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A metalloproteinase-9 polymorphism which affects its expression is associated with increased risk for oral squamous cell carcinoma
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EJSO 34 (2008) 450e455
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A metalloproteinase-9 polymorphism which affects its expression is associated with increased risk for oral squamous cell carcinoma E. Vairaktaris a,*, S. Vassiliou a, E. Nkenke b, Z. Serefoglou a, S. Derka a, C. Tsigris d, A. Vylliotis a, C. Yapijakis a, F.W. Neukam b, E. Patsouris c a
Department of Oral and Maxillofacial Surgery, University of Athens Medical School, ‘‘Attikon’’ Hospital, Rimini 1, Athens, GR-12462, Greece b Department of Oral and Maxillofacial Surgery, Universita¨t Erlangen, Klinik und Poliklinik f}ur Mund-, Kiefer-, Gesischtschirurgie, Glueckstrasse 11, Erlangen D-91054, N}urnberg, Germany c Department of Pathology, University of Athens Medical School, Mikras Asias 75, Athens GR-11527, Greece d First Department of Surgery, University of Athens Medical School, ‘‘Laikon’’ General Hospital, Mikras Asias 75, Athens GR-11527, Greece Accepted 27 March 2007 Available online 11 May 2007
Abstract Aim: In light to recently found contribution of factors associated with angiogenesis, thrombosis and inflammation to carcinogenesis, we investigated the possible association of metalloproteinase-9 (MMP-9) with increased risk of oral cancer. Methods: In DNA samples of 152 patients with oral squamous cell carcinoma and 162 healthy controls of comparable ethnicity, age and sex, we studied the 1562 C/T polymorphism in the MMP-9 gene promoter, which affects its transcription. Results: The detected frequency for the high expression T allele in the patients’ group was significantly increased in comparison to that of the control group (22% versus 15%, respectively; P < 0.05). This difference was due to the relative increase of C/T heterozygotes in the group of patients, in comparison to controls (P < 0.05, 95% OR 1.92, CI 1.21e3.06). The same pattern of significance was observed between controls and the subgroups of patients with initial (I & II) stages of cancer, without positive family history of cancer or thrombophilia, with smoking and alcohol abuse habits. Conclusions: The investigated MMP-9 polymorphism has a strong association with increased risk for developing oral cancer in a subset of the general population. These results are in accordance to previous studies of constitutive expression and secretion of MMP-9 in invasive oral carcinoma cell lines. The observation that T allele carriers have an increased risk for developing oral cancer only in initial stages, but not in advanced ones, may be due to the role of MMP-9 in the inhibition of angiogenesis by generating angiostatin from plasminogen. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Oral cancer; Metalloproteinase-9; Polymorphism; Angiogenesis; Oncogenesis; Tumor progression
Introduction Oral squamous cell carcinoma (OSCC), the most common form of oral cancer, is a multistep process implicated in which are not only environmental factors (such as smoking and alcohol abuse) but also genetic alterations in oncogenes and tumor suppressor genes.1 OSCC is a major cause of cancer morbidity and mortality worldwide, and despite therapeutic developments, it is characterized by low survival rate.1 Recently, factors associated with angiogenesis, thrombosis and inflammation have also been correlated with increased risk for oral cancer.2e5 Metalloproteinase-9 * Corresponding author. Tel.: þ30 210 6443035; fax: þ30 210 6443803. E-mail address: [email protected] (E. Vairaktaris). 0748-7983/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2007.03.024
(MMP-9, also known as gelatinase B) is one such factor implicated in thrombosis, inflammation, angiogenesis and oncogenesis.6e9 MMP-9 belongs to the large group of secreted proteinases that require zinc for their catalytic activity.10 Their proteolytic activity is controlled by changes in the delicate balance between the expression and synthesis of MMPs and their major endogenous inhibitors, tissue inhibitors of matrix metalloproteinases.11 MMP-9 is a collagenase type IV, which degrades the extracellular matrix and basement membrane components, a very crucial stage in tumor cell invasion and metastasis.12 On the other hand, there is evidence that MMP-9 may play a significant role in angiogenesis.13e17 Some reports have implicated both MMP-9 and MMP-2 in the induction of early angiogenic events,
E. Vairaktaris et al. / EJSO 34 (2008) 450e455
but it is also known that MMP-9 degrades plasminogen and generates angiostatin, an inhibitor of endothelial cell proliferation and angiogenesis.13e17 MMP-9 is expressed in low levels under physiological conditions but it is increased dramatically in several malignancies including oral, esophageal, breast, renal, colorectal carcinomas and melanomas.18e24 In particular, a study of oral squamous cell carcinoma cell lines suggested that constitutive expression and secretion of MMP-9 is an important determinant of the invasive phenotype by activation of the protein-kinase-C pathway.25 Another study indicated that MMP-9 expression in vitro is modulated by integrin avb6, which is expressed only in oral carcinoma cells and not in the normal oral epithelium.26 The human MMP-9 gene is located on chromosome 20q12.2e13.1 and it contains 10 sequence variants in tight linkage disequilibrium across its entire length.27,28 A functional C to T single nucleotide polymorphism located in the promoter region (position 1562) is known to affect transcription.28 The less common T allele has been found to confer a 2-fold higher gene expression than the C allele.28 The frequency of the T allele in Caucasian and Asian populations is about 12e15%.31e33 The frequency of the high expression T allele has been found to be increased in patients with invasive gastric cancer, while no difference was detected between the 1562 C/T polymorphism and colorectal, ovarian and non-small cell lung carcinoma.30,32e34 In light