Potential conflict of interest: Nothing to report.
Hepatitis B x antigen (HBxAg) is a trans-activating protein that contributes to liver cancer, in part, by altering the expression of cellular genes. However, few natural effectors of HBxAg have been identified. Hence, HBxAg positive and negative HepG2 cells were prepared and analyzed by PCR select cDNA subtraction. The results identified elevated vascular endothelial growth factor receptor-3 short form splice variant (VEGFR-3S) expression in HBxAg positive compared to negative cells. Normally, VEGFR-3 activates Akt signaling in lymphatic endothelial cells, resulting in lymphangiogenesis. In contrast, the results here show that the expression of VEGFR-3S is up-regulated in >75% of HBxAg positive hepatocellular carcinoma (HCC) nodules. VEGFR-3S up-regulation correlates with the expression of HBxAg, is associated with decreased survival in tumor bearing patients, and when over-expressed in HepG2 cells, strongly stimulated cell growth in culture, in soft agar, and accelerated tumor formation in a ligand independent manner. VEGFR-3S siRNA partially blocked the ability of HBxAg to promote hepatocellular growth. In conclusion, HBxAg may short circuit VEGFR-3S signaling in liver cancer. Blocking VEGFR-3S signaling may be effective in preventing tumor development and/or prolonging survival in tumor bearing patients. (HEPATOLOGY 2007;45:1390–1399.)
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Chronic hepatitis B virus infection is associated with the development of hepatitis, cirrhosis and HCC and constitutes a major public health problem worldwide.1, 2 HBxAg participates in the pathogenesis of HCC.3, 4 For example, high levels of intrahepatic HBxAg directly correlate with the intensity of liver disease.5, 6 HBxAg also transforms cells in vitro,7 while sustained high levels of HBxAg in transgenic mice lead to HCC.8 HBxAg is a trans-activating protein that alters gene expression by binding to nuclear transcription factors, and by stimulating cytoplasmic signaling pathways that promote cell growth and survival.3, 4, 9, 10 HBxAg also binds to and inactivates or down-regulates tumor suppressors and senescence-related factors.11 Despite this work, few natural effectors of HBxAg that promote the development of HCC have been identified.
VEGFR-3 is a receptor tyrosine kinase that is expressed in lymphatic endothelial cells.12 Binding of VEGFR-3 to VEGF-C or VEGF-D stimulate lymphangiogenesis,13 while in carcinogenesis, the production of VEGFs by tumors promote metastases and result in decreased survival.14 Elevated VEGF has been found in patients with HCC.15, 16 VEGFR-3 is also expressed in tumor cells from several tumor types,14, 17 including HCC,15 implying the existence of an autocrine/paracrine loop that promotes tumor development independent of lymphangiogenesis.14 In HCC, elevated VEGFR-3 is associated with portal vein invasion of tumors, increased hepatic tumor recurrence, and shorter survival,15 suggesting that VEGFR-3 is important in the pathogenesis of HCC.
VEGFR-3 signaling promotes cell survival under oxidative stress.18 Under these conditions, VEGFR-3 becomes tyrosine phosphorylated by activated src kinases. Src kinases are activated by HBxAg19 resulting in cell cycle progression in early HCC.20, 21 VEGFR-3 and HBxAg also activate phosphoinositol 3-kinase (PI3K),13, 22 which promotes HCC development and metastasis.23 Importantly, VEGFR-3 consists of VEGFR-3L (long) and VEGFR-3S(short) isoforms, the latter being a splice variant that lacks 65 carboxy-terminal amino acids.24 Both isoforms become phosphorylated within their carboxy-terminal regions. VEGFR-3L then mediates growth in soft agar and tumorigenicity in nude mice.25 VEGFR-3S is over-expressed in several tumor types,26 although its function(s) and signaling pathways are not well described. Phosphorylation of VEGRF-3L at Y1337 induces transformation,25 while phosphorylation of Y1230 and Y1231 in both forms contribute to VEGFR-3 dependent proliferation, migration and survival by activation of Akt.27 In this report, HBxAg selectively up-regulates VEGFR-3S, which promotes hepatocellular growth and tumorigenesis associated with Akt activation.
