Expression of betacellulin and epidermal growth factor receptor in hepatocellular carcinoma: implications for angiogenesis

Human Pathology (2006) 37, 1324 – 1332

www.elsevier.com/locate/humpath

Original contribution

Expression of betacellulin and epidermal growth factor receptor in hepatocellular carcinoma: implications for angiogenesisB Woo Sung Moon MD, PhDa,d,*, Ho Sung Park MDa,d, Ki Hoon Yu PhDa,d, Min Young Park MSa,d, Kyung Ryoul Kim MSa,d, Kyu Yun Jang MD, PhDa,d, Jong Suk Kim MD, PhDb,d, Baik Hwan Cho MD, PhDc,d a

Department of Pathology, Research institute of Clinical Medicine, Chonbuk National University, Medical School, Jeonju 560-181, South Korea b Department of Biochemistry, Research institute of Clinical Medicine, Chonbuk National University, Medical School, Jeonju 560-181, South Korea c Department of Surgery, Research institute of Clinical Medicine, Chonbuk National University, Medical School, Jeonju 560-181, South Korea d Center for Healthcare Technology Development, Jeonju 561-756, South Korea Received 3 November 2005; revised 13 March 2006; accepted 27 April 2006

Keywords: Carcinoma; Hepatocellular neovascularization; Pathologic betacellulin receptor; Epidermal growth factor

Summary Hepatocellular carcinoma (HCC) is becoming one of the common malignant tumors worldwide and is characterized by high vascularity. Angiogenesis (formation of new microvessels) is critical for growth and progression of various human solid tumors. Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family, and its signal action is mediated through EGF receptors (EGFR). In this study, to understand the role of BTC in relation to EGFR in HCC, we examined localization, expression, and involvement in angiogenesis of BTC and EGFR. The results revealed that expression of BTC, EGFR, and tumor growth factor–a messenger RNA in HCC was increased by 80%, 60%, and 40%, respectively, as compared with those in the nontumorous tissues. Increased expression of BTC protein was observed in 31 (61%) of 51 HCC specimens, and the level of tumor growth factor–a protein was higher in 17 (33%) of 51 HCC specimens than in nonmalignant hepatocytes. Betacellulin was predominantly expressed in HCC cells, whereas EGFR was observed in sinusoidal endothelial cells of HCC in 25 tumors (49%). Betacellulin was secreted in all 4 examined HCC cell lines. The HCC specimens showing positive EGFR expression in tumor endothelial cells had a significantly higher microvessel density than those without EGFR expression ( P b .005). A strong correlation was found between BTC

B

This work was supported by the Regional Research Centers Program of the Korean Ministry of Education and Human Resources Development through the Center for Healthcare Technology Development, Jeonju, South Korea, and a research fund of Chonbuk National University Hospital Research Institute of Clinical Medicine, Jeonju, South Korea. * Corresponding author. Department of Pathology, Chonbuk National University, Medical School, Jeonju-si, Jeollabuk-do 560-181, South Korea. E-mail address: [email protected] (W. S. Moon). 0046-8177/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.04.022

Expression of BTC and EGFR in HCC

1325

expression in cancer cells and EGFR expression in tumor endothelial cells ( P b .001). These findings suggest that overexpression of BTC by HCC cells and EGFR by tumor endothelial cells enhance vascularity in a paracrine manner. D 2006 Elsevier Inc. All rights reserved.