of the above, the purpose of this study was to investigate whether the 1562 C/T polymorphism is associated with oral cancer, by genotyping patients with oral squamous cell carcinoma and healthy controls representing the general population. Methods Subjects under study The individuals under study included 314 unrelated Greeks and Germans, consisting of 152 patients with oral cancer and 162 healthy blood donors (controls) of equivalent ethnicity, age and gender. The patients were mostly men (N ¼ 120) and their age ranged between 40 and 84 years (mean ¼ 58.4 years). The sex ratio (N ¼ 123 men) and the age of the controls (range 31e83 years; mean ¼ 54.7 years) were comparable to those of the patients. Included in this study were patients who had developed oral squamous cell carcinoma and were operated recently or up to a decade ago. In addition to clinical presentation, a biopsy with pathological diagnosis of tumor stages IeIV and a family history regarding cancer and thrombophilia were available. Fifty-eight of them (38.2%) had one or two first-degree relatives with any type of cancer and their age range (mean ¼ 58.8 years) did not differ from the whole group of patients. Furthermore, 26 patients (17.1%) had one or two first-degree relatives with idiopathic thrombosis and an earlier age range (mean ¼ 56.4 years), but again with no
451
significant difference with the whole group. Fourteen patients (9.2%) had a positive family history for both cancer and thrombophilia (mean age ¼ 56.4 years). Nearly all patients (93.6%) were smokers consuming at least 10 cigarettes per day. About one third of patients (32.05%) were ethanol abusers consuming at least three glasses of alcoholic drinks per day. Most of the participants in the study worked in a low-risk environment, with the exception of one patient and three controls who worked in chemical factories. No data were available on healthy controls regarding their family history or smoking and alcohol consumption habits. Molecular analysis Blood samples were collected from patients and controls after informed consent. DNA was isolated from blood with the use of NucleonÔ kit (Amersham). Molecular detection of the 1562 C/T polymorphism in the MMP-9 gene was performed by restriction fragment length polymorphism typing, as previously described.30 This involved a combination of PCR amplification and digestion with restriction endonuclease Sph I followed by gel electrophoretic analysis. The generated PCR product of 435 bp was cleaved by restriction enzyme Sph I into two fragments of 247 bp and 188 bp only if the T allele was present, while the C allele was not cleaved by the enzyme and kept the original size of 435 bp. Statistical analysis Statistical analysis was performed using SASÒ software (version 9.0; SAS Institute Inc.). The frequencies of alleles and genotypes of the whole group or subgroups of patients were compared to the respective frequencies of the control group using the Fisher’s exact test and odds ratios, while all genotype distributions were according to HardyeWeinberg estimates. The significance level was set at P < 0.05 and the results are presented as hazard ratios using the Maentele Haenzel method with 95% confidence intervals (CI). Results Genotype and allele frequencies of studied controls The prevalence of detected MMP-9 genotypes in healthy controls (representing the general population) and patients with oral cancer is shown in Table 1, after bilateral statistical analysis. The data for the two populations under study (Greek and German healthy controls) were analyzed together, since there were no significant differences in genotype and allele frequencies of the (1562 C/T) polymorphism among the two populations. The ‘‘high expression’’ T allele frequency in the control group was 15% and the carrier frequency was 30% (observed genotypes TT ¼ 0, CC ¼ 114, CT ¼ 48, Table 1). The (1562 C/T) genotype distributions were as expected in HardyeWeinberg equilibrium in the control
E. Vairaktaris et al. / EJSO 34 (2008) 450e455
28% (P < 0.05)
group, as well as in the whole group and subgroups of patients.
30% (P < 0.05)
19% (N.S.)
Comparisons between patients and controls
Statistical difference of frequencies observed in patients and their subgroups in comparison to controls (P-values). N.S.: not significant P-value.
30% (N.S.) 22% (P < 0.05) 15% (N.S.) 22% (P < 0.05) T allele frequency
15%
25% (P < 0.05)
19% (N.S.)
17% (N.S.)
25% (P < 0.05)
23% (P < 0.05)
0 20 26 46 0 64 38 102 0 20 30 50 0 4 6 10 0 80 62 142 0 66 60 126 0 18 8 26 0 46 48 94 0 84 68 152 TT CC CT Total
0 114 48 162
0 42 42 84
0 42 26 68
0 38 20 58
Patients with positive family history of thrombosis Patients without positive family history of cancer Patients with positive family history of cancer Patients with cancer stages III & IV Patients with cancer stages I & II Patients Controls Prevalence of T allele
Table 1 Prevalence of MMP-9 T allele and carrier frequencies in healthy controls and patients with oral cancer
Patients without positive family history of thrombosis
Patients with tobacco abuse
Patients without tobacco abuse
Patients with alcohol abuse
Patients without alcohol abuse
Patients with tobacco and alcohol abuse
452
