Luciferase gene under the control of the HRE from the VEGF promoter (49) . Consistent with the changes in HIF-1{alpha} protein levels, the hypoxia-induced HIF-1 transcriptional activation was enhanced 2-fold by DHT and was inhibited by flutamide (Fig. 2B)Citation . Because HIF-1{alpha} protein levels are very low in LNCaP cells under normoxia, HIF-1 transcriptional activity under normoxia measured by the reporter gene assay was undetectable (Fig. 2B)Citation . HIF-1{alpha} was also induced by low concentration (0.1 nM) of R1881, a nonmetabolizable synthetic androgen, and this induction was inhibited by the antiandrogen bicalutamide (Casodex; Fig. 2CCitation ).



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  		Fig. 1. Activation of AR stimulates HIF-1{alpha} expression in LNCaP cells. A, LNCaP cells were cultured in androgen-free medium for 24 h. They were then treated with vehicle (0.1% DMSO and 0.1% ethanol), 1 nM DHT, or 1 µM flutamide under normoxic conditions for the indicated time (days). The cells were harvested, and NEs and CEs were prepared for Western blotting with antibody to HIF-1{alpha} and VEGF, respectively. The blots were stripped and reprobed with HIF-2{alpha} or TOPO-I and actin, respectively. The culture medium from the above incubations were analyzed for PSA (B) and VEGF proteins (C) as described under "Materials and Methods;" bars, ±SD.

 


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  		Fig. 2. Antiandrogens inhibit androgen-activated HIF-1. A, LNCaP cells were treated with vehicle, 1 nM DHT, 1 µM flutamide, or 1 nM DHT plus 1 µM flutamide for 2 days under normoxic conditions. The cells were harvested, and whole cellular extract was prepared for Western blotting with anti-HIF-1{alpha}, and reblotting with anti-AR and antiactin antibodies. B, LNCaP cells transiently transfected with pBI-GL V6L were treated with vehicle, 1 nM DHT, 1 µM flutamide, or 1 nM DHT plus 1 µM flutamide for 2 days under normoxic and hypoxic conditions. Luciferase reporter activity was measured in the whole cellular extract 16 h later. Luciferase activity represents arbitrary units per µg protein in each assay point. Columns, means; bars, ±SD; n = 3; *, ** P < 0.05 compared with normoxic control and with DHT under hypoxia, respectively. C, LNCaP cells were treated with vehicle, R1881, or R1881 combined with bicalutamide at the indicated concentrations for 2 days under normoxic conditions. The cells were harvested, and NEs were prepared for Western blotting with antibodies to HIF-1{alpha} and TOPO-I.

 
Androgen Induces HIF-1{alpha} through a Translation-dependent Pathway.
To better understand the processes involved in HIF-1{alpha} induction in response to androgen treatment, we investigated the effect of DHT and flutamide on the amount of HIF-1{alpha} mRNA. LNCaP cells were treated with DHT or flutamide, total RNA was extracted, and Northern blot analysis was performed using a HIF-1{alpha} cDNA probe (Fig. 3A)Citation . We found neither DHT nor flutamide treatment changed significantly HIF-1{alpha} mRNA levels, suggesting that androgens do not regulate HIF-1{alpha} mRNA transcription. In contrast, the amount of GAPDH mRNA, which is a HIF-1 regulated gene (52, 53, 54) , was increased by DHT treatment suggesting the coactivation of HIF-1 transcriptional function in response to DHT (Fig. 3A)Citation . To analyze the effect of androgen on HIF-1{alpha} protein synthesis, we performed a time course of HIF-1{alpha} disappearance in the presence of the protein translation inhibitor, CHX (Fig. 3B)Citation . Within 15 min of CHX incubation HIF-1{alpha} protein fell to undetectable levels after DHT treatment. We compared the effect of DHT on HIF-1{alpha} with CoCl2 and EGF. CoCl2 mimic hypoxia and are known to block HIF-1{alpha} degradation but have no effect on HIF-1{alpha} synthesis (55) . As expected in cells exposed to CoCl2, HIF-1{alpha} level remained almost stable for 60 min, despite the lack of ongoing protein synthesis (Fig. 3B)Citation . On the other hand, EGF has been shown in previous studies to regulate HIF-1{alpha} through PI3K/AKT pathway mainly by enhancing the efficiency of HIF-1{alpha} mRNA translation (29, 30, 31) . Similar to EGF, the induction of HIF-1{alpha} by DHT was completely blocked by CHX within 15 min (Fig. 3C)Citation . Together, these results suggest that androgens increases HIF-1{alpha} levels through a translation-dependent pathway.