1. Introduction Hepatocellular carcinoma (HCC) is becoming one of the most common malignant tumors worldwide and is characterized by its high vascularity. Angiogenesis, the formation of new microvessels, is critical for the growth and progression of various human solid tumors because it enables the delivery of oxygen and nutrients [1,2]. Growth factors regulate the development of cancer through several mechanisms. These include uncontrolled cell growth that is attributable to the autocrine production of growth factors by the cancer cells and the stimulation of tumor neovascularization because of paracrine stimulation of the endothelial cells by the angiogenic growth factors secreted by the cancer cells [2-6]. The development of blood vessels in a tumor is regulated by the production of several growth factors and growth inhibitors [2,5]. Enhanced expression of various angiogenic factors in human cancers is directly correlated with increased neovascularization, as is measured by the microvessel density (MVD), within the tumor [7-9]. Moreover, the density of the microvessels in the areas of the most intense neovasularization has been demonstrated to be an independent prognostic marker for HCC [9,10]. The ErbB family of type I receptor tyrosine kinases has 4 members: epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 [11,12]. All the ErbB family members share common features including an extracellular ligandbinding domain, a transmembrane portion, and an intracellular tyrosine kinase domain. Originally, the epidermal growth factor (EGF) system was considered to include only 1 receptor (EGFR/HER1) and 1 ligand (EGF) yet. Five additional ligands have been identified during the last decade. These ligands include tumor growth factor (TGF)–a, amphiregulin, betacellulin (BTC), heparin-binding EGF-like growth factor, and epiregulin. Binding of any of the 6 ligands to EGFR induces a specific dimerization between 1 of the 4 receptors, and an intracellular signaling pathway is thereby activated that eventually leads to cell division and differentiation. Aberrant expression of the EGF family members has been implicated in the accelerated growth or initiation of various tumors [12], and recently, antibodies directed against EGFR have been introduced for the treatment of those cancers expressing EGFR [13]. Betacellulin is a member of the EGF family; it efficiently activates all 4 members of the ErbB receptor family, and it binds to EGFR with an affinity similar to that of EGF [14]. Most tumors of an epithelial origin, including HCC, express

multiple ErbB receptors, and they coexpress 1 or more of the EGF-related ligands [12,13,15,16]; this suggests that autocrine or paracrine receptor activation plays a major role in tumor cell proliferation. A recent study has shown that BTC could be a strong angiogenic factor because it induces cell survival, DNA synthesis, migration, and tubule formation in endothelial cells [17]. However, the expression of BTC and EGFR in HCC and its potential relationship to angiogenesis is unknown. The aim of the present study was to correlate the expressions of BTC and TGF-a and their receptor with the rate of proliferation and the degree of angiogenesis, with a special emphasis being placed on the possible stimulation of angiogenesis by the paracrine loops.

2. Materials and methods 2.1. Patients and specimens This study was approved by the Human Ethics Committee of Chonbuk National University Medical School. We retrospectively studied HCC specimens that were obtained from 51 patients who underwent surgical resection between 1990 and 1999 at the Chonbuk National University Hospital. Of the 51 patients with HCC, 38 were men and 13 were women. The mean age of patients was 54 years (age range, 29-76 years). The mean size of tumor was 4.4 cm (size range, 1.2-16 cm). Grade of the HCC differentiation was classified into 2 groups: low-grade, including Edmondson grades 1 and 2, high-grade, including Edmondson grades 3 and 4. The histologic features of the tumor and adjacent nontumorous liver are summarized in Table 1. Scoring of the nontumorous liver followed modified histologic activity index grading by Ishak [18]. Ten pairs of fresh HCC specimens and their adjacent nontumor liver tissues were obtained and preserved in liquid nitrogen for reverse transcriptase–polymerase chain reaction (RT-PCR).

2.2. Hepatocellular carcinoma cell lines The human HCC cell lines HLE and Huh-7 were purchased from the Health Science Research Resources Bank (Osaka, Japan), and HepG2 was purchased from the American Type Culture Collection (Manassas, VA). In addition, we used the sarcomatoid HCC cell line, designated as SH-J1, which was established by our efforts [19]. The HepG2, HLE, and Huh-7 cell lines were cultured according to the cell bank’s instructions.

