Tuberculosis and Respiratory Diseases

The Korean Academy of Tuberculosis and Respiratory Diseases (대한결핵및호흡기학회)
권호 표지

Identification of DNA Methylation Markers for NSCLC Using Hpall-Mspl Methylation Microarray

Hpall-Mspl Methylation Microarray를 이용한 비소세포폐암의 DNA Methylation Marker 발굴

  • Kwon, Mi Hye (Departments of Internal Medicine, Konyang University School of Medicine) ;
  • Lee, Go Eun (Departments of Internal Medicine, Konyang University School of Medicine) ;
  • Kwon, Sun Jung (Departments of Internal Medicine, Konyang University School of Medicine) ;
  • Choi, Eugene (Departments of Internal Medicine, Konyang University School of Medicine) ;
  • Na, Moon Jun (Departments of Internal Medicine, Konyang University School of Medicine) ;
  • Cho, Hyun Min (Departments of Chest Surgery, Konyang University School of Medicine) ;
  • Kim, Young Jin (Departments of Chest Surgery, Konyang University School of Medicine) ;
  • Sul, Hye Jung (Departments of Pathology, Konyang University School of Medicine) ;
  • Cho, Young Jun (Departments of Radiology, Konyang University School of Medicine) ;
  • Son, Ji Woong (Departments of Internal Medicine, Konyang University School of Medicine)
  • 권미혜 (건양대학교병원 호흡기내과) ;
  • 이고은 (건양대학교병원 호흡기내과) ;
  • 권선중 (건양대학교병원 호흡기내과) ;
  • 최유진 (건양대학교병원 호흡기내과) ;
  • 나문준 (건양대학교병원 호흡기내과) ;
  • 조현민 (건양대학교병원 흉부외과) ;
  • 김영진 (건양대학교병원 흉부외과) ;
  • 설혜정 (건양대학교병원 병리학과) ;
  • 조영준 (건양대학교병원 방사선학과) ;
  • 손지웅 (건양대학교병원 호흡기내과)
  • Received : 2008.10.09
  • Accepted : 2008.11.19
  • Published : 2008.12.30
Download PDF
⟨ Previous Next ⟩

Abstract

Background: Epigenetic alterations in certain genes are now known as at least important as genetic mutation in pathogenesis of cancer. Especially abnormal hypermethylation in or near promoter region of tumor suppressor genes (TSGs) are known to result in gene silencing and loss of gene function eventually. The authors tried to search for new lung cancer-specific TSGs which have CpG islands and HpaII sites, and are thought to be involved in carcinogenesis by epigenetic mechanism. Methods: Tumor tissue and corresponding adjacent normal tissue were obtained from 10 patients who diagnosed with non small cell lung cancer (NSCLC) and underwent surgery in Konyang university hospital in 2005. Methylation profiles of promoter region of 21 genes in tumor tissue & non-tumor tissue were examined with HpaII-MspI methylation microarray (Methyl-Scan DNA chip$^{(R)}$, Genomic tree, Inc, South Korea). The rates of hypermethylation were compared in tumor and non-tumor group, and as a normal control, we obtained lung tissue from two young patients with pneumothorax during bullectomies, methylation profiles were examined in the same way. Results: Among the 21 genes, 10 genes were commonly methylated in tumor, non-tumor, and control group. The 6 genes of APC, AR, RAR-b, HTR1B, EPHA3, and CFTR, among the rest of 11 genes were not methylated in control, and more frequently hypermethylated in tumor tissue than non-tumor tissue. Conclusion: In the present study, HTR1B, EPHA3, and CFTR are suggested as possible novel TSGs of NSCLC by epigenetic mechanism.

