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Carcinogenesis Advance Access originally published online on November 4, 2007
Carcinogenesis 2008 29(1):44-51; doi:10.1093/carcin/bgm232
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A hypoxia-independent up-regulation of hypoxia-inducible factor-1 by AKT contributes to angiogenesis in human gastric cancer

Byung Lan Lee1,2,{dagger}, Woo Ho Kim2,3,{dagger}, Jieun Jung1, Sung Jin Cho1, Jong-Wan Park4, Jihyen Kim5, Hee-Yong Chung6, Mee Soo Chang3 and Seon Young Nam7,*

1 Department of Anatomy
2 Cancer Research Institute
3 Department of Pathology
4 Department of Pharmacology, Seoul National University College of Medicine, Seoul 110-799, Korea
5 Korean Minjok Leadership Academy, Gangwon-do, Korea
6 Department of Microbiology, Hanyang University College of Medicine, Seoul 133-791, Korea
7 Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd, Seoul 132-703, Korea

* To whom correspondence should be addressed. Tel: +82 2 3499 6663; Fax: +82 2 3499 6669; Email: seonynam{at}khnp.co.kr


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Underlying mechanisms involved in the activation of hypoxia-inducible factor-1 (HIF-1) in cancer cells are diverse and cell type specific. Although both HIF-1{alpha} and AKT (protein kinase B) have been implicated in gastric tumor promotion and angiogenesis, it remains unclear whether HIF-1 mediates the role of AKT in terms of promoting vascular endothelial growth factor (VEGF) expression. The present study was performed to investigate the correlation between HIF-1{alpha} activation and AKT activation in gastric cancer using human gastric cancer specimens, in vitro cell experiments and in vivo animal experiments. Immunohistochemistry performed on tissue array slides containing 268 surgical specimens of gastric carcinomas showed immunoreactivity for HIF-1{alpha} in 29% of samples. Moreover, HIF-1{alpha} was positively associated with phosphorylated AKT (pAKT) (P = 0.002) or VEGF (P = 0.002), and the immunoreactivities of pAKT and VEGF were positively correlated (P < 0.001). Western blot analysis and reverse transcription–polymerase chain reaction in cell experiments revealed that the over-expression of constitutively active AKT (CA-AKT) promotes the expressions of HIF-1{alpha} protein and VEGF messenger ribonucleic acid in Seoul national university (SNU)-216 and SNU-668 gastric cancer cells under normoxic conditions, whereas kinase-dead mutant of AKT down-regulated these expressions under the same conditions. Xenografts in nude mice derived from stable gastric cancer cells over-expressing CA-AKT showed higher tumor incidence, larger tumor volumes, higher microvessel density and stronger HIF-1{alpha} immunoreactivity than those derived from vector control cells. Thus, we propose that the hypoxia-independent promotion of the AKT–HIF-1{alpha}–VEGF pathway contributes, at least in part, to gastric cancer tumorigenesis and angiogenesis.

Abbreviations: CA-AKT, constitutively active AKT; HIF-1{alpha}, hypoxia-inducible factor-1{alpha}; KD-AKT, kinase-dead mutant of AKT; mRNA, messenger ribonucleic acid; MVD, microvessel density; pAKT, phosphorylated AKT; PI3K, phosphatidylinositol-3 kinase; VEGF, vascular endothelial growth factor; AKT, protein kinase B; SNU, Seoul national university; GSK, glycogen synthase kinase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates blood vessel formation by affecting the expression of vascular endothelial growth factor (VEGF) (1). Moreover, HIF-1 is critically required for oxygen transport, glycolysis and glucose uptake (2). Since HIF-1 is a heterodimeric transcription factor composed of oxygen-dependent HIF-1{alpha} and constitutively expressed HIF-1β subunits, HIF-1 transcriptional activity is largely determined by regulated expression of the HIF-1{alpha} subunit (2). HIF-1{alpha} is stabilized under hypoxic conditions, whereas, under normoxic conditions, it is hydroxylated at proline residues in its oxygen-dependent degradation domain and then binds with the von-Hippel–Lindau protein-containing complex, which targets HIF-1{alpha} for ubiquitination and subsequent 26S-proteasomal degradation (3,4).

