|Year : 2020 | Volume
| Issue : 1 | Page : 144-149
Transforming growth factor-β1 gene polymorphism as a potential risk factor in Turkish patients with laryngeal squamous cell carcinoma
Candan Demiröz Abakay1, Mehrdad Pashazadeh2, Elif Ardahanli3, Haluk Barbaros Oral4
1 Department of Radiation Oncology, Faculty of Medicine; Department of Microbiology, Immunology Division, Health Science Institute, Bursa Uludag University, Bursa, Turkey
2 Department of Microbiology, Immunology Division, Health Science Institute; Department of Immunology, Faculty of Medicine, Bursa Uludag University, Bursa, Turkey
3 Department of Immunology, Faculty of Medicine; Department of Immunology, Health Science Institute, Bursa Uludag University, Bursa, Turkey
4 Department of Immunology, Faculty of Medicine, Bursa Uludag University, Bursa, Turkey
|Date of Submission||14-Aug-2019|
|Date of Acceptance||12-Nov-2019|
|Date of Web Publication||29-Apr-2020|
Haluk Barbaros Oral
Department of Immunology, Faculty of Medicine, Bursa Uludag University, Gorukle Campus 16059, Nilufer, Bursa
Source of Support: None, Conflict of Interest: None
Introduction: Laryngeal cancer is the most common head-and-neck malignancies with more than 20% of all cases. The vast majority of tumors are squamous cell carcinoma (SCC). Several genes encoding different cytokines may play crucial roles in host susceptibility to cancer because cytokine production capacity varies among individuals and depends on cytokine gene polymorphisms.
Materials and Methods: The association between cytokine gene polymorphisms with primary laryngeal SCC was investigated. DNA samples were obtained from a Turkish population of eighty patients with primary cancer and fifty healthy controls.
Results: All genotyping (interferon-gamma, transforming growth factor-β1 [TGF-β1], tumor necrosis factor-alpha [TNF-α], interleukin [IL]-6, and IL-10) experiments were performed using polymerase chain reaction sequence-specific primers. When compared to the healthy controls, the frequencies of TGF-β1 codon 25 (rs1800471) GC genotype and 25 C allele were significantly more common in the patient group.
Conclusions: These results suggest that TGF-β1 gene polymorphisms may affect host susceptibility to laryngeal cancer.
Keywords: Cytokine, genotyping, larynx cancer, single-nucleotide gene polymorphism, transforming growth factor-beta
|How to cite this article:|
Abakay CD, Pashazadeh M, Ardahanli E, Oral HB. Transforming growth factor-β1 gene polymorphism as a potential risk factor in Turkish patients with laryngeal squamous cell carcinoma. J Can Res Ther 2020;16:144-9
|How to cite this URL:|
Abakay CD, Pashazadeh M, Ardahanli E, Oral HB. Transforming growth factor-β1 gene polymorphism as a potential risk factor in Turkish patients with laryngeal squamous cell carcinoma. J Can Res Ther [serial online] 2020 [cited 2021 Aug 5];16:144-9. Available from: https://www.cancerjournal.net/text.asp?2020/16/1/144/283318
| > Introduction|| |
Laryngeal squamous cell carcinoma (SCC) is the most common malignancy of head-and-neck cancers (HNC), and the major causative factors include excessive tobacco and alcohol consumption and/or exposure to environmental risk factors such as air pollution. However, the fact that a small part of the laryngeal cancer patients are the ones who are not exposed to any of these risk factors suggests that genetic susceptibility is also involved in the etiopathogenesis of this disease. Indeed, genetic susceptibility to SCC of the larynx was well documented., The disease is much more common in the male gender. The highest incidence of laryngeal cancer occurs between the fifth and seventh decades of life. Cytokines participate in mediating many of the effector phases of immune and inflammatory responses. T helper cells, according to cytokine synthesis profiles, can be considered as T helper cell type 1 (Th1) promoting cell-mediated immunity (interleukin [IL]-2 and interferon gamma [IFN-γ]), T helper cell type 2 (Th2) leading to humoral immunity (IL-4, IL-5, IL-10, and