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The Clinicopathological Significance of Heparanase and Basic Fibroblast Growth Factor Expressions in Hepatocellular Carcinoma

The Second Department of Surgery, Shimane Medical University, Izumo 693-8501, Japan

	ABSTRACT

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Heparan sulfate plays an essential role for insolubility of the components of extracellular matrix and represents a storage depot for various growth factors. Therefore, heparanase produced by a given tumor may facilitate tumor invasiveness and angiogenesis through the release of heparan sulfate-bound growth factors. Although the growth factors responsible for angiogenesis in hepatocellular carcinoma (HCC) have recently been investigated, the clinicopathological significance of heparanase in connection with basic fibroblast growth factor (bFGF) expression in HCC has not been evaluated so far.

Fifty-five patients who had undergone hepatic resection for HCC without preoperative treatment were included in the present study. Expression of heparanase mRNA was evaluated by reverse transcription-PCR, and bFGF was examined by Western blotting using a monoclonal antibody. Tumor angiogenesis was evaluated by immunostaining with a factor VIII-related monoclonal antibody. Expression of heparanase mRNA was detected in 47% of HCCs and was significantly correlated with larger tumor size (P = 0.01), presence of portal vein invasion (P = 0.01), and higher overall tumor invasiveness (P = 0.02). Moreover, its expression was correlated with tumor microvessel density (MVD; P = 0.02). There was a direct correlation between the levels of bFGF proteins and MVD in HCCs (P = 0.0001), and, furthermore, concomitant expression of bFGF and heparanase was associated with higher tumor MVD as compared with expression of either factor alone (P = 0.01).

In conclusion, the expression of heparanase in HCC enhances growth, invasion, and angiogenesis of the tumor, and bFGF seems to be a potent angiogenic factor for HCC.

	INTRODUCTION

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Tumor cell invasion and metastasis is the hallmark of malignant disease and the greatest impediment to cancer cure. Two essential processes required for metastasis are neoangiogenesis and tumor cell invasion of the basement membrane and extracellular matrix (1) . H-S² is an essential component of the extracellular matrices of most tissues and is also a prominent component of blood vessels, which is essential for insolubility of the extracellular components, cell adhesion, and locomotion (2, 3, 4, 5, 6, 7, 8, 9, 10) . Accordingly, cleavage of H-S by heparanase enzyme may play a decisive role in extravasation and invasion of tumor cells. So far, heparanase activity has been detected in various tumors and was found to correlate with their metastatic potentials (2 , 11, 12, 13, 14, 15) . Meanwhile, heparanase may also contribute to angiogenesis by releasing the H-S-bound growth factors such as bFGF (6) . However, because the characterization and cloning of the enzyme has remained elusive until the recent reports of Hullet et al. (2) and Vlodavsky et al. (6) , studies aiming at detection and evaluation of heparanase production and its in vivo biological role in patients with different malignancies, including HCC, has been hindered. Moreover, many study groups including our institute have evaluated the role of different growth factors aiming at elucidation of the biological predictor of angiogenesis in HCC. However, neither of these studies has identified such factors (16, 17, 18) .

bFGF is a potent angiogenic growth factor that requires heparin or H-S for its biological activity mediated through tyrosine kinase signaling (19, 20, 21, 22, 23) . The activity of bFGF is stringently controlled because it can be inactive in normal tissues and becomes activated on tissue injury, inflammation, and tumor invasion (24) . Heparanase enzyme possesses the ability to activate bFGF through structural modulation of the cell surface H-S proteoglycan (25) . Accordingly, heparanase and bFGF could play complementary biological activity in tumor angiogenesis and invasion. To our knowledge, expression of heparanase and its biological role in connection with bFGF expression in HCC have not been evaluated so far. In the present study, we tried to find out whether both bFGF and heparanase were directly correlated to angiogenesis in HCC, whether heparanase expression was associated with the degree of tumor invasiveness or not, and whether the coexpression of heparanase and bFGF enhances the tumor angiogenesis compared with expression of either factor alone.