In human tumors, HIF-1{alpha} can also be over-expressed under non-hypoxic conditions, such as the activation of protein kinase B (AKT) in prostate cancer cells (5), the loss of von-Hippel–Lindau protein in renal cancer cells (3) and the increased activity of reactive oxygen species in gastric cancer cells (6). Moreover, HIF-1{alpha} over-expression has been detected immunohistochemically in various cancer types, including breast, oropharyngeal, nasopharyngeal, prostate, brain, lung, head, neck and stomach cancer (7), and has been associated with tumor aggressiveness, vascularity, treatment failure and mortality (8–11). In addition, it was found that xenograft tumor growth and angiogenesis are also dependent on HIF-1 activity and HIF-1{alpha} expression (12–14).

AKT (protein kinase B) is a homologue of the transforming viral oncogene v-akt and is activated by phosphatidylinositol (3,4,5)-triphosphate that is generated by activated phosphatidylinositol-3 kinase (PI3K) (15). Immunoreactivity for phosphorylated AKT (pAKT) (the activated form of AKT) is up-regulated in various human tumors, e.g. colon, ovary, pancreatic and stomach cancers (16,17). Like HIF-1, AKT induces VEGF to promote angiogenesis and enhances glucose metabolism, tumor malignancy and metastasis (18–21). Thus, the PI3K–AKT and HIF pathways share many features. Of the non-hypoxic routes that induce HIF-1{alpha} over-expression, the PI3K–AKT pathway has received much recent attention. However, whether PI3K–AKT is involved in HIF-1 pathway regulation, under what conditions, and to what extent PI3K–AKT regulates HIF-1 activity remain controversial (22,23). Indeed, in various cancers, it has been reported that VEGF expression (regulated by PI3K–AKT) appears to occur via both HIF-1-dependent (24–26) and -independent (27,28) mechanisms. Thus, the effect of the PI3K–AKT pathway on HIF-1{alpha} activity and VEGF expression mediated by HIF-1{alpha} appears to be cell type specific.

Regarding gastric cancer, HIF-1{alpha} (29,30) and AKT (31,32) have been reported to be over-expressed in surgical specimens. However, to date, it remains unclear whether HIF-1{alpha} and AKT expressions are correlated in gastric cancer. We know of only two studies on the correlation between HIF-1{alpha} and AKT in gastric cancer cells, and these studies were performed in vitro and produced contradictory findings concerning the effect of AKT inhibition on HIF-1{alpha} expression (32,33). In one of those papers, Kobayashi et al. (32) reported that treatment of gastric cancer cells with LY294002 (a PI3K inhibitor) did not change the expressions of HIF-1{alpha} protein or VEGF messenger ribonucleic acid (mRNA), whereas Ardyanto et al. (33) found that LY294002 dose dependently inhibited HIF-1{alpha} protein expression in gastric cancer cells.

In the present study, we immunohistochemically evaluated the expressions of nuclear HIF-1{alpha}, pAKT and VEGF proteins in 268 surgically excised human gastric carcinoma tissues on tissue array slides and searched for relations between the three. In addition, we performed cell culture and animal studies after establishment of stable SNU gastric cancer cell lines expressing constitutively active AKT (CA-AKT) or kinase-dead mutant of AKT (KD-AKT).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Patients and tissue array methods
A total of 268 surgically resected human gastric cancer specimens were obtained from the Department of Pathology, Seoul National University College of Medicine from 1 January to 30 June 1995 and six paraffin array blocks were prepared by Superbiochips Laboratories (Seoul, Korea), as described previously (34). Briefly, core tissue biopsies (2 mm in diameter) were taken from individual paraffin-embedded gastric tumors (donor blocks) and arranged in a new recipient paraffin block (tissue array block) using a trephine apparatus. As we have reported previously (35), the staining results of the different intratumoral areas of gastric carcinomas in these tissue array blocks showed an excellent agreement. A core was chosen from each case for analysis. We defined an adequate case as a tumor occupying >10% of the core area. Sections of 4 µm thickness were cut from each tissue array block, deparaffinized and dehydrated. Immunostaining results were considered to be positive if ≥10% (for AKT and VEGF) or ≥5% (for nuclear HIF-1{alpha}) of the neoplastic cells were stained. The nature of correlations between the expressions of pAKT, HF-1{alpha} and VEGF were then determined. This protocol was reviewed and approved by the Institutional Review Board of Seoul National University (Approval No. C-0603-162-170).

