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Clinical Cancer Research Vol. 12, 6910-6919, December 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Regulation of Angiogenesis by Id-1 through Hypoxia-Inducible Factor-1{alpha}–Mediated Vascular Endothelial Growth Factor Up-regulation in Hepatocellular Carcinoma

Terence K. Lee1, Ronnie T.P. Poon1, Anthony P. Yuen1, Ming Tat Ling2, Xiang Hong Wang2, Yong Chuan Wong1, Xin Yuan Guan3, Kwan Man1, Zao You Tang4 and Sheung Tat Fan1

Authors' Affiliations: Departments of 1 Surgery, 2 Anatomy, and 3 Clinical Oncology, The University of Hong Kong, Hong Kong, China and 4 Department of Surgery, Zhongshan Hospital, Fudan University, Shanghai, China

Requests for reprints: Ronnie T.P. Poon, Department of Surgery, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, China. Phone: 852-2855-3641; Fax: 852-2817-5475; E-mail: poontp{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Metastasis is commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Being an important angiogenic factor, vascular endothelial growth factor (VEGF) plays a central role in HCC growth and metastasis. Recently, Id-1 (inhibitor of differentiation/DNA synthesis) has been suggested to be a key factor in cancer progression but the molecular mechanism remains unknown.

Experimental Design: We first showed that overexpression of Id-1 was correlated with HCC metastasis (P < 0.001) and its expression was significantly correlated with VEGF expression by tissue microarray. By ectopic transfection of Id-1 into HCC cells, Id-1 was able to induce VEGF secretion through activation of VEGF transcription.

Results: Increased VEGF secretion in Id-1 transfectants induced morphologic change and proliferation of human umbilical vascular endothelial cell resulting in promotion of angiogenesis. Id-1 induced transcriptional activation of VEGF by stabilizing hypoxia-inducible factor-1{alpha} protein. Down-regulation of Id-1 by antisense approach led to suppression of hypoxia-inducible factor-1{alpha}–mediated VEGF production. In addition, Id-1 suppression resulted in retardation of cell invasion through down-regulation of VEGF.

Conclusions: Id-1 is a novel angiogenic factor for HCC metastasis and down-regulation of Id-1 may be a novel target to inhibit HCC metastasis through suppression of angiogenesis.

Hepatocellular carcinoma (HCC) is a highly malignant tumor, which is characterized by active neovascularization (1). It is the fifth most common malignancy worldwide and the second leading cause of cancer death in Hong Kong (Hong Kong Hospital Authority, Hong Kong Cancer Registry, 2001; refs. 2, 3). The poor prognosis of HCC is commonly associated with propensity for vascular invasion and metastasis (3). It has been shown previously that angiogenesis makes significant contribution to tumor growth, invasiveness, and metastatic potential of HCC (4–6). Thus far, there are limited data on the role of various proangiogenic factors in HCC (7). Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that plays a central role in angiogenesis in various cancer types (8). Previous studies from our group have reported that VEGF is a potent angiogenic factor leading to HCC invasiveness and metastasis (9, 10). However, the mechanisms by which VEGF is regulated are far from understood.

Id-1 (inhibitor of differentiation/DNA synthesis) protein belongs to the Id family of helix-loop-helix proteins (11). These proteins act as dominant inhibitors of basic helix-loop-helix transcription factors by forming transcriptionally inactive heterodimers (11). Id-1 has been shown to play a central role in cell proliferation (12), differentiation, and senescence (13, 14). Overexpression of Id-1 has been reported in several types of primary tumors, including breast (15), pancreatic (16), prostate (17), cervical (18), and colorectal adenocarcinoma (19). Apart from its oncogenic function, elevated Id-1 expression was correlated with malignant progression of human cancers (20). For example, Id-1 was overexpressed in highly aggressive but not the nonaggressive breast cancer cells (15). One of the distinct characteristics of the aggressive cancer cells is the propensity to vascular invasion and metastasis. Poor vascularization resulting from generation of Id-1 and Id-3 double knockout mice is a convincing evidence showing the central role of Id-1 overexpression in tumor metastasis (21). Loss of Id-1 has been shown to lead to down-regulation of several proangiogenic genes, such as intergrin {alpha}6 (22). Hence, a positive role of Id-1 in tumor metastasis through promoting angiogenesis was suggested. However, the exact role of Id-1 in angiogenesis and the regulatory mechanisms involved are still unclear. Given the central role of VEGF in cancer angiogenesis, it is of great interest to elucidate whether overexpression of Id-1 promotes cancer angiogenesis by interacting with VEGF expression.