	PATIENTS AND METHODS

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Patients.
Fifty-five patients (43 males and 12 females) underwent curative hepatic resection for HCC between March 1996 and December 1999, and were included in the present study. None of the patients had received preoperative chemo- or embolic therapy. The patient’s ages ranged from 32 to 77 years (61 ± 7 years, mean ± SD). Liver cirrhosis was detected in 37 patients, and the remaining 18 patients had chronic hepatitis. The etiologies of underlying liver diseases were hepatitis C in 26 patients, hepatitis B in 17, mixed viral infection in 3, alcoholic cirrhosis in 2, cirrhosis of nonidentified etiology in 2 patients, and cryptogenic hepatitis in 5 patients. Thirty-five patients were in Child’s class A, 16 in class B, and four patients in class C. The degree of HCC invasiveness was verified according to our invasiveness scoring system (26) .

Tissue Selection.
HCC tissues from all of the patients were selected from the most viable areas of tumor immediately after surgical resection. This aimed at excluding areas of tissue necrosis and hemorrhages, which may influence the density of microvessels as well as the quality and the quantity of the extracted RNA and protein. For selection of surrounding liver tissues, specimens were obtained from tissues at a clear distance from the tumor edge (>1 cm), if there was no evidence of nearby tumor invasion. Twenty-three surrounding liver tissues were included in the current study. The normal liver tissues were obtained from nine patients with no evidence of liver disease and with informed consent. All of them were HCV- and HBV-negative serologically. Five patients were operated on for early gastric cancer, two for esophageal cancer, one for colon cancer, and two for chronic cholelithiasis. None of the patients had hepatic metastasis or liver tumors during, and for at least one year after, surgery. Tissues were snap-frozen immediately after the resection and kept at -80°C until the time of the experiment.

RNA Extraction and cDNA Synthesis.
Fifty to 100 mg of tumor or normal liver tissue were used for total RNA isolation using TRIzol reagent (Life Technologies, Inc., Rockville, MD), according to the recommendations of the manufacturer. First-strand cDNA was synthesized by priming 1 µg of total RNA with oligo(dT)₁₆ primer in a 20-µl reverse transcription mixture containing 4 µl of 5x first-strand buffer, 2 µl of dNTP mix containing 25 mM each deoxynucleotid triphosphate base (Pharmacia Biotech, Tokyo, Japan), 2 µl of 0.1 M dithiothreitol, and 200 units of Moloney murine leukemia virus RT (Life Technologies, Inc.).

PCR Amplification of Heparanase and ß-Actin Genes.
The resulting cDNA was used for PCR amplification using Taq polymerase (Takara Biochemicals, Ohotsu, Japan). The sequence of the oligonucleotides used for heparanase was: forward, 5'-TTC GAT CCC AAG AAG GAA TCA AC-3'; and reverse, 5'-GTA GTG ATG CCA TGT AAC TGA ATC-3' (6) . The primers used for ß-actin had the following sequence: forward, 5'-GCT CTC TTC CAA CCT TCC TT-3'; and reverse, 5'-TGG AAG GTG GAC AGC GAG GC-3', as generated by the Oligo 4.0 S computer software. The PCR conditions included initial denaturation at 96°C for 2 min, followed by 28 cycles of amplification with subsequent denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension for 1 min at 72°C. Eight µl of the PCR product underwent electrophoresis using 1.5% agarose and was visualized by UV absorption and ethidium bromide.