IL-13), or Th17 (IL-17, IL-21, IL-22, and IL-26) playing critical functions in the development of autoimmunity and allergic reactions as well as cancer., T regulatory (Tr) cell is another Th cell type that is involved in the regulation of Th1, Th2, and Th17 type responses. Therefore, the balance among Th cells and Tr cells is extremely important for the development and progression of tumors. The in - vitro maximal capacity of the immune cells to produce different cytokines in response to mitogen stimulation was shown to vary between individuals. Such differences can be attributed to several molecular mechanisms including variations in transcription, translation, and secretion pathways., Recently, an additional potential mechanism was described involving conservative mutation within cytokine coding regions and nucleotide variations within more pronounced regulatory regions., Genetic polymorphisms potentially affecting production levels of certain cytokines may be important determinants of disease risk and severity or protection for several conditions in which the immune system plays significant roles, such as malignancies. Eventually, many studies have reported that there is an association between cytokine gene polymorphisms and the development of certain infectious diseases, allergies, autoimmune disorders, and cancers. In some studies, these genetic polymorphisms are shown to affect the overall expression and secretion of cytokines both in vitro and sporadically in vivo systems. However, a multicentric study using an identical in - vitro cell culture system and study population has to be set up to analyze the relationship between certain cytokine gene polymorphisms and in - vitro cytokine production because there are also some contradictory results obtained from other studies. The aim of the present study was to investigate whether there are associations between cytokine gene polymorphism profile and the risk of laryngeal SCC development. We used a panel of pro-inflammatory (tumor necrosis factor alpha [TNF-α, IL-6, and IFN-γ) and anti-inflammatory (transforming growth factor-beta 1 [TGF-β1] and IL-10) cytokines that are known to be involved in tumor immunity.
| > Materials and Methods|| |
A total of eighty male patients with laryngeal SCC from the Department of Radiation Oncology, Uludaǧ University School of Medicine, were enrolled in this study. Diagnosis of laryngeal SCC was confirmed by histopathological examination. The control group comprised geographically and racially matched fifty male healthy blood donors.
The study was approved by the Ethical Committee of Bursa Uludag University, and all participants gave written informed consent.
DNA isolation and cytokine genotyping
Genomic DNA was extracted from whole ethylenediaminetetraacetic acid-treated blood with the NucleoSpin® Blood DNA isolation kit (Machery Nagel, Duren, Germany) according to the manufacturer's instructions. Single-nucleotide polymorphisms (SNPs) were analyzed in five cytokines for genotype assignment. The presence of a G or A nucleotide in position 308 of the promoter region was analyzed for TNF-α. Two single-nucleotide mutations in the coding region were surveyed for TGF-β1 codon 10 (+869, rs1800470, formerly rs1982073), can be either T or C, and codon 25 (+915, rs1800471), either C or G. Three different polymorphisms were analyzed for the IL-10 promoter region: position −1082 (rs1800896) (G vs. A), position 819 (rs1800871) (C vs. T), and position −592 (rs1800872) (A vs. C). The presence of a single-nucleotide modification in position −174 (rs2069840) was examined for IL-6 promoter. An additional coding sequence mutation (T vs. A) at position +874 (rs2430561) was analyzed for IFN-γ. Cytokine genotypes were determined using polymerase chain reaction (PCR) sequence-specific primers method by a commercially available kit (One lambda, Inc., Canoga Park, CA, USA) in accordance with the manufacturer's instructions. The DNA extractions and PCR amplifications were performed by a technician blinded to the study groups.