Recently, our group has reported that Id-1 overexpression induced HCC cell proliferation through inactivation of p16/RB pathway (23). However, the relationship between Id-1 expression and HCC invasiveness and metastasis has not been studied. In this study, we first showed significant up-regulation of Id-1 in human metastatic HCCs compared with primary ones by tissue microarray (TMA), and its expression was significantly correlated with VEGF expression. Ectopic Id-1 expression promotes HCC angiogenesis through hypoxia-inducible factor-1{alpha} (HIF-1{alpha})–mediated VEGF promoter activation in PLC cell. Down-regulation of Id-1 expression by antisense approach significantly inhibits HCC angiogenesis both in vitro and in vivo. This line of evidence strongly provides an insight on the novel regulatory mechanism of VEGF in HCC by Id-1. In addition, down-regulation of Id-1 may provide a novel target to inhibit HCC angiogenesis and metastasis.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient samples. Samples were obtained with informed consent from 10 healthy liver transplant donors and 80 patients undergoing hepatectomy for HCC from 1997 to 2000 in the Department of Surgery, The University of Hong Kong (Hong Kong, China). Tissue specimens from TMA were obtained from 60 patients who underwent hepatectomy for HCC between 1995 and 1999 in Eastern Hepatobiliary Surgery Hospital (Shanghai, China) and subsequently developed intrahepatic or extrahepatic metastases. Matched pairs of primary and metastatic HCC samples were obtained for the TMA.

Construction of TMA. The HCC TMA was constructed as described previously (24). Briefly, all tissue samples embedded in paraffin for array studies were sectioned freshly and stained with H&E. The representative regions of lesion were reviewed carefully and defined by two pathologists. Based on the clinicopathologic information, specimens were grouped in tissue cylinders and a diameter of 0.6 mm was taken from the selected regions of the donor block and then punched precisely into a recipient paraffin block using a tissue array instrument (Beecher Instruments, Silver Spring, MD). Five micrometer of consecutive sections of the microarray blocks were made with a microtome. Finally, a TMA section with 60 pairs of primary and matched metastatic HCC samples (including 31 intrahepatic and 29 extrahepatic metastases) was constructed.

Immunostaining. Formalin-fixed and paraffin-embedded sections with a thickness of 4 µm were dewaxed in xylene and graded alcohols, hydrated, and washed in PBS. After pretreatment in a microwave oven [12 minutes in sodium citrate buffer (pH 6)], the endogenous peroxidase was inhibited by 0.3% H2O2 for 30 minutes, and the sections were incubated with 10% normal goat serum for 30 minutes. Primary antibodies [rabbit polyclonal anti-Id-1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-VEGF (R&D Systems, Inc., Minneapolis, MN), and rat anti-mouse CD34 (Biogenex, San Ramon, CA)] were applied overnight in a moist chamber at 4°C, respectively. A standard avidin-biotin peroxidase technique (DAKO, Carpinteria, CA) was applied. Briefly, biotinylated goat anti-rabbit immunoglobulin, goat anti-mouse and goat anti-rat immunoglobulin, and avidin-biotin peroxidase complex were applied for 30 minutes each, with 15-minute washes in PBS. The reaction was finally developed by DAKO Liquid DAB+ Substrate Chromogen System (DAKO). Cytoplasmic expression of Id-1 and VEGF was determined by two independent observers (K.M. and M.T.L.) who assessed semiquantitatively the percentage of stained tumor cells as well as staining intensity according to Birner et al. (25).

Quantitation of VEGF and CD34 expression. VEGF expression was classified as strong if >30% of the cells were stained positive and weak if <30% of the cells were stained positive. Tissue sections were immunostained with rat anti-mouse CD34 monoclonal antibody (Biogenex). At low-power field (x40), the tissue sections were screened, and five areas with the most intense neovascularization (hotspots) were selected. Microvessel counts of these areas were done at high-power fields (x200). To reduce observer-related variation, counting of the microvessels was done with a computer image analyzer (MetaMorph Imaging System version 3.0, Universal Imaging, West Chester, PA), which is an integrated system of a Windows-based software especially designed for immunohistochemical analysis. Any positively stained endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessels and tumor cells and connective tissues was counted as one microvessel, irrespective of the presence of a vessel lumen. An automated microvessel count per field was computed in each hotspot, and the mean microvessel count of the five most vascular areas was taken as the microvessel density, which was expressed as the absolute number of microvessels per high-power fields. Evaluation of the microvessel count was assessed by two independent observers (K.M. and M.T.L.).

Hypoxic treatment. Cells were dispensed into a 100-mm culture dishes. The dishes were placed in a sealed hypoxia chamber (Billups-Rothenberg, Del Mar, CA) equilibrated with a humidified 5% CO2 atmosphere or with certified gas containing 0.1% O2, 5% CO2, and 94% N2.

Plasmids and reagents. pcDNA-Id-1 was a gift from Dr. Eiji Hara (Kyoto Prefectural University of Medicine, Kyoto, Japan) and pBabe-As-Id-1 was constructed as described (26). Recombinant human VEGF (rhVEGF) was purchased from R&D Systems. Flk-1 kinase inhibitor (40 mmol/L; SU1498) and VEGF-neutralizing antibody were purchased from Calbiochem (San Diego, CA) and R&D Systems, respectively.

Cell lines. Human HCC cell lines MHCC-97L and MHCC-97H (from Liver Cancer Institute, Fudan University, Shanghai, China; ref. 27), Huh-7 (a gift from Dr. H. Nakabayashi, Hokkaido University School of Medicine, Sapporo, Japan; ref. 28), Hep3B (American Type Culture Collection, Manassas, VA), PLC (Japanese Cancer Research Bank, Tokyo, Japan), H2P, and H2M (24) were maintained in DMEM with high glucose (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies), 100 mg/mL penicillin G, and 50 µg/mL streptomycin (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO2.