Western Blotting Evaluation of bFGF.
Tissues were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 400 mM NaCl, 1 mM EDTA, and aprotinin, at final concentration of 2 µg/ml. Nuclei and cell debris were removed by centrifugation at 16,000 x g for 10 min at 4°C. After the measurement of the protein contents in the supernatant extract, a total of 100 µg of protein were loaded and separated by 10% SDS/PAGE and then were transferred to nitrocellulose membrane (Millipore Corporation, Danvers, MA). Blocking of the membrane was performed in 2% skim milk. The Western blots were probed using a monoclonal mouse antibody raised against recombinant bFGF of human origin [b-FGF (Ab-3); Oncogene, Cambridge, England]. The second antibody used for development was a horseradish peroxidase-labeled rabbit antimouse IgG (1:1000) (Medical & Biological Laboratories Co. LTD.). The immunoreactive proteins were visualized by enhanced chemiluminescence using the ECL Western blot detection system (Amersham Pharmacia Biotech, Buckinghamshire, England) according to the recommendations of the manufacturer. All of the blots were performed including an HCC sample, which was used as a positive control for each reaction and also was used to stabilize the quantification among different blots.

Immunostaining for Factor VIII.
Using paraffin-embedded tissue sections, immunohistochemistry was performed according to the avidin-biotin peroxidase complex method, as described previously (16) . Briefly, deparaffinized rehydrated sections were treated in 0.1% trypsin (Sigma Chemical Company, St. Louis, MO) for 30 min at room temperature. Staining for vascular endothelial cells were obtained by mouse monoclonal antihuman von Willebrand factor at a final dilution of 1:50 (factor VIII-related antibody; Dako A/S, Glostrup, Denmark). The incubation time for the first antibody was 1 h at room temperature. The steps of immunohistochemistry were conducted using Histofine, SAB(M) kit (Nichirei Corporation, Tokyo, Japan) according to the recommendations of the manufacturer and as mentioned previously (16) .

Evaluation of MVD.
Evaluation of MVD was performed as mentioned previously (16) . Briefly, after microscopic screening for tumor areas of highest MVD at x40, counting the vessels in five areas with highest MVD under x200 magnification was performed by two independent observers without knowledge of the patient’s background. The mean value of the vessels counted in five fields by the two observers was considered the MVD for the tumor. Generally, there was no interobserver significant differences, and in cases with wide differences, MVD were reevaluated by a third observer until the three observers reached an agreement.

Statistical Analysis.
Statistical comparisons for significance between nominal variables were evaluated by the ² test and the Fisher’s exact probability test. Student’s t test was used when comparing two means. Simple regression analysis was used to test the correlation between two continuous variables. Statistical analyses were performed using Stat View 4.51 computer software (Abacus Concepts, Inc., Berkeley, CA) and Ps of less than 0.05 were considered statistically significant.

	RESULTS

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Expression and Clinical Significance of Heparanase.
Heparanase mRNA was not detectable in any of the normal liver samples after 28 cycles of amplification. Surrounding liver tissues generally had either a faint or nondetectable heparanase expression; however, six specimens of surrounding tissues had a clear expression comparable to those in positive HCC (Fig. 1) .

View larger version (27K): [in this window] [in a new window]   Fig. 1. Detection of heparanase mRNA by RT-PCR in surrounding liver (S) and HCC (T); ß-actin was used as a positive control.

Twenty-six (47%) HCCs were heparanase-positive and 29 (53%) tumors had a nondetectable level of heparanase mRNA. Expression of heparanase was significantly more frequent in tumors larger than 5 cm (Fisher’s test, P = 0.01). Heparanase expression was also associated with a higher degree of tumor invasiveness. Portal vein invasion was significantly higher in heparanase-positive tumors compared with heparanase-negative tumors (Fisher’s test, P = 0.01). Furthermore, besides portal vein invasion, heparanase-positive HCCs had more than one feature reflecting tumor invasiveness, including absence of tumor capsule, capsule invasion, portal vein invasion, intrahepatic metastasis, hepatic venous invasion, and serosal invasion, when evaluated for the overall degree of tumor invasiveness. Eighty-five % of heparanase-positive HCCs were classified as moderately invasive (n = 6) or highly invasive tumors (n = 16). The values were significantly higher compared with those for heparanase-negative tumors (Fisher’s test, P = 0.02). However, there was no significant correlation between heparanase expression and the presence of tumor capsule, capsule invasion, or intrahepatic metastasis when they were evaluated as separate events (Table 1) . The incidence of heparanase expression was significantly higher in HCV-related HCC (n = 16 versus 10) compared with HCV-negative patients (n = 10 versus 17; Fisher’s test, P = 0.047).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Detection of bFGF protein by Western blotting using monoclonal antibody in surrounding liver (S), HCC (T), and the standard sample (Stand.).