1326 Table 1 Age (y) Sex Etiology

Nontumor

W. S. Moon et al. The clinicopathologic features of 51 cases N50 V50 Male Female HBV HCV Alcohol Unknown Piecemeal necrosis

Focal lytic necrosis

Portal inflammation

Fibrosis

Tumor

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 5 6

N5 cm V5 cm Low-grade High-grade Vascular invasion

33 18 38 13 31 5 5 10 2 19 20 10 8 24 16 3 11 23 17 0 0 9 7 12 6 17 26 25 33 18 10

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus.

transferred into a polymerase chain reaction mixture for the EGFR, TGF-a, and BTC. The reaction mixture consisted of 50 mmol/L KCl, 10 mmol/L Tris-HCl, 2 mmol/L MgCl2, 1.3 pmol/L primers, and 1 U Taq DNA polymerase in a total volume of 50 lL. Specific primers for the EGFR, TGF-a, and BTC were designed as follows: the EGFR upper primer was 5V-ATGTCCGGGAACACAAAGAC-3V; the EGFR lower primer was 5V-TTCCGTCATATGGCTTGGAT-3V; the TGF-a upper primer was 5V-TCAGTTCTGCTTCCATGCAACC-3V; the TGF-a lower primer was 5V-TTTCTGAGTGGCAGCAAGCG-3V; the BTC upper primer was 5V-TTCACTGTGTGGTGGCAGATGG-3V; and the BTC lower primer was 5V- ACAGCATGTGCAGACACCGATG-3V. Polymerase chain reaction amplifications were performed as follows: for the EGFR, denaturation was done at 958C for 25 seconds, annealing was done at 608C for 35 seconds, and polymerization was done at 728C for 35 seconds; for the TGF-a, denaturation was done at 948C for 60 seconds, annealing was done at 608C for 60 seconds, and polymerization was done at 728C for 60 seconds; for the BTC, denaturation was done at 948C for 60 seconds, annealing was done at 578C for 60 seconds, and polymerization was done at 728C for 60 seconds. All the experiments were performed using conditions that were optimized for linear amplification. The EGFR was amplified for 25 cycles, the TGF-a for 30 cycles, and the BTC for 35 cycles. The polymerase chain reaction products were then subjected to electrophoresis on a 1.5% agarose gel in a Trisacetate EDTA buffer containing 1 lg/mL ethidium bromide, and the results were photographed. For the semiquantitative analysis of the messenger RNA (mRNA), densitometric analysis was done. The density for mRNA against b-actin was calculated.

2.3. RNA preparation and messenger RNA analysis by RT-PCR

2.4. Immunohistochemistry

The total cellular RNA was extracted from 10 pairs of fresh HCCs and their adjacent nontumor liver tissues with using TRIzol Reagent (Life Technologies, Inc, Carlsbad, CA), according to the manufacturer’s instruction. The RNA was further purified using chloroform treatment, and it was precipitated using 95% ethanol before the complementary DNA synthesis. The quality of the isolated RNA was verified by electrophoresis on 1.0% agarose-formaldehyde gels, and its quantity was determined by measuring its absorbance at 260- and 280-nm wavelengths. The singlestranded complementary DNA was synthesized using a RTPCR kit (GeneAmp RNA-PCR kit, Perkin-Elmer, Foster, CA). Briefly, the RNA (5 lg) was incubated in a reaction mixture containing 5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl, 1 mmol/L dNTP, 2.5 mmol/L random hexamers, and 2.5 U/mL MULV reverse transcriptase in 20 lL for 10 minutes at room temperature for the extension of the hexameric primers, for 1 hour at 428C for the annealing, and then for 10 minutes at 958C to denature the enzyme. The reverse-transcribed mixture (4 lL) was