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  		Fig. 3. Androgen induces HIF-1{alpha} through a translation-dependent pathway. A, LNCaP cells were treated with vehicle, 1 nM DHT, or 1 µM flutamide for 2 days under normoxic conditions. The cells were harvested and total RNA was prepared for Northern blot analysis of HIF-1{alpha}, GAPDH, and ß-actin. B, HIF-1{alpha} expression was induced by the exposure of LNCaP cells to DHT (1 nM) or CoCl2 (150 µM) under normoxia. After 4 h, CHX was added to a final concentration of 10 µg/ml, and the cells were harvested after being incubated for the indicated time in the presence of CHX and the inducer. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} and actin. C, LNCaP cells were treated with vehicle, DHT (1 nM), or EGF (100 µg/ml) under normoxic conditions. After 48 h, 10 µg/ml CHX was added, and the cells were incubated in the presence of CHX for 15 min. The cells were then harvested and NEs were prepared for Western blotting using antibodies to HIF-1{alpha} and TOPO-I.

 
Androgen Induces HIF-1{alpha} through an Autocrine PI3K/AKT-dependent Pathway.
We evaluated whether the effect of androgens on HIF-1{alpha} is mediated directly by the activation of the AR or is a consequent event of AR-downstream proliferative effects involving growth factors and their signaling pathways. To test that the effect of androgens on HIF-1 is mediated by the AR, we treated the hormone-insensitive PC-3 cells with DHT and flutamide. Neither DHT nor flutamide affected the steady state levels of HIF-1{alpha} protein in direct comparison of PC-3 cells with LNCaP cells (Fig. 4A)Citation . This result suggests that the effect of androgens on HIF-1{alpha} is mediated by the activation of the AR itself by androgens.



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  		Fig. 4. LNCaP conditioned media induce HIF-1{alpha} in PC-3 cells. A, LNCaP and PC-3 cells were treated with vehicle, 1 nM DHT, or 1 µM flutamide for 2 days under normoxic conditions. The cells were harvested, and NEs were prepared for Western blotting with anti-HIF-1{alpha} and reblotting with anti-TOPO-I antibodies. B, LNCaP cells were treated with vehicle, 1 nM DHT, or 1 µM flutamide for 2 days under normoxic conditions. Subsequently, conditioned media (CM) from each condition were removed and transferred to PC-3 cells that were grown for 24 h in androgen-free medium before the addition of CM. After incubation overnight, the cells were harvested, and NEs were prepared for Western blotting with anti-HIF-1{alpha} and reblotting with anti-TOPO-I antibodies (top panel). The medium was obtained from the LNCaP cells treated in the manner indicated above each lane, Densitometry of the developed blots was performed, and the ratios between the density of the HIF-1{alpha} and the loading control TOPO-I were determined (bottom panel); bars, ±SD. C, PC-3 cells were incubated for overnight with androgen-free medium containing the indicated percentages of CM from untreated or DHT-treated LNCaP cells (top panel). PC-3 cells were incubated overnight with DHT-treated LNCaP CM (100%), which were incubated for 4 h at room temperature with the indicated amounts of antibody to rhEGF before the addition to PC-3 cells (bottom panel). Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} and actin. The CM from untreated and DHT-treated LNCaP cells were analyzed for VEGF (D) and PSA (E) proteins. Columns, means; bars, ±SD; n = 2; * P < 0.05.

