Department of Obstetrics and Gynecology, Okayama University Medical School, Okayama, Japan
Received 13 March 2003; revised 24 May 2003; accepted 4 June 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endoglycosidic heparanase degrades heparan sulfate glycosaminoglycans, and may be important in cancer invasion and metastasis, although its expression in human cervical cancer has not been characterized.
Materials and methods:
Heparanase association with clinicopathological features related to prognostic significance was examined in patients presenting with invasive cervical cancer. Gene expression of heparanase was assessed by RTPCR in 10 normal cervix and 92 invasive cervical cancer samples.
Results:
Heparanase mRNA expression was not detected in any of the normal cervix specimens, but was significantly higher in advanced-stage tumors (P = 0.026). In cases treated with radical hysterectomy and pelvic lymphadenectomy, heparanase mRNA expression was significantly higher in tumors exhibiting lymph-vascular space invasion (P = 0.01). A significant relationship was found between microvessel counts and heparanase mRNA expression (P = 0.035). The disease-free and overall survival rates of patients exhibiting heparanase mRNA expression were significantly lower than those of patients lacking heparanase mRNA expression (P = 0.019 and 0.017, respectively). Furthermore, multivariate analysis showed that heparanase mRNA expression was an independent prognostic factor for both disease-free and overall survival.
Conclusions:
These findings provide evidence that heparanase expression can serve as an indicator of aggressive potential and poor prognosis in cervical cancer. Consequently, heparanase inhibitor will be a novel candidate for therapeutic intervention in this disease.
Key words: angiogenesis, cervical cancer, heparanase, survival
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heparan sulfate proteoglycans (HSPG), in which heparan sulfate glycosaminoglycans (HS-GAG) chains are covalently attached, are classified into several families according to the amino acid sequence of the core protein, such as syndecans and perlecan [1, 2]. Syndecans, cell surface HSPG, participate in cellcell and cellextracellular matrix (ECM) interactions [1, 3]. Syndecan-1 is the most studied member of the syndecan family. Decreased expression of syndecan-1 has been reported to correlate with tumorigenicity, tumor invasion and progression [46]. In fact, decreased expression of syndecan-1 has been reported in invasive cervical cancers [7]. Syndecan-1 binds to various ECM components, such as collagen and fibronectin, via its HS-GAG, and most of its biological functions are considered to be associated with this process [13]. On the other hand, perlecan, a basement membrane-bound HSPG, is one of the main components of the basement membrane. In carcinomas, basement membranes often separate tumor cells from the surrounding tissue, and represent the main barrier limiting tumor invasion [8]. Furthermore, the penetration of the basement membranes in blood or lymphatic vessels is an essential step in the process of metastasis [9].
Endoglycosidic heparanase, which cleaves HS-GAG at only a few sites, degrades HS-GAG into fragments of appreciable size [10]. The cDNA sequence of the heparanase gene was recently reported, and its mRNA found to be preferentially expressed in various human tumor tissues, including cervical cancer [1113]. Heparanase activity has been shown to correlate with the metastatic potential of mouse melanoma and lymphoma cell lines [14, 15]. Furthermore, heparanase may contribute to angiogenesis by releasing HSPG-binding angiogenic factors [16].
Heparanase may be important in cancer invasion and metastasis, although its expression in human cervical cancer has not previously been characterized. Therefore, we investigated the expression of heparanase mRNA in 10 normal cervix, five microinvasive and 87 invasive cervical cancer specimens using semi-quantitative RTPCR. Moreover, the association between heparanase mRNA expression and clinicopathological features including microvessel counts and patient prognosis was determined.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological cell types of tumors were assigned according to the World Health Organization (WHO) classification: 58 were classified as squamous cell carcinoma, 22 as adenocarcinoma and 12 as adenosquamous carcinoma. Staging was reviewed based on the International Federation of Gynecology and Obstetrics (FIGO) staging system: five, 27, 44, 12 and four were categorized as stage IA1, IB, II, III and IV, respectively. The median age at the time of treatment was 50 years (range 2690). Radical hysterectomy and pelvic lymphadenectomy were performed on 64 subjects with stage IBIIB disease but otherwise in good physical condition. Patients displaying lymph node metastasis, parametrial involvement, deep stromal invasion or marked lymph-vascular space involvement were treated with adjuvant external whole-pelvic irradiation (50 Gy) or adjuvant combination chemotherapy. Total abdominal hysterectomy was performed on five subjects with stage IA1 disease. The remaining 23 patients were treated primarily with radiotherapy or concurrent chemoradiotherapy. Disease-free and overall survival were defined as the interval from the initial therapy to clinically or radiologically proven recurrence and death, respectively. The end of the follow-up study for analysis was 31 March 2002. The median duration of follow-up was 36 months (range 383). In addition, 10 normal cervical specimens were obtained from patients exhibiting benign gynecological disease.