Cell transfection. PLC/PRF/5 was obtained from the Japanese Cancer Research Bank. The cells were transfected with 2 µg plasmid DNA of either Id-1 or pcDNA3.1(–) (kindly provided by Prof. Y.C. Wong, The University of Hong Kong) containing the entire coding of Id-1 or expression vector pcDNA3.1(–) alone using Fugene 6 according to the manufacturer's protocol (Boehringer Mannheim GmbH, Mannheim, Germany). After 48 hours, the medium was replaced with fresh DMEM with geneticin (G418) at 1 mg/mL. After 2 weeks of clonal selection, all the clones were grown in the presence of G418 at 0.4 mg/mL to ensure stable transfection. For suppression of Id-1 by antisense approach, retrovirus carrying the empty vector or the full-length Id-1 gene was generated from the packaging cell line PG13 and used to transfect the MHCC-97H cells. After 48 hours, the medium was replaced with fresh DMEM with geneticin (G418) at 1 mg/mL.

Luciferase promoter assay. MHCC-97H cells (5 x 104 per well) were plated into 24-well culture plates and allowed to grow for 24 hours. pGL3-V109, pGL3-V411, and pGL3-V2274 (these three VEGF promoters were kindly provided by Dr. K. Xie of University of Texas, Houston, TX) and pRL-CMV-Luc were cotransfected with either the pcDNA-Id-1 or pcDNA into the cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN). Cells were lysed 48 hours after transfection and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). For measuring VEGF promoter activity of the stable pBabe-As-Id-1 transfectants and pBabe vector control, the cells were cotransfected with pGL3-V2274 and pRL-CMV-Luc vector. Firefly luciferase activity was measured at 48 hours after transfection and the reading was then normalized with the Renilla luciferase activity, which served as internal control for transfection efficiency. Each experiment was done at least thrice in duplicate wells and each data point represented the mean and SD. The percentage increase in luciferase activity of the pcDNA-Id-1-transfected cells with various VEGF promoter or the MHCC-97H-pBabe-As-Id-1 transfectants was calculated relative to that of the vector controls. The mean percentage increase (or decrease) in luciferase activity was presented as the final results and the SD of the means was used as error bars.

Secretory VEGF protein quantitation by ELISA. An equal number of PLC and PLC-Id-1 transfectants (clones 1-4); MHCC-97H-pBabe and MHCC-97H-pBabe-As-Id-1 transfectants; and the vector control cells (2 x 105 cells per well) were plated in six-well plates in DMEM containing 10% fetal bovine serum. The cells were allowed to grow for 24 hours until they reached approximately 60% to 70% confluency. The growth medium was then removed and replaced with fresh DMEM containing 1% fetal bovine serum. The cells were incubated for further 24 hours until they reached ~80% confluency. The medium was then harvested and filtered for measurement of secretory VEGF. The remaining cells were collected and the viable cells were counted. VEGF present in the growth medium was measured using a Quantikine Human VEGF ELISA kit according to the manufacturer's instructions (R&D Systems). The concentration of VEGF was measured as picograms per milliliter (pg/mL) in the growth medium and the results were then calculated as picograms of secreted VEGF per cell. Each experiment was done at least thrice and the mean concentration of VEGF secretion was presented as the final result. SD of the means was used as error bars.

Capillary tube formation assay. PLC, PLC-pcDNA3.1, and PLC-Id-1 transfectants were plated into six-well plates (2 x 105 cells per well) in DMEM containing 10% FCS. After 48 hours, the medium was removed and fresh medium (DMEM) was added. The cells were allowed to grow for 24 hours until they were ~80% confluent. The growth medium was collected and filtered for the treatment of human umbilical vascular endothelial cells (HUVEC). HUVECs at passage 4 (1 x 104 per well) were seeded onto three-dimensional collagen gel (1:10, collagen gel/1x PBS; Chemicon International, Temecula, CA) in 96-well plates in the growth medium of PLC, PLC-pCDNA, and PLC-Id-1 transfectants. DMEM (1% fetal bovine serum) containing rhVEGF (20 ng/mL; R&D Systems) and DMEM only were also used to culture HUVECs as positive and negative controls, respectively. Tube formation in each treatment was inspected under an inverted light microscope after incubation for 10 hours at 37°C and images were captured at a single level beneath the monolayer.

Bromodeoxyuridine staining. HUVECs (5,000 per well) were grown on chamber slides in EGM2 medium for 24 hours. The culture medium was removed and replaced with growth medium of PLC and its transfectants, 20 ng/mL rhVEGF in DMEM with 1% FCS, EGM2, and DMEM containing 1% FCS, respectively, for incubation of 24 hours. The treatment medium was then removed and EGM2 was added for incubation for another 24 hours. The cells were then stained with bromodeoxyuridine (10 µmol/L) for 2 hours and stained with FITC antibody against bromodeoxyuridine as described (29). At least 1,000 cells were counted in each experiment and results were presented as percentage of bromodeoxyuridine-positive cells.