View larger version (133K): [in this window] [in a new window]   Fig. 3. Examples of MVD as evaluated by immunostaining with monoclonal antihuman von Willebrand factor (factor VIII). Microvessels were seen mainly in the intra- and pretumor connective tissue septa (A; x40) and in the tumor parenchyma (B; x200).

View larger version (19K): [in this window] [in a new window]   Fig. 4. The correlation between MVD and the protein levels of bFGF in HCC as evaluated by the simple regression model. Tumor MVD was directly correlated with the level of bFGF in HCC with adjusted r2 = 0.23, correlation coefficient = 0.496, and two-tailed probability; P = 0.0001.

Furthermore, the effect of heparanase and bFGF in the MVD of HCC in patients with liver cirrhosis was evaluated. Despite the small number of patients in these statistical analyses (n = 37), the level of bFGF showed a modest significant linear correlation with the MVD in HCC of such patients (adjusted r² = 0.086; correlation coefficient = 0.334; and 2-tailed P = 0.043). Moreover, tumors with positive heparanase expression were associated with higher MVD [high MVD (n = 15) versus low MVD (n = 3)] compared with that in heparanase-negative HCCs [high MVD (n = 10) versus low MVD (n = 9; Fisher’s test, P = 0.049]. This indicated that the presence of liver cirrhosis per se did not influence the angiogenic role of bFGF or of heparanase.

	DISCUSSION

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H-S protoglycans are present in the basement membrane of every vascularized organ and in the tumor stroma of several human cancers (27) . It is expressed intensely in the liver sinusoids and in the blood vessels of portal triads (28) . A major function of the proteoglycans is attributed to the properties of H-S (29) , which is essential for insolubility of the extracellular components, cell adhesion, and locomotion (6, 7, 8, 9, 10) . H-S also works as a storage depot for active growth factors, of which bFGF is the most extensively studied (30) . Thus, acquisition of heparanase and splitting of H-S by a given tumor would offer such a tumor two essential features of malignancy: (a) solubilization of the other extracellular matrix constituents facilitating tumor invasion through blood vessels and tissues; and (b) releasing and activation of H-S-binding growth factors and, hence, enhancing the tumor angiogenesis.

Our results showed that heparanase mRNA was detectable in 47% of the HCCs according to the method of Vlodavsky et al. (6) and that its expression was closely associated with a higher degree of HCC invasiveness. Eighty-five % of the heparanase-positive tumors were classified as highly invasive (61%) or moderately invasive (24%) tumors, which indicates the presence of at least two features reflecting tumor invasion, such as portal vein invasion, intrahepatic metastasis, capsule invasion, hepatic vein invasion, and serosal invasion (26) . There was also a direct correlation between portal vein invasion and heparanase expression.

Moreover, heparanase expression was directly correlated with tumor size. Generally, the tumor size reflects tumor growth that is the outcome of many integrated factors, including the availability of enough nutritional support through abundant blood supply (angiogenesis) and of proliferation stimuli from active growth factors. Heparanase may influence the bioavailability of different growth factors including FGFs (31, 32, 33, 34) , VEGF (35) , HGF (36, 37) , and PDGF (38) , which are stored in H-S and possess H-S-binding sequences. It is quite reasonable to assume that the release of such growth factors may influence tumor growth and angiogenesis. In the present study, we tested whether heparanase could influence tumor vascularity or not, and whether there was a direct correlation between heparanase expression and angiogenesis in HCC. Tumors with positive heparanase expression had a significantly higher MVD compared with heparanase-negative tumors. Accordingly, we can conclude that heparanase expression has an axial role not only in the tumor growth and invasion but also in the angiogenesis of HCC.

