Abstract
Background/Aim: This study aimed to determine the role of the peroxisome proliferator-activated receptor-gamma (PPARg) C161T genotype and allele frequencies in predisposition to colorectal cancer (CRC).
Patients and Methods: PPARg C161T (His447His; rs3856806) gene polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism analysis in patients with CRC (n=101) and controls (n=238).
Results: The T161 allele (CT+TT genotypes) of PPARg C161T polymorphism was associated with CRC development (p<0.001; OR=3.239, 95%CI=1.997-5.252). Subgroup analysis showed that the T161 allele was associated with a 3.056-fold increased risk for colon cancer (CC) (p<0.001; 95%CI=1.709-5.464) and 3.529-fold increased risk for rectal cancer (RC) (p<0.001; 95%C=1.784-6.981). Frequencies of the T161 allele were also higher in total CRC and CC patients with poorly differentiated tumors (p<0.001, c2=30,601, OR=3.109; 95%CI=1.970-4.906 and p<0.001, Fisher exact test, respectively).
Conclusion: PPARg T161 allele carriers have increased risk for developing CRC.
Keywords: PPARg C161T polymorphism, colorectal cancer, Turkish population
Colorectal cancer (CRC) occurs over a long period (1). Previous reports suggest that several single nucleotide polymorphisms (SNPs) in the genome could influence the development of sporadic CRC (2). In addition, CRC susceptibility genes are not completely determined (3) and remain to be identified.
Peroxisome proliferator-activated receptor g (PPARg) is a member of the nuclear receptor superfamily and is a ligand-activated transcription factor. Genetic variants of PPARg gene have been suggested to be risk factors for cancer (4). One of the identified common structural SNPs in the PPARg2 gene is a silent C-to-T substitution at position 161 in exon 6 (C161T) (rs3856806, His447His, His449His or H449H or C1431T or c.1347C>T (5,6). Even though there is evidence that PPARg polymorphisms may increase CRC risk, the results are inconsistent (7-13). Of these studies, only a few have investigated the PPARg C161T polymorphism in CRC (7,9,14). Jiang et al. found that the CT+TT genotypes were associated with a 1.61-fold increased risk of CRC development in the Indian population (7). Li et al. reported that the risk of CRC development in non-smoking, non-drinking Eastern Chinese Han males over 61 years old with a BMI≥24 kg/m2 was increased with PPARG rs3856806 C>T polymorphism (14), whereas no increase in CRC risk related to C161T polymorphism was observed in the Japanese population (9). In addition, a meta-analysis (15) showed that the C161T polymorphism had a significant protective effect against CRC. However, no protective effect of this polymorphism against CRC was observed in Asians (15). Furthermore, the results of a recent meta-analysis do not support the association between CRC risk and PPARg C161T polymorphism because of limited data (16). In another recent meta-analysis, it was emphasized that more studies are needed to confirm the relationship between PPARg C161T SNP and CRC (16). Therefore, we aimed to determine the effect of the C161T polymorphism of the PPARg gene on CRC risk.
Patients and Methods
Study groups. Three hundred and thirty-nine individuals were included in the study. Among them, 101 were patients with CRC (60 had colon cancer and 41 had rectal cancer) admitted to the Gastrointestinal Surgery Department, Istanbul University, Faculty of Medicine and 238 healthy controls. Informed consent was obtained from all patients and the control group. Ethical permission was granted by the Istanbul University Ethical Committee.
PPAR gamma C161T mutation analysis. Genomic DNA was extracted from peripheral whole blood samples (17). Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were performed using Eco72I restriction enzyme to determine PPARg genotype. The PCR product of the PPARg rs3856806 (C>T) (200 bp) was not digested with Eco72I in the presence of the T161 allele.
Statistical analysis. Statistical analyses were performed using the R (R Core Team, 2017, Vienna, Austria) program. p-Values less than 0.05 were considered to be statistically significant.
Results
Characteristics of the study group. The characteristics of the patients with CRC and healthy controls are presented in Table I. There was no significant difference in the baseline characteristics between patients and controls (p>0.05) (Table I).
Table I. Characteristics of study groups.
Age values of the study groups are presented as mean±standard deviation (X±SD), remaining values are given as a percentage (%). CRC: Colorectal cancer (Total patient group); CC: colon cancer; RC: rectal cancer.