The detected allele and carrier frequencies for the high expression T allele in the patients’ group (22% and 45%, respectively) were significantly increased in comparison to that of the control group (P < 0.05, Table 1). This statistical difference was due to the relative increase of CT heterozygotes in the group of patients, in comparison to controls (genotypes TT ¼ 0, CC ¼ 84, CT ¼ 68, Table 1). The same pattern of significantly increased frequencies of T allele and CT heterozygotes, compared to controls, was also observed in the following subgroups of patients (Table 1): (a) with cancer stages I & II (P < 0.05), (b) without positive family history of cancer (P < 0.05), (c) without positive family history of thrombosis (P < 0.05), (d) with tobacco abuse (P < 0.05), (e) with alcohol abuse (P < 0.001) and (g) with alcohol and tobacco abuse (P < 0.05). Finally, in comparison to controls no significant differences in genotype or allele frequencies were detected in subgroups of patients with advanced cancer stages (III & IV), with positive family history of cancer or thrombosis, as well as without tobacco or alcohol abuse (Table 1). Compared to individuals with the CC genotype, the relative risk (OR) for CT heterozygotes to develop oral cancer was 1.92 (95% CI 1.21e3.06). Interestingly, CT individuals have an even higher relative risk for developing early stages of OSCC (OR 2.37, 95% CI 1.38e4.09). Finally, there were no significant differences due to categorizations of patients in regard to gender, age and age at onset of oral cancer. Discussion MMP-9 and tumor MMP-9 is a zinc-dependent proteinase, which is involved in numerous physiological and pathological processes. Due to its ability to degrade the extracellular matrix and the basement membrane components, it seems to play a crucial role in tumor progression.12 Although MMP-9 is expressed in low levels under physiological conditions, its expression is highly increased in many malignancies, including oral squamous cell carcinoma.18e24 The MMP-9 gene contains 10 sequence polymorphic variants which are in tight linkage disequilibrium, and one of them, the functional 1562 C/T polymorphism has been recognised to affect gene transcription.28 The promoter activity of the MMP-9 gene is increased considerably when the less common, ‘‘high expression’’ Tallele is present.28 Findings of this study In light of the above, the purpose of this study was to investigate the genotypes and allele frequencies of the
E. Vairaktaris et al. / EJSO 34 (2008) 450e455
1562 C/T polymorphism in a cohort of 152 patients with oral cancer in comparison to 162 healthy controls of equivalent age, sex and ethnicity. Although the previously observed high levels of MMP-9 in oral carcinomas might be attributed to a secondary effect of some altered mechanism(s), or even to the tumor micro-environment, we hypothesized that endogenous high MMP-9 expression might confer a genetic predisposition for oral cancer.35 In order to investigate the putative effect of genetically determined high MMP-9 expression on risk for oral cancer, the 1562 C/T polymorphism was examined only in normal blood cells and not in cancer cells of patients. Despite the modest number of studied individuals, the obtained results revealed a strong association of the examined MMP-9 polymorphism with increased risk of oral cancer. In comparison to controls, a significant increase of C/T heterozygotes was observed not only in the whole group of patients, but in the subgroups of patients with initial cancer stages, without family history of cancer or thrombosis, with smoking or/and alcohol abuse habits. Accordingly, the carrier and allele frequencies for the high expression T were increased in the whole group of patients and in all the above-mentioned subgroups.
Implications of findings The results of this study, which implicate in oral oncogenesis the higher gene expression of MMP-9 conferred by the T allele, are in accordance to previous reports that constitutive expression and secretion of MMP-9 is an important determinant of the malignant phenotype in oral squamous cell carcinoma.25,26 The interesting observation of the present study that T allele carriers have an increased risk for developing oral cancer only in initial stages, but not in advanced ones, may be due to the significant role of MMP-9 in the inhibition of angiogenesis by generating angiostatin.17 A similar finding was reported in patients with breast cancer, who seem to have a better prognosis if they carry the T allele.29 The prophylactic role of high MMP-9 levels in advanced stages of OSCC at first seems to be puzzling. Some older studies have implicated high expression of both MMP-9 and MMP-2 in initial stages of angiogenesis involving endothelial morphogenesis and tube formation in normal veins as well as in certain carcinomas.13e16 Nevertheless, a recent study in OSCC has provided evidence that the role of MMP-9 in the matrix degradation is minimal compared to MMP-2, which seems to be the main enzyme of gelatinolysis, invasion and metastasis in the oral region.36 MMP-9 is highly expressed in OSCC and produced in an inactive proform, which needs to be converted into its active form in order to degrade the matrix.36 Interestingly, the ratio of activated form/proform of MMP-9 is very low in OSCC, thus its role in matrix degradation is minimal.36
453
Conversion from proform into the active form of MMP9 may involve plasminogen, as a result of the interaction of tumor with stromal fibroblast cells.25,35 In the presence of plasminogen, MMP-9 is activated and in turn converts plasminogen into angiostatin, one of the most potent inhibitors of angiogenesis.17 Therefore, the role of MMP-9 in advanced stages of cancer may indeed be inhibitory. In support to this notion, we previously found that a polymorphic allele that results in high levels of plasminogen activator inhibitor-1 (PAI-1) is associated only with initial stages of OSCC.4 The inhibition of plasminogen activators in early cancer stages results in high levels of plasminogen (since it is not converted into plasmin) and consequently in high levels of active MMP-9, which facilitates tumor development by degradation of extracellular matrix. On the contrary, in advanced OSCC stages PAI-1 levels are low, plasminogen is converted to plasmin and MMP-9 proform remains inactive, therefore, the level of MMP-9 gene expression is not associated with tumor progression and metastasis. This notion is also in accordance to previously observed in vitro inhibition of angiogenesis by PAI-1.37 The fact that the high expression T allele is increased only in patients without a positive family history of cancer probably indicates that the MMP-9 polymorphism is associated with only a few types of malignancies. In accordance to this notion, the frequency of the T allele has been found to be increased only in patients with invasive gastric cancer, while no such difference was detected in patients with colorectal, ovarian and