RNA preparation of samples and RTPCR
Total RNA was prepared from each specimen using an RNeasy Total RNA kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. Tissues exhibiting RNA characterized by high quality 18S and 28S bands on ethidium bromide-stained gels were preferentially selected.
Reverse transcription was conducted according to the Thermoscript RTPCR System (Life Technologies, Rockville, MD, USA) protocol for reverse transcription of 3 µg total RNA. Transcribed products were subjected to PCR for heparanase (sense primer, 5'-TTCGATCCCAAGAAGGAATCAAC-3'; antisense primer, 5'-GTAGTGATGCCATGTAACTGAATC-3') [13] and ß-actin (sense primer, 5'-CTCACCATGGATGATGATAT-3'; antisense primer, 5'-TGGGTCATCTTCTCGCGGTT-3') in a PCR mixture described elsewhere [17]. Heparanase cDNA amplification was initiated with denaturation for 3 min at 94°C followed by 30 cycles of 1 min denaturation at 94°C, annealing at 60°C for 1 min, followed by extension at 72°C for 1 min. The PCR profile for ß-actin consisted of an initial denaturation of 3 min at 94°C, followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C and a 1 min extension at 72°C. The PCR mixture was maintained at 72°C for 15 min for final extension. Resultant PCR products were then electrophoresed through a 2% agarose gel and stained with ethidium bromide. UV-illuminated gels were photographed using Polaroid Type 667 films. Photographs were quantitated using an image scanner GT-9500 (Epson, Suwa, Japan) and analyzed with Basic Quantifier software (BioImage, Ann Arbor, MI, USA).
In order to obtain semi-quantification of heparanase mRNA levels, cDNA amounts were corrected using ß-actin as the internal standard. For this reason, a technique based on the competitive PCR approach employing a non-homologous internal standard was utilized (COMPETITOR, Competitive DNA Construction Kit; Takara, Kyoto, Japan). cDNAs derived from samples were co-amplified in the presence of serial dilutions of ß-actin COMPETITOR. The point of equal intensity between the bands of ß-actin COMPETITOR and the cDNA template was then determined. cDNAs in the presence of 1 x 104 copies of ß-actin were subsequently used in the amplification of heparanase genes. PCR products derived from heparanase genes were assigned to strongly positive (2+), positive (+), or weak or negative () heparanase expressing groups according to band intensity.
Immunohistochemical staining for microvessels
Expression of factor VIII-related antigen was assessed in 64 formalin-fixed, paraffin-embedded sections obtained at the time of radical surgery by the avidinbiotin complex (ABC) procedure. Anti-factor VIII-related monoclonal antibody (Dakopatts, Copenhagen, Denmark) was utilized as a primary antibody. The entire tumorous lesion was scanned under low-power magnification in order to select regions displaying the most intense vascularization. The number of microvessels was recorded by counting any positively stained endothelial cell or endothelial cell clusters as a single, countable microvessel in a 100x microscopic field (0.618 mm2) in 10 neovascularized areas. The mean of the top three counts was used as the microvessel count for each case.
Statistical analyses
Univariate analysis included the 2-test, Spearman rank correlation test and MannWhitney U-test. Survival rates were calculated by the KaplanMeier method and differences were examined by the log-rank test. Furthermore, the prognostic factors for disease-free and overall survival were determined by stepwise Coxs univariate and multivariate proportional hazard models. These analyses were performed utilizing the StatView v. 5.0 software (Abacus Concepts, Berkeley, CA, USA). P values <0.05 were considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Via HS-GAG attached to the extracellular domain, syndecan-1 binds many ECM molecules and plays a role as a matrix anchor. Reduced tumor cell adhesion is prerequisite for invasion and metastasis. In fact, decreased expression of syndecan-1 has been reported in invasive cervical cancers [6]. Mikami et al. [20] reported that a reduction in HS-GAG expression was more pronounced than that of core protein in invasive esophageal carcinomas. These findings suggest that cleavage of HS-GAG from syndecan-1 core protein, which is mediated by heparanase, is very important for reduced tumor cell adhesion and accelerated tumor cell invasion. In the present study, we demonstrated that heparanase mRNA expression is significantly higher in invasive cervical cancer at the advanced stage. All cases with distant metastasis expressed heparanase mRNA, whereas this mRNA was not detectable in the cases with microinvasive cancer. The present data are compatible with the reports of positive correlation between heparanase expression and tumor progression in cases of bladder, colon, pancreatic, hepatocellular, oral, gastric and esophageal cancers [18, 2025]. These findings suggest that heparanase expression may contribute to the progression of malignant solid tumors.