HBxAg, hepatitis B x antigen; VEGFR-3S, vascular endothelial growth factor receptor 3 short form; HCC, hepatocellular carcinoma; VEGFR-3, vascular endothelial growth factor receptor 3; VEGFR-3L, vascular endothelial growth factor receptor 3 long form; CAT, chloramphenicol acetyltransferase; ISH: in situ hybridization; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; RACE, rapid amplification of cDNA ends; PCNA, proliferating cell nuclear antigen; MTT, modified tetrazolium salt assay; NT, nontumor; T, tumor.
Materials and Methods
PCR Select cDNA Subtraction of RNAs from HepG2X and HepG2CAT Cells.
HepG2X and HepG2CAT cultures were established and subjected to PCR select cDNA subtraction as described.28 The resulting PCR fragments were cloned, sequenced, and compared to existing sequences within GenBank. One of these fragments, encoding VEGFR-3, was chosen for further characterization.
Forty paired tumor/nontumor samples came from Chinese patients who underwent surgery at the Eastern Hepatobiliary Hospital in Shanghai. Fourteen additional paired tumor/nontumor samples were obtained from South African (Black) patients who underwent surgery at the University of Witwatersrand in Johannesburg. Additional characteristics of these populations have been published.28 Formalin fixed, paraffin embedded tissues, fresh frozen blocks, and −80°C snap-frozen paired liver and tumor samples were collected, used for diagnostic purposes, and then used here. Uninfected human liver from 2 individuals, and paired tumor/nontumor samples from 6 patients with hepatitis C virus related HCC, were available as controls. This work was approved by the Institutional Review Boards at each site and provided with informed consent.
In situ Hybridization (ISH).
The VEGFR-3 cDNA fragment obtained from subtractive hybridization was used as a probe for ISH on cells and fresh frozen tissue samples, as described.28
Detection of VEGFR-3 mRNA.
Northern blotting with RNA isolated from tissue culture cells was conducted as reported.28 Hybridization was conducted under stringent conditions with the VEGFR-3 cDNA fragment obtained above radiolabeled with α-32P-dCTP (NEN, Boston, MA) by random priming (Prime-A-Gene labeling kit, Promega, Madison, WI). Glyceraldehyde 3- phosphate dehydrogenase (G3PDH) was used for normalization.
Cloning and Sequencing of Full-length VEGFR-3L and VEGFR-3S cDNAs.
To obtain full-length clones of the VEGFR-3 isoforms, rapid amplification of cDNA ends (RACE) was used. The primer for 5′ RACE PCR was 5′-ACTGTCTGTCTGGTTGTCCACAGAGCC-3′ while the primer for 3′ RACE PCR was 5′-GAGACCCCAAGGCGAGACCTGCATTCT-3′ (for VEGFR-3S) and 5′-GACCTGGCCAGAATGTGGCTGTGA-3′ (for VEGFR-3L). These primers were used with the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). Human placental cDNA was used as the template. PCR products were cloned into pT7blue vector (Novagen, Madison, WI) and sequenced. Appropriate 3′ and 5′ gene specific fragments were subcloned into pSLXCMV, and the integrity of the full-length clone verified by DNA sequencing at the Nucleic Acid Facility of Thomas Jefferson University.
Detection of VEGFR-3 and HBxAg.
The amino acid sequence of VEGFR-3L was analyzed in the PEPTIDESTRUCTURE and PLOTSTRUCTURE programs to identify hydrophilic regions, which were then made by solid phase peptide synthesis at the Kimmel Cancer Center of Thomas Jefferson University. Peptides 1, 2, and 3 were from residues 265-284 (NH2-DWDYPGKQ-AERGKWVPERRSC-COOH), 472–490 (NH2-RSLRRRQQQDLMPQCRDWR-COOH) and 713–728 (NH2-KDERLLEEKSGVDLADSC-COOH) that included sequences shared by VEGFR-3L and VEGFR-3S. Peptide 4 was from VEGFR-3L specific residues 1333–1349 (NH2-CHDEESPESLEGYESNY-COOH). Corresponding peptide antibodies were raised in New Zealand white rabbits.29 A mixture of these antibodies (each at a dilution of 1:1,000) was used for staining using the DAKO EnVision Plus System (DAKO, Carpenteria, CA).30 Controls included staining with preimmune serum and preincubation of primary antibodies with an excess (25 μg) of the corresponding synthetic peptide(s) prior to staining.