Statistical analysis was performed by Epi Info version 3.2.2 (Centers for Disease Control and Prevention, Atlanta GA, USA). The distribution of cytokine gene polymorphisms was compared between patients with laryngeal SCC and healthy controls by Chi-square or Fisher's exact test. The data were analyzed for appropriateness between the observed and expected genotype values and their fit to Hardy–Weinberg equilibrium (HWE) by Arlequin version 3.5 Software. P < 0.05 was considered significant. Odds ratios (OR) and 95% confidence intervals (CI) were also calculated in case Chi-square or Fisher's exact test was significant. In addition, significant probability values obtained were corrected for multiple testing (Bonferroni correction; Pc ), because of small sample size in our study.
| > Results|| |
As more than 95% of our patients are males, we only included male patients and controls in our study. The average age of the patients was 59.30 (± 9.44) years, with a range from 42 to 86 years. The average age of the controls was 58.50 (± 8.66), with a range from 44 to 77 years.
The staging of the tumor according to the TNM system, regarding the local extension of the tumor at the time of diagnosis, was as follows: Stage T1 in 24 patients (30.0%), Stage T2 in 7 (8.8%), Stage T3 in 23 (28.7%), and finally, Stage T4 in 26 (32.5%) patients. The patients with carcinoma in situ were not included in this study.
The distribution of the cytokine genotypes among the patients with laryngeal SCC and healthy controls and ORs of significantly different cytokine polymorphisms are summarized in [Table 1] and [Table 2], respectively. TNF-α −308 TGF-β1 codon 25, IL-10 1082, IL-10 −819, IL-10 −592, IL-6 −174 genotype frequencies were in HWE both for patients and controls, whereas genotype frequencies of TGF-β1 codon 10 and IFN-γ +874 were not in HWE for controls and patients, respectively. In some cases, deviations from HWE are due to assay failures. This is very unlikely that genotyping of these SNPs was performed in triplicates. Because these results indicated that deviations from HWE were not due to an assay failure, genotype data for TGF-β1 codon 10 and IFN-γ +874 were included in the analyses.
|Table 1: Cytokine gene polymorphisms among laryngeal squamous cell carcinoma patients and healthy controls|
Click here to view
|Table 2: Genotypic and allele frequencies of transforming growth factor-β1 codon 10 polymorphisms in eighty patients and healthy controls|
Click here to view
The frequency of TGF-β1 codon 10–25 T/C-G/C was significantly higher in the patient group when compared with controls (20.5% vs. 2.0%, P : 0.003, OR: 12.65 [CI 1.62–98.69]) [Table 1]. When TGF-β1 codon 10 and codon 25 genotypes were evaluated separately, TGF-β1 codon 10 TC genotype and TGF-β1 codon 25 GC genotypes were significantly higher in comparison to those of controls (55.1% vs. 34.0, P = 0.029 and 20.5% vs. 4.0%, P = 0.009, respectively) [Table 2]. However, statistical significance for TGF-β1 codon 10 TC genotype was lost when the P value was adjusted by Bonferroni correction ( Pc = 0.089). In addition, analysis of allele frequencies showed that the frequency of TGF-β1 codon 25 C allele was significantly higher in the patients when compared to the controls (16% vs. 2%, P : 0.012, OR: 5.6) [Table 2].
On the other hand, lower frequency of IFN-γ +874 AA (low producing) genotype was observed in the patient group in comparison to controls (15.0% vs. 30.0%, P = 0.048, OR: 0.041 [CI 0.17–0.97]) [Table 1] and [Table 3]. Statistical significance for this genotype was lost when the P value was adjusted by Bonferroni correction ( Pc = 0.122).
|Table 3: Cytokine gene polymorphisms and their associated phenotypes[10,11] among laryngeal squamous cell carcinoma patients and healthy controls|
Click here to view
Genotype or allele frequencies for the other cytokine gene polymorphisms did not differ significantly between the patient and control groups [Table 1].