 
Because HIF-1{alpha} is regulated by extracellular peptide growth factors under normoxic conditions (29, 30, 31) , we hypothesized that activation of AR could involve pathways upstream to HIF-1, such as PI3K/AKT signaling pathway. To test this hypothesis, we treated PC-3 cells with conditioned medium from LNCaP cells treated with vehicle, DHT, or flutamide. As shown in Fig. 4BCitation , the "conditioned" culture medium taken from LNCaP treated with DHT induced a modest increase in HIF-1{alpha} protein levels, whereas flutamide inhibited HIF-1{alpha} in ~70% compared with the control (Fig. 4B)Citation . This result suggests that conditioned medium contained soluble factors such as growth factors, secreted by LNCaP cells that effected HIF-1{alpha} levels in PC-3 cells. It seems that these "factors" were also present in the conditioned medium taken from control LNCaP cells, because HIF-1{alpha} levels were modestly enhanced by conditioned medium from DHT-treated cells but were inhibited by flutamide to an extent lesser than the control (Fig. 4B)Citation . The effect of conditioned medium from both untreated and DHT-treated LNCaP cells on HIF-1{alpha} was dose-dependent (Fig. 4Citation C, top panel). Investigators have found that androgen stimulation of PCA cells in vitro leads to increased levels of secreted growth factors and their receptors including EGF (56 , 57) . On the basis of these reported findings, we anticipated that one of the soluble factors in LNCaP conditioned medium and that affects HIF-1{alpha} in PC-3 cells could be EGF. As depicted in Fig. 4CCitation (bottom panel) HIF-1{alpha} levels in PC-3 cells incubated with conditioned medium from LNCaP cells treated with DHT were inhibited by increasing concentration of a blocking monoclonal antibody to rhEGF that neutralizes EGF bioactivity (R & D Systems, Inc.). This result suggests that a significant part of HIF-1{alpha} induction in PC-3 cells by LNCaP conditioned medium is derived from EGF activity. As a control for androgen response we measured VEGF (Fig. 4D)Citation and PSA (Fig. 4E)Citation levels in LNCaP conditioned medium used in PC-3 cells.

To test whether the up-regulation of HIF-1{alpha} by DHT is dependent on PI3K, we studied the effect of two inhibitors of tyrosine kinase receptor/PI3K, LY294002, and wortmannin in LNCaP cells. LY294002 completely blocked HIF-1{alpha} protein in DHT-treated LNCaP cells similar to those treated with EGF (Fig. 5A)Citation as well as inhibited the DHT transactivation of HIF-1-dependent reporter gene (Fig. 5B)Citation . Similar results were obtained by wortmannin (data not shown). Interestingly, flutamide failed to inhibit HIF-1{alpha} levels after EGF treatment (Fig. 5A)Citation suggesting that the effect of AR activation on HIF-1 is upstream to EGF activity. To additionally confirm that the effect of LY294002 in LNCaP cells is because of PI3K pathway, we studied the phosphorylation status of AKT forms. As was shown previously, AKT was constitutively activated as a result of a frameshift mutation in the PTEN gene (58) , but its phosphorylation was totally blocked by LY294002 in LNCaP cells in comparison with NIH-3T3 cells as a control (Fig. 5CCitation , top panel). Neither DHT nor flutamide affected the activated AKT (Fig. 5CCitation , bottom panel). On the other hand, EGF enhanced the activation of AKT, which was entirely blocked by the addition of LY294002 but not by flutamide (Fig. 5CCitation , bottom panel). Furthermore, LY294002 blocked the phosphorylation of AKT in the presence of DHT, indicating that the inhibitory effect of LY294002 on DHT-induced HIF-1 is mediated through PI3K/AKT pathway. The effect of LY294002 was specific on PI3K because it had no effect on the EGF-activated forms of MAPK (Fig. 5D)Citation . In summary, DHT induces HIF-1{alpha} by increasing secretion of at least EGF, which through an autocrine mechanism activates PI3K/AKT pathway.



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  		Fig. 5. Androgens activate HIF-1 through a PI3K/AKT-dependent pathway. A, LNCaP cells were treated in the presence of the following additions: vehicle, 1 nM DHT, or 100 ng/ml EGF combined with 1 µM flutamide or 20 µM LY294002 as indicated on the top of each lane. NEs were prepared for Western blotting with anti-HIF-1{alpha} and anti-TOPO-I. B, LNCaP cells transiently transfected with pBI-GL V6L were treated with vehicle or 1 nM DHT in the presence or absence of 20 µM LY294002 under hypoxic conditions. Luciferase reporter activity was then measured. Columns, means; bars, ±SD; n = 3; * P < 0.05. C, LNCaP cells were stimulated for 15 min with EGF (100 ng/ml) or DHT (1 nM) with or without pretreatment for 45 min with LY294002 (20 µM) or flutamide (1 µM) as indicated on the top of each lane. Whole cell extracts were prepared and analyzed by Western blotting using antibodies to phosphorylated AKT, phosphorylated p44/p42 MAPK, and total AKT. Whole cell extracts of NIH-3T3 cells untreated or treated with 50 ng/ml of platelet-derived growth factor (PDGF) were used as a control.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The aim of this study was to test the hypothesis that HIF pathway is a direct downstream target of androgen action, and HIF-1, HIF-2, or both regulate androgen-induced VEGF expression in PCA cells.