non-small cell lung carcinoma.30,32e34 Furthermore, the observation that the T allele is increased only in patients without a positive family history of thrombophilia possibly suggests that the MMP-9 polymorphism is not associated with inherited predisposition for thrombosis. Although the role of MMP-9 in risk for thrombosis is still controversial, our results are in accordance with previous studies in which the 1562 C/T polymorphism was not correlated with the severity of coronary atherosclerosis or myocardial infarction.28,31 Although the role of alcohol in oral carcinogenesis seems to be synergistic, the fact that T allele frequency was significantly higher only in patients who over-consume alcohol seems to underline the combined effect of high MMP-9 levels and ethanol. The same significant increase of T alleles was also observed in smoking patients, but the small amount of available non-smoking patients who were included in this study does not allow safe conclusions to be drawn regarding the combined effect of tobacco and high MMP-9 levels. The synergistic effect of smoking and heavy alcohol intake was also significantly associated with an increase of T alleles, although the respective subgroup of patients was rather small. In conclusion, the investigated MMP-9 polymorphism has a strong association with increased risk for developing oral cancer in a subset of the general population. This
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illustrates the great importance of genetic association studies, which may ultimately result in presymptomatic testing and preventive measures safeguarding the health status and lives of certain at risk individuals in the general population. Conflict of interest There are no financial or personal relationships of authors with other people or organizations that could inappropriate influence their work. Acknowledgements This work was co-funded by the European Social Fund and National Resources (EPEAEK II ‘‘Pythagoras’’ 70/3/ 7391) grant to E.V. References 1. McDowell JD. An overview of epidemiology and common risk factors for oral squamous cell carcinoma. Otolaryngol Clin of North Am 2006; 39:277–94. 2. Tsai MH, Chen WC, Chen HY, Tsai FJ. Urokinase gene 30 -UTR T/C polymorphism is associated with oral cancer. J Clin Lab Anal 2004;18: 276–9. 3. Vairaktaris E, Yapijakis C, Derka S, et al. Association of platelet Ia polymorphism with minor increase of risk for oral cancer. Eur J Surg Oncol 2006;32:455–7. 4. Vairaktaris E, Yapijakis C, Serefoglou Z, et al. Plasminogen activator inhibitor-1 polymorphism is associated with increased risk for oral cancer. Oral Oncol 2006;42:888–92. 5. Vairaktaris E, Yapijakis C, Serefoglou Z, et al. The interleukin-8 (-251A/T) polymorphism is associated with increased risk for oral squamous cell carcinoma. Eur J Surg Oncol 2007;33:504–7. 6. Alvarez B, Ruiz C, Chacon P, et al. Serum values of metalloproteinase-2 and metalloproteinase-9 as related to unstable plaque and inflammatory cells in patients with greater than 70% carotid artery stenosis. J Vasc Surg 2004;40:469–75. 7. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflammation and cancer: how hot is the link? Biochem Pharmacol 2006;72: 1605–21. 8. Ruokolainen H, Pa¨a¨kko¨ P, Turpeenniemi-Hujanen T. Expresssion of matrix metalloproteinase-9 in head and neck squamous cell carcinoma: a potential marker for prognosis. Clin Cancer Res 2004;10: 3110–6. 9. John A, Tuszynski G. The role of matrix metalloproteinases in tumour angiogenesis and tumour metastasis. Pathol Oncol Res 2001;7:14–23. 10. Nagase H, Woessner JF. Matrix metalloproteinases. J Biol Chem 1999; 274:21491–4. 11. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta 2000;1477:267–83. 12. Stetler-Stevenson WG. The role of matrix metalloproteinases in tumour invasion, metastasis, and angiogenesis. Surg Oncol Clin N Am 2001;10:383–92. 13. Schnaper HW, Grant DS, Stetler-Stevenson WG, et al. Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. Cell Physiol 1993;156:235–46. 14. Iurlaro M, Loverro G, Vacca A, et al. Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with upgrading and myometrial invasion in endometrial carcinoma. Eur J Clin Invest 1999;29:793–801.
15. Koivunen E, Arap W, Valtanen H, et al. Tumor targeting with a selective gelatinase inhibitor. Nat Biotechnol 1999;17:768–74. 16. Eccles SA, Box GM, Court WJ, et al. Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the matrix metalloproteinase inhibitor batimastat (BB-94). Cancer Res 1996; 56:2815–22. 17. Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 1997;272:28823–5. 18. Duffy MJ, Maguire TM, Hill A, et al. Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res 2000;2:252–7. 19. Ohashi K, Nemoto T, Nakamura K, Nemori R. Increased expression of matrix metalloproteinase 7 and 9 membrane type 1-matrix metalloproteinase in esophageal squamous cell carcinomas. Am Cancer Society 2000;88:2201–9. 20. Kallakury BV, Karikehalli S, Haholu A, Sheehan CE, Azumi N, Ross JS. Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma. Clin Cancer Res 2001;7:3113–9. 21. Franchi A, Santucci M, Masini E, Sardi I, Paglierani M, Gallo O. Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck. Am Cancer Society 2002;96:1902–10. 22. Tsai CH, Hsieh YS, Yang SF, Chou MY, Chang YC. Matrix metalloproteinase 2 and matrix metalloproteinase 9 expression in human oral squamous cell carcinoma and the effect of protein kinase C inhibitors: preliminary observations. Oral Surg Oral Med Oral Pathol 2003;95: 710–6. 23. Shellman YG, Makela M, Norris DA. Induction of secreted matrix metalloproteinase-9 activity in human melanoma cells by extracellular matrix proteins and cytokines. Melanoma Res 2006;16:207–11. 24. Kim TD, Song KS, Li G, et al. Activity and expression of urokinasetype plasminogen activator and matrix metalloproteinases in human colorectal cancer. MBC Cancer 2006;6:211 [Epub ahead of print]. 25. Juarez J, Clayman G, Nakajima M, et al. Role and regulation of expression of 92-kDa type-IV collagenase (MMP-9) in 2 invasive squamous-cell-carcinoma cell lines of the oral cavity. Int J Cancer 1993;55:10–8. 