Proliferation was evaluated by staining with mouse anti-human proliferating cell nuclear antigen (PCNA) (PC-10, Santa Cruz Biotech, Santa Cruz, CA; 1:800 dilution). The PCNA staining score (Table 1) was based upon counting 1000 nuclei.
Table 1. Summary of HBxAg, PCNA and VEGFR-3 Expression in Tumor and Nontumor Liver
ISH and immunohistochemical (IHC) staining is estimated as follows: 0: no signal; 1: signal in <10% of cells; 2: signal in 10%–25% of cells; 3: signal in 25%–50% of cells; 4: signal in >50% of cells. For HBxAg and VEGFR-3, staining is scored in hepatocytes. For PCNA staining: 1: signal in ≤1% of cells; 2: signal in 2%–25% of cells; 3: signal in >25% of cells.
For western blotting, 100 μg of protein from each sample was analyzed by SDS/PAGE on 8% running gels and transferred to Immobilon-P membrane (Millipore, Bedford, MA).28 The primary antibody was a mixture of anti-VEGFR-3 peptides 1–3, each used at 1:400 dilution or anti-HBx.30 Anti-Akt, anti-phospho-Akt (ser473), anti-phospho-PTEN (ser380), and anti-phospho-raf (ser259) were obtained from the phosphor-Akt Pathway Sampler Kit (Cell Signaling Technology, Beverly, MA) and all used at 1:1000 dilution. The secondary antibody was horseradish peroxidase–conjugated goat anti-rabbit Ig (Santa Cruz Biotechnology). Preimmune serum was used as a negative control. As a positive control, the peptides used for immunization were spotted near the edge of the membranes following the transfer step. The results were visualized using the enhanced chemiluminescence detection system (Amersham, Uppsala, Sweden). β-actin was used as an internal control.
Construction of VEGFR-3 Over-expressing HepG2 and Control Cells.
The transduction and maintenance of HepG2 cells was carried out as described.28 Cell viability was estimated by trypan blue staining, and by the modified tetrazolium salt (MTT) assay (Cell Titer 96 Non-radioactive Cell Proliferation Assay, Promega).
Growth of Cells in Soft Agar and Tumorigenicity in Nude Mice.
To test for growth in soft agar, 1 × 104 cells/well were seeded in triplicate into 6-well plates, grown for 21 days, and counted under code using an inverted microscope.
For tumorigenicity assays, 3 groups of 10 nude mice each were injected subcutaneously at a single site with 5 × 106 cells. Tumor onset was scored visually and by palpitation at the sight of injection independently by 2 trained lab personnel. Tumor sizes were determined by wet weight at the time of death (6 weeks) except for mice that became moribund earlier. Tumors were verified as being HCC by hematoxylin and eosin staining. These experiments were approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University.
VEGF-C and anti-VEGF-C.
Human VEGF-C (having the cys156ser mutation) and anti-human VEGF-C (clone 193208) were purchased from R&D Systems (Minneapolis, MN). The VEGF-C mutant eliminated binding to VEGFR-2 but retained binding and agonist activity to VEGFR-3.
Akt inhbitor IV, which targets the ATP binding site of a kinase upstream of Akt, but downstream of PI3K, was purchased from Calbiochem (San Diego, CA).
The relationship between HBxAg and VEGFR-3 obtained by ISH and immunohistochemistry was determined using 2 × 2 comparisons in the Chi square (χ2) test. Statistical significance was observed when P < 0.05. The relationship between VEGFR-3 expression and survival was determined using the SPSS 10.0 for windows program package (SPSS Inc., Chicago, IL). Survival curves were plotted using the Kaplan-Meier method, and statistical differences between life tables were determined by a log-rank test. P < 0.05 denoted a statistically significant difference. The mean difference between colonies (in soft agar), or cell cycle phase for HepG2X, HepG2-VEGFR-3S, HepG2-VEGFR-3L, and HepG2CAT cells was determined by the Student t test. A significant relationship was indicated when P < 0.05.