For immunohistochemical staining, the DAKO Envision system that used dextran polymers conjugated with horseradish peroxidase (DAKO, Carpinteria, CA), was used to avoid any endogenous biotin contamination. Briefly, after deparaffinization, the tissue sections were treated with a microwave antigen retrieval procedure in 0.01 mol/L sodium citrate buffer for 10 minutes. After blocking the endogenous peroxidase, the sections were incubated with Protein Block Serum-Free (DAKO) at room temperature for 10 minutes to block the nonspecific staining, and then the sections were incubated for 2 hours at room temperature with anti-BTC (1:50, Santa Cruz Biotechnology, Santa Cruz, CA), TGF-a (1:100, Santa Cruz Biotechnology), EGFR (1:1000, Sigma, Saint Louis, MO), CD34 (an endothelial cell marker in HCC) (1:50, DAKO), and proliferating cell nuclear antigen (PCNA) (1:50, DAKO) antibodies. The peroxidase activity was detected with the enzyme substrate 3-amino-9-ethyl carbazole. For the negative controls, the sections were treated the same way except that they were incubated with Tris-buffered saline without

Expression of BTC and EGFR in HCC

1327 the liquid substrate-chromogen solution. After quenching the enzyme reaction, the slides were incubated in doublestain block at room temperature for 3 minutes to block endogenous phosphatase. The slides were then incubated with anti-EGFR antibody (1:200, Sigma) for 2 hours at room temperature. After washing, the slides were incubated with labeled polymer-alkaline phosphatase antimouse antibody for 30 minutes at room temperature. Fast red substrate-chromogen solution was used for the visualization of EGFR antibody. The sections were counterstained with Mayer’s hematoxylin. For double immunostaining of CD34 and EGFR, we used the same method previously described [20].

Fig. 1 Expression of BTC, TGF-a and EGFR mRNA in HCC. Consistent with the immunohistochemistry results, the overexpression of BTC, EGFR, and TGF-a mRNA was detected in 80%, 60%, and 40% of the tumor specimens (T), respectively, as compared with the corresponding nontumorous adjacent liver tissues (N).

the primary antibody. For the semiquantification of the BTC and TGF-a expressions, a scoring system was developed by multiplying the intensity of the staining by the area that was stained. The intensity of the cell staining was graded according to the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; and 3+, marked staining. The area of staining was evaluated using the following scale: (0), less than 10% of the cells stained positive; (1+), 10% to 30% of the cells stained positive; and (2+), 30% to 70% of the cells stained positive; (3+), more than 70% of the cells stained positive. The maximum combined score was 9, and the minimum combined score was zero. Because the nonmalignant hepatocytes can also express BTC and TGF-a, the signals in the tumor cells that were at least 1 scale increment (+1) stronger than that of the nonmalignant hepatocytes were defined as positive. The PCNA labeling index (PCNA-LI) was defined as the percentage of nuclei with positive PCNA staining in the total number of tumor cells or in the endothelial cells that were counted, respectively. 2.4.1. Double immunohistochemical stain To determine the relationship between BTC and EGFR expression in HCC, we performed double immunostaining for BTC and EGFR on the same specimens. The tissue sections were deparaffinized, and the slides were treated with the microwave antigen retrieval procedure in 0.01 mol/L sodium citrate buffer for 10 minutes. After blocking the endogenous peroxidase, the sections were incubated with Protein Block Serum-Free (DAKO) at room temperature for 10 minutes to block the nonspecific staining, and then the sections were incubated for 2 hours at room temperature with anti-BTC antibody (1:50, Santa Cruz Biotechnology). After washing, the sections were incubated with a biotin-conjugated secondary antigoat antibody at room temperature for 30 minutes and, finally, with peroxidase-conjugated streptavidin at room temperature for 30 minutes. Peroxidase activity was detected with