When high-, intermediate-, and low-producing genotypes were compared, no statistical difference in genotype frequencies was detected [Table 3].
| > Discussion|| |
TGF-β1 is a member of the TGF-β superfamily of cytokines and that is predominantly secreted by Tregs. TGF-β1 can regulate both the immune system and cellular functions including cell differentiation and proliferation; extracellular matrix production; binding of ligand to a heterodimeric receptor; and initiation of signals that regulate growth, apoptosis, and angiogenesis.,, It induces mesenchymal transition, antagonizes IL-2 functions, and induces immune responses. Therefore, it promotes tumor progression, escape, invasion, and metastasis.,, Recent studies have shown that SNP of TGF-β1 is associated with susceptibility to a large range of cancers, including lung cancer, prostate cancer, gastric cancer, and hepatocellular cancer. Clinical studies have also been conducted to explore the association between SNPs in TGF-β1 and HNC susceptibility.,, TGF-β1 may contribute to the aggressive behavior of cancers through the local and systemic immunosuppression effect., Furthermore, increased plasma levels of TGF-β1 were described as a tumor marker and prognostic factor in HNCs including laryngeal SCC. These findings suggest that TGF-β1 may be useful in the early detection of laryngeal SCC recurrence or to control the success of immunochemotherapy. Though there are studies showing that the C allele in −509 C/T polymorphism (rs1800469) could increase the level of TGF-β1 expression in serum and in nasopharyngeal carcinoma cell lines,, Shi et al . failed to identify a significant association between TGF-β1 −509 C/T polymorphism and HNC risk in all the alleles or genotype models.
In terms of TGF-β1 codon 10 gene polymorphism, statistically significant associations were observed in the allelic model (C vs. T: OR = 1.351, 95% CI: 1.030–1.772, P = 0.030), homozygote model (CC vs. TT: OR = 1.585, 95% CI: 1.026–2.449, P = 0.038), and dominant model (CT/CC vs. TT: OR = 1.398, 95% CI: 1.008–1.937, P = 0.044) of TGF-β1 codon 10 polymorphism (rs1982073). These data suggest that the C allele and the CC genotype can increase the risk of HNC. This may be associated with an increased TGF-β1 level in carriers of the C allele, which might lead to a slightly attenuated immune function and a further rise of risk in developing HNC., One meta-analysis found weaker document for TGFβ1 codon 10 polymorphism and breast cancer risk, but another meta-analysis, based on thirty studies, conducted a subgroup analysis to explore the association between TGFβ1 codon 10 polymorphisms and HNC risk and found that this SNP was associated with an increased risk of HNC. On the other hand, Khaali et al . found that in the North African sample, the TGF-β1 −509 C/T polymorphisms did not substantially influence HNC susceptibility.
In our study, it was shown that TGF-β1 codon gene polymorphism may be associated with increased risk for laryngeal SCC. However, further studies in larger sample sizes are needed to confirm that observation.
Regarding TGF-β1 codon 25 polymorphism, no significant association was observed in all of the previous studies. In contrast, we found that TGF-β1 codon 25 C allele seemed to be the risk factor for laryngeal SCC development in our study. In this study, we investigated the potential associations between cytokine gene polymorphisms and laryngeal SCC. Our data suggest that the presence of TGF-β1 codon 10–25 T/C-G/C genotype and TGF-β1 codon 25 C allele may increase the risk for developing laryngeal SCC.
Polymorphism analysis of IFN-γ, an important cytokine in antitumor host defense, showed no statistically significant genotype difference at position +874 in Turkish HNC patients in the present study. This result was similar to a renal cell carcinoma study originating from the same ethnic group. The therapeutic potential of TNF-α and TGF-β antagonists and their receptor antagonists in cancer therapy has been investigated, and some promising data have been accumulated.,, Therefore, it would be useful to know the genetic characteristics of cancer patients prior to deciding the therapeutic use of these cytokines themselves or of antibodies which could be specific to each ethnic group and cancer types because the production of these cytokines is regulated by genetic polymorphisms.
| > Conclusions|| |
We have demonstrated that there is a significant association between TGF-β1 gene polymorphisms and laryngeal SCC, and these polymorphisms may be valuable predictor determinants for the development of laryngeal SCC. As our study population was small in this study, larger studies are necessary to investigate the independence of these polymorphisms involved in the oncogenesis of laryngeal SCC.