PPARg C161T polymorphism. Genotype and allele frequencies were not observed to significantly differ among patient subgroups (colon and rectal cancer patients) (p>0.05). Hardy-Weinberg equilibrium for PPARg C161T genotypes was obtained for genotypic distributions of patients with CRC and controls (p=0.262 and p=0.902, respectively) (Table II).
Table II. Frequencies of PPARγ C161T genotype distributions in colorectal cancer patients and controls.
The data represent percentages. *p<0.001. CRC: Colorectal cancer (Total patient group); CC: colon cancer; RC: rectal cancer.
The T161 allele (CT+TT genotypes) was found at higher frequency [p<0.001, odds ratio (OR)=3.239, 95% confidence intervals (95%CI)=1.997-5.252] and the C allele (CT+CC genotypes) was observed at lower frequency (p=0.022, OR=0.945, 95%CI=0.899-1.004) in the group of patients with CRC compared to those in the control group (Table II). Subgroup analysis showed that the T161 allele (CT+TT) had a higher frequency in colon cancer patients (p=0.001). The T161 allele was associated with a 3.056-fold colon cancer risk (OR=3.056, 95%CI=1.709-5.464) (Table II). In patients with rectal cancer, the C allele (CC+CT) was found to be lower (p=0.021, OR=0.258, 95%CI=0.076-0.876), and the T161 allele was increased compared to the controls (p=0.001, OR=3.529, 95%CI=1.784-6.981) (Table II).
When we compared the clinicopathological characteristics of all patients, the frequency of the T161 allele (CT+TT genotypes) was increased in the total patient group (p<0.001, OR=3.109, 95%CI=1.970-4.906) and in colon cancer patients (p<0.001, Fisher exact test) with poorly differentiated tumors (Table III). All colon cancer patients with poorly differentiated tumors had the T161 allele.
Table III. Comparison of the characteristic of patients with PPARγ C161T alleles.
All data represent percentages. *p-values less than 0.05 are considered as significant. Bold values indicate statistical significance.
In addition, colon cancer patients with the T161 allele had larger tumor size (T3+T4) (p=0.008, OR=2.20, 95%CI=1.336-3.623) and higher positive nodal status (N1+N2) (p=0.008, OR=2.20, 95%CI=1.336-3.623) (Table III). In addition, the T161 allele was associated with a positive nodal status in rectal cancer patients (p=0.013, Fisher exact test, OR=2.063, 95%CI=1.451-2.932) (Table III).
Discussion
The functional role of C/T substitution at position 161 in exon 6 (C161T) of the PPARg is unclear. C/T substitution might produce a new cryptic splice donor site, change the stability of mRNA, or affect leptin expression and TT genotypes (6,7), Activation of PPAR by ligand binding triggers the transcription of genes that play a role in cellular development, differentiation, and carcinogenesis. However, evidence concerning the relationship between PPARg C161T polymorphism and CRC is still limited. Only a few studies have investigated the PPARg C/T substitution in CRC (7,9,14). The PPARg C161T polymorphism has been shown to increase CRC risk in the Indian (7) and Eastern Chinese Han populations (14). However, no association was observed in the Japanese population (9). These studies suggest the important role of epigenetic factors in the development of CRC. Jiang et al. (7) reported a 1.61-fold increased risk for CRC in T161 allele carriers. In our study, T161 allele carriers had a 3.056-fold increased risk of CRC. Other studies have mainly focused on the effects of PPARg Pro12Ala polymorphism (8,10,13,18,19), and their results are also controversial.
In this study, we examined the relation between the PPARg C161T polymorphism and CRC risk. We demonstrated that the PPARg C161T gene polymorphism was related to the CRC development and differentiation in CRC patients. Individuals carrying the T161 allele had an increased risk for CRC. Moreover, a higher number of T161 allele carriers was observed among CRC patients and colon cancer patients with poorly differentiated tumors (p<0.001). Additionally, the T161 allele was associated with both larger tumor size (T3+T4) in colon cancer patients (p=0.008) and positive nodes in colon (p=0.008) and rectal cancer patients (p=0.013).
Siezen et al. (20) showed that the CT genotype (OR=0.63, 95%CI=0.45-0.89) and the T161 allele were associated with a decreased risk of adenomas (20). Another study on CRC patients without a K-ras mutation reported a high frequency of C161T minor alleles and suggested a role of PPARg gene in CRC development (21). In a haplotype analysis, Siezen et al. showed that the Pro12Ala major allele and the C161T minor allele strengthened the association between C161T and adenomas (20). Moreover, they suggested that the combination of these two SNPs had a functional effect either on protein function or on the amount of protein present (20).