Reverse transcription-PCR. Total RNA was isolated using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen) and then amplified by PCR with Id-1 and VEGF primers: Id-1, 5-CCGGCAAGACAGCGAGCGGTGCG-3 (forward) and 5-GGCGCTGATCTCGCCGTTGAGGG-3 (reverse; ref. 12) and VEGF, 5-TTCTGTATCAGTCTTTCCTGG-3 (forward) and 5-CGAAGTGGTGAAGTTCATGGA-3 (reverse; ref. 30). PCR cycling protocol was as follows: 30 cycles for 10 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 55°C, 1 minute at 72°C, and 10 minutes at 72°C. 18S was amplified as an internal control. The PCR products were electrophoresed on 2% agarose gel and analyzed using a gel documentation system.

Western blotting. The cells were lysed and protein extraction was done. The samples were separated in 10% sodium dodecyl-sulfate acrylamide gel and electrophoretically transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, United Kingdom). The membrane was blotted with 10% nonfat milk, washed, and then probed with Id-1, actin, and HIF-1{alpha} (Santa Cruz Biotechnology) and VEGF (R&D Systems). After washing, the membrane was incubated with horseradish peroxidase–conjugated rabbit anti-mouse antibody (Amersham) and then visualized by enhanced chemiluminescence plus according to the manufacturers' protocol.

Invasion assay. Invasion assays were done with 24-well BioCoat Matrigel invasion chambers (Becton Dickinson, Bedford, MA) using 5 x 104 cells in serum-free DMEM and plated onto either control or Matrigel-coated filters. Conditioned medium from MHCC-97H-pBabe or MHCC-97H-pBabe-As-Id-1 was placed in the lower chambers as chemoattractant. After 22 hours in culture, cells were removed from the upper surface of the filter by scraping with a cotton swab. Cells that invaded through the Matrigel and were adherent to the bottom of the membrane were stained with crystal violet solution. The cell-associated dye was eluted with 10% acetic acid and its absorbance at 595 nm was determined. Each experiment was done in triplicates and the mean values ± SE are presented.

Approximately 1 x 107 cells in 0.1 mL culture medium were injected s.c. into the right flank of the four-week-old male BALB/c-nu/nu mice nude mice. Each group contains five nude mice inoculated with either MHCC-97H-pBabe or MHCC-97H-pBabe-As-Id-1. After 25 days, the tumor was taken out and the tumor volume was measured as (L x W2 / 2).

Statistical analysis. Continuous data were expressed as median and range and compared between groups using the Mann-Whitney U test. Categorical variables were compared using the {chi}2 test (or Fisher's exact test where appropriate). Pearson test was used for bivariate correlation comparison. All statistical analyses were done using a statistical software (SPSS 9.0 for Windows, SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Id-1 overexpression was correlated significantly with metastasis and VEGF expression in human HCC tumor samples. We evaluated 80 samples of HCC and their corresponding nontumor tissues by immunostaining. In normal liver, no cytoplasmic expression of Id-1 in hepatocytes was observed (Fig. 1A ). However, in nontumor liver (cirrhotic liver and chronic hepatitis), absent to weak cytoplasmic expression of Id-1 was observed (Fig. 1A). In HCC samples, cytoplasmic expression of Id-1 was not found in 10 (12.9%) cases, weak in 30 (33.3%) cases, and moderate to strong in 40 (50.0%) cases. In 60 pairs of primary HCC and their matched metastatic tumors, strong Id-1 expression could be detected in 25 of 59 (42%) and 53 of 59 (89.8%), respectively. Id-1 expression was significantly associated with HCC metastasis (P < 0.001). To assess the possible correlation of Id-1 expression with angiogenesis, we evaluated protein expression of an important angiogenic factor VEGF in another TMA specimen. In 60 pairs of primary HCC and their matched metastatic tumors, strong VEGF expression could be detected in 20 of 59 (33.9%) and 45 of 59 (76.2%), respectively. It was found that Id-1 was positively correlated with VEGF expression (r = 0.47; P < 0.001; Fig. 1B).


Figure 1
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  		Fig. 1. A, correlation of Id-1 expression with metastasis by clinical samples. Immunostaining analysis showing no Id-1 expression in normal liver (a), weak expression in nontumorous liver (b), and strong expression in tumor case (c). Overview of a tissue array section containing 60 pairs of primary HCC and their matched metastatic tumors showing Id-1 expression (d). Immunostaining of Id-1 in two pairs of HCC (e and f). Higher magnification of one pair of a primary and its matched metastatic HCC (case 21) from the area of the boxed in (g and h). B, the correlation between Id-1 and VEGF was shown in consecutive sections of HCCs. Case 11 showed strong immunostaining of Id-1 and VEGF. In case 18, both cases showed negative for Id-1 and VEGF. Arrows, nuclear expression of lymphocyte.

