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Annals of Surgical Oncology 10:297-304 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

Quantitative Analysis of Heparanase Gene Expression in Esophageal Squamous Cell Carcinoma

Masahide Ikeguchi, MD, Kenji Fukuda, MD, Ken-ichi Yamaguchi, MD, Akira Kondo, MD, Shun-ichi Tsujitani, MD and Nobuaki Kaibara, MD

From the Divisions of Operating Room (MI) and Surgical Oncology (KF, KY, AK, ST, NK), Faculty of Medicine, Tottori University, Yonago, Japan.

Correspondence: Address correspondence and reprint requests to: Masahide Ikeguchi, MD, Division of Operating Room, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago 683-8504, Japan; Fax: 81-859-34-8095; E-mail: masaike{at}grape.med.tottori-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Heparan sulfate proteoglycans, the main components of the extracellular matrix, are recognized as important components of signal transduction and play an important role in tumor progression. Heparanase (hep) degrades heparan sulfate proteoglycans, but the clinical importance of hep is unclear. In this study, we investigated the clinicopathologic importance of hep messenger RNA (mRNA) expression in esophageal squamous cell carcinoma (ESCC).

Methods: Fresh tumors and noncancerous epithelia were obtained from 57 ESCC patients after esophagectomy. Expression levels of hep and glyceraldehyde-3-phosphate dehydrogenase mRNA were quantitatively analyzed by real-time reverse transcriptase-polymerase chain reaction. Apoptotic cancer cells and microvessel density were evaluated immunohistochemically.

Results: The relative hep mRNA expression level (hep:glyceraldehyde-3-phosphate dehydrogenase ratio) in ESCC was lower than in noncancerous tissue (P < .001). Tumor hep expression decreased according to tumor progression and correlated with the occurrence of apoptotic cancer cells, but not with tumor microvessel density. Moreover, low hep expression correlated with poor patient survival.

Conclusions: Reduced hep mRNA expression might result in abnormal cell growth and correlate with ESCC progression.

Key Words: Apoptosis • Esophageal squamous cell carcinoma • Heparan sulfate proteoglycans • Heparanase • Real-time reverse transcriptase-polymerase chain reaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors; it has a dismal prognosis, and lymph node metastasis has been reported to have a strong prognostic effect for patients with ESCC.1 Tumor cell invasion and subsequent distant spread via blood and lymph vessels are critical steps in tumor progression. In the process of cancer cell invasion and metastasis, cancer cells produce enzymes that degrade basement membranes and extracellular matrix (ECM),2 of which heparan sulfate proteoglycans (HSPGs) are a ubiquitous component.

Heparanase (endo-ß-D-glucuronidase) degrades the heparan sulfate side chains of HSPGs and thus degrades HSPGs. This heparanase activity is reported to be essential in the disassembly of the ECM by invading cells, particularly metastatic tumor cells and leukocytes entering inflammatory sites.3 Recently, protein or messenger RNA (mRNA) expression of heparanase has been identified in various cancer cells, and the overexpression of heparanase protein or mRNA in tumor cells has been reported to correlate with the metastatic potential of tumor cells in vitro and in vivo4,5 and with poor patient prognosis.6,7