This study was supported by a grant from Uludag University, Bursa/Turkey (grant number OUAP(T)-2013/25). Furthermore, part of the study was supported by Turkish Society for Radiation Oncology.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Brugere J, Guenel P, Leclerc A, Rodriguez J. Differential effects of tobacco and alcohol in cancer of the larynx, pharynx, and mouth. Cancer 1986;57:391-5.
Foulkes WD, Brunet JS, Sieh W, Black MJ, Shenouda G, Narod SA. Familial risks of squamous cell carcinoma of the head and neck: Retrospective case-control study. BMJ 1996;313:716-21.
Bardakci F, Canbay E, Degerli N, Coban L, Canbay EI. Relationship of tobacco smoking with GSTM1 gene polymorphism in laryngeal cancer. J Cell Mol Med 2003;7:307-12.
Unal M, Tamer L, Akbaş Y, Pata YS, Vayisoglu Y, Degirmenci U, et al
. Genetic polymorphism of N-acetyltransferase 2 in the susceptibility to laryngeal squamous cell carcinoma. Head Neck 2005;27:1056-60.
Cattaruzza MS, Maisonneuve P, Boyle P. Epidemiology of laryngeal cancer. Eur J Cancer B Oral Oncol 1996;32B: 293-305.
Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al
. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235-8.
Maruyama T, Kono K, Mizukami Y, Kawaguchi Y, Mimura K, Watanabe M, et al
. Distribution of Th17 cells and FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, tumor-draining lymph nodes and peripheral blood lymphocytes in patients with gastric cancer. Cancer Sci 2010;101:1947-54.
Stummvoll GH, DiPaolo RJ, Huter EN, Davidson TS, Glass D, Ward JM, et al
. Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells. J Immunol 2008;181:1908-16.
Bidwell J, Keen L, Gallagher G, Kimberly R, Huizinga T, McDermott MF, et al
. Cytokine gene polymorphism in human disease: On-line databases. Genes Immun 1999;1:3-19.
Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro
production of IFN-gamma correlates with CA repeat polymorphism in the human IFN-gamma gene. Eur J Immunogenet 1999;26:1-3.
Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al
. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369-76.
Hollegaard MV, Bidwell JL. Cytokine gene polymorphism in human disease: On-line databases, Supplement 3. Genes Immun 2006;7:269-76.
Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997;34:391-9.
Warlé MC, Farhan A, Metselaar HJ, Hop WC, Perrey C, Zondervan PE, et al
. Are cytokine gene polymorphisms related to in vitro
cytokine production profiles? Liver Transpl 2003;9:170-81.
Massagué J. TGFβ signalling in context. Nat Rev Mol Cell Biol 2012;13:616-30.
HayGlass KT, Wang M, Gieni RS, Ellison C, Gartner J. In vivo
direction of CD4 T cells to Th1 and Th2-like patterns of cytokine synthesis. Adv Exp Med Biol 1996;409:309-16.
Gaur P, Singh AK, Shukla NK, Das SN. Inter-relation of Th1, Th2, Th17 and Treg cytokines in oral cancer patients and their clinical significance. Hum Immunol 2014;75:330-7.
Wrzesinski SH, Wan YY, Flavell RA. Transforming growth factor-beta and the immune response: İmplications for anticancer therapy. Clin Cancer Res 2007;13:5262-70.
Jakowlew SB. Transforming growth factor-beta in cancer and metastasis. Cancer Metastasis Rev 2006;25:435-57.
Fan H, Yu H, Deng H, Chen X. Transforming growth factor-β1 rs1800470 polymorphism is associated with lung cancer risk: A meta-analysis. Med Sci Monit 2014;20:2358-62.