Identification and Cloning of VEGFR-3.
Whole cell RNA was extracted from HepG2X and HepG2CAT cultures and subjected to PCR select cDNA subtraction.28 A cDNA fragment (≈1.5 kb) from an up-regulated mRNA in HepG2X cells was sequenced. Compared to entries in GenBank, it had extensive homology with VEGFR-3. When ISH was performed to verify differential expression, there was hybridization in the cytoplasm of HepG2X cells (Fig. 1A), but little signal in HepG2CAT cells (Fig. 1B) or in HepG2X cells hybridized with an irrelevant probe (Fig. 1C). Northern blotting showed bands at 4.6 kb and 5.5 kb in HepG2X (Fig. 1D, lane 1) and HepG2CAT cells (Fig. 1D, lane 2). The ratio of 4.6kb:5.5kb bands was 7.7 + 1.4 in HepG2X cells and 0.39 + 0.11 in HepG2CAT cells. The levels of 5.5 kb RNA in HepG2X cells was roughly half that in HepG2CAT, while the levels of the 4.6 kb RNA was greater than 19-fold higher in HepG2X compared to control cells. Previous work showed that these RNAs were splice variants encoding the long or short isoforms of VEGFR-3.31 To verify this, western blotting was performed. When anti-VEGFR-3L was used, similar levels of long form were in HepG2X and HepG2CAT cells (Fig. 1E, upper portion of blot), but when anti-VEGFR-3L+S was used, HepG2X cells showed 9.2 + 1.0 fold greater signal (Fig. 1E, lane 1) compared to HepG2CAT cells (Fig. 1E, lane 2), suggesting up-regulation of VEGFR-3S in HepG2X cells.
Full-length VEGFR-3 cDNA was then obtained using RACE PCR. VEGFR-3 cDNA was 4,765 bp long, which encoded VEGFR-3S. The nucleic acid sequence was 99.6% homologous and amino acid sequence 99.8% homologous to GenBank entry AR201982, with 7 codons in Genbank different from that of VEGFR-3 cDNA sequenced here (gly24asp, arg745pro, asn752arg, ala753pro, his890gln, leu1128val, and arg1146his). The basis for these differences is unknown, although the sequence herein may be a liver specific isoform of VEGFR-3. VEGFR-3S (GenBank no. AY233382) was 1,298 amino acids with a deduced molecular weight of 145,613 daltons. VEGFR-3L had this same sequence with an additional 65 amino acids on the carboxy-terminus. VEGFR-3L was 1,364 amino acids long with a deduced molecular weight of 152,396 daltons (GenBank no. AY233383).
Expression of VEGFR-3 in HCC and Nontumor Liver.
To determine whether VEGFR-3 was up-regulated in vivo, ISH was performed on fresh-frozen tissue sections. In HCC, ISH signals were observed in 10 of 14 (71%) South African and in 32 of 40 (80%) Chinese patients (Table 1). When adjacent nontumor liver was analyzed, 8 of 14 (57%) South African and 25 of 40 (63%) Chinese patients had detectable but weaker signals (Table 1). Uninfected liver showed faint ISH signals in less than 10% of the cells, while an irrelevant probe on HCC tissues resulted in no signal (data not shown). Hence, the number of ISH positive patients and proportion of positive cells was greater in HCC compared to nontumor liver.
ISH results were then verified by imunohistochemistry. Anti-VEGFR-3L+S staining significantly correlated with ISH in HCC tissue from South African (χ2 = 10.1; P < 0.005) and Chinese (χ2 = 14.4; P < 0.001) patients (Table 1). Significant relationships were also observed in nontumor liver from South African (χ2 = 5.09; P < 0.025) and Chinese (χ2 = 11.2; P < 0.001) patients, although the expression was faint and in relatively few cells compared to tumor. These observations provide cross-validation of ISH and staining, and suggest that VEGFR-3 is up-regulated at both the RNA and protein levels in HCC.
Relationship Between VEGFR-3 and HBxAg Expression In Vivo.