Our results indicate that androgens and antiandrogens regulate HIF-1{alpha} expression, HIF-1 transcriptional activity leading to the secretion of VEGF (Figs. 1Citation and 2Citation ). HIF-2{alpha} was not affected by androgens or antiandrogens in the LNCaP system. DHT and flutamide did not affect HIF-1{alpha} mRNA levels (Fig. 3A)Citation . Using the protein translation inhibitor CHX, we found that the induction of HIF-1{alpha} by DHT is likely dependent on a translation pathway rather than affecting the rate of HIF-1{alpha} protein stability or degradation (Fig. 3B)Citation . Given HIF-1{alpha} signaling by FRAP/mTOR (29, 30, 31) , we suspect that DHT via FRAP/mTOR is increasing the rate of ribosomal recruitment to mRNA and, thus, enhancing synthesis of new HIF-1{alpha} protein molecules (27) .

HIF-1 regulation was restricted only to androgen-responsive LNCaP cells but not to the hormone-irresponsive PC-3 cells. Interestingly, when PC-3 cells were treated with increasing amounts of conditioned medium from LNCaP cells, HIF-1{alpha} protein was induced in a dose-dependent manner (Fig. 4C)Citation . Most importantly, conditioned medium from LNCaP cells treated with flutamide inhibited HIF-1{alpha} expression compared with the other conditioned medium (Fig. 4B)Citation . This implied that the LNCaP cells secrete factors under the influence of the androgen and that these factors, rather than the androgen, resulted in the increased expression of HIF-1{alpha}. Because HIF-1{alpha} expression, particularly under normoxic conditions, is under the control of the receptor tyrosine kinase/PI3K/AKT signaling pathway (29, 30, 31, 32) , we anticipated that part of the factors effecting HIF-1{alpha} levels in the conditioned medium are likely growth factors such as EGF. Indeed, the stimulatory effect of the conditioned medium from LNCaP cells on HIF-1{alpha} in PC-3 cells was inhibited when conditioned medium were pretreated with antibody to rhEGF (Fig. 4B)Citation . These results suggest that an extracellular autocrine growth factor effect is involved in DHT stimulation of HIF-1{alpha}. Moreover, we observed that the DHT-induced HIF-1{alpha} and HIF-1 activity is totally inhibited by LY294002, whereas the EGF-induced HIF-1{alpha} was not affected by flutamide (Fig. 5)Citation . These responses were accordingly correlated with the phosphorylation status of AKT.

Hydroxyflutamide, an active metabolite of flutamide has been found previously to enhance AR translocation and to promote AR transcriptional activity in LNCaP cells as an AR agonist, because there is a mutation in the LNCaP AR gene (59 , 60) . As a positive control, we found that flutamide induced a small increase of PSA expression at 48-h time point, whereas DHT enhanced PSA expression by >3-fold (Fig. 1B)Citation . A similar dynamic pattern of expression was also observed on VEGF (Fig. 1C)Citation and on HIF-1{alpha} (Fig. 1A)Citation at the 48-h time point. Interestingly, when 1 µM flutamide was combined with 1 nM DHT, flutamide had an antagonistic effect on HIF-1, probably by competing with DHT as a weak agonist (Fig. 2)Citation . These effects are similar to what was reported previously with EGF; androgens induce a marked increase of extracellular EGF secretion, whereas this induction is blocked by the antiandrogen hydroxyflutamide in LNCaP cells (56) . In addition to the increase in EGF release after androgen stimulation, both EGFR number and receptor binding affinity are also increased (57) . Furthermore, EGF was reported recently to greatly enhance the expression of VEGF in androgen-independent PCA cell lines, PC-3 and DU-145 cells (61) . Taken together, our results and findings published previously lead us to propose a new model explaining the transcription factor cross-talk between AR and HIF-1 intracellular signaling pathways (Fig. 6)Citation . Androgen ligand would activate the AR and lead to proliferative effects on LNCaP cells, and consequently stimulates growth factor secretory responses (62 , 63) including growth factors and their receptors such as EGF (56 , 57) . As a result, these growth factors like EGF, bind via cognate receptors activating tyrosine kinase/PI3K/AKT/FRAP pathway and enhance HIF-1{alpha} protein synthesis under normoxic conditions as described previously in PCA and other in vitro systems (29, 30, 31, 32) . As a consequence, activation of HIF-1 drives the expression of genes involved in angiogenesis, survival, energy metabolism, and proliferation (27 , 64) .



