The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran

Document Type : Original article


1 Department of Biochemistry, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran

2 Autoimmune Diseases Research Center, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran

3 Autophagy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

4 Colorectal Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

5 Autophagy Research Center, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran


To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3′UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3′UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.


  1. Vaiopoulos AG, Athanasoula KC, Papavassiliou AG. Epigenetic modifications in colorectal cancer: molecular insights and therapeutic challenges. Biochim Biophys Acta 2014;1842: 971-980.
  2. Abbasi M, Asgari S, Pirdehghan A, Sedighi Pashaki AA, Esna-Ashari F. Survival rate of colorectal cancer and its effective factors in Iran. Acta Medica Iranica 2021;59:290.
  3. Saadati HM, Okhovat B, Khodamoradi F. Incidence and risk factors of colorectal cancer in the Iranian population: a systematic review. J Gastrointest Cancer 2021;52:414-421.
  4. Crea F, Nobili S, Paolicchi E, Perrone G, Napoli C, Landini I, Danesi R, Mini E. Epigenetics and chemoresistance in colorectal cancer: an opportunity for treatment tailoring and novel therapeutic strategies. Drug Resist Updat 2011;14:280-296.
  5. Akhavan-Niaki H, Samadani AA. DNA methylation and cancer development: molecular mechanism. Cell Biochem Biophys 2013;67:501-513.
  6. Li SY, Rong M, Iacopetta B. Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in human breast cancer. Oncol Rep 2006;15:221-225.
  7. Horie N, Aiba H, Hojo H, Takeishi K. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct funct 1995;20:191-197.
  8. Marsh S, Collie-Duguid ES, Li T, Liu X, McLeod HL. Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations. Genomics 1999;58:310-312.
  9. Mandola MV, Stoehlmacher J, Zhang W, Groshen S, Yu MC, Iqbal S, Lenz HJ, Ladner RD. A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics 2004;14:319-327.
  10. Ulrich CM, Bigler J, Velicer CM, Greene EA, Farin FM, Potter JD. Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev 2000;9:1381-1385.
  11. Ulrich CM, Bigler J, Bostick R, Fosdick L, Potter JD. Thymidylate synthase promoter polymorphism, interaction with folate intake, and risk of colorectal adenomas. Cancer Res 2002;62:3361-3364.
  12. Mund C, Lyko F. Epigenetic cancer therapy: Proof of concept and remaining challenges. Bioessays 2010;32:949-957.
  13. Ting AH, Jair KW, Suzuki H, Chiu Yen RW, Baylin SB, Schuebel KE. Mammalian DNA methyltransferase 1: inspiration for new directions. Cell Cycle 2004;3:1024-1026.
  14. Bardhan K, Liu K. Epigenetics and colorectal cancer pathogenesis. Cancers (Basel) 2013;5: 676-713.
  15. Zhang J, Yang C, Wu C, Cui W, Wang L. DNA methyltransferases in cancer: biology, paradox, aberrations, and targeted therapy. Cancers (Basel) 2020;12:2123.
  16. Weisenberger DJ, Liang G, Lenz HJ, DNA methylation aberrancies delineate clinically distinct subsets of colorectal cancer and provide novel targets for epigenetic therapies. Oncogene 2018;37:566-577.
  17. Wilson AJ, Byun DS, Popova N, Murray LB, L'Italien K, Sowa Y, Arango D, Velcich A, Augenlicht LH, Mariadason JM. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. J Biol Chem 2006;281:13548-13558.
  18. Wilson AJ, Byun DS, Nasser S, Murray LB, Ayyanar K, Arango D, Figueroa M, Melnick A, Kao GD, Augenlicht LH, Mariadason JM. HDAC4 promotes growth of colon cancer cells via repression of p21. Mol Biol Cell 2008;19:4062-4075.
  19. Chalkiadaki A, Guarente L. The multifaceted functions of sirtuins in cancer. Nat Rev Cancer 2015;15:608-624.
  20. Li Y, Seto E. HDACs and HDAC inhibitors in cancer development and therapy. Cold Spring Harb Perspect Med 2016;6:a026831.
  21. Zhai X, Gao J, Hu Z, Tang J, Qin J, Wang S, Wang X, Jin G, Liu J, Chen W, Chen F, Wang X, Wei Q, Shen H. Polymorphisms in thymidylate synthase gene and susceptibility to breast cancer in a Chinese population: a case-control analysis. BMC Cancer 2006;6:138.
  22. Wang L, Miao X, Tan W, Lu X, Zhao P, Zhao X, shan Y, Li H, Lin D. Genetic polymorphisms in methylenetetrahydrofolate reductase and thymidylate synthase and risk of pancreatic cancer. Clin Gastroenterol Hepatol 2005;3:743-751.
  23. Nosho K, Shima K, Irahara N, Kure S, Baba Y, Kirkner GJ, Chen L, Gokhale S, Hazra A, Spiegelman D, Giovannucci EL, Jaenisch R, Fuchs CS, Ogino S. DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin Cancer Res 2009;15:3663-3671.
  24. Ibrahim AE, Arends MJ, Silva AL, Wyllie AH, Greger L, Ito Y, Vowler SL, Huang THM, Tavare' S, Murrell A, Brenton JD. Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut 2011;60:499-508.
  25. Steine EJ, Ehrich M, Bell GW, Raj A, Reddy S, Oudenaarden AV, Jaenisch R, Linhart HG. Genes methylated by DNA methyltransferase 3b are similar in mouse intestine and human colon cancer. J Clin Invest 2011;121:1748-1752.