2.5. The BTC and EGFR expressions in the HCC cell lines as accessed by Western blotting We examined the BTC and EGFR levels in the 4 different human HCC cell lines by performing Western blotting: the HepG2, HLE, HuH-7 and SH-J1 cell lines. Four different antibodies for EGFR (A204, Sigma; H11, DAKO; E30, DAKO; SC-03, Santa Cruz, CA) were used to examine the expression of EGFR in HCC cell lines. Briefly, the cells were lysed in a buffer solution containing 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaC1, 0.5% Nonidet P-40, 1 mmol/L phenylmethylsulfonyl fluoride, 2 lg/mL leupeptin, 2 lg/mL aprotinin, 5 mmol/L sodium fluoride and 1 mmol/L sodium orthovanadate. The protein concentration of the lysates was determined by performing bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Equal amounts of protein (150 lg) were subjected to 8% sodium dodecyl sulfate– polyacrylamide gel electrophoresis, and then the proteins were transferred to nitrocellulose membranes. The membranes were incubated with BTC and EGFR antibodies at room temperature for 1 hour. The membranes were then washed and incubated with the corresponding anti-IgG peroxidase conjugates at room temperature for 1 hour. The signal of the bound antibodies was visualized by chemiluminescence (Amersham Life Science, Arlington Heights, IL). The membranes were stripped and reprobed with monoclonal anti–b-actin antibody (Sigma, St Louis, MO) as a control for the protein loading and transfer. The A431 cell is known to be positive for BTC and EGFR, and it was used as a positive control. Quantification of the data was Table 2 The density for mRNA against b-actin in tumor samples and normal tissues BTC TGF-a EGFR

N T N T N T

Mean F SE

Pa

0.34 0.56 0.48 0.55 0.41 0.53

.005

F F F F F F

0.03 0.05 0.06 0.08 0.03 0.07

Abbreviations: N, normal tissue; T, tumor tissue. a Student t test.

.500 .124

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W. S. Moon et al.

Fig. 2 Immunohistochemistry for BTC and EGFR. A, BTC expression in HCC tumor cells (T, red brown signals) with no staining on the adjacent nontumorous hepatocytes (NT) (BTC). B, High magnification. Strong cytoplasmic staining for BTC in the tumor cells (BTC). C, High-magnification EGFR staining in the tumor endothelial cells in same area (EGFR). D, In double immunostaining, BTC (brown) expressed strongly in the cytoplasm of tumor cells, and EGFR (red) expressed in the tumor endothelial cells (BTC and EGFR). Note the close spatial relationship between the BTC in the tumor cells and the EGFR in the endothelial cells.

performed using ImageQuant software (Molecular Dynamics, Piscataway, NJ). Each signal was normalized against the corresponding b-actin signal.

2.6. The BTC expression in the conditioned medium of the HCC cell lines To determine whether BTC is secreted by the HCC cells, we performed immunoblotting analysis of the BTC level in the conditioned media. Twenty-four hours after plating, the cultures were washed 4 times with phosphate-buffered saline, and then each culture was incubated with 4 mL of serum-free medium for 6 hours. After 72-hour incubation, the conditioned medium was collected from the cultures, and it was centrifuged at 2000g to remove the cells. The conditioned medium was centrifuged at 10 000g for 10 minutes in a microfuge, and then it was concentrated 10-fold by using a Centricon-10 microconcentrator (Millipore, Billerica, MA). Samples having an equal protein load were analyzed by Western blotting to determine the expression of BTC in the conditioned medium.

Fig. 3 Western blotting for BTC and EGFR in the HCC cell lines. Different levels of BTC were observed in the 4 examined HCC cell lines’ lysates and the conditioned media. Note the absence of EGFR expression in all 4 examined HCC cell lines.

Expression of BTC and EGFR in HCC

1329

2.8. Statistical analysis The comparisons between the expressions of BTC, TGFa, EGFR, and the MVD were made using the Mann-Whitney rank sum test. The comparisons between the expressions of BTC and TGF-a and PCNA-LI were made using the Student t test. Correlations between the expressions of BTC and TGF-a in the cancer cells and the EGFR in the tumor endothelium were tested by the v 2 test. The comparisons between the expressions of BTC, TGF-a, EGFR, and the prognostic factors such as size, degree of differentiation, and vascular invasion, were tested by the v 2 test. The results are presented as means F SE for the MVD and the PCNA-LI. P values less than .05 were considered as significant. Fig. 4 Western blotting for EGFR in the HCC cell lines using 4 different antibodies. None of the 4 HCC cell lines expressed EGFR protein.