연구배경: 유전자의 후생적인 변화(epigenetic alteration)는 악성종양의 병인론에 있어서 유전자 변이와 동등한 위치를 점하고 있다. 특히 종양억제 유전자의 전사 촉진(promoter) 부위에 발생하는 비정상적인 메칠화(methylation)는 유전자의 발현을 침묵화(silencing)하고, 결과적으로 유전자의 기능 소실을 일으키게 된다. 저자들은 CpG island와 HpaII site를 가지고 있으며 암화 과정에 관여할 것으로 생각되는 유전자에 대하여 HpaII-MspI methylation microarray를 이용하여 새로운 종양억제 유전자를 발굴하고자 하였다. 방 법: 2005년 건양대학교 병원에서 수술한 비 소세포성 폐암 환자 10명에서 폐암조직과 상응하는 암 주변의 정상조직을 얻었으며, HpaII-MspI methylation microarray (Methyl-Scan DNA chip$^{(R)}$, Genomic tree, Inc, South Korea)를 이용하여 21개의 유전자에 대하여 DNA methylation profile을 분석하였다. 각각의 유전자에서 메칠화된 정도를 두 그룹에서 비교하였고, 정상 대조군으로 두 명의 젊고 건강한 기흉 환자에서 수술한 폐 조직에 대하여 methylation profile을 분석하였다. 결 과: 21개의 대상 유전자 중 10개의 유전자에서 폐암조직, 폐암 주변 정상 조직, 대조군에서 모두 공통적으로 과메칠화 되었고, 나머지 11개의 유전자 중 APC, AR, RAR-b, HTR1B, EPHA3, CFTR의 6개의 유전자에서 대조군에서 메칠화가 없으며, 폐암조직에서 폐암 주변 정상 조직에 비하여 더 빈번하게 과메칠화 되었다. 결 론: HTR1B, EPHA3, CFTR은 비소세포 폐암에서 후생적 변화로 발생하는 새로운 종양억제 유전자의 후보 유전자로서의 가능성이 있을 것으로 생각한다.