To determine whether VEGFR-3 is up-regulated in vivo, consecutive tissue sections were stained for VEGFR-3 and for HBxAg. Co-staining was observed in HCC from 6 of 14 (43%) South African patients (χ2 = 5.83; P < 0.02), and in 28 of 40 (70%) Chinese patients (χ2 = 13.3; P < 0.001). In nontumor liver, co-staining was seen in 9 of 14 (64%) South African patients (χ2 = 0.32; P > 0.5), and in 15 of 40 (38%) Chinese patients (χ2 = 2.2; P > 0.1) (Table 1). VEGFR-3 and HBxAg staining was cytoplasmic in HCC cells (Fig. 2A and 2B, respectively). Staining with preimmune rabbit serum was negative (Fig. 2C). No signal was observed without secondary antibody, or when staining was conducted after preincubation of the primary antibodies with the synthetic peptides used for immunization (data not shown). Weak VEGFR-3 staining, in less than 10% of cells, was found in the cytoplasm of hepatocytes from uninfected liver (Fig. 2D) and in both tumor and nontumor liver from 6 of 6 patients with hepatitis C virus associated HCC (data not shown, but similar to Fig. 2D). No membranous staining for VEGFR-3 was observed in any liver or tumor sections. Hence, HBxAg is associated with up-regulated VEGFR-3 expression in HCC but not in nontumor liver.
Relationship Between VEGFR-3 and PCNA In Vivo.
Among Chinese patients, there was a correlation between VEGFR-3 and PCNA in tumor (χ2 = 20.83; P < 0.001) but not in nontumor liver (χ2 = 0.005; P > 0.9). Among South African patients, the two markers also correlated in tumors (χ2 = 5.83; P < 0.02) but not in nontumor liver (χ2 = 0.31; P > 0.5) (Table 1). Hence, elevated VEGFR-3 is associated with increased tumor growth.
VEGFR-3S is Up-regulated in HCC.
Table 1 and Fig. 2 show that up-regulated VEGFR-3L+S is mostly in HCC cells. When staining was repeated with anti-VEGFR-3L, weak signals were observed in most HCC (Fig. 2F) and in nontumor liver (Fig. 2G). In contrast, northern blotting showed that VEGFR-3S mRNA predominated in tumor, while VEGFR-3L mRNA was more abundant in nontumor liver (Fig. 2H). When western blotting was conducted with anti-VEGFR-3L, the levels were low and similar in paired tumor and nontumor liver (Fig. 2I). However, when anti-VEGFR-3L+S was used, strong signals were observed in tumors, and weaker or no signals in liver (Fig. 2I), suggesting that VEGFR-3S is selectively up-regulated in HCC. These results were similar to those in cell culture (Fig. 1).
Importantly, up-regulated VEGFR-3S in HCC was inversely related to patient survival (Fig. 3). Among Chinese patients, the mean survival time for those with VEGFR-3 positive tumors was 22 months, while for those with VEGFR-3 negative tumors, it was greater than 60 months (P < 0.001). The combined up-regulation of VEGFR-3 and PCNA suggest that reduced patient survival is associated with a more rapid tumor growth.
VEGFR-3 Stimulates Cell Growth in Tissue Culture.
HepG2-VEGFR-3L and HepG2-VEGFR-3S cells were prepared, the levels of VEGFR-3 verified by western blotting, and growth was then assayed relative to HepG2CAT and HepG2X cells. Compared to HepG2CAT cells (Fig. 4A, lane 1), the levels of VEGFR-3 were 4.6 ± 0.8 fold higher in HepG2-VEGFR-3L cells (Fig. 4A, lane 2) and 5.3 ± 1.4 fold higher in HepG2-VEGFR-3S cells (Fig. 4A, lane 3). VEGFR-3S stimulated the growth of HepG2 cells in medium containing 10% serum (Fig. 4B) but not in serum free medium (Fig. 4C), while VEGFR-3L did not (Fig. 4B,C). This was verified by flow cytometry. At 24 hours after the release of synchronized cultures, 38.3% of HepG2X cells were in S phase compared to 21.5% of HepG2CAT cells (P < 0.01) (Fig. 4D,E), 31.4% of HepG2-VEGFR-3S (Fig. 4F) and 19.1% of HepG2-VEGFR-3L cells (Fig. 4G). No evidence of DNA degradation was observed (Fig. 4D–G), suggesting that VEGFR-3S and HBxAg stimulated cell cycle progression.