A meta-analysis showed that the C161T polymorphism had a significant protective effect against CRC (15), but another meta-analysis reported that there was no protective effect of this polymorphism against CRC in Asians (15). Kuriki et al. (9) also studied the effects of PPARg Pro12Ala and C161T polymorphisms on CRC risk and reported that ProPro+ (CT+TT) genotype carriers with a high animal and herbal fat intake had a marginally increased risk for CRC compared to those with the ProPro and ProPro+ CC genotype in the Japanese population. In contrast to our results, PPARg C161T polymorphism was not shown to affect cancer risk in the Japanese population (9). The discrepancy between our results and those of the Japanese population might result from epigenetic differences in the etiology of CRC development in these populations.
This study has two imitations. The first is the size of the study groups. The second is that the functional role of the C/T substitution of the PPARg gene was not examined. Further analysis in population-based studies is reqired for elucidating the role of PPARg C161T polymorphism in the development of CRC.
In conclusion, the PPARg C161T polymorphism may be associated with CRC development and tumor differentiation, suggesting the prognostic importance of this gene polymorphism. A study with a larger sample is required to determine its role in the development and differentiation of CRC.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Authors’ Contributions
EC: Design, Writing, Management, Analysis, Conclusion. OKG and BCT: Performing experiments, Analysis. EC-TB-NS: Samples’ support and clinical data. HYE and TI: Design and Management.
Acknowledgements
The present study was supported by a grant from the Scientific Research Projects Coordination Unit of Istanbul University (Project No: 6669).
References
- 1.Weitz J, Koch M, Debus J, Höhler T, Galle PR, Büchler MW. Colorectal cancer. Lancet. 2005;365(9454):153–165. doi: 10.1016/S0140-6736(05)17706-X. [DOI] [PubMed] [Google Scholar]
- 2.Butterly LF, Goodrich M, Onega T, Greene MA, Srivastava A, Burt R, Dietrich A. Improving the quality of colorectal cancer screening: assessment of familial risk. Dig Dis Sci. 2010;55(3):754–760. doi: 10.1007/s10620-009-1058-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Potter JD. Colorectal cancer: molecules and populations. J Natl Cancer Inst. 1999;91(11):916–932. doi: 10.1093/jnci/91.11.916. [DOI] [PubMed] [Google Scholar]
- 4.Krishnan A, Nair SA, Pillai MR. Biology of PPAR gamma in cancer: a critical review on existing lacunae. Curr Mol Med. 2007;7(6):532–540. doi: 10.2174/156652407781695765. [DOI] [PubMed] [Google Scholar]
- 5.Ding H, Chen Y, Qiu H, Liu C, Wang Y, Kang M, Tang W. PPARG c.1347C>T polymorphism is associated with cancer susceptibility: from a case-control study to a meta-analysis. Oncotarget. 2017;8(60):102277–102290. doi: 10.18632/oncotarget.20925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Meirhaeghe A, Fajas L, Helbecque N, Cottel D, Lebel P, Dallongeville J, Deeb S, Auwerx J, Amouyel P. A genetic polymorphism of the peroxisome proliferator-activated receptor gamma gene influences plasma leptin levels in obese humans. Hum Mol Genet. 1998;7(3):435–440. doi: 10.1093/hmg/7.3.435. [DOI] [PubMed] [Google Scholar]
- 7.Jiang J, Gajalakshmi V, Wang J, Kuriki K, Suzuki S, Nakamura S, Akasaka S, Ishikawa H, Tokudome S. Influence of the C161T but not Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma on colorectal cancer in an Indian population. Cancer Sci. 2005;96(8):507–512. doi: 10.1111/j.1349-7006.2005.00072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Landi S, Moreno V, Gioia-Patricola L, Guino E, Navarro M, de Oca J, Capella G, Canzian F, Bellvitge Colorectal Cancer Study Group Association of common polymorphisms in inflammatory genes interleukin (IL)6, IL8, tumor necrosis factor alpha, NFKB1, and peroxisome proliferator-activated receptor gamma with colorectal cancer. Cancer Res. 2003;63(13):3560–3566. [PubMed] [Google Scholar]