 
Id-1 overexpression was correlated with metastatic potential in various HCC cell lines. In Fig. 1, we found that Id-1 expression was correlated with metastasis and VEGF expression in HCC clinical samples. To further confirm this relationship, we used six HCC cell lines with various metastatic potential. Both Id-1 mRNA and protein levels were found to be up-regulated in metastatic HCC cell lines (H2M, MHCC-97L, and MHCC-97H) when compared with primary nonmetastatic ones (PLC, Huh-7, and H2P; Fig. 2 ). Accompanied with the increased Id-1 expression, both VEGF mRNA and protein expressions were also up-regulated (Fig. 2). Both Id-1 and VEGF were up-regulated in metastatic HCC cell lines at both mRNA and protein levels.


Figure 2
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  		Fig. 2. A and B, correlation of Id-1 with VEGF in HCC cell lines. By reverse transcription-PCR, both Id-1 and VEGF mRNAs were up-regulated in metastatic HCC cell lines when compared with the primary ones. D and E, by Western blot, Id-1 and VEGF proteins were also found to be up-regulated in metastatic HCC cell lines. The expression levels of Id-1 and VEGF correlated with both mRNA and protein levels in HCC cell lines.

 
Ectopic introduction of Id-1 led to up-regulation of VEGF in HCC cell. Given the significant correlation between Id-1 and VEGF, we then investigated whether Id-1 increased metastatic potential through up-regulation of the angiogenic factor VEGF in HCC cells. In this study, we transfected full-length Id-1 cDNA into PLC to examine the effect on VEGF at mRNA and protein levels. After transfection, four stable clones and pool of clones (~100 clones) were isolated. Different Id-1 protein expression was observed in four Id-1 transfectants (clones 1–4) and pool of Id-1 transfectants (Fig. 3A ). With Id-1 transfection, up-regulation of VEGF protein level was observed when compared with empty vector control. Increase in VEGF expression was correlated with Id-1 protein expression (Fig. 3A). Ectoptic Id-1 introduction increased not only VEGF protein expression but also VEGF mRNA level (Fig. 3B). By ELISA, there was approximately 35% to 168% increase in VEGF protein secretion in Id-1 transfectants and pool of clones when compared with the empty vector control (Fig. 3C).


Figure 3
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  		Fig. 3. Id-1 increased VEGF secretion in HCC cell lines. A, Id-1 expression in PLC-pCDNA3.1 and Id-1 transfectants. Accompanied with the increased Id-1 protein level, VEGF protein level was also up-regulated. B, by reverse transcription-PCR, Id-1 activated transcriptional level of VEGF. Secretory VEGF was significantly increased in Id-1 transfectants. C, VEGF protein concentration was measured by ELISA method and results were presented as pictogram per cell. Amount of VEGF secretion correlated Id-1 expression in Id-1 transfectants.

 
Effect of secretory VEGF on angiogenesis in HUVEV cells. We then investigated the effect of VEGF protein secreted by Id-1 transfectants on HUVECs in terms of angiogenic and proliferation abilities. As shown in Fig. 4A , HUVECs showed elongated morphology with network structure after culturing the cells in the medium of Id-1 transfectants and pool of Id-1 clones for 10 hours in capillary tube formation assay when compared with scattered and round-shaped morphology in DMEM or medium in PLC parental cell or PLC-pcDNA3.1. Elongated network structure was also observed in the same cells treated with rhVEGF (20 ng/mL; Fig. 4A). Furthermore, the proliferation rate of HUVECs in medium of Id-1 transfectants was approximately 1- to 3-fold higher than HUVECs cultured in the medium of PLC parental or PLC-pcDNA cells. Increased proliferation was observed in medium supplemented with 20 ng/mL rhVEGF (Fig. 4B). The above showed that secretory VEGF by Id-1 PLC transfectants and pool of Id-1 clones was able to promote angiogenesis as well as proliferation of HUVECs. To further confirm that increased VEGF secretion by Id-1 transfection was responsible for the angiogenic effect on HUVECs, we treated HCC cell culture medium with an inhibitor of VEGF receptor Flk-1, SU1498, and VEGF-neutralizing antibody. The addition of SU1498 (40 µmol/L) and VEGF-neutralizing antibody (0.1 µg/mL) in the culture medium of rhVEGF and Id-1 transfectants led to disappearance of the elongated morphology (Fig. 4C) and decreased proliferation rate of HUVECs (Fig. 4D).


Figure 4
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  		Fig. 4. Id-1 induced angiogenic effect on HUVECs. A, morphologic changes in HUVECs cultured in medium of PLC, PLC-pcDNA, and Id-1 transfectants (clones 1-4) and pool of Id-1 clones. HUVECs treated with DMEM only and rhVEGF (20 ng/mL) were used as negative and positive control, respectively. HUVECs showed elongated morphology after culturing the cells in the medium of Id-1 transfectants for 10 hours in capillary tube formation assay when compared with scattered and round-shaped morphology in DMEM or medium in PLC parental cell or PLC-pcDNA3.1. Elongated network structure was also observed in the same cells treated with (20 ng/mL). B, the effect of cell culture medium of Id-1 transfectants on HUVEC cell proliferation. The proliferation rate of HUVECs in medium of Id-1 transfectants was higher than HUVECs cultured in the medium of PLC parental or PLC-pcDNA cells. Increased proliferation was observed in medium supplemented with 20 ng/mL rhVEGF. The growth medium from Id-1 transfectants induced proliferation and angiogenesis in HUVECs at a similar level to that of rhVEGF. Angiogenic effect of Id-1 on HUVECs was reversed by addition of Flk-1 inhibitor and VEGF-neutralizing antibody into the culture medium of rhVEGF and Id-1 transfectants. Treatment of HUVECs with SU1498 (40 µmol/L) and VEGF-neutralizing antibody (0.1 µg/mL) led to suppression of capillary tube formation ability (C) and proliferation rate (D) of HUVECs.