VEGFR-3S Promotes Growth in Soft Agar and Tumor Formation in Nude Mice.
Both HBxAg and VEGFR-3S stimulated growth in soft agar more than 8-fold above background (P < 0.001), while VEGFR-3L over-expressing cells did not (Table 2). HBxAg and VEGFR-3S also accelerated the appearance and size of tumors recovered from nude mice (Table 3). VEGFR-3L also accelerated tumor onset, but the effect was weak (Table 3). Hence, VEGFR-3S and HBxAg stimulated anchorage-independent growth and tumor formation.
Table 2. Growth of HepG2 Cells Over-expressing VEGFR-3L or VEGFR-3S in Soft Agar
The column to the right of “onset of tumor” lists the P values calculated from comparisons of HepG2X or HepG2-VEGFR-3 cell cultures to the HepG2CAT control.
The column to the right of “average size of tumor” compares the tumor sizes for each culture with that of the HepG2CAT cells.
43 ± 3
0.8 ± 0.4
30 ± 4
P < 0.01
1.6 ± 0.3
P < 0.01
13 ± 1
P < 0.001
2.8 ± 0.5
P < 0.005
35 ± 4
P < 0.05
1.2 ± 0.3
P > 0.5
Effect of VEGFR-3 siRNA on the Growth of HepG2X Cells.
Given that HBxAg promotes growth by many pathways,3, 4 it is not clear that elevated VEGFR-3S is rate limiting. To test this, HepG2X cells were transiently transfected with VEGFR-3 specific or irrelevant siRNAs. Two VEGFR-3 specific siRNAs reduced VEGR-3S protein levels by 4–6 fold (Fig. 4H, lanes 4 and 5), while controls were not inhibitory (Fig. 4H, lanes 1–3). When growth was assayed, only cells transfected with VEGFR-3 specific siRNAs were inhibited (Fig. 4I, curves 4 and 5). Hence, up-regulated expression of VEGFR-3S contributes importantly to how HBxAg stimulates cell growth.
Pathways Associated with VEGFR-3 Signaling.
Given that VEGFR-3 and HBxAg may signal through Akt,22, 32 experiments were designed to ask whether HBxAg activation of Akt was mediated through up-regulation of VEGFR-3S. Western blotting showed no difference in the total levels of Akt in the cultures tested (Fig. 5A). However, activated (phosphorylated) Akt was observed in HepG2X and HepG2-VEGFR-3S cells (Fig. 5B). Given that PTEN blocks Akt by inhibiting PI3K, levels of phosphorylated (inactive) PTEN were assayed. Inactivation of PTEN was observed in HepG2X and HepG2-VEGFR-3S cells (Fig. 5C). Since ras also stimulates Akt through phosphorylated raf, the latter was assayed. The results showed that HBxAg and VEGFR-3S stimulated raf phosphorylation by 3-5 fold (Fig. 5D). Hence, VEGFR-3S stimulates Akt signaling by inhibition of PTEN and activation of ras. None of these changes were observed in HepG2CAT or HepG2-VEGFR-3L cells (Fig. 5B–D). Importantly, Akt inhibitor IV blocked the ability of HBxAg and VEGFR-3S to stimulate growth (Fig. 5F), thereby showing the relevance of Akt activation to the growth and survival of HepG2X cells through up-regulation of VEGFR-3S.
Treatment of HepG2X Cells with VEGF-C or Anti-VEGFR-3.
The cytoplasmic staining of VEGFR-3 in HCC cells (Fig. 2) suggests that signaling may be ligand independent. To test this, HepG2X cells were treated with exogenous VEGF-C. The results showed modest stimulation of growth by exogenous VEGF-C in serum free medium on days 2 and 3 (Fig. 6A,B) and mild suppression of growth on day 1 after addition of anti-VEGF-C (Fig. 6C,D), suggesting ligand dependent signaling. However, western blotting of cell lysates demonstrated VEGF-C (and VEGF-D) (data not shown), suggesting that signaling may also be activated by autocrine stimulation. This combined data is compatible with activated VEGFR-3 signaling through both ligand dependent and independent mechanisms.