2.7. Quantification of the MVD For the measurements of immunoreactivity for CD34, the 4 most highly vascularized areas were selected under 200 magnification. The MVD was expressed as the percentage of the total CD34 stained area (the endothelial cells of the sinusoid and portal blood vessels in the tumor) per the total evaluated section area [21]. For the measurements of the percentage of the CD34 vessels expressing EGFR, the 10 most highly vascularized areas were selected under 400 magnification. Using a double immunostaining and an image analysis system (Soft Imaging System GmbH, Lakewood, CO), the CD34-positive areas and EGFR-positive areas could be calculated by their different immunostaining colors.

3. Results 3.1. Expression of BTC, TGF-a a , and EGFR in the HCC specimens We first evaluated the expression of the BTC, TGF-a and EGFR mRNA in 10 pairs of HCC tissue and their corresponding nontumorous liver tissue by performing RTPCR. The representative gel indicating the BTC, EGFR, and TGF-a mRNA expressions is shown in Fig. 1. The expression of BTC, EGFR, and TGF-a mRNA in the HCCs was increased by 80%, 60%, and 40%, respectively, as compared with those of the corresponding nontumorous liver tissues (Fig. 1, Table 2). The density for mRNA against b-actin showed significant increase of the BTC mRNA in tumor samples than normal tissues (Table 2). Second, we performed immunohistochemistry to verify the expression and localization of the BTC, TGF-a, and EGFR protein. A representative immunostaining is shown in Fig 2. The expression of BTC was increased in 31 (61%) of the 51

Fig. 5 Immunohistochemistry for PCNA and CD34. A, Strong positive reaction for PCNA in the tumor cells as well as in the endothelial cells (arrows) (PCNA). B, Double-staining of CD34 (brown) and EGFR (red) represents the high MVD in the BTC and EGFR positive HCC (CD34 and EGFR).

1330

W. S. Moon et al.

Table 3 Correlation of the MVD (expressed as percentage of the total microvessel area per the total area of four 200 microscopic fields) and the expression of growth factors No. of HCC specimens BTC Positive Negative TGF-a Positive Negative EGFR in tumor EC Positive Negative

MV area (%) (mean F SE) in tumor

Table 4 Correlation between the expression of BTC or TGF-a in HCC and the EGFR expression in the tumor endothelial cells

Pa

31 20

5.1 F 0.5 4.1 F 0.7

.086

17 34

5.0 F 0.9 4.6 F 0.4

.636

25 26

6.0 F 0.7 3.4 F 0.3

.003

EGFR positive EGFR negative Total P no. in tumor in tumor endothelium endothelium 21 4 25

10 16 26

31 20 51

.0008a

TGF-a–positive 9 TGF-a–negative 16 Total no. 25

8 18 26

17 34 51

.692b

BTC-positive BTC-negative Total no.

a

Abbreviations: MV, microvessel; EC, endothelial cell. a Mann-Whitney rank sum test.

HCC specimens, and the expression of TGF-a was enhanced in 17 (33%) of the 51 HCC specimens, compared with the nonmalignant hepatocytes. Both BTC and TGF-a were predominantly expressed in the HCC cells with intense labeling of the cytoplasm (Fig. 2A and B). All of the HCC tumor tissue samples were negative for EGFR in the cancer cells. There was no immunohistochemical expression on macrophages and stromal cells. However, there was strong EGFR immunoreactivity observed in the HCC sinusoidal endothelial cells in 25 tumors (49%) (Fig. 2C), and in 4 cirrhotic liver specimens. The BTC protein expression in the tumor cells also had a close spatial association with the EGFR expression in the tumor endothelial cells (Fig. 2D).