Furthermore, we demonstrated that heparanase mRNA expression is significantly higher in cases where lymph-vascular space involvement was in evidence. Mikami et al. [20] reported that heparanase expression correlates with the incidence of lymphatic invasion and venous involvement in invasive esophageal carcinomas. Tang et al. [25] reported that heparanase expression correlates with lymphatic and venous invasion in gastric carcinomas. In addition, intense heparanase immunostaining was observed in breast carcinoma cells that had entered the circulation, and in lymph node metastases [26]. HSPG are found in the subendothelial basement membrane of capillaries [27]. Therefore, cleavage of HSPG may enable tumor cells to invade through blood vessel walls.
In addition, heparanase may contribute to angiogenesis by releasing HSPG-binding angiogenic factors, such as basic fibroblast growth factors, vascular endothelial growth factor (VEGF), platelet-derived endothelial growth factor, interleukin-8, hepatocyte growth factor and transforming growth factor-ß [16]. In a previous study, we showed that the expression of VEGF is involved in the promotion of angiogenesis in cervical cancer [28]. We also demonstrated in the present study that heparanase gene expression correlates with tumor vascularity in invasive cervical cancer. In accordance with this, heparanase expression has been reported to be correlated with increased microvessel counts in bladder and esophageal cancers [20, 21].
Heparanase mRNA expression was examined to the correlation with prognosis in invasive cervical cancer. Heparanase is a significant prognostic factor in bladder, pancreatic and gastric cancers [21, 23, 25], which is consistent with the present study regarding the association of heparanase mRNA expression with poor prognosis. Furthermore, heparanase mRNA was an independent prognostic factor both in progression-free and overall survival in the present study. Thus, heparanase expression will most likely be a useful prognostic factor in patients presenting with invasive cervical cancer. However, a study involving a large cohort of patients is required to confirm this possibility.
Sulfated polysaccharides such as heparin, dextran sulfate and xylan sulfate are very effective inhibitors of tumor metastasis, although it was originally thought that the antimetastatic properties of these compounds were due to their anticoagulant activity. Recent studies have revealed that the antimetastatic activity of the sulfated polysaccharides correlates with their ability to inhibit the enzyme heparanase rather than their potency as anticoagulants [29]. Although the existence of a heparanase inhibitor has been found in murine melanoma cells, an inhibitor molecule has not yet been isolated [30]. Heparanase inhibitor may be an important strategy for antimetastatic drugs in invasive cervical cancer.
In conclusion, our findings provide evidence that heparanase expression can serve as an indicator representing aggressive potential and poor prognosis in cervical cancer. Heparanase inhibitor will be a novel candidate for therapeutic intervention in this disease.
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Yanagishita M, Hascall VC. Cell surface heparan sulfate proteoglycans. J Biol Chem 1992; 267: 94519454.
3. Carey D. Syndecans: multifunctional cell-surface corecepters. Biochem J 1997; 327: 116.[ISI][Medline]
4. Liebersbach BF, Sanderson RD. Expression of syndecan-1 inhibits cell invasion into type I collagen. J Biol Chem 1994; 269: 2001320019.