Cai Q, Tang Y, Zhang M, Shang Z, Li G, Tian J, et al
. TGFβ1 Leu10Pro polymorphism contributes to the development of prostate cancer: Evidence from a meta-analysis. Tumour Biol 2014;35:667-73.
Chang WW, Zhang L, Su H, Yao YS. An updated meta-analysis of transforming growth factor-β1 gene: Three polymorphisms with gastric cancer. Tumour Biol 2014;35:2837-44.
Lu WQ, Qiu JL, Huang ZL, Liu HY. Enhanced circulating transforming growth factor beta 1 is causally associated with an increased risk of hepatocellular carcinoma: A Mendelian randomization meta-analysis. Oncotarget 2016;7:84695-704.
Hu S, Zhou G, Zhang L, Jiang H, Xiao M. The effects of functional polymorphisms in the TGFβ1 gene on nasopharyngeal carcinoma susceptibility. Otolaryngol Head Neck Surg 2012;146:579-84.
Carneiro NK, Oda JM, Losi Guembarovski R, Ramos G, Oliveira BV, Cavalli IJ, et al
. Possible association between TGF-β1 polymorphism and oral cancer. Int J Immunogenet 2013;40:292-8.
Hsu HJ, Yang YH, Shieh TY, Chen CH, Kao YH, Yang CF, et al
. TGF-β1 and IL-10 single nucleotide polymorphisms as risk factors for oral cancer in Taiwanese. Kaohsiung J Med Sci 2015;31:123-9.
Tang B, Vu M, Booker T, Santner SJ, Miller FR, Anver MR, et al
. TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression. J Clin Invest 2003;112:1116-24.
Wei YS, Zhu YH, Du B, Yang ZH, Liang WB, Lv ML, et al
. Association of transforming growth factor-beta1 gene polymorphisms with genetic susceptibility to nasopharyngeal carcinoma. Clin Chim Acta 2007;380:165-9.
Niu H, Niu Z, Zhang XL, Chen ZL. Absence of association between transforming growth factor B1 polymorphisms and gastric cancer: A meta-analysis. DNA Cell Biol 2012;31:706-12.
Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 2003;3:807-21.
Shi Q, Wang X, Cai C, Yang S, Huo N, Liu H. Association between TGF-β1 polymorphisms and head and neck cancer risk: A meta-analysis. Front Genet 2017;8:169.
Cox A, Dunning AM, Garcia-Closas M, Balasubramanian S, Reed MW, Pooley KA, et al
. A common coding variant in CASP8 is associated with breast cancer risk. Nat Genet 2007;39:352-8.
Gu YY, Wang H, Wang S. TGF-β1 C-509T and T869C polymorphisms and cancer risk: A meta-analysis. Int J Clin Exp Med 2015;8:17932-40.
Khaali W, Moumad K, Ben Driss EK, Benider A, Ben Ayoub W, Hamdi-Cherif M, et al
. No association between TGF-β1 polymorphisms and risk of nasopharyngeal carcinoma in a large North African case-control study. BMC Med Genet 2016;17:72.
Brandacher G, Winkler C, Schroecksnadel K, Margreiter R, Fuchs D. Antitumoral activity of interferon-gamma involved in impaired immune function in cancer patients. Curr Drug Metab 2006;7:599-612.
Baştürk B, Yavaşçaoǧlu I, Vuruşkan H, Göral G, Oktay B, Oral HB. Cytokine gene polymorphisms as potential risk and protective factors in renal cell carcinoma. Cytokine 2005;30:41-5.
Akhurst RJ. TGF-beta antagonists: Why suppress a tumor suppressor? J Clin Invest 2002;109:1533-6.
Flanders KC, Burmester JK. Medical applications of transforming growth factor-beta. Clin Med Res 2003;1:13-20.
Balkwill F. TNF-alpha in promotion and progression of cancer. Cancer Metastasis Rev 2006;25:409-16.
[Table 1], [Table 2], [Table 3]