However, HSPGs have an important function as cell-surface receptors and interact with growth factors and cytokines. Basic fibroblast growth factor (bFGF) acts in cell growth and development,8 and the biological activity of bFGF is mediated by interaction with transmembrane receptor tyrosine kinases (RTKs).9 Basic FGF binds to HSPGs on the cell surface, whereas the stable binding of bFGF to RTKs and signaling requires the presence of HSPGs.10 This ternary complex of bFGF/HSPG/RTK plays an important role in malignant tumor progression.11 Overexpression of heparanase may destroy the cell-surface bFGF/HSPG/RTK complex and downregulate tumor cell growth signals. Thus, the role of heparanase in malignant tumors remains unclear. In this study, to evaluate the clinicopathologic significance of heparanase gene expression in ESCC, we analyzed heparanase mRNA expression levels in carcinomas and in noncancerous tissue by using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Messenger RNA Extraction From Tissue
We obtained tumors and noncancerous esophageal epithelia from 57 patients with ESCC. These 57 patients had undergone esophagectomy between 1993 and 2000. Informed consent was obtained from all patients for the subsequent use of their resected tissues, and this study conformed to the ethical standards of the World Medical Association Declaration of Helsinki. Tissue samples of approximately .1 g were collected immediately after resection of the specimen. Noncancerous tissues were obtained from regions distant from the tumors. Half of the tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections (4 µm thick) were prepared for hematoxylin and eosin staining for histopathologic diagnosis and for immunohistochemical staining. The other half of the tissue was stored at -80°C until needed. Before the study began, histopathologic examination confirmed that no cancer cells had contaminated the noncancerous tissues. Total RNA from tissues and EC-GI-10 cells (the human ESCC cell line, purchased from Riken Gene Bank [Tsukuba Science City, Japan]) was isolated with RNeasy Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA was eluted with 50 µl of diethylpyrocarbonate water. RNA concentrations were determined by spectrophotometry. Complementary DNA (cDNA) was synthesized with 1 µg of total RNA with Ready-to-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Real-Time Quantitative RT-PCR Assay
The following primers and TaqMan probes (Roche, Somerville, NJ) were used for heparanase: forward primer, 5'-TCACCATTGACGCCAACCT-3'; reverse primer, 5'-CTTTGCAGAACCCAGGAGGAT-3'; and probe, 5'-6-carboxy-fluorescein (FAM)-CCACGGACCCGCGGTTCCT-3'-6-carboxy-tetramethyl-rhodamine (TAMRA).12 For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), they were forward primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; reverse primer, 5'-GAAGATGGTGATGGGATTTC-3'; and probe, 5'-FAM-CAAGCTTCCCGTTCTCAGCC-3'-TAMRA.13 AmpliTaq (Roche) DNA polymerase extended the primer and displaced the TaqMan probe through its 5'/3' exonuclease activity. The TaqMan probes were labeled with a reporter fluorescent dye (FAM) at the 5' end and a quencher fluorescent dye (TAMRA) at the 3' end. When the probe was intact, the fluorescence emission of the reporter was quenched, and no signal was emitted. Nuclease degradation of the hybridization probe removed the quenching effect of TAMRA. The FAM fluorescent emission increased at 517 and 554 nm.

Quantification of gene expression was performed with a real-time quantitative RT-PCR Gene Amp 5700 Sequence Detection System (PerkinElmer Applied Biosystems, Foster City, CA), which uses the 5' nuclease activity of Taq polymerase to detect PCR amplicons.14,15 The PCR solution (50 µl) was composed of 1 µl of cDNA solution, 5 pmol of the forward and reverse primers, 10 pmol of the TaqMan probe, and TaqMan Universal PCR Master Mix. PCR was performed after incubation at 50°C for 2 minutes and denaturing at 95°C for 10 minutes, 45 cycles of 95°C for 15 seconds, and 61°C for 1 minute.

Quantification of Gene Expression
For each reaction tube, the fluorescence signal of the reporter dye (FAM) was divided by the fluorescence signal of the passive reference dye (TAMRA) to obtain a ratio defined as the normalized reporter signal. The threshold line was set at a ratio of .05.14 The point at which the amplification plot crossed this threshold was defined as Ct, which represented the cycle number at this point. Standard curves for GAPDH and heparanase were generated by using serial dilution (containing 160, 80, 40, and 20 ng) of total RNA derived from the EC-GI-10 cell line (Fig. 1). The plots represented the log of the input amount (log nanograms of total starting RNA) as the x-axis and Ct as the y-axis. Equations were derived from the lines of the calibration curves.14 The two formulas for log nanograms of heparanase and GAPDH were as follows: heparanase, y = 32 - 3.4x (r2 = .999); GAPDH, y = 26.3 - 4.5x (r2 = .991) (Fig. 2). For each of the experimental samples, the amount of heparanase and GAPDH mRNA was determined from the standard curves. Total RNA concentration from tissue is determined by spectrophotometry; however, RNA will be degraded during RNA extraction or cDNA synthesis. Thus, the precise amount of total RNA added to each reaction mix is difficult to assess. The heparanase gene expression level in tissue obtained by real-time RT-PCR may not reveal the real heparanase gene expression level. Therefore, to know the real expression level of the heparanase gene, it is necessary to normalize heparanase mRNA expression by an internal control gene expression level. In this study, the normalized amount of heparanase mRNA was determined by dividing the amount of heparanase mRNA by the amount of GAPDH mRNA for each sample.