26. Thomas GJ, Lewis MP, Hart IR, Marshall JF, Speight PM. avb6 integrin promotes invasion of squamous carcinoma cells through upregulation of matrix metalloproteinase-9. Int J Cancer 2001;92:641–50. 27. Zhang B, Henney A, Eriksson P, Hamsten A, Watkins H, Ye S. Genetic variation at the matrix metalloproteinase-9 locus on chromosome 20q12.2-13.1. Hum Genet 2004;105:418–23. 28. Zhang B, Ye S, Herrmann SM, et al. Functional polymorphism in the regulatory region of gelatinase B gene in relation to severity of coronary atherosclerosis. Circulation 1999;99:1788–94. 29. Grieu F, Li WQ, Iacopetta B. Genetic polymorphisms in the MMP-2 and MMP-9 genes and breast cancer phenotype. Breast Cancer Res Treat 2004;88:197–204. 30. Wang Y, Fang S, Lizhen Wei, et al. No association between the C-1562T polymorphism in the promoter of matrix metalloproteinase-9 gene and non-small cell lung carcinoma. Lung Cancer 2005;49:155–61. 31. Polla¨nen PJ, Lehtima¨ki T, Mikkelsson J, et al. Matrix metalloproteinase 3 and 9 gene promoter polymorphisms: joint action of two loci as a risk factor for coronary artery complicated plaques. Atherosclerosis 2005;180:73–8. 32. Matsumura S, Oue N, Nakayama H, et al. A single nucleotide polymorphism in the MMP-9 promoter affects tumor progression and invasive phenotype of gastric cancer. J Cancer Res Clin Oncol 2005;131: 19–25. 33. Li Y, Jin X, Kang S, et al. Polymorphisms in the promoter regions of the matrix metalloproteinases-1, -3, -7, and -9 and the risk of epithelial ovarian cancer in China. Gynecol Oncol 2006;101:92–6.
E. Vairaktaris et al. / EJSO 34 (2008) 450e455 34. Elander N, Soderkvist P, Fransen K. Matrix metalloproteinase (MMP)1, -2, -3 and -9 promoter polymorphisms in colorectal cancer. Anticancer Res 2006;26:791–5. 35. Ikebe T, Nakayama H, Shinohara M, Shirasuna K. NF-kB involvement in tumor-stroma interaction of squamous cell carcinoma. Oral Oncol 2004;40:1048–56.
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36. Kato K, Hara A, Kuno T, et al. Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity. J Cancer Res Clin Oncol 2005;131:340–6. 37. Schnaper HW, Barnathan ES, Mazar A, et al. Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways. Cell Physiol 1995;165:107–18.
EJSO 34 (2008) 450e455
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A metalloproteinase-9 polymorphism which affects its expression is associated with increased risk for oral squamous cell carcinoma E. Vairaktaris a,*, S. Vassiliou a, E. Nkenke b, Z. Serefoglou a, S. Derka a, C. Tsigris d, A. Vylliotis a, C. Yapijakis a, F.W. Neukam b, E. Patsouris c a
Department of Oral and Maxillofacial Surgery, University of Athens Medical School, ‘‘Attikon’’ Hospital, Rimini 1, Athens, GR-12462, Greece b Department of Oral and Maxillofacial Surgery, Universita¨t Erlangen, Klinik und Poliklinik f}ur Mund-, Kiefer-, Gesischtschirurgie, Glueckstrasse 11, Erlangen D-91054, N}urnberg, Germany c Department of Pathology, University of Athens Medical School, Mikras Asias 75, Athens GR-11527, Greece d First Department of Surgery, University of Athens Medical School, ‘‘Laikon’’ General Hospital, Mikras Asias 75, Athens GR-11527, Greece Accepted 27 March 2007 Available online 11 May 2007
Abstract Aim: In light to recently found contribution of factors associated with angiogenesis, thrombosis and inflammation to carcinogenesis, we investigated the possible association of metalloproteinase-9 (MMP-9) with increased risk of oral cancer. Methods: In DNA samples of 152 patients with oral squamous cell carcinoma and 162 healthy controls of comparable ethnicity, age and sex, we studied the 1562 C/T polymorphism in the MMP-9 gene promoter, which affects its transcription. Results: The detected frequency for the high expression T allele in the patients’ group was significantly increased in comparison to that of the control group (22% versus 15%, respectively; P < 0.05). This difference was due to the relative increase of C/T heterozygotes in the group of patients, in comparison to controls (P < 0.05, 95% OR 1.92, CI 1.21e3.06). The same pattern of significance was observed between controls and the subgroups of patients with initial (I & II) stages of cancer, without positive family history of cancer or thrombophilia, with smoking and alcohol abuse habits. Conclusions: The investigated MMP-9 polymorphism has a strong association with increased risk for developing oral cancer in a subset of the general population. These results are in accordance to previous studies of constitutive expression and secretion of MMP-9 in invasive oral carcinoma cell lines. The observation that T allele carriers have an increased risk for developing oral cancer only in initial stages, but not in advanced ones, may be due to the role of MMP-9 in the inhibition of angiogenesis by generating angiostatin from plasminogen. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Oral cancer; Metalloproteinase-9; Polymorphism; Angiogenesis; Oncogenesis; Tumor progression
Introduction Oral squamous cell carcinoma (OSCC), the most common form of oral cancer, is a multistep process implicated in which are not only environmental factors (such as smoking and alcohol abuse) but also genetic alterations in oncogenes and tumor suppressor genes.1 OSCC is a major cause of cancer morbidity and mortality worldwide, and despite therapeutic developments, it is characterized by low survival rate.1 Recently, factors associated with angiogenesis, thrombosis and inflammation have also been correlated with increased risk for oral cancer.2e5 Metalloproteinase-9 * Corresponding author. Tel.: þ30 210 6443035; fax: þ30 210 6443803. E-mail address: [email protected] (E. Vairaktaris). 0748-7983/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2007.03.024