Cell lines and culture conditions
Two human gastric cancer cell lines, SNU-216 and SNU-668, were obtained from the Korean Cell Line Bank (Seoul, Korea). Both cell lines were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Life Technologies) and maintained in a 37°C humidified incubator containing 95% air and 5% CO2.

Retroviral infection of human gastric cancer cell lines and generation of stable cells
The control retroviral vector MFG.EGFP.IRES.puro has been described previously (36). MFG.EGFP.IRES.puro and the retroviral vectors MFG.CA-AKT.IRES.puro and MFG.KD-AKT.IRES.puro (containing CA-AKT or KD-AKT, respectively) were generated and infected into SNU cell lines, as described previously (37). Pooled puromycin-resistant cells were used for subsequent analyses.

Immunoprecipitation and in vitro AKT kinase assays
To confirm the AKT activities in the stable SNU-216 or SNU-668 gastric cancer cells over-expressing CA-AKT or KD-AKT, AKT kinase assays were performed using AKT kinase assay kits (Cell Signaling Technology, Denver, MA) according to the manufacturer’s instructions. Cells were lysed in 1 ml of ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, equal amounts of cellular proteins were incubated with agarose beads crosslinked with anti-AKT antibody at 4°C overnight. Immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer [25 mM Tris (pH 7.5), 5 mM β-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4 and 10 mM MgCl2]. Then, in vitro AKT kinase assays were performed using equal amounts of glycogen synthase kinase 3{alpha}/β (GSK-3{alpha}/β) fusion protein (1 µg) provided in the above-mentioned assay kit as AKT substrate. Phosphorylation of GSK-3{alpha}/β, which represents relative AKT kinase activity, was assessed by western blotting using anti-pGSK-3{alpha}/β antibody. Its band densities were determined by Scion Image analysis (Scion Corporation, Frederick, MD).

Immunoblotting
Cells were lysed with 1x Laemmli lysis buffer [2.4 M glycerol, 0.14 M Tris (pH 6.8), 0.21 M sodium dodecyl sulfate, 0.3 mM bromophenol blue] and then boiled for 10 min. Protein contents were measured using BCA Protein Assay Reagent (Pierce, Rockford, IL). Samples were diluted with 1x lysis buffer containing 1.28 M β-mercaptoethanol, and equal amounts of protein were loaded onto 8% sodium dodecyl sulfate–polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose membranes, and membranes were then blocked with 7.5% non-fat dry milk in phosphate-buffered saline-Tween-20 (0.1%, v/v) at 4°C overnight. They were then incubated with mouse anti-HIF-1{alpha} antibody (BD Transduction Laboratories, San Diego, CA) for 3 h and horseradish peroxidase-conjugated anti-mouse immunoglobulin G for 1 h. Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Reverse transcription–polymerase chain reaction
Total RNA was extracted using TRIzol (Gibco BRL Life Technologies) according to the manufacturer’s instructions and converted to complementary DNA using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) using oligo(deoxythymidine) primers (Novagen, Milwaukee, WI). To detect VEGF mRNA, 5 µl of the resultant complementary DNA was added to 50 µl of a polymerase chain reaction mixture containing 1x polymerase chain reaction buffer, 2.5 units Taq DNA polymerase, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates and 20 pmol of each specific primer. The following specific primers were used: VEGF, 5'-CCATGAACTTTCTGCTGTCTT-3' and 5'-TCGATCGTTCTGTATCAGTCT-3' and glyceraldehyde-3-phosphate dehydrogenase, 5'-TGCCGTCTAGAAAAACCTGC-3' and 5'-ACCCTGTTGCTGTAGCCAAA-3'. The polymerase chain reaction conditions used were as follows: 1 cycle at 94°C for 2 min and then 35 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min and a final extension for 10 min at 72°C. Products were separated in 1.5% agarose gel containing 0.5 µg/ml ethidium bromide.