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  		FIG. 1. Amplification plots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and heparanase mRNA. Serial dilutions of total RNA (a, 160 ng; b, 80 ng; c, 40 ng; d, 20 ng) derived from EC-GI-10 cells were amplified. The horizontal line at Rn = .05 is the threshold for detection. Rn = ratio of the normalized reporter signal.

 


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  		FIG. 2. The calibration curves of the plots represent the log of the input amount (log nanograms of total starting RNA of EC-GI-10 cells: a, 160 ng; b, 80 ng; c, 40 ng; and d, 20 ng) as the x-axis and threshold cycle (Ct) as the y-axis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (left) and heparanase (right). Equations were derived from the lines of the calibration curves.

 
Immunohistochemistry
The sliced sections of paraffin-embedded tumor tissues were dewaxed and incubated with the primary antibodies. A polyclonal rabbit anti-single-stranded DNA (diluted 1/200; Dako Japan Co., Ltd., Kyoto, Japan) was used to detect the apoptotic cells, and a monoclonal antibody against CD34 (QB-END/10, diluted 1/25; Novocastra Laboratories, Newcastle, UK) was used to detect microvessels. To determine the average numbers of apoptotic cancer cells, 20 microscopic fields were monitored randomly in each sample, and 1000 to 2000 cells were examined by an independent observer (K.F.). The results were expressed as the apoptotic index (AI; percentage of immunostained cells). In addition, in each case intratumoral microvessel density (MVD) was expressed as the number of microvessels per square millimeter.16

Patients
The subjects included 54 men and 3 women, with age at the time of surgery being 65.2 ± 8.9 years (mean ± SD; range, 45–84 years). The patients’ clinical records and histopathologic diagnoses were fully reviewed. Tumors were staged according to the tumor-node-metastasis system as stage I (n = 8), II (n = 18), III (n = 26), or IV (n = 5). None of the patients received preoperative chemotherapy or radiotherapy. Transthoracic esophagectomy was performed on 36 patients by right-sided anterolateral thoracotomy and laparotomy. Intrathoracic and perigastric lymph nodes were dissected during this procedure. Transhiatal esophagectomy without thoracotomy was performed on 19 patients. Lower esophagectomy through the transabdominal approach was performed on two patients. Curative esophagectomy was performed on 45 patients, and noncurative esophagectomy was performed on 12 (liver metastasis, n = 1; extended lymph node metastasis, n = 3; local invasion, n =8). All patients were followed up until April 2002. The mean follow-up period was 29.8 months (range, 1–101 months). Causes of death were determined from clinical findings. Nineteen patients were alive in April 2002, and 38 had died. Eight patients died from diseases other than ESCC (four from operative complications; the in-hospital mortality rate was 7%), whereas 30 died from recurrence or relapse of ESCC.

Statistics
The relative heparanase mRNA expression levels among clinicopathologic parameters were evaluated by the Mann-Whitney U-test and the Kruskal-Wallis test. The relationship between relative heparanase mRNA levels and AIs or MVDs in ESCC was evaluated statistically by using Spearman’s rank correlation test. Survival rates were calculated with the Kaplan-Meier method. The log-rank test was used for comparisons of two survival curves. P values of <.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quantitative Heparanase mRNA Expression
The results from patient samples were plotted on the standard curve. The estimated amounts of heparanase and GAPDH mRNA were calculated as described previously, and heparanase:GAPDH ratios were indicated to note relative heparanase gene expression in each sample. The mean heparanase:GAPDH mRNA ratio of the 57 tumors was 7.9 (SD, 7.0; range, .8–32.3; median, 5.6), and the mean heparanase:GAPDH mRNA ratio of 57 noncancerous tissues was 18.7 (SD, 15.9; range, 1.2–104.8; median, 14.5). The average heparanase:GAPDH mRNA ratio of the noncancerous tissues was significantly higher than that of the ESCCs (P < .001; Fig. 3). Figure 4 shows a typical case in which the GAPDH mRNA levels of the tumor and the noncancerous tissue were almost the same, whereas the heparanase mRNA expression level of the tumor was lower than that of the noncancerous tissue.



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  		FIG. 3. The average heparanase:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio of the noncancerous tissues (mean ± SD; 18.7 ± 15.9) was significantly higher (P < .001) than that of the esophageal squamous cell carcinoma tissues (mean ± SD; 7.9 ± 7).