In the present study, heparanase expression was significantly higher in HCV-related HCCs compared with that in HCV-negative patients. Moreover, high levels of heparanase were detected in six surrounding livers, of which five patients were HCV positive. It is possible to assume that HCV enhances heparanase expression that may be involved in the HCV-related pathological and malignant changes in the liver. However, this possibility is to be further evaluated in the near future.

Despite evaluation of the role of bFGF in different human tumors (39, 40, 41, 42, 43) , its correlation with angiogenesis in HCC was not evaluated except for the study of Mise et al. (44) . In their study, mRNA levels of bFGF were not correlated with tumor angiographic findings. We have previously demonstrated that different angiographic findings were not directly correlated with the MVD in HCC, because the degree of tumor stain is likely to be influenced by different factors, such as arterial supply, specificity of the canulation, lymphatic and venous drainage, and the amount of the contrast materials, besides the MVD (16) . Accordingly, we used MVD as a direct index for tumor neovascularization. In the present study, the level of bFGF protein was significantly higher in HCC compared with that in the surrounding and normal livers, indicating that its up-regulation was involved in the tumor biology. Moreover, bFGF protein levels were directly correlated with the MVD of HCC. bFGF induces neovascularization through various mechanisms including a potent mitogenic effects on the vascular and capillary endothelial cells (19 , 22) , stimulation of endothelial migration and capillary formation, and production of plasminogen activators, proteases that are involved in the invasive property of endothelial cells during angiogenesis (45) .

It has been reported that the potency of bFGF for stimulating angiogenesis in vitro is superior to that of VEGF in terms of molecular weight (46) . In their study, they showed that both factors together have a potent synergetic action in angiogenesis. In that sense, bFGF could be expected to exert more potent angiogenic response than does VEGF, because, besides its independent angiogenic potency, it retains the ability to up-regulate VEGF secretion (47) . This may partially explain the lack of direct correlation between VEGF and angiogenesis in HCC in the previous studies (16 , 18) and the presence of positive correlation between bFGF and HCC angiogenesis in the present study.

In the current study, there was an obvious synergetic effect of heparanase and bFGF tumor expression. Coexpression of both factors was associated with higher MVD compared with expression of either factor alone. Such synergetic action may be attributed to the direct ability of heparanase to solubilize the components of extracellular matrix, which enhances endothelial cell migration during neovascularization. On the other hand, heparanase increases the biological activity of bFGF as well as of other H-S-bound growth factors like VEGF, PDGF, and HGF (31, 32, 33, 34, 35, 36, 37, 38) , which are expected to further enhance the angiogenic effects.

In conclusion, our study demonstrated the biological importance of heparanase expression in HCC. The expression of heparanase was found to influence different malignant behaviors in HCC including growth, invasion, and angiogenesis. Moreover, we elucidated that bFGF was a significant angiogenic factor for HCC and that coexpression of both bFGF and heparanase showed a synergetic effect on angiogenesis in HCC. These findings may suggest that targeting the activity of heparanase may be a beneficial antitumor therapy for HCC.

	FOOTNOTES

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

¹ To whom requests for reprints should be addressed, at Second Department of Surgery, Shimane Medical University, Izumo 693-8501, Japan. Phone: 81-853-20-2232; Fax: 81-853-20-2229.

² The abbreviations used are: H-S, heparan sulfate; FGF, fibroblast growth factor; bFGF, basic FGF; HCC, hepatocellular carcinoma; MVD, microvessel density; VEGF, vascular endothelial growth factor; HCV, hepatitis C virus; HBV, hepatitis B virus; HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor.

Received 9/ 5/00; revised 12/ 7/00; accepted 1/ 2/01.

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