  26. Sarabi MM, Naghibalhossaini F. Association of DNA methyltransferases expression with global and gene‐specific DNA methylation in colorectal cancer cells. Cell Biochem Funct 2015;33:427-433.
  27. Tatar M, Varedi M, Naghibalhossaini F. Epigenetic effects of blackberry extract on human colorectal cancer cells. Nutr Cancer 2022;74:1446-1456.
  28. Quintero-Ramos A, Gutie'rrez-Rubio SA, Toro-Arreola AD, Franco-Topete RA, Oceguera-Villanueva A, Jime'nez-Pe'rez LM, Castro-Cervantes JM, Barraga'n-Ruiz A, Va'zquez-Camacho JG, Daneri-Navarro A. Association between polymorphisms in the thymidylate synthase gene and risk of breast cancer in a Mexican population. Genet Mol Res 2014;13: 8749-8756.
  29. Mokarram P, Naghibalhossaini F, Firoozi MS, Hosseini SV, Izadpanah A, Salahi H, Malek-Hosseini SA, Talei A, Mojallal M. Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status. World J Gastroenterol 2008;14:3662.3671.
  30. Lenz HJ. Pharmacogenomics and colorectal cancer. Ann Oncol 2004;15 Suppl 4:iv173-7.
  31. Ulrich CM, Robien K, McLeod HL. Cancer pharmacogenetics: polymorphisms, pathways and beyond. Nat Rev Cancer 2003;3:912-920.
  32. Zinn RL, Pruitt K, Eguchi S, Baylin SB, Herman JG. hTERT is expressed in cancer cell lines despite promoter DNA methylation by preservation of unmethylated DNA and active chromatin around the transcription start site. Cancer Res 2007;67:194-201.
  33. Tatar M, Bagheri Z, Varedi M, Naghibalhosseini F. Blackberry extract inhibits telomerase activity in human colorectal cancer cells. Nutr Cancer 2019;71:461-471.
  34. Guilleret I, Benhattar J. Unusual distribution of DNA methylation within the hTERT CpG island in tissues and cell lines. Biochem Biophys Res Commun 2004;325:1037-1043.
  35. Zheng S, Chen P, McMillan A, Lafuente A, Lafuente MJ, Ballesta A, Trias M, Wiencke JK. Correlations of partial and extensive methylation at the p14 ARF locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors. Carcinogenesis 2000;21:2057-2064.
  36. Xu XL, Yu J, Zhang HY, Sun MH, Gu J, Du X, Shi DR, Wang P, Yang ZH, Zhu JD. Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World J Gastroenterol 2004;10:3441-3454.
  37. Vlaykova T, Mitkova A, Stancheva G, Kadiyska T, Gulubova M, Yovchev Y, Cirovski G, Chilingirov P, Damyanov D, Kremensky I, Mitev V, Kaneva R. Microsatellite instability and promoter hypermethylation of MLH1 and MSH2 in patients with sporadic colorectal cancer. J BUON 2011;16:265-273.
  38. Shima K, Nosho K, Baba Y, Cantor M, Meyerhardt JA, Giovannucci EL, Fuchs CS, Ogino S. Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review. Int J Cancer 2011;128:1080-1094.
  39. Nezamalhosseini Mojarad E, Kuppen PJK, Asadzadeh Aghdaei H, Zali MR. The CpG island methylator phenotype (CIMP) in colorectal cancer. Gastroenterol Hepatol Bed Bench 2013; 6:120-128.
  40. Christou N, Perraud A, Blondy S, Jauberteau MO, Battu S, Mathonnet M. E‑cadherin: A potential biomarker of colorectal cancer prognosis. Oncol Lett 2017;13:4571-4576.
  41. Luczak MW, Jagodziński PP. The role of DNA methylation in cancer development. Folia Histochem Cytobiol 2006;44:143-154.
  42. Ropero S, Esteller M. The role of histone deacetylases (HDACs) in human cancer. Mol Oncol 2007;1:19-25.
  43. Schnekenburger M, Diederich M. Epigenetics offer new horizons for colorectal cancer prevention. Curr Colorectal Cancer Rep 2012;8:66-81.
  44. Schmidt WM, Sedivy R, Forstner B, Steger GG, Zochbauer-Muller S, Mader RM. Progressive up‐regulation of genes encoding DNA methyltransferases in the colorectal adenoma‐carcinoma sequence. Mol Carcinog 2007;46:766-772.
  45. Jones PA. The DNA methylation paradox. Trends Genet 1999;15:34-37.
  46. Kang MY, Lee BB, Kim YH, Chang DK, Park SK, Chun HK, Song SY, Park J, Kim DH. Association of the SUV39H1 histone methyltransferase with the DNA methyltransferase 1 at mRNA expression level in primary colorectal cancer. Int J Cancer 2007;121:2192-2197.
  47. Kanai Y, Ushijima S, Kondo Y, Nakanishi Y, Hirohashi S. DNA methyltransferase expression and DNA methylation of CPG islands and peri‐centromeric satellite regions in human colorectal and stomach cancers. Int J Cancer 2001;91:205-212.
  48. Gu Y, Yang P, Shao Q, Liu X, Xia S, Zhang M, Xu H, Shao Q. Investigation of the expression patterns and correlation of DNA methyltransferases and class I histone deacetylases in ovarian cancer tissues. Oncology letters 2013;5:452-458.
  49. Jin F, Dowdy SC, Xiong Y, Eberhardt NL, Podratz KC, Jiang SW. Up-regulation of DNA methyltransferase 3B expression in endometrial cancers. Gynecol Oncol 2005;96:531-538.
  50. Weichert W, Roske A, Niesporek S, Noske A, Buckendahl AC, Dietel M, Gekeler V, Boehm M, Beckers T, Denkert C. Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo. Clin Cancer Res 2008;14:1669-1677.
  51. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Danenberg PV, Laird PW. CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. Cancer Res 1999;59:2302-2306.