Assessment of cell growth
Cells were seeded into 24-well plates at an appropriate cell density. Cell numbers were measured in triplicate wells of three separate experiments performed on the indicated days after plating as described previously (37). Briefly, cells were stained with 0.2% crystal violet in 20% methanol and dissolved in 1% sodium dodecyl sulfate, and absorbance was measured at 570 nm using an ELISA reader (Bio-Rad, Hercules, CA). Absorbance in each well was expressed as fold differences versus the corresponding vector controls.

Mouse xenograft model
Six-week-old female athymic nude mice (BALB/cSlc-nu) were purchased from SLC (Hamamatsu, Shizuoka, Japan). All animal procedures were performed in accord with the procedures described in the Seoul National University Laboratory Animal Maintenance Manual. Tumors were established by injecting 1 x 107 gastric cancer cells in 100 µl of matrigel (provided by Prof. Hynda K.Kleinman, George Washington University, Washington, DC) subcutaneously into both flanks of each mouse (SNU-668) or into one flank of each mouse (SNU-216 and SNU-668). Tumors were measured on alternate days using a caliper, and tumor volumes were calculated using the formula: (length x width x height) x ({pi}/6). After killing, tumor xenografts were removed and prepared for immunohistochemistry or western blotting.

Tumor histology and immunohistochemistry
Tissue specimens from the surgical samples of gastric cancer and from tumors xenografted into nude mice were fixed with 10% neutral-buffered formalin, and 4 µm paraffin sections were then prepared. One section was stained with hematoxylin and eosin for histologic assessment, and the other sections were immunostained using a streptavidin peroxidase procedure after microwave antigen retrieval. The primary antibodies used were anti-phospho-AKT (1:50, Ser473, New England Biolabs, Beverly, MA), anti-AKT (1:200, New England Biolabs), anti-HIF-1{alpha} (1:50, provided by Dr Jong-Wan Park in Seoul National University), anti-VEGF (1:200, Santa Cruz, Santa Cruz, CA) and anti-CD31 (1:100, Santa Cruz). Visualization was performed using diaminobenzidine. In order to verify the specificity of anti-HIF-1{alpha} antibody, SNU-668 cells were exposed to either hypoxic or normoxic conditions for 24 h, detached from culture surfaces, fixed in formalin, embedded in paraffin, sectioned and immunostained for HIF-1{alpha}. All immunostained sections were then lightly counterstained with Mayer’s hematoxylin. Throughout the above analyses, controls were prepared by omitting the primary antibody.

Quantification of microvessel density in xenograft tumors
Microvessel densities (MVDs) were determined by light microscopy/optical image analysis after immunostaining xenograft tumor sections with anti-CD31 antibody as described previously (14). The three most highly vascularized areas in areas of tumors near the tumor–normal tissue interface were selected. Photographs of CD31-immunopositive vessels in tumor sections were taken under a light microscope and the cross-sectional areas of CD31-immunopositive structures (i.e. vessel areas) were quantified by capturing images, converting them to grayscale and analyzing CD31-stained areas using NIH Image Analysis software (version 1.62; National Institutes of Health, Bethesda, MD) after setting one consistent intensity threshold for all slides. Then, CD31-positive areas were expressed as pixels squared per high-power field and were measured for all tumors.