5. Leppa S, Mali M, Miettinen HM, Jalkanen M. Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells. Proc Natl Acad Sci USA 1992; 89: 932936.[Abstract]
6. Inki P, Stenback F, Talve L, Lakanen M. Immunohistochemical localization of syndecan in mouse skin tumors induced by UV irradiation. Loss of expression associated with malignant transformation. Am J Pathol 1991; 139: 13331340.[Abstract]
7. Inki P, Joensuu H, Grenman R et al. Immunohistochemical localization of syndecan-1 in normal and pathological human cervix. J Pathol 1994; 172: 349355.[ISI][Medline]
8. Timple R. Structure and biological activity of basement membrane proteins. Eur J Biochem 1989; 180: 487502.[Abstract]
9. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 1991; 64: 327336.[ISI][Medline]
10. Pikas DS, Li JP, Vlodavsky I, Lindahl U. Substrate specificity of heparanase from human hepatoma and platelets. J Biol Chem 1998; 273: 1877018777.
11. Hulett MD, Freeman C, Hamdorf BJ et al. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med 1999; 5: 803809.[CrossRef][ISI][Medline]
12. Toyoshima M, Nakajima M. Human heparanase. Purification, characterization, cloning, and expression. J Biol Chem 1999; 274: 2415324160.
13. Vlodavsky I, Friedmann Y, Elkin M et al. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med 1999; 5: 793802.[CrossRef][ISI][Medline]
14. Nakajima M, Irimura T, Di Ferrante N, Nicolson G. Heparan sulfate degradation: relation to tumor invasion and metastatic properties of mouse B 16 melanoma sublines. Science 1983; 220: 611613.[ISI][Medline]
15. Vlodavsky I, Fuks Z, Bar-Ner M et al. Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: relationship to tumor cell metastasis. Cancer Res 1983; 43: 27042711.[Abstract]
16. Vlodavsky I, Korner G, Ishai-Michaeli R et al. Extracellular matrix-resident growth factors and enzymes: possible involvement in tumor metastasis and angiogenesis. Cancer Metastasis Rev 1990; 9: 203226.[ISI][Medline]
17. Seki A, Kodama J, Miyagi Y et al. Amplification of the mdm-2 gene and p53 abnormalities in uterine sarcomas. Int J Cancer 1997; 73: 3337.[CrossRef][ISI][Medline]
18. Ikuta M, Podyma KA, Maruyama K et al. Expression of heparanase in oral cancer cell lines and oral cancer tissues. Oral Oncol 2001; 37: 177184.[CrossRef][ISI][Medline]
19. Vlodavsky I, Goldshmidt O, Zcharia E et al. Mammalian heparanase: involvement in cancer metastasis, angiogenesis and normal development. Cancer Biol 2002; 12: 121129.[CrossRef]
20. Mikami S, Ohashi K, Usui Y et al. Loss of syndecan-1 and increased expression of heparanase in invasive esophageal carcinomas. Jpn J Cancer Res 2001; 92: 10621073.[ISI][Medline]
21. Gohji K, Hirano H, Okamoto M et al. Expression of three extracellular matrix degradative enzymes in bladder cancer. Int J Cancer 2001; 95: 295301.[CrossRef][ISI][Medline]
22. Friedmann Y, Vlodavsky I, Aingorn H et al. Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Am J Pathol 2000; 157: 11671175.
23. Koliopanos A, Friess H, Kleff J et al. Heparanase expression in primary and metastatic pancreatic cancer. Cancer Res 2001; 61: 46554659.
24. El-Assai ON, Yamanoi A, Ono T et al. The clinicopathological significance of heparanase and basic fibroblast growth factor expression in hepatocellular carcinoma. Clin Cancer Res 2001; 7: 12991305.
25. Tang W, Nakamura Y, Tsujimoto M et al. Heparanase: a key enzyme in invasion and metastasis of gastric carcinoma. Mod Pathol 2002; 15: 593598.[ISI][Medline]
26. Zcharia E, Metzger S, Chajek-Shaul T et al. Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis. J Mammary Gland Biol Neoplasia 2001; 6: 311322.[CrossRef][ISI][Medline]
27. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 2001; 108: 341347.
28. Kodama J, Seki N, Tokumo K et al. Vascular endothelial growth factor is implicated in early invasion of cervical cancer. Eur J Cancer 1999; 35: 485489.[CrossRef][ISI][Medline]
29. Parish CR, Freeman C, Hulett MD. Heparanase: a key enzyme involved in cell invasion. Biochim Biophys Acta 2001; 1471: M99M108.[CrossRef][ISI][Medline]
30. Keren Z, Leland F, Nakajima M, LeGrue SJ. Inhibition of experimental metastasis and extracellular matrix degradation by butanol extracts from B16-F1 murine melanoma. Cancer Res 1989; 49: 295300.[Abstract]