3.2. Epidermal growth factor receptor is not overexpressed in HCC, and BTC is secreted by the HCC tumor cells To verify the above observations that BTC is expressed in HCC, whereas EGFR is expressed in the endothelial cells, we examined the BTC and EGFR levels in 4 different human HCC cell lines by performing Western blotting. Consistent with the immunohistochemistry analyses, different levels of BTC expression were observed in the 4 HCC cell lines that we examined, but EGFR protein was not expressed in all 4 of the HCC cell lines (Fig. 3). Furthermore, BTC was observed in the conditioned media that was collected from these cells, and this suggested that BTC was secreted from the HCC cells (Fig. 3). None of the 4 HCC cell lines expressed EGFR protein using 4 different antibodies against EGFR (Fig. 4).

3.3. Evaluation of the PCNA-LI and the MVD in the HCC specimens The PCNA-LI of the HCC cells ranged 8% to 86% with an average 40.7%. In addition to the tumor cells, the

b

v 2 Test ( v 2 = 11.1). v 2 Test.

PCNA-LI of the tumor endothelial cells also showed strong nuclear staining for PCNA, ranging from 3% to 13% with an average of 8.5%, and this indicated the proliferation of the endothelial cells (Fig. 5A). CD34 was expressed in the endothelium lining of the sinusoids in the tumor tissue, but it was not expressed in the normal liver tissue (Fig. 5B). The MVD of the HCC tumors ranged from 0.1% to 16.3%, with an average of 4.8% F 3.1%. Some of the cirrhotic nodules showed a focal or peripheral pattern of sinusoidal CD34 expression. The percentage of the CD34 vessels expressing EGFR was measured to be 38.3 F 2.1%.

3.4. Statistical results The specimens that showed a positive EGFR expression in the tumor endothelial cells had a significantly higher MVD than those specimens that displayed an absent of EGFR expression ( P = .003). The BTC- and TGFa–positive tumors showed a higher MVD than the BTCand TGF-a–negative tumors, but this was without statistical significance (Table 3). The putative combination, EGFRpositive in endothelial cell/BTC-positive in tumor cell specimens showed a higher MVD (5.7 F 0.7) than for BTC alone (5.1 F 0.5). The coexpression of the BTC protein in the cancer cells and EGFR in the tumor endothelial cells was present in 21 (41%) of the 51 cases. A strong correlation was found between the BTC expression in the cancer cells and the EGFR expression in the Table 5 Correlation of PCNA-LI and the expression of growth factors

BTC TGF-a a

Positive Negative Positive Negative

Student t test.

No. of HCC specimens

PCNA-LI in tumor (mean F SE)

Pa

31 20 17 34

42.3 38.1 42.0 40.0

.451

F F F F

3.2 4.9 4.5 3.4

.742

Expression of BTC and EGFR in HCC Table 6

1331

Correlation between the expressions of BTC, TGF-a, EGFR, and the prognostic factors Pa

BTC Size Grade Cirrhosis Vessel invasion

N5 cm V5 cm Low-grade High-grade Positive Negative Positive Negative

(+)

( )

15 16 22 9 11 23 5 26

9 11 10 10 6 11 7 13

.813 .131 .834 .121

Pa

TGF-a (+)

( )

9 8 10 7 3 7 5 12

15 19 25 9 14 27 3 31

.552 .286 .803 .057

Pa

EGFR (+)

( )

16 9 18 7 8 13 4 21

9 17 15 11 9 21 6 20

.036 .285 .546 .525

NOTE. (+), positive; ( ), negative. a v 2 Test.

tumor endothelial cells (v 2 = 11.1, P = .0008) (Table 4). The BTC and TGF-a–positive tumors showed a higher PCNA-LI than did the BTC and TGF-a–negative tumors; however, there was no significant difference between either the BTC expression or the TGF-a expression and the PCNA-LI of tumor cells (Table 5). There was no significant difference between either the BTC expression or the TGF-a expression and the prognostic factors, but expression of the EGFR was significantly related with the tumor size (Table 6).