 


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  		FIG. 4. A typical case. The amplification curves of glyceraldehyde-3-phosphate dehydrogenase messenger RNA (mRNA) from the tumor (line a) and the noncancerous esophageal epithelium (line b) are almost the same level, whereas the amplification curve of heparanase mRNA from tumor (line d) is obviously lower than that of heparanase mRNA from noncancerous epithelium (line c). Rn = ratio of the normalized reporter signal.

 
Clinicopathologic Parameters and Heparanase:GAPDH mRNA Ratios
Correlations between clinicopathologic parameters and heparanase:GAPDH mRNA ratios are listed in Table 1. Heparanase mRNA expression did not correlate with tumor differentiation, whereas the expression level was significantly reduced according to tumor progression.


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  		TABLE 1. Clinicopathologic findings of patients and tumor heparanase:GAPDH mRNA ratios
 
Correlation Between Tumor Heparanase mRNA Expression and AI or MVD
The mean AI of the 57 tumors was 1.9% (SD, 2.7%; range, .2%–20.4%; median, 1.2%). The mean AI of 8 stage I tumors was 3.7, of 18 stage II tumors was 1.9, of 26 stage III tumors was 1.4, and of 5 stage IV tumors was 1.2. Even though the difference was not significant (P = .856), the mean AI of tumors decreased according to increasing tumor stages. The mean MVD of tumors was 300.4/mm2 (SD, 129.2/mm2; range, 36–668/mm2; median, 304/mm2). The mean MVD of 8 stage I tumors was 361.8/mm2, of 18 stage II tumors was 305.5/mm2, of 26 stage III tumors was 259.1/mm2, and of 5 stage IV tumors was 374.8/mm2. No significant difference was detected (P = .103). A significant positive correlation was detected between heparanase:GAPDH mRNA ratios and AIs in 57 tumors by Spearman’s rank correlation test ({rho} = .356; P = .008; Fig. 5), whereas no significant correlation was observed between heparanase:GAPDH mRNA ratios and MVDs in tumors ({rho} = -.034; P = .8). Also, no significant correlation was detected between AIs and MVDs in tumors ({rho} = -.05; P = .705). These findings indicate that the occurrence of spontaneous apoptosis in tumor cells and heparanase mRNA expression may decrease according to the tumor progression in ESCC and that this phenomenon may not correlate with tumor microangiogenesis.



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  		FIG. 5. Correlation between heparanase:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratios and apoptotic indexes in 57 tumors. A significant positive correlation was detected ({rho} = .356; P = .008). The correlation line was y = .01 + .236x (r2 = .365).

 
Prognostic Significance of Heparanase mRNA Expression in ESCC
In the 53 surviving patients (4 patients died from operative complications), adjuvant radiotherapy was performed for 36 (total dose was 30–50 Gy). The remaining 17 were excluded from adjuvant radiotherapy because of old age, cardiopulmonary complications, or an early tumor stage. The 5-year survival rate of the 36 patients who underwent adjuvant radiotherapy (32.8%) was not different from that of the 17 patients who did not (45.8%; P = .3317). The 53 patients were divided into two subgroups (high heparanase mRNA expression group: heparanase:GAPDH mRNA ratio >5.6, n = 24; low heparanase mRNA expression group: heparanase:GAPDH mRNA ratio <=5.6, n = 29) according to the median heparanase:GAPDH mRNA ratios of the 57 tumors. The disease-specific 5-year survival rate of the 24 patients with high heparanase mRNA expression tumors (57.8%) was significantly better than that of the 29 patients with low heparanase mRNA expression (19.7%; P = .008).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HSPGs are complex glycosaminoglycans consisting of polysaccharide chains of up to 400 modified sugar residues in length. HSPGs are an essential component of the extracellular matrices of most tissues and are prominent components of blood vessels. Malignant tumor cells invade the surrounding matrix and penetrate the basement membrane in a process that is a fundamental characteristic of cancer cells. To degrade extracellular HSPGs, cells express the enzyme heparanase, an endoglycosidase that cleaves HSPG chains at a limited number of sites along the polysaccharide chain.17 Heparanase activity has been reported to correlate with the metastatic potential of tumor cells in an animal model.5 In human cancers, Ikuta et al.18 reported that the incidence of cancer metastasis in patients with oral cancer correlated with a high level of heparanase mRNA expression. In addition, Koliopanos et al.12 reported that there was a significant correlation between enhanced heparanase mRNA expression and shorter postoperative patient survival in pancreatic cancer. These observations strongly suggested that enhanced heparanase mRNA expression in tumors correlated with the metastatic potential of tumor cells.