Statistical analyses
For tissue array analysis, statistical analyses were conducted using SPSS version 11.0 statistical software program (SPSS, Chicago, IL), and the chi-squared test or the Fisher’s exact test (two sided) was used to determine the correlation between the expression of HIF-1{alpha} and that of other proteins. For the animal and cell experiments, data were analyzed using SAS software (version 8.1; SAS Institute, Cary, NC) and the two-tailed Student’s t-test was used to determine the significances of the results. P values of <0.05 were considered statistically significant for all statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Expression of HIF-1{alpha} in relation to the expressions of pAKT and VEGF in human gastric carcinoma tissues
Immunohistochemistry revealed that HIF-1{alpha} was expressed in both the nuclei and cytoplasm of tumor cells (Figure 1A). Cells showing distinct nuclear staining, regardless of the presence of cytoplasmic staining, were considered to express activated HIF-1{alpha}. Positive immunoreactivity for nuclear HIF-1{alpha} was found in 79 of 268 (29%) surgical gastric cancer specimens. In addition, pAKT (Figure 1B) and VEGF (Figure 1C) were found in 78 and 40% of gastric cancer specimens, respectively. To confirm the specificity of HIF-1{alpha} antibody, SNU-668 human gastric cancer cells were exposed to either hypoxia or normoxia for 24 h. Immunohistochemistry for HIF-1{alpha} demonstrated that hypoxic cells (Figure 1J) express stronger and more frequent nuclear immunoreactivity than normoxic cells (Figure 1K).


Figure 1
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  		Fig. 1. Representative immunohistochemical features of proteins in gastric cancer tissues (A–I) and cells (J–L). Positive (A–C) versus negative (D–F) examples of gastric cancer for HIF-1{alpha}(A and D), pAKT (B and E) and VEGF (C and F). (G) A normal adjacent tumor section in an array slide stained for HIF-1{alpha}. (H and I) Negative controls of gastric cancer treated without the primary antibodies. (J and K) Hypoxic (J) and normoxic (K) SNU-668 cells stained for HIF-1{alpha}. (L) A negative control cell-block section treated without primary antibody. Bars in (A–F) and (J–L) = 20 µm. Bars in (G–I) = 50 µm.

 
Data concerning correlations between HIF-1{alpha} expression and those of other HIF-1{alpha}-associated proteins are summarized in Table I. Nuclear HIF-1{alpha} expression was found to be significantly and positively correlated with pAKT expression (P = 0.002) and VEGF expression (P = 0.002). Moreover, a positive correlation was found between pAKT and VEGF (P < 0.001).


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  		Table I. Relationships between the expressions of nuclear HIF-1{alpha}, pAKT and VEGF in human gastric carcinomas

 
Effect of AKT activation on HIF-1{alpha} protein and VEGF mRNA expression and cell proliferation in gastric cancer cells in vitro
Since AKT is a well-known oncogene that induces HIF-1 activation under non-hypoxic conditions (38), cell experiments were performed to investigate whether the above relation holds under normoxic conditions. Initially, we established stable cell lines from SNU-216 and SNU-668 cells infected with retroviral vectors containing enhanced green fluorescent protein (control vector), CA-AKT or KD-AKT (Figure 2). In vitro AKT kinase assays were then performed using 1 µg of GSK-3 fusion protein as substrate, which showed that AKT activities were significantly increased or decreased in CA-AKT or KD-AKT stable transfectants, respectively (Figure 2A and B).


Figure 2
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  		Fig. 2. AKT activity in SNU-216 cells (left panels) and SNU-668 cells (right panels) infected with retroviral vectors containing enhanced green fluorescent protein, CA-AKT or KD-AKT. (A) Activations of AKT in control vector transfectants, CA-AKT stable transfectants and KD-AKT stable transfectants were determined using AKT kinase assays with equal amounts of GSK-3{alpha}/β fusion protein (1 µg) as substrate and western blotted using pGSK-3{alpha}/β antibody. (B) The band densities of pGSK-3{alpha}/β, which represents relative AKT kinase activity, in stable transfectants were measured by Scion Image analysis and compared with those of the vector control. Values represent means ± standard deviations (n = 5) and were determined using the Student’s t-test. *P < 0.001 and **P < 0.005 versus SNU-216.Vector control. {dagger}P < 0.001 versus SNU-668.Vector control.