 
Id-1 activated VEGF promoter activity by stabilizing HIF-1{alpha} protein in HCC cell. In Fig. 3B, we found that Id-1 up-regulated VEGF at both mRNA and protein levels. Therefore, we hypothesized that increased VEGF expression in Id-1 transfectants was due to gene activation at transcription level rather than protein stabilization. To verify this hypothesis, we then cotransfected the pcDNA3.1-Id-1 together with a luciferase reporter harboring three different lengths of VEGF promotes (pG3-V109, pG3-V411, and pG3-V2274) and generated transient transfectants. We found that VEGF promoter activity was increased approximately from 0.5- to 2-fold in pG3-V109 and pG3-V411, respectively, in Id-1 transfectants when compared with the vector control (Fig. 5A ). For pG3-V2274, VEGF promoter that contained HIF-1{alpha} binding site, the promoter activity significantly increased up to 9-fold, suggesting the importance of HIF-1{alpha} in Id-1-induced VEGF up-regulation. To confirm this observation, Western blot was done to examine HIF-1{alpha} expression in various Id-1 transfectants. HIF-1{alpha} protein level was increased ~8-fold in Id-1 transfectants (clone 4) when compared with empty vector control (Fig. 5B). However, there is no change in the HIF-1{alpha} mRNA level (Fig. 5B). To examine whether Id-1 played a role in hypoxia-induced angiogenesis through increase in HIF-1{alpha} protein stabilization, we monitored the levels of Id-1-induced HIF-1{alpha} protein synthesis addition of cycloheximide to block HIF-1{alpha} protein synthesis. Rapid decay of HIF-1{alpha} was observed after 20 minutes and undetectable in normoxic condition in empty vector control cell (Fig. 5C). In contrast, Id-1-induced HIF-1{alpha} protein level in normoxia remained strong even 60 minutes after cycloheximide (Fig. 5C).


Figure 5
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  		Fig. 5. Id-1 induced HIF-1{alpha}-mediated VEGF activation. A, the VEGF promoter with various lengths (pG3-V109, pG3-V411, and pG3-V2274) was used for examination of the promoter activity on Id-1 activation. Maximal promoter activation was found in pG3-V2274 that contains HIF-1{alpha} binding site; the promoter activity significantly increased up to 9-fold. B, by Western blot, consistent with promoter assay, HIF-1{alpha} protein was up-regulated in Id-1 transfectants when compared with empty vector control. However, the mRNA level remained unchanged (boxed). C, to examine whether Id-1 induced stability of HIF-1{alpha} protein, we monitored the levels of Id-1-induced HIF-1{alpha} protein synthesis by cycloheximide. After cycloheximide treatment, the cells were further incubated under normoxic condition. Rapid decay of HIF-1{alpha} was observed for 20 minutes and undetectable for 60 minutes in normoxic condition for empty vector control. In contrast, Id-1 induced HIF-1{alpha} protein level in normoxia remained strong even 60 minutes after cycloheximide treatment.

 
Antisense-Id-1 inhibited HCC cell invasion through down-regulation of VEGF expression. To evaluate the tumor growth and angiogenesis of Id-1-expressing cells in vivo, 1 x 106 cells of PLC-pcDNA or PLC-Id-1 (clone 4) were injected into the nude mice s.c. The tumor volumes of pcDNA-Id-1- and pcDNA-transfected PLC cells were 1,859 ± 143 mm3 and 1,123 ± 89 mm3, respectively. pcDNA-Id-1-transfected cells showed significant increase in tumor volume when compared with vector control in vivo (P = 0.000154). Increased cytoplasmic VEGF expression and CD34 expression were also found when compared with empty vector control (data not shown). To further confirm that Id-1 induced HCC cell invasion through promotion of angiogenesis, we transfected MHCC-97H with pBabe-As-Id-1 and one stable clone and pool of clones (150-200) were isolated. By Western blot, we confirmed suppression of Id-1 expression by pBabe-As-Id-1 (Fig. 6A ). On suppression of Id-1 by pBabe-As-Id-1, both mRNA and protein levels of VEGF decreased (Fig. 6A). Accompanied with decrease in VEGF protein level in MHCC-97H, secretory VEGF protein was shown to decrease as well (data not shown). To further investigate whether the decrease in VEGF protein was due to inactivation of transcription level, we used the VEGF promoter assay. We transfected pG3-V2274 into pBabe-As-id-1 and pBabe transfectants and found that a significant decrease in VEGF promoter activity was observed in pBabe-As-id-1 transfectants (up to 85% decrease) and pool of Id-1 clones (~45% decrease) when compared with vector control. Accompanied with decreased in VEGF promoter activity, Id-1 suppression led to down-regulation of HIF-1{alpha} protein by 7-fold (Fig. 6B). By Matrigel invasion assay, ~65% decrease in cell invasion was observed in pBabe-As-id-1 MHCC-97H cell and 22% decrease in pool of Id-1 clones when compared with empty vector control (Fig. 6C). Cell invasion in pBabe-As-id-1 MHCC-97H cell could be elevated by addition of exogenous rhVEGF (40 ng/mL; Fig. 6C). This showed that Id-1 induced HCC cell metastasis through promotion of HCC invasion. When a high number of stable pBabe-As-id-1 clone and pBabe MHCC-97H cells were injected into the nude mice s.c., tumor formation was recorded after 25 days of tumor inoculation. As shown in Fig. 6D, a significant decrease in tumor volume was observed in pBabe-As-id-1 transfectants when compared with the empty vector control (P = 0.000857). The tumor volumes of pBabe-As-id-1- and pBabe-transfected MHCC-97H cells were 415 ± 41 mm3 and 962 ± 39 mm3, respectively. When the tumors were observed phenotypically, pBabe-As-id-1 formed tumor was less vascularized when compared with the empty vector control (Fig. 6D). Consistently, we found weak cytoplasmic VEGF expression in pBabe-As-id-1 tumor when compared with strong VEGF expression in vector control by immunohistochemical staining. In addition, CD34 expression was also significantly decreased in pBabe-As-id-1 tumor when compared with empty vector control (P = 0.00171; Fig. 6E).