However, our results in ESCC are completely opposite from those of previous reports. In this study, we found that the relative heparanase mRNA expression levels in tumors were significantly lower than those in noncancerous epithelia in ESCC. Moreover, the tumor heparanase mRNA expression level decreased according to increasing depth of tumor invasion. These findings indicate that the heparanase mRNA expression level may be lost during tumor progression in ESCC. Ogawa et al.19 established rat hepatocellular carcinoma cell lines with a high metastatic potential and found decreased heparanase mRNA expression in one cell line, which showed high lung metastasis when it was injected into nude mice subcutaneously. These negative results indicate that the biological importance of heparanase mRNA expression in cancer cells may be more complex.

Recently, the biological importance of HSPGs has been reported. Cell-surface HSPGs bind to bFGF and its receptors, and this ternary complex plays an important role in signal transduction.11 Kuniyasu et al.20 found that heparan sulfate treatment increased the invasive activity of colon cancer cell lines. In addition, Liu et al.21 reported that heparinase III, which cleaves at the undersulfated regions of HSPGs, treatment reduced both the tumor volume and the number of lung metastases in B16BL6 melanoma cell-injected mice. These findings indicate that HSPGs may play an important role in tumor progression or metastasis and that overexpressed heparanase may destroy this bFGF/HSPG/RTK complex, resulting in tumor cell progression being suppressed.

Recently, it was reported that heparanase promoted tumor angiogenesis in vivo.22 It was hypothesized that heparanase degrades the connection between HSPGs and bFGF or vascular endothelial growth factor in ECM and that these free bFGF or vascular endothelial growth factors in ECM may show strong angiogenic activity. In a clinical sample, El-Assal et al.23 reported that overexpressed heparanase mRNA was significantly correlated with high tumor microvessel density in hepatocellular carcinoma. However, we could not find any significant correlation between tumor heparanase mRNA expression levels and tumor MVDs in ESCC. Angiogenesis requires the interaction of various factors. In the angiogenic process, heparanase may play a role, but it will be difficult to estimate the importance of heparanase for tumor angiogenesis in clinical samples.

Surprisingly, Ginath et al.24 reported that a high concentration of heparanase protein was detected by immunohistochemistry in apoptotic cells in ovarian cancer. They concluded that apoptosis might be the main mechanism releasing heparanase from cells to the extracellular space. However, we found a significant positive correlation between heparanase mRNA expression levels and the percentages of apoptotic cancer cells in ESCC. This indicates a direct interaction between heparanase mRNA expression and the occurrence of apoptosis in cells. Liu et al.21 reported that B16BL6 melanoma cells, injected subcutaneously in mice, showed a significant increase of apoptotic cells after heparinase III treatment. This phenomenon suggests that the disruption of the HSPG/bFGF/receptor complex by heparanase may induce a reduction of the growth signal through the bFGF pathway and that such cells may fall into apoptosis. Thus, heparanase may control the growth signal transduction, and heparanase mRNA expression may be essential for normal cell turnover. Also, reduced heparanase mRNA expression may result in abnormal cell growth and thus correlate with tumor progression.

In previous studies, to evaluate expression levels of heparanase mRNA, a traditional RT-PCR method was used.23 RT-PCR assay is an easy method for detecting heparanase mRNA expression in tissue. However, the expression levels of GAPDH (internal control) mRNA have been reported to be quite different among samples, even if the same amounts of total RNA were used.25 This phenomenon might be overlooked in Northern blot or traditional RT-PCR assay. Thus, the results obtained from an RT-PCR method may not reveal the true tissue heparanase mRNA expression level. In this study, we used a quantitative real-time RT-PCR method to evaluate the relative expression levels of heparanase mRNA in ESCC. The normalized amount of heparanase gene expression was determined by dividing the amount of heparanase mRNA by the amount of GAPDH mRNA for each sample. Thus, by using this new method, we could evaluate heparanase mRNA expression levels in tissues more accurately than by using the usual RT-PCR method.


   Footnotes
 
Heparanase messenger RNA (mRNA) expression correlates with the occurrence of apoptosis, and reduced heparanase mRNA expression might result in abnormal cell growth and correlate with esophageal cancer progression.

Received for publication May 20, 2002. Accepted for publication October 8, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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