 
We then examined whether the positive correlations observed between pAKT, HIF-1{alpha} and VEGF found in human gastric cancer tissue specimens exist under normoxic conditions in gastric cancer cells. Western blotting showed that CA-AKT or KD-AKT increased or decreased HIF-1{alpha} protein expression, respectively, in stable transfectants derived from SNU-216 or SNU-668 cells (Figure 3A). CA-AKT or KD-AKT was also found to increase or decrease VEGF mRNA expression, respectively, in both cell lines (Figure 3B). These findings suggest that AKT regulates VEGF expression by up-regulating HIF-1{alpha} under normoxic condition.


Figure 3
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  		Fig. 3. Effect of AKT activation on the expressions of HIF-1{alpha} protein and VEGF mRNA and on the proliferations of SNU-216 (left panels) and SNU-668 cells (right panels). (A) Levels of HIF-1{alpha} (MW 120 kDa) and β-actin proteins were determined by western blotting. (B) Levels of VEGF and glyceraldehyde-3-phosphate dehydrogenase mRNA were monitored by reverse transcription–polymerase chain reaction. Product sizes were 500 and 650 bp for VEGF and 233 bp for glyceraldehyde-3-phosphate dehydrogenase. (C) Cell growths were analyzed using crystal violet on the indicated days, and absorbance was measured. Values represent the means ± standard deviations of triplicate samples in three separate experiments. Data were analyzed using the Student’s t-test. *P < 0.005 and **P < 0.05 versus SNU-216.Vector control. {dagger}P < 0.001 versus SNU-668.Vector control.

 
Next, we examined the role of AKT in the growths of SNU-216 and SNU-668 cells. CA-AKT expression in both cell lines significantly enhanced cell growth, whereas KD-AKT over-expression in SNU-216 cells reduced cell growth (Figure 3C).

AKT activation enhanced tumor growth, HIF-1{alpha} activation and MVD in the nude mouse xenograft model
Since it has been shown that inhibiting constitutive HIF-1{alpha} protein expression decreases tumor growth and angiogenesis in gastric cancer xenografts (14), we established gastric carcinoma xenografts derived from injected stable SNU gastric cancer cells over-expressing either CA-AKT (SNU-216 and SNU-668) or KD-AKT (SNU-216) and examined the effects of AKT activation on tumor growth, HIF-1{alpha} activation and angiogenesis.

CA-AKT over-expression in SNU-216 cells caused a remarkable increase in the efficacy of tumor take-up to 100%, but SNU-668 cells showed no difference (Figure 4A and B). In contrast, KD-AKT-expressing SNU-216 cells showed no tumor formation. Among tumor-bearing mice, those injected with CA-AKT cells had significantly greater mean tumor volumes than those injected with vector control cells (Figure 4C). Taken together, these results suggest that AKT activation increases tumorigenicity and growth.


Figure 4
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  		Fig. 4. Effect of CA-AKT over-expression on differently sized xenografted tumors in nude mice. SNU-668.Vector, SNU-668.CA-AKT, SNU-216.Vector, SNU-216.CA-AKT or SNU-216.KD-AKT transfectants were injected subcutaneously into flanks of BALB/c nude mice. Animals were killed 25 days after implanting SNU-668 transfectants or 72 days after implanting SNU-216 transfectants. (A) Representative mice bearing xenograft tumors derived from stable SNU-216 transfectants. Photographs were taken after killing. The arrows indicate xenografted tumors in mouse flanks. (B) Numbers of mice showing tumor formation and times of tumor appearance. (C) Tumor growth curve. Data are presented as means ± standard deviations and were analyzed using the Student’s t-test. {dagger}P < 0.05 versus SNU-216.Vector control. *P < 0.001 versus SNU-668.Vector control.