- 9.Kuriki K, Hirose K, Matsuo K, Wakai K, Ito H, Kanemitsu Y, Hirai T, Kato T, Hamajima N, Takezaki T, Suzuki T, Saito T, Tanaka R, Tajima K. Meat, milk, saturated fatty acids, the Pro12Ala and C161T polymorphisms of the PPARgamma gene and colorectal cancer risk in Japanese. Cancer Sci. 2006;97(11):1226–1235. doi: 10.1111/j.1349-7006.2006.00314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Murtaugh MA, Ma KN, Caan BJ, Sweeney C, Wolff R, Samowitz WS, Potter JD, Slattery ML. Interactions of peroxisome proliferator-activated receptor {gamma} and diet in etiology of colorectal cancer. Cancer Epidemiol Biomarkers Prev. 2005;14(5):1224–1229. doi: 10.1158/1055-9965.EPI-04-0681. [DOI] [PubMed] [Google Scholar]
- 11.McGreavey LE, Turner F, Smith G, Boylan K, Timothy Bishop D, Forman D, Roland Wolf C, Barrett JH, Colorectal Cancer Study Group No evidence that polymorphisms in CYP2C8, CYP2C9, UGT1A6, PPARdelta and PPARgamma act as modifiers of the protective effect of regular NSAID use on the risk of colorectal carcinoma. Pharmacogenet Genomics. 2005;15(10):713–721. doi: 10.1097/01.fpc.0000174786.85238.63. [DOI] [PubMed] [Google Scholar]
- 12.Koh WP, Yuan JM, Van Den Berg D, Ingles SA, Yu MC. Peroxisome proliferator-activated receptor (PPAR) gamma gene polymorphisms and colorectal cancer risk among Chinese in Singapore. Carcinogenesis. 2006;27(9):1797–1802. doi: 10.1093/carcin/bgl001. [DOI] [PubMed] [Google Scholar]
- 13.Slattery ML, Curtin K, Wolff R, Ma KN, Sweeney C, Murtaugh M, Potter JD, Levin TR, Samowitz W. PPARgamma and colon and rectal cancer: associations with specific tumor mutations, aspirin, ibuprofen and insulin-related genes (United States) Cancer Causes Control. 2006;17(3):239–249. doi: 10.1007/s10552-005-0411-6. [DOI] [PubMed] [Google Scholar]
- 14.Lin J, Chen Y, Tang WF, Liu C, Zhang S, Guo ZQ, Chen G, Zheng XW. PPARG rs3856806 C>T polymorphism increased the risk of colorectal cancer: a case-control study in Eastern Chinese Han Population. Front Oncol. 2019;9:63. doi: 10.3389/fonc.2019.00063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Liang X, Fan X, Tan K, Zhang L, Jian L, Yu L. Peroxisome proliferators-activated receptor gamma polymorphisms and colorectal cancer risk. J Cancer Res Ther. 2018;14(Supplement):S306–S310. doi: 10.4103/0973-1482.235346. [DOI] [PubMed] [Google Scholar]
- 16.Xu W, Li Y, Wang X, Chen B, Liu S, Wang Y, Zhao W, Wu J. PPARgamma polymorphisms and cancer risk: a meta-analysis involving 32,138 subjects. Oncol Rep. 2010;24(2):579–585. [PubMed] [Google Scholar]
- 17.Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Theodoropoulos G, Papaconstantinou I, Felekouras E, Nikiteas N, Karakitsos P, Panoussopoulos D, Lazaris ACh, Patsouris E, Bramis J, Gazouli M. Relation between common polymorphisms in genes related to inflammatory response and colorectal cancer. World J Gastroenterol. 2006;12(31):5037–5043. doi: 10.3748/wjg.v12.i31.5037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Slattery ML, Murtaugh MA, Sweeney C, Ma KN, Potter JD, Caan BJ, Samowitz W. PPARgamma, energy balance, and associations with colon and rectal cancer. Nutr Cancer. 2005;51(2):155–161. doi: 10.1207/s15327914nc5102_5. [DOI] [PubMed] [Google Scholar]
- 20.Siezen CL, van Leeuwen AI, Kram NR, Luken ME, van Kranen HJ, Kampman E. Colorectal adenoma risk is modified by the interplay between polymorphisms in arachidonic acid pathway genes and fish consumption. Carcinogenesis. 2005;26(2):449–457. doi: 10.1093/carcin/bgh336. [DOI] [PubMed] [Google Scholar]
- 21.Tomita S, Kawamata H, Imura J, Omotehara F, Ueda Y, Fujimori T. Frequent polymorphism of peroxisome proliferator activated receptor gamma gene in colorectal cancer containing wild-type K-ras gene. Int J Mol Med. 2002;9(5):485–488. [PubMed] [Google Scholar]