4. Discussion This study demonstrated that (1) the expression of BTC and EGFR in the HCC specimens was increased; (2) BTC was predominantly expressed in the HCC cells whereas EGFR was exclusively expressed in the sinusoidal endothelial cells of the HCC specimens; (3) the expression of EGFR in the endothelial cells of the HCC specimens was strongly correlated with the increased MVD of the tumors; and (4) BTC was secreted in all of the examined 4 HCC cell lines. In addition, the TGF-a and BTC expressions were not significantly correlated with the MVD by itself. However, for the putative paracrine combination of EGFR/BTC expression, we found a higher MVD than for the BTC expression alone. Betacellulin is a member of the EGF family, and it was originally identified as a growth-promoting factor of a mouse pancreatic b-cell carcinoma (insulinoma) cell line [14], and it has since been identified in humans [22]. Betacellulin efficiently activates all 4 members of the c-erbB receptor family, and it binds to EGFR with an affinity that is similar to that of EGF [14]. Thus, BTC produces its physiologic and pathologic effects through EGFR. As shown in pancreatic and colon carcinoma models [23,24], tumor-associated endothelial cells express EGFR, and this is involved in tumor progression and tumor angiogenesis. However, the effect of EGFR and its

ligand, BTC, on the angiogenesis of HCC has not been fully characterized. In this study, we found strong EGFR immunoreactivity in the sinusoidal endothelial cells of the HCC, and there was significant correlation between the EGFR expression and the high MVD that is observed in HCC specimens. Previous studies have demonstrated that the increased expression of EGFR in endothelial cells is associated with angiogenesis [25,26], and this is in agreement with our findings. Strong EGFR immunoreactivity in the vascular endothelial cells of meningioma has been reported, and this suggests that EGFR might participate in the angiogenesis of meningioma [26]. Our results have also shown a close association among the expressions of BTC in tumor cells and EGFR in the endothelial cells of HCC, and an increased MVD. Furthermore, the tumor endothelial cells showed strong nuclear staining for PCNA, which indicated the proliferation activity of the endothelial cells. A recent study has reported that BTC exerts angiogenic activity through the sequential activation of the EGFRs, mitogen-activated protein kinase, and phosphatidyl inositol 3V-kinase/Akt in endothelial cells [17]. Taken together, we can propose that the overexpression of BTC by the HCC cells and the overexpression of EGFR by the tumor endothelial cells enhance the tumor vascularity in a paracrine manner. It has been reported that 47% to 68% of the human HCC tissue samples expressed immunoreactive EGFR [27,28]. In contrast, the present study has revealed that immunoreactive EGFR is absent in all of the HCCs we studied, as well as in the HCC cell lines that we examined. However, our results are in agreement with those recently reported by other researchers [29]. Nakopoulou et al [29] have reported that only 3 of 71 cases of HCC showed a heterogenous membrane expression of EGFR with using mouse monoclonal anti-EGFR antibody (Biomaker). Mutation of the kinase domain of the EGFR does not play a significant role in the tumorigenesis of HCC [30]. A possible explanation of this discrepancy may be due to the different criteria for

1332 positivity and the different antibodies and detection methods that were used for different studies. In conclusion, our results suggest that the coexpression of BTC in tumor cells and its receptor EGFR in the tumor endothelial cells may play a role for tumor angiogenesis in HCC via paracrine mechanisms. This study may provide scientific rationale for exploring therapeutic approaches that can target EGFR tyrosine kinase in HCC.

Acknowledgment We thank Dr M. J. Im for her discussion and editorial suggestions.

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