 
Immunostaining was then performed to investigate whether HIF-1{alpha} mediates the effect of AKT activation on angiogenesis in tumors derived from SNU-668 cells (Figure 5). Immunoreactivity of nuclear HIF-1{alpha} was significantly enhanced in tumor tissues derived from SNU-668.CA-AKT cells (Figure 5A, panel f) as compared with those derived from vector control cells (Figure 5A, panel c). Moreover, immunostaining of serial sections revealed the co-localization of pAK and nuclear HIF-1{alpha} immunoreactivity in identical cells (Figure 5A, panels g and h), reflecting a positive correlation between HIF-1{alpha} activation and AKT activation in individual tumor cells.


Figure 5
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  		Fig. 5. Effect of CA-AKT over-expression on HIF-1{alpha} in differently sized (A and B) or similarly sized (C–E) xenografted tumors derived from SNU-668.Vector (panels Aa–c and panels Ca–c) or SNU-668.CA-AKT (panels Ad–h and panels Cd–f) transfectants. (A and C) Representative immunohistochemical features for pAKT (panels Ab, e and g and panels Cb and e) and HIF-1{alpha} (panels Ac, f and h and panels Cc and f). (A) Serial sections (b and c, e and f) were obtained from paraffin blocks containing xenografted tumors, immunostained with an antibody against pAKT or HIF-1{alpha} and counterstained with Meyer’s hematoxylin. High-power views of the boxed areas in e and f are shown in g and h, respectively, and show the co-localizations of HIF-1{alpha} and pAKT immunoreactivities in identical cells. (B and D) The frequencies of HIF-1{alpha}-immunopositive cells in differently sized (B) and similarly sized (D) xenografted tumors were quantified by counting >600 cells per slide under a light microscope, and then calculating the ratio of tumor cells with nuclear staining to total cells. (E) A representative western blotting result showing the effect of CA-AKT over-expression on HIF-1{alpha} expression. NC, negative control treated without the primary antibodies (panels Aa and d, panels Ca and d). *P < 0.05 versus SNU-668.Vector control. Bars = 50 µm.

 
Furthermore, to exclude the possibility that this difference in HIF-1{alpha} immunoreactivity is correlated with the size of xenograft tumors rather than AKT activity, we compared HIF-1{alpha} expression in three sets of similarly sized xenograft tumors: the sizes of CA-AKT tumors were 334, 352.7 and 423 mm3 and the sizes of corresponding vector control tumors were 335, 352.7 and 427 mm3, respectively. Immunostaining and western blotting results for HIF-1{alpha} showed that CA-AKT over-expression increased the frequencies of immunoreactive cells (Figure 5C and D) and HIF-1{alpha} protein expression (Figure 5E) in similar size tumors.

Tumor sections were also immunostained with antibody against CD31. CA-AKT tumors showed markedly enhanced CD31 immunoreactivity as compared with vector control tumors (Figure 6A). Optical image analysis showed that CA-AKT tumors were characterized by a dramatic increase in CD31-immunopositive vessel area compared with vector controls (Figure 6B). Taken together, these findings indicate that CA-AKT increases tumor vascularization via HIF-1{alpha} activation in gastric cancer.


Figure 6
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  		Fig. 6. Effect of CA-AKT over-expression on MVD in xenografted tumors derived from SNU-668.Vector or SNU-668.CA-AKT transfectants. (A) Tumor sections were immunostained for CD31. (B) The areas of blood vessels immunostained for CD31 were quantified. Bars represent means and lower or upper 95% confidence intervals. *P < 0.005 versus SNU-668.Vector control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Angiogenesis is an important aspect of tumor growth and progression, and it has become evident that a detailed understanding of the mechanisms underlying angiogenesis is of therapeutical use for cancers that require neovascularization. Despite the fact that gastric cancer is a major cause of death in Asia and that tumor angiogenesis is considered the most important predictor of overall survival in gastric cancer (39), little is known about the molecular mechanism underlying gastric cancer angiogenesis.