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  		Fig. 6. A proposed model of androgen-driven PI3K signal transduction to HIF-1. DHT binds and activates the AR in an LNCaP cell. This results in proliferation and an increase in secretory responses including EGF secretion. Activation of growth factor receptors drives the synthesis of HIF-1{alpha} protein through PI3K/AKT/FRAP pathway, and then transcription of VEGF, survival genes, and so forth. ARE, androgen response elements.

 
The fact that the effect of androgens on HIF-1 activity is indirect and is regulated via a tyrosine kinase/PI3K pathway provides additional rationale for targeting HIF-1{alpha} or HIF-1 upstream pathways such as blockade of EGFR (65) especially in androgen-independent PCA. This is supported by data reported recently showing the antiangiogenic and antitumor activity of EGFR blockade either by the anti-EGFR antibody ImClone C225 (IMC-C225; Ref. 66 ) or the selective EGFR tyrosine kinase inhibitor ZD1839 (67) .

Androgen regulation of HIF-1 occurs under normoxic conditions but can be additionally enhanced under hypoxia (Fig. 2BCitation ; Fig. 4DCitation ). This provides additional evidence that synergistic interactions exist between PI3K/AKT pathway activation and hypoxic pathway activation in PCA to regulate HIF-1{alpha} and drive angiogenesis (29) . Antiandrogens are major antineoplastic drugs for advanced PCA. Our data suggest part of their clinical activity could involve reduction in HIF-1 transcriptional activation of VEGF and, thus, reducing angiogenic potential of androgen-sensitive clones.


   ACKNOWLEDGMENTS
 
We thank Dr. Jay N. Umbreit (Winship Cancer Institute, Department of Hematology and Oncology, Emory University, Atlanta, GA) and the honorable Hamilton Jordan (Georgia Cancer Coalition, Atlanta, GA) for critical discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Prostate Cancer Specialized Programs of Research Excellence Grant CA-58236 (to J. W. S.), Avon Foundation (to J. W. S. and H. Z.), and CaP CURE Foundation (to J. W. S.). Back

2 To whom requests for reprints should be addressed, at 1365 Clifton Road, NE, Suite B4100, Atlanta, GA 30322. Phone: (404) 778-5177; Fax: (404) 778-5048; E-mail: jonathan_simons{at}emoryhealthcare.org Back

3 The abbreviations used are: PCA, prostate cancer; VEGF, vascular endothelial growth factor; HIF-1, hypoxia-inducible factor 1; EGF, epidermal growth factor; PI3K, phosphatidylinositol 3'-kinase; DHT, dihydrotestosterone; CHX, cycloheximide; R1881, methyltrienolone; AR, androgen receptor; TOPO-I, topoisomerase I; rhEGF, recombinant human EGF; MAPK, mitogen-activated protein kinase; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; PSA, prostate-specific antigen; HRE, hypoxia response element; AKT, protein kinase B; mTOR, mammalian target of rapamycin; FRAP, FKBP (FK506-binding protein) and rapamycin-associated protein; EGFR, epidermal growth factor receptor; NE, nuclear extract; CE, cytoplasmic extract. Back

Received 11/13/02; revised 1/24/03; accepted 2/ 4/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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Commentary

HIF-{alpha}, a Gender Independent Transcription Factor
Quynh-Thu Le and Amato J. Giaccia
Clin. Cancer Res. 2003 9: 2391-2393. [Full Text] [PDF]



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