(MMP-9, also known as gelatinase B) is one such factor implicated in thrombosis, inflammation, angiogenesis and oncogenesis.6e9 MMP-9 belongs to the large group of secreted proteinases that require zinc for their catalytic activity.10 Their proteolytic activity is controlled by changes in the delicate balance between the expression and synthesis of MMPs and their major endogenous inhibitors, tissue inhibitors of matrix metalloproteinases.11 MMP-9 is a collagenase type IV, which degrades the extracellular matrix and basement membrane components, a very crucial stage in tumor cell invasion and metastasis.12 On the other hand, there is evidence that MMP-9 may play a significant role in angiogenesis.13e17 Some reports have implicated both MMP-9 and MMP-2 in the induction of early angiogenic events,
E. Vairaktaris et al. / EJSO 34 (2008) 450e455
but it is also known that MMP-9 degrades plasminogen and generates angiostatin, an inhibitor of endothelial cell proliferation and angiogenesis.13e17 MMP-9 is expressed in low levels under physiological conditions but it is increased dramatically in several malignancies including oral, esophageal, breast, renal, colorectal carcinomas and melanomas.18e24 In particular, a study of oral squamous cell carcinoma cell lines suggested that constitutive expression and secretion of MMP-9 is an important determinant of the invasive phenotype by activation of the protein-kinase-C pathway.25 Another study indicated that MMP-9 expression in vitro is modulated by integrin avb6, which is expressed only in oral carcinoma cells and not in the normal oral epithelium.26 The human MMP-9 gene is located on chromosome 20q12.2e13.1 and it contains 10 sequence variants in tight linkage disequilibrium across its entire length.27,28 A functional C to T single nucleotide polymorphism located in the promoter region (position 1562) is known to affect transcription.28 The less common T allele has been found to confer a 2-fold higher gene expression than the C allele.28 The frequency of the T allele in Caucasian and Asian populations is about 12e15%.31e33 The frequency of the high expression T allele has been found to be increased in patients with invasive gastric cancer, while no difference was detected between the 1562 C/T polymorphism and colorectal, ovarian and non-small cell lung carcinoma.30,32e34 In light of the above, the purpose of this study was to investigate whether the 1562 C/T polymorphism is associated with oral cancer, by genotyping patients with oral squamous cell carcinoma and healthy controls representing the general population. Methods Subjects under study The individuals under study included 314 unrelated Greeks and Germans, consisting of 152 patients with oral cancer and 162 healthy blood donors (controls) of equivalent ethnicity, age and gender. The patients were mostly men (N ¼ 120) and their age ranged between 40 and 84 years (mean ¼ 58.4 years). The sex ratio (N ¼ 123 men) and the age of the controls (range 31e83 years; mean ¼ 54.7 years) were comparable to those of the patients. Included in this study were patients who had developed oral squamous cell carcinoma and were operated recently or up to a decade ago. In addition to clinical presentation, a biopsy with pathological diagnosis of tumor stages IeIV and a family history regarding cancer and thrombophilia were available. Fifty-eight of them (38.2%) had one or two first-degree relatives with any type of cancer and their age range (mean ¼ 58.8 years) did not differ from the whole group of patients. Furthermore, 26 patients (17.1%) had one or two first-degree relatives with idiopathic thrombosis and an earlier age range (mean ¼ 56.4 years), but again with no
451
significant difference with the whole group. Fourteen patients (9.2%) had a positive family history for both cancer and thrombophilia (mean age ¼ 56.4 years). Nearly all patients (93.6%) were smokers consuming at least 10 cigarettes per day. About one third of patients (32.05%) were ethanol abusers consuming at least three glasses of alcoholic drinks per day. Most of the participants in the study worked in a low-risk environment, with the exception of one patient and three controls who worked in chemical factories. No data were available on healthy controls regarding their family history or smoking and alcohol consumption habits. Molecular analysis Blood samples were collected from patients and controls after informed consent. DNA was isolated from blood with the use of NucleonÔ kit (Amersham). Molecular detection of the 1562 C/T polymorphism in the MMP-9 gene was performed by restriction fragment length polymorphism typing, as previously described.30 This involved a combination of PCR amplification and digestion with restriction endonuclease Sph I followed by gel electrophoretic analysis. The generated PCR product of 435 bp was cleaved by restriction enzyme Sph I into two fragments of 247 bp and 188 bp only if the T allele was present, while the C allele was not cleaved by the enzyme and kept the original size of 435 bp. Statistical analysis Statistical analysis was performed using SASÒ software (version 9.0; SAS Institute Inc.). The frequencies of alleles and genotypes of the whole group or subgroups of patients were compared to the respective frequencies of the control group using the Fisher’s exact test and odds ratios, while all genotype distributions were according to HardyeWeinberg estimates. The significance level was set at P < 0.05 and the results are presented as hazard ratios using the Maentele Haenzel method with 95% confidence intervals (CI). Results Genotype and allele frequencies of studied controls The prevalence of detected MMP-9 genotypes in healthy controls (representing the general population) and patients with oral cancer is shown in Table 1, after bilateral statistical analysis. The data for the two populations under study (Greek and German healthy controls) were analyzed together, since there were no significant differences in genotype and allele frequencies of the (1562 C/T) polymorphism among the two populations. The ‘‘high expression’’ T allele frequency in the control group was 15% and the carrier frequency was 30% (observed genotypes TT ¼ 0, CC ¼ 114, CT ¼ 48, Table 1). The (1562 C/T) genotype distributions were as expected in HardyeWeinberg equilibrium in the control
E. Vairaktaris et al. / EJSO 34 (2008) 450e455
28% (P < 0.05)
group, as well as in the whole group and subgroups of patients.