Figure 6
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  		Fig. 6. Antisense-Id-1 inhibited HCC cell metastasis through VEGF down-regulation. A, Id-1 expression in MHCC-97H-pBabe and MHCC-97H-pBabe-As-Id-1 and pool of pBabe-As-Id-1 clones were shown by Western blot. Suppression of Id-1 not only decreased VEGF mRNA and protein levels. B, HIF-1{alpha} protein was decreased significantly in MHCC-97H-pBabe-As-Id-1 cell but not in pool of pBabe-As-Id-1 clones by Western blot. C, both MHCC-97H-pBabe-As-Id-1 and MHCC-97H-pBabe-As-Id-1 cells showed decreased invasion when compared with MHCC-97H-pBabe and parental MHCC-97H. Cell invasion in pBabe-As-id-1 MHCC-97H cell and pool of MHCC-97H-pBabe-As-Id-1 cells could be elevated by the addition of exogenous rhVEGF (20 ng/mL). D, when the cells were inoculated into the nude mice s.c., decreased tumor volume was observed in pBabe-As-id-1-bearing mice when compared with empty vector control. When the tumor was excised, pBabe-As-id-1 MHCC-97H tumor showed less vascularization when compared with empty vector control. E, by immunostaining, cytoplasmic Id-1 and VEGF protein expression was decreased in pBabe-As-id-1 MHCC-97H. Antisense Id-1 also suppressed CD34 expression. Suppression of Id-1 resulted in decrease of CD34 expression from 49.3 ± 3.2 to 19 ± 4.2.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
HCC metastasis is commonly associated with poor prognosis because of the lack of effective systemic chemotherapy (3). Recently, Id-1 has been found to be up-regulated in various tumors (17, 18), and a previous study from our group suggested that Id-1 acts as an oncogene that promotes tumorigenesis through inactivation of tumor suppressor genes, such as p16INK4a (23). However, the role of Id-1 in HCC tumor progression is unclear. In this study, we first reported that Id-1 overexpression in tumor tissues correlated with HCC metastasis. This result confirmed the finding of previous in vitro study, which showed correlation of Id-1 with aggressiveness of breast cancer cells (15).