In this report, HBxAg correlated with the selected up-regulation of VEGFR-3S in HepG2 cells (Fig. 1) and in HCC (Fig. 2, Table 1), suggesting that VEGFR-3S is a natural effector of HBxAg. Although VEGFR-3 is elevated in other tumor types,14, 31, 33 this is the first report where VEGFR-3 up-regulation is associated with the “oncogene” of a human cancer virus.
The findings that HBxAg and VEGFR-3S promote HepG2 growth and cell cycle progression (Fig. 4), growth in soft agar (Table 2), and tumorigenesis (Table 3), suggest that VEGFR-3S may carry out some of the functions of HBxAg. This is supported by the fact that VEGFR-3 siRNAs partially inhibited the ability of HBxAg to promote hepatocellular growth (Fig. 4H–I). The finding that VEGFR-3S activates Akt (Fig. 5), through the stimulation of ras and by PTEN inactivation, suggests how VEGFR-3S may promote tumorigenesis. The fact that HBxAg also stimulates ras10 and Akt,22 while blocking PTEN,34 implies that these events may be carried out by VEGFR-3S. This is further supported by the finding that inhibition of Akt partially blocks the ability of HBxAg and VEGFR-3S to stimulate growth (Fig. 5F). Hence, up-regulation of VEGFR-3S in HCC highlights the centrality of constitutive PI3K/Akt signaling in anchorage independent growth (Table 2) and spread of HCC.23 This is underscored by the strong inverse correlation between VEGFR-3 over-expression in HCC and the duration of survival post-tumor diagnosis (Fig. 3).
Many tumors up-regulate VEGFR-3, VEGF-C and/or VEGF-D, suggesting that they set up autocrine loops that promote growth.14 In HCC, the lack of membranous VEGFR-3 (Fig. 2), and that exogenous VEGF-C or anti-VEGF-C had a small impact on HepG2X growth (Fig. 6), suggest that stimulated cell growth may be largely ligand independent. In chronic liver disease, oxidative stress triggered by cytotoxic T cells and cytokines promote HBxAg and VEGFR-3 activities.18, 35 Activated HBxAg stimulates src kinase signaling. The latter promotes tyrosine phosphorylation (activation) of VEGFR-3, which then stimulates PI3K.22 Since activated PI3K is important for HCC development and metastases,23 HBxAg may short circuit normal VEGFR-3 signaling by up-regulating VEGFR-3S and constitutively activating the latter through tyrosine phosphorylation. Ligand independent proliferation, associated with cytoplasmic VEGFR-3 localization, would be advantageous for tumor cells arising from hypoxic cirrhotic nodules in the chronically infected liver. Hence, it is likely that both ligand dependent and independent signaling mechanisms may be operative.
The stimulation of ras and inhibition of PTEN by VEGFR-3S and by HBxAg22 results in the activation of Akt (Fig. 5), which promotes cell survival by inhibiting apoptosis. Activated Akt blocks glycogen synthase kinase 3β activity, resulting in the stabilization of β-catenin and cell cycle progression. The latter is up-regulated by HBxAg.36 Activated Akt also blocks BAD, resulting in the release of the anti-apoptotic Bcl-xL. The latter could also be stimulated by ras, through ERK1/2, both of which are also turned on by HBxAg.37 Activated Akt promotes p53 degradation by phosphorylating (activating) mdm-2. HBxAg also activates mdm-2.38 Hence, HBxAg up-regulated VEGFR-3S may constitutively activate PI3K/Akt signaling that promotes tumorigenesis in chronically infected livers.
Finally, the results of this study suggest that VEGFR-3S may be an important therapeutic target in tumor bearing patients. For example, a protein tyrosine kinase inhibitor of all VEGFRs is in Phase III clinical trials for metastatic colorectal cancer,39 while thienopyrimidine ureas have shown potent activities against VEGFRs.40 Gefitinib and erlotinib also show promising results in clinical trials,41 and other classes of small molecule VEGFR antagonists are under development,42 suggesting that they may be useful for many patients with HCC.