30% (P < 0.05)
19% (N.S.)
Comparisons between patients and controls
Statistical difference of frequencies observed in patients and their subgroups in comparison to controls (P-values). N.S.: not significant P-value.
30% (N.S.) 22% (P < 0.05) 15% (N.S.) 22% (P < 0.05) T allele frequency
15%
25% (P < 0.05)
19% (N.S.)
17% (N.S.)
25% (P < 0.05)
23% (P < 0.05)
0 20 26 46 0 64 38 102 0 20 30 50 0 4 6 10 0 80 62 142 0 66 60 126 0 18 8 26 0 46 48 94 0 84 68 152 TT CC CT Total
0 114 48 162
0 42 42 84
0 42 26 68
0 38 20 58
Patients with positive family history of thrombosis Patients without positive family history of cancer Patients with positive family history of cancer Patients with cancer stages III & IV Patients with cancer stages I & II Patients Controls Prevalence of T allele
Table 1 Prevalence of MMP-9 T allele and carrier frequencies in healthy controls and patients with oral cancer
Patients without positive family history of thrombosis
Patients with tobacco abuse
Patients without tobacco abuse
Patients with alcohol abuse
Patients without alcohol abuse
Patients with tobacco and alcohol abuse
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The detected allele and carrier frequencies for the high expression T allele in the patients’ group (22% and 45%, respectively) were significantly increased in comparison to that of the control group (P < 0.05, Table 1). This statistical difference was due to the relative increase of CT heterozygotes in the group of patients, in comparison to controls (genotypes TT ¼ 0, CC ¼ 84, CT ¼ 68, Table 1). The same pattern of significantly increased frequencies of T allele and CT heterozygotes, compared to controls, was also observed in the following subgroups of patients (Table 1): (a) with cancer stages I & II (P < 0.05), (b) without positive family history of cancer (P < 0.05), (c) without positive family history of thrombosis (P < 0.05), (d) with tobacco abuse (P < 0.05), (e) with alcohol abuse (P < 0.001) and (g) with alcohol and tobacco abuse (P < 0.05). Finally, in comparison to controls no significant differences in genotype or allele frequencies were detected in subgroups of patients with advanced cancer stages (III & IV), with positive family history of cancer or thrombosis, as well as without tobacco or alcohol abuse (Table 1). Compared to individuals with the CC genotype, the relative risk (OR) for CT heterozygotes to develop oral cancer was 1.92 (95% CI 1.21e3.06). Interestingly, CT individuals have an even higher relative risk for developing early stages of OSCC (OR 2.37, 95% CI 1.38e4.09). Finally, there were no significant differences due to categorizations of patients in regard to gender, age and age at onset of oral cancer. Discussion MMP-9 and tumor MMP-9 is a zinc-dependent proteinase, which is involved in numerous physiological and pathological processes. Due to its ability to degrade the extracellular matrix and the basement membrane components, it seems to play a crucial role in tumor progression.12 Although MMP-9 is expressed in low levels under physiological conditions, its expression is highly increased in many malignancies, including oral squamous cell carcinoma.18e24 The MMP-9 gene contains 10 sequence polymorphic variants which are in tight linkage disequilibrium, and one of them, the functional 1562 C/T polymorphism has been recognised to affect gene transcription.28 The promoter activity of the MMP-9 gene is increased considerably when the less common, ‘‘high expression’’ Tallele is present.28 Findings of this study In light of the above, the purpose of this study was to investigate the genotypes and allele frequencies of the
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1562 C/T polymorphism in a cohort of 152 patients with oral cancer in comparison to 162 healthy controls of equivalent age, sex and ethnicity. Although the previously observed high levels of MMP-9 in oral carcinomas might be attributed to a secondary effect of some altered mechanism(s), or even to the tumor micro-environment, we hypothesized that endogenous high MMP-9 expression might confer a genetic predisposition for oral cancer.35 In order to investigate the putative effect of genetically determined high MMP-9 expression on risk for oral cancer, the 1562 C/T polymorphism was examined only in normal blood cells and not in cancer cells of patients. Despite the modest number of studied individuals, the obtained results revealed a strong association of the examined MMP-9 polymorphism with increased risk of oral cancer. In comparison to controls, a significant increase of C/T heterozygotes was observed not only in the whole group of patients, but in the subgroups of patients with initial cancer stages, without family history of cancer or thrombosis, with smoking or/and alcohol abuse habits. Accordingly, the carrier and allele frequencies for the high expression T were increased in the whole group of patients and in all the above-mentioned subgroups.