Angiogenesis is fundamental to tumor metastasis (9, 10). Angiogenesis is regulated by various angiogenic factors, such as interleukin-8 (31), migration inhibitory factor (32), transforming growth factor-ß (33), and VEGF (7), of which VEGF seems to play a central role. VEGF is a reliable marker for HCC angiogenesis (10). Previously, impaired angiogenesis has been reported in Id-1-deficient mice as well as breast cancer xenografts (34). With this observation, we hypothesized that Id-1 might enhance HCC metastasis through promotion of angiogenesis. From TMA, we found significant correlation between Id-1 protein expression and VEGF (r = 0.47; P < 0.001). The correlation of Id-1 and VEGF was further confirmed by an in vitro system showing increased VEGF expression in metastatic cell lines showing high Id-1 expression when compared with primary nonmetastatic ones. These in vitro and human tissue data have suggested that Id-1 might promote angiogenesis through interaction with VEGF. To confirm the direct link between Id-1 and VEGF protein, we transfected Id-1 full-length cDNA into PLC cell. Interestingly, ectopic Id-1 introduction increased not only VEGF protein expression but also mRNA level. Accompanied with increase in the VEGF protein level, increased secretory VEGF was observed in culture medium of four Id-1 transfectants when compared with empty vector control. Secretory VEGF produced by Id-1 transfectants in the culture medium was able to induce angiogenesis, indicating that VEGF produced by Id-1 transfectants was functionally active and was able to promote endothelial proliferation and form new blood vessels. This result was further confirmed by addition of SU1498 and VEGF-neutralizing antibody into culture medium of Id-1 transfectants and rhVEGF showing disappearance of elongated structure and proliferation rate of HUVECs. The increase in VEGF protein in Id-1 transfectants is due to activation of VEGF promoter as evidenced by promoter assay. From the promoter assay, we found that HIF-1{alpha} is an important site for Id-1-induced VEGF activation as promoter activity was maximal in VEGF promoter harboring HIF-1{alpha} binding site. HIF-1{alpha} is a heterodimeric transcription factor composed of two basic helix-loop-helix subunits (35). HIF-1{alpha} is a major regulator of VEGF contributing to angiogenesis (36). In tumors under hypoxia condition, the VEGF protein level was up-regulated on enhanced HIF-1{alpha} stability (37). From Western blot in Fig. 5B, we found up-regulation of HIF-1{alpha} in various Id-1 transfectants. These data suggested that Id-1 induced VEGF up-regulation through HIF-1{alpha}. Increased HIF-1{alpha} protein expression rather than mRNA level can be attributed to either enhanced protein stability or protein synthesis. The mechanism for Id-1-induced HIF-1{alpha} protein expression was further confirmed by investigating the decay of hypoxia-stabilized HIF-1{alpha} protein after returning to normoxia condition in PLC cells. HIF-1{alpha} protein stability in cycloheximide-treated cells showed that the degradation of HIF-1{alpha} protein was decreased in Id-1-transfected cells when compared with control under hypoxia condition, supporting the finding that induction of HIF-1{alpha} protein level by Id-1 activation was due to enhanced HIF-1{alpha} protein stability. Many reports have shown the potential role of signal transducer and activator of transcription 3 in HIF-1{alpha}-mediated VEGF expression (38, 39). Interestingly, we found that signal transducer and activator of transcription 3 was activated in the Id-1-overexpressing cells by Western blot (data not shown). Therefore, Id-1 might promote angiogenesis in HCC through HIF-1{alpha}-mediated VEGF up-regulation in the signal transducer and activator of transcription 3–dependent pathway.

Suppression of Id-1 expression in MHCC-97H cell by the antisense approach decreased VEGF mRNA and protein levels in pBabe-As-Id-1-transfected MHCC-97H cell. Id-1-activated VEGF in the transcriptional level was confirmed by decreased promoter activity in pBabe-As-Id-1-transfected MHCC-97H cell. Accompanied with decreased promoter activity, a dramatic decrease in the HIF-1{alpha} protein level was also observed in antisense-suppressed MHCC-97H cell. A recent study on breast cancer showed that down-regulation of Id-1 using the antisense approach was able to prevent breast cancer cells from metastasizing to the lungs of experimental animals (32). In this study, we further showed that suppression of Id-1 by antisense approach also decreased HCC cell invasiveness as shown in our matrix gel invasion assay. Interestingly, we observed that HCC cell invasiveness was regulated by VEGF. This mechanism was confirmed by addition of exogenous rhVEGF to pBabe-As-Id-1-transfected MHCC-97H cells. After rhVEGF addition, cell invasiveness of pBabe-As-Id-1-transfected MHCC-97H cell increased. This mechanism was confirmed by increased cell invasion after addition of exogenous rhVEGF to pBabe-As-Id-1-transfected MHCC-97H cells. In our unpublished data, MHCC-97H was found to be VEGF receptor-1 and VEGF receptor-2 positive. Recent studies showed that VEGF-1 on tumor cell might promote migration and invasion (40, 41). Therefore, it suggested that VEGF produced by pBabe-As-Id-1 transfectants might increase HCC invasion by activation of VEGF-1 receptor.

To observe the effect of Id-1 suppression in HCC cells in vivo, we inoculated a high number of pBabe-As-Id-1-transfected MHCC-97H and MHCC-97H-pBabe cells into the nude mice s.c. After 25 days, we found a decreased tumor volume in pBabe-As-Id-1-transfected MHCC-97H tumor when compared with the empty vector control. The decrease in tumor volume was associated with decreased angiogenesis in tumor as evidenced by altered VEGF and CD34 expression in pBabe-As-Id-1-transfected MHCC-97H cells. This result confirmed the role of angiogenesis in tumor growth and progression (42). Taken together, the above data suggested a novel regulatory mechanism of VEGF by Id-1 leading to HCC progression. Recently, there have been reports showing Id-1 to be involved in early HCC carcinogenesis (43, 44). Instead of viewing this as a mutually exclusive event, we speculated that this may provide additional evidence to our finding that Id-1 is involved in HCC angiogenesis and angiogenesis plays a crucial role in tumor devilment in the early stage.

In summary, we first showed that Id-1 is a novel angiogenic factor in HCC metastasis. Id-1 promoted HCC angiogenesis through HIF-1{alpha}-mediated VEGF activation. Our results not only provide a molecular basis for the role of Id-1 in HCC metastasis but also suggest a novel therapeutic target for the treatment of metastatic HCC.


   Footnotes
 
Grant support: Sun Chieh Yeh Research Foundation for Hepatobiliary and Pancreatic Surgery of The University of Hong Kong.

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.

Received 3/ 1/06; revised 8/22/06; accepted 9/21/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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