Keywords

References

  1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66. https://doi.org/10.3322/canjclin.57.1.43
  2. Ellis JR, Gleeson FV. Lung cancer screening. Br J Radiol 2001;74:478-85. https://doi.org/10.1259/bjr.74.882.740478
  3. Marcus PM. Lung cancer screening: an update. J Clin Oncol 2001;19:83S-6S.
  4. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415-28. https://doi.org/10.1038/nrg816
  5. Worm J, Guldberg P. DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy. J Oral Pathol Med 2002;31:443-9. https://doi.org/10.1034/j.1600-0714.2002.00034.x
  6. Baylin SB. DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2005;2 Suppl 1:S4-11. https://doi.org/10.1038/ncponc0052
  7. Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001;61:3225-9.
  8. Zöchbauer-Müller S, Minna JD, Gazdar AF. Aberrant DNA methylation in lung cancer: biological and clinical implications. Oncologist 2002;7:451-7. https://doi.org/10.1634/theoncologist.7-5-451
  9. Palmisano WA, Divine KK, Saccomanno G, Gilliland FD, Baylin SB, Herman JG, et al. Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 2000;60:5954-8.
  10. Park JC, Chae YK, Son CH, Kim MS, Lee J, Ostrow K, et al. Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer. Biochem Biophys Res Commun 2008;365:221-6. https://doi.org/10.1016/j.bbrc.2007.10.144
  11. Gu J, Berman D, Lu C, Wistuba II, Roth JA, Frazier M, et al. Aberrant promoter methylation profile and association with survival in patients with non-small cell lung cancer. Clin Cancer Res 2006;12:7329-38. https://doi.org/10.1158/1078-0432.CCR-06-0894
  12. Harden SV, Tokumaru Y, Westra WH, Goodman S, Ahrendt SA, Yang SC, et al. Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients. Clin Cancer Res 2003;9:1370-5.
  13. Kim DH, Kim JS, Ji YI, Shim YM, Kim H, Han J, et al. Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer. Cancer Res 2003;63:3743-6.
  14. Adorján P, Distler J, Lipscher E, Model F, Müller J, Pelet C, et al. Tumour class prediction and discovery by microarray- based DNA methylation analysis. Nucleic Acids Res 2002;30:e21. https://doi.org/10.1093/nar/30.1.21
  15. Shi H, Maier S, Nimmrich I, Yan PS, Caldwell CW, Olek A, et al. Oligonucleotide-based microarray for DNA methylation analysis: principles and applications. J Cell Biochem 2003;88:138-43. https://doi.org/10.1002/jcb.10313
  16. Yan PS, Chen CM, Shi H, Rahmatpanah F, Wei SH, Caldwell CW, et al. Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res 2001;61:8375-80.
  17. Fukasawa M, Kimura M, Morita S, Matsubara K, Yamanaka S, Endo C, et al. Microarray analysis of promoter methylation in lung cancers. J Hum Genet 2006;51:368-74. https://doi.org/10.1007/s10038-005-0355-4
  18. Hatada I, Kato A, Morita S, Obata Y, Nagaoka K, Sakurada A, et al. A microarray-based method for detecting methylated loci. J Hum Genet 2002;47:448-51. https://doi.org/10.1007/s100380200063
  19. Hou P, Ji M, He N, Lu Z. Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI. Biochem Biophys Res Commun 2004;314:110-7. https://doi.org/10.1016/j.bbrc.2003.12.063
  20. Takai D, Yagi Y, Wakazono K, Ohishi N, Morita Y, Sugimura T, et al. Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis. Oncogene 2001;20:7505-13. https://doi.org/10.1038/sj.onc.1204940
  21. Kim JS, Han J, Shim YM, Park J, Kim DH. Aberrant methylation of H-cadherin (CDH13) promoter is associated with tumor progression in primary nonsmall cell lung carcinoma. Cancer 2005;104:1825-33. https://doi.org/10.1002/cncr.21409
  22. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A. The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet 1998;103:96-101. https://doi.org/10.1007/s004390050790
  23. Toyooka KO, Toyooka S, Virmani AK, Sathyanarayana UG, Euhus DM, Gilcrease M, et al. Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas. Cancer Res 2001;61:4556-60.
  24. Kim DS, Kim MJ, Lee JY, Kim YZ, Kim EJ, Park JY. Aberrant methylation of E-cadherin and H-cadherin genes in nonsmall cell lung cancer and its relation to clinicopathologic features. Cancer 2007;110:2785-92. https://doi.org/10.1002/cncr.23113
  25. Bunn PA Jr, Helfrich BA, Brenner DG, Chan DC, Dykes DJ, Cohen AJ, et al. Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo. Clin Cancer Res 1998;4:2849-58.
  26. Cohen AJ, Franklin WA, Magill C, Sorenson J, Miller YE. Low neutral endopeptidase levels in bronchoalveolar lavage fluid of lung cancer patients. Am J Respir Crit Care Med 1999;159:907-10. https://doi.org/10.1164/ajrccm.159.3.9806062
  27. Kristiansen G, Schluns K, Yongwei Y, Dietel M, Petersen I. CD10 expression in non-small cell lung cancer. Anal Cell Pathol 2002;24:41-6.
  28. Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science 2001;293:1068-70. https://doi.org/10.1126/science.1063852
  29. Sachan M, Raman R. Developmental methylation of the regulatory region of HoxB5 gene in mouse correlates with its tissue-specific expression. Gene 2006;380:151-8. https://doi.org/10.1016/j.gene.2006.05.029
  30. Yung RL, Julius A. Epigenetics, aging, and autoimmunity. Autoimmunity 2008;41:329-35. https://doi.org/10.1080/08916930802024889
  31. Tsoli E, Gorgoulis VG, Zacharatos P, Kotsinas A, Mariatos G, Kastrinakis NG, et al. Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas. Mol Med 2001;7:418-29.
  32. Virmani AK, Tsou JA, Siegmund KD, Shen LY, Long TI, Laird PW, et al. Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Cancer Epidemiol Biomarkers Prev 2002;11:291-7.
  33. Winters ZE. P53 pathways involving G2 checkpoint regulators and the role of their subcellular localisation. J R Coll Surg Edinb 2002;47:591-8.
  34. Zöchbauer-Müller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res 2001;61:249-55.
  35. Tsou JA, Hagen JA, Carpenter CL, Laird-Offringa IA. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene 2002;21:5450-61. https://doi.org/10.1038/sj.onc.1205605
  36. Jacquot J, Tabary O, Le Rouzic P, Clement A. Airway epithelial cell inflammatory signalling in cystic fibrosis. Int J Biochem Cell Biol 2008;40:1703-15. https://doi.org/10.1016/j.biocel.2008.02.002
  37. Cantin AM, Bilodeau G, Ouellet C, Liao J, Hanrahan JW. Oxidant stress suppresses CFTR expression. Am J Physiol Cell Physiol 2006;290:C262-70. https://doi.org/10.1152/ajpcell.00070.2005
  38. Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene 2001;20:2390-400. https://doi.org/10.1038/sj.onc.1204383
  39. Karamouzis MV, Konstantinopoulos PA, Papavassiliou AG. The activator protein-1 transcription factor in respiratory epithelium carcinogenesis. Mol Cancer Res 2007; 5:109-20. https://doi.org/10.1158/1541-7786.MCR-06-0311
  40. Brantley DM, Cheng N, Thompson EJ, Lin Q, Brekken RA, Thorpe PE, et al. Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo. Oncogene 2002;21:7011-26. https://doi.org/10.1038/sj.onc.1205679