Implications of findings The results of this study, which implicate in oral oncogenesis the higher gene expression of MMP-9 conferred by the T allele, are in accordance to previous reports that constitutive expression and secretion of MMP-9 is an important determinant of the malignant phenotype in oral squamous cell carcinoma.25,26 The interesting observation of the present study that T allele carriers have an increased risk for developing oral cancer only in initial stages, but not in advanced ones, may be due to the significant role of MMP-9 in the inhibition of angiogenesis by generating angiostatin.17 A similar finding was reported in patients with breast cancer, who seem to have a better prognosis if they carry the T allele.29 The prophylactic role of high MMP-9 levels in advanced stages of OSCC at first seems to be puzzling. Some older studies have implicated high expression of both MMP-9 and MMP-2 in initial stages of angiogenesis involving endothelial morphogenesis and tube formation in normal veins as well as in certain carcinomas.13e16 Nevertheless, a recent study in OSCC has provided evidence that the role of MMP-9 in the matrix degradation is minimal compared to MMP-2, which seems to be the main enzyme of gelatinolysis, invasion and metastasis in the oral region.36 MMP-9 is highly expressed in OSCC and produced in an inactive proform, which needs to be converted into its active form in order to degrade the matrix.36 Interestingly, the ratio of activated form/proform of MMP-9 is very low in OSCC, thus its role in matrix degradation is minimal.36
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Conversion from proform into the active form of MMP9 may involve plasminogen, as a result of the interaction of tumor with stromal fibroblast cells.25,35 In the presence of plasminogen, MMP-9 is activated and in turn converts plasminogen into angiostatin, one of the most potent inhibitors of angiogenesis.17 Therefore, the role of MMP-9 in advanced stages of cancer may indeed be inhibitory. In support to this notion, we previously found that a polymorphic allele that results in high levels of plasminogen activator inhibitor-1 (PAI-1) is associated only with initial stages of OSCC.4 The inhibition of plasminogen activators in early cancer stages results in high levels of plasminogen (since it is not converted into plasmin) and consequently in high levels of active MMP-9, which facilitates tumor development by degradation of extracellular matrix. On the contrary, in advanced OSCC stages PAI-1 levels are low, plasminogen is converted to plasmin and MMP-9 proform remains inactive, therefore, the level of MMP-9 gene expression is not associated with tumor progression and metastasis. This notion is also in accordance to previously observed in vitro inhibition of angiogenesis by PAI-1.37 The fact that the high expression T allele is increased only in patients without a positive family history of cancer probably indicates that the MMP-9 polymorphism is associated with only a few types of malignancies. In accordance to this notion, the frequency of the T allele has been found to be increased only in patients with invasive gastric cancer, while no such difference was detected in patients with colorectal, ovarian and non-small cell lung carcinoma.30,32e34 Furthermore, the observation that the T allele is increased only in patients without a positive family history of thrombophilia possibly suggests that the MMP-9 polymorphism is not associated with inherited predisposition for thrombosis. Although the role of MMP-9 in risk for thrombosis is still controversial, our results are in accordance with previous studies in which the 1562 C/T polymorphism was not correlated with the severity of coronary atherosclerosis or myocardial infarction.28,31 Although the role of alcohol in oral carcinogenesis seems to be synergistic, the fact that T allele frequency was significantly higher only in patients who over-consume alcohol seems to underline the combined effect of high MMP-9 levels and ethanol. The same significant increase of T alleles was also observed in smoking patients, but the small amount of available non-smoking patients who were included in this study does not allow safe conclusions to be drawn regarding the combined effect of tobacco and high MMP-9 levels. The synergistic effect of smoking and heavy alcohol intake was also significantly associated with an increase of T alleles, although the respective subgroup of patients was rather small. In conclusion, the investigated MMP-9 polymorphism has a strong association with increased risk for developing oral cancer in a subset of the general population. This
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illustrates the great importance of genetic association studies, which may ultimately result in presymptomatic testing and preventive measures safeguarding the health status and lives of certain at risk individuals in the general population. Conflict of interest There are no financial or personal relationships of authors with other people or organizations that could inappropriate influence their work. Acknowledgements This work was co-funded by the European Social Fund and National Resources (EPEAEK II ‘‘Pythagoras’’ 70/3/ 7391) grant to E.V. References 1. McDowell JD. An overview of epidemiology and common risk factors for oral squamous cell carcinoma. Otolaryngol Clin of North Am 2006; 39:277–94. 2. Tsai MH, Chen WC, Chen HY, Tsai FJ. Urokinase gene 30 -UTR T/C polymorphism is associated with oral cancer. J Clin Lab Anal 2004;18: 276–9. 3. Vairaktaris E, Yapijakis C, Derka S, et al. Association of platelet Ia polymorphism with minor increase of risk for oral cancer. Eur J Surg Oncol 2006;32:455–7. 4. Vairaktaris E, Yapijakis C, Serefoglou Z, et al. Plasminogen activator inhibitor-1 polymorphism is associated with increased risk for oral cancer. Oral Oncol 2006;42:888–92. 5. Vairaktaris E, Yapijakis C, Serefoglou Z, et al. The interleukin-8 (-251A/T) polymorphism is associated with increased risk for oral squamous cell carcinoma. Eur J Surg Oncol 2007;33:504–7. 6. Alvarez B, Ruiz C, Chacon P, et al. Serum values of metalloproteinase-2 and metalloproteinase-9 as related to unstable plaque and inflammatory cells in patients with greater than 70% carotid artery stenosis. J Vasc Surg 2004;40:469–75. 7. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflammation and cancer: how hot is the link? Biochem Pharmacol 2006;72: 1605–21. 8. Ruokolainen H, Pa¨a¨kko¨ P, Turpeenniemi-Hujanen T. Expresssion of matrix metalloproteinase-9 in head and neck squamous cell carcinoma: a potential marker for prognosis. Clin Cancer Res 2004;10: 3110–6. 9. John A, Tuszynski G. The role of matrix metalloproteinases in tumour angiogenesis and tumour metastasis. Pathol Oncol Res 2001;7:14–23. 10. Nagase H, Woessner JF. Matrix metalloproteinases. J Biol Chem 1999; 274:21491–4. 11. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta 2000;1477:267–83. 12. Stetler-Stevenson WG. The role of matrix metalloproteinases in tumour invasion, metastasis, and angiogenesis. Surg Oncol Clin N Am 2001;10:383–92. 13. Schnaper HW, Grant DS, Stetler-Stevenson WG, et al. Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. Cell Physiol 1993;156:235–46. 14. Iurlaro M, Loverro G, Vacca A, et al. Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with upgrading and myometrial invasion in endometrial carcinoma. Eur J Clin Invest 1999;29:793–801.
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