Angiogenesis is regulated by multiple factors and many of these factors may individually predict MVD (39). Recently, it has become evident that the mechanism underlying the activation of HIF-1 in various cancer cells depends on cancer type (38). With respect to gastric cancer, previous studies have shown that HIF-1{alpha} inhibition reduces angiogenesis and tumor growth in xenografted gastric tumors (14). Moreover, it was found that the over-expression of HIF-1{alpha} is significantly correlated with VEGF protein expression, and this over-expression is a prognostic factor in patients with gastric cancer (9,30). AKT inhibition was also found to decrease VEGF expression in human gastric cancer and to reduce tumor growth in xenografted gastric tumors (32). However, at present, it is not known whether HIF-1{alpha} mediates the role of AKT in VEGF expression in gastric cancer, as in vitro studies using LY294002 (a PI3K inhibitor) have produced contradictory results (32,33).

In the present study, immunohistochemical tissue array analysis showed that HIF-1{alpha} was constitutively expressed in 29% of the 268 surgical samples of gastric carcinomas and that this was positively correlated with the expressions of pAKT (P = 0.002) and VEGF (P = 0.002). Moreover, a positive relationship was found between pAKT and VEGF (P < 0.001). In xenografted gastric tumors, we used stable cell lines infected with retroviruses over-expressing CA-AKT or KD-AKT, which could prove to be a powerful tool for obtaining further insight into the role of AKT in the regulation of HIF-1 activity in the gastric cancer cells. We found that the over-expressions of CA-AKT and KD-AKT increased and decreased, respectively, tumor take-up and growth and angiogenesis, as was manifested by increased MVD and HIF-1{alpha} immunoreactivity. These findings indicate that HIF-1{alpha} acts downstream of AKT in the AKT pathway that induces VEGF expression. Moreover, using serial sections of xenograft tumors, we observed the co-localization of HIF-1{alpha} and pAKT immunoreactivity in the identical tumor cells, which confirms the existence of a close relationship between HIF-1{alpha} and pAKT. Thus, the present study, as far as we are aware, provides the first evidence of the positive regulation of HIF-1{alpha} activation by constitutive pAKT in human gastric cancer angiogenesis.

In addition, we used a cell culture system and found that CA-AKT and KD-AKT in gastric cancer cells increased or decreased, respectively, the expressions of HIF-1{alpha} protein and VEGF mRNA under normoxic conditions. These findings are consistent with previously published reports, which showed that the PI3K–AKT pathway regulates the expressions of VEGF and HIF-1 and angiogenesis under normoxic conditions in breast cancer cells, glioblastoma cells, prostate cancer cells and colon cancer cells (38,40–42). Thus, the AKT–HIF-1–VEGF pathway might also exist in gastric cancer cells under non-hypoxic conditions. Furthermore, this notion is substantiated by the findings of a previously study (14), in which the authors suggested that HIF-1{alpha} is constitutively over-expressed in gastric tumor cells by hypoxia-independent mechanisms.

In conclusion, the results obtained from human gastric cancer specimens, gastric tumor xenografts and cell experiments indicate that HIF-1{alpha} mediates AKT-induced angiogenesis by increasing the expressions of VEGF and MVD and that this occurs under non-hypoxic conditions. Thus, HIF-1{alpha} and pAKT may be candidate molecular targets for gastric cancer therapy, and in particular, blocking the AKT–HIF-1{alpha} pathway with appropriate inhibitors might be useful therapeutically for treating gastric carcinoma.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Korea Research Foundation (KRF-2003-003-E00163); second stage Brain Korea 21 Project in 2006 (J.J. and S.J.C.).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Superbiochip Laboratories for their technical assistance and Dr H.J. Shu, Y.H. Kim, Y.J. Cho and Y.S. Ko for their contribution.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received April 12, 2007; revised September 19, 2007; accepted October 19, 2007.


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