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癌症分子流行病學及其在肺癌之研究

癌症分子流行病學及其在肺癌之研究. 王憶卿 成大藥理所 ycw5798@mail.ncku.edu.tw. 1. ★. ★. ★. 2. 3. ★. ★. 4. 5. . 6. Examples of polymorphic genes ~ Growth control and motility control genes ~. 7. ~ DNA repair genes ~. ★. 8. ★ Single polymorphism often plays a minor effect on disease formation.

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癌症分子流行病學及其在肺癌之研究

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  1. 癌症分子流行病學及其在肺癌之研究 王憶卿 成大藥理所 ycw5798@mail.ncku.edu.tw 1

  2. ★ ★ ★ 2

  3. 3

  4. ★ ★ 4

  5. 5

  6. 6

  7. Examples of polymorphic genes ~ Growth control and motility control genes ~ 7

  8. ~ DNA repair genes ~ ★ 8 ★ Singlepolymorphism often plays a minor effect on disease formation.

  9. Combination effects of multiple polymorphic genes Not including 1=> significant value ★ 9

  10. Racial differences Statistical power also is affected by the total number of individual included in the study cohort. 10

  11. Polymorphism vs. survival 11

  12. Free circulating DNA in the plasma/serum/sputum (A) dead cancer cells release their nucleic acids to blood (B) cancer cells extravasate into blood and then release their nucleic acids 12

  13. 13

  14. (26%) 14

  15. 15

  16. 16

  17. ◄ ◄ 17

  18. 18

  19. Most of p53 mutations are missense mutations (base substitution). 19

  20. 19 19-1

  21. 肺癌之基因體及外遺傳不穩定性及其臨床應用分析肺癌之基因體及外遺傳不穩定性及其臨床應用分析 Genetic and Epigenetic Instabilities and Their Clinical Implications in Lung Cancer 20

  22. Epigenetic alterations Chromatin modifications DNA methylation RNA interference DNA alterations (mutations, deletion, gene rearrangements, etc.) Normal DNA (Healthy individual) Mutant DNA (Disease individual) Genetic alterations 21

  23. Loose chromatin Epigenetic controls Yes gene expression No gene expression Histone acetylation DNA methyaltion Compact chromatin 22

  24. Modified two-hit model for tumor suppressor gene 23

  25. Secular trend of age-adjusted lung cancer mortality rate per 100,000 population from 1964 to 2005 in Taiwan by sexes. ----------------------------------------------------------------------------------- Year Aged adjusted Male-to-female Male Female ratio ----------------------------------------------------------------------------------- 1964~1968 10.35 5.84 1.77 1969~1973 13.97 6.68 2.09 1974~1978 16.65 8.29 2.01 1979~1983 21.79 10.42 2.09 1984~1988 24.91 12.22 2.04 1989~1992 27.24 11.19 2.43 1993~1995 32.41 14.35 2.26 1995~1998 36.07 16.06 2.25 1998~2002 41.12 19.38 2.12 2003~2005 44.03 19.91 2.21 ----------------------------------------------------------------------------------- Lung cancer is the leading cause of cancer death in both male and female in Taiwan. 24

  26. Others 10% Others 9% SCLC 8% SCLC 3% Adeno- carcinomas 38% Adeno- carcinomas 63% Squamous carcinomas 45% Squamous carcinomas 22% Female Male Distinct epidemiological data of lung cancer in Taiwan compared to other countries in the world • Highest rate of increase in cancer mortality • M/F ratio around 2 : 1 for 30 years (M/F ratio of smokers around 60% : 4%; 15 : 1) • High incidence of adenocarcinoma 25

  27. Cancer Res., 58:328-333, 1998. 26

  28. 27

  29. MSI Genomic Instability 28

  30. Microsatellite instability (MSI) ~ Microsatellites ~ 1、Highly polymorphic short tandem repeats : for example, (CA)n, (CAG)n, (GATA)n. 2、Widely distributed on genome maps: fairly even intervals of about 105 bp in mammalian genomes. Normal Lung Tumor Lung MSI+ 28-1

  31. Clin. Cancer Res., 6: 1639, 2000 MSI plays a significant role in NSCLC tumorigenesis in Taiwan and may involve in intragenic deletion of the p53 gene. Clinical features of MSI-positive and MSI-negative patients Characteristics Patient No. + (%) -- (%) P value Overall 68 28 (41.2) 40 (58.8) Age (mean+SD) 68+9 69+8 0.986 Sex Male 59 23 (39.0) 36 (61.0) 0.347 Female 9 5 (55.6) 4 (44.4) Smoker 51 22 (43.1) 29 (56.9) 0.569 Nonsmoker 17 6 (35.3) 11 (64.7) Tumor stage I+II 42 19 (45.2) 23 (54.8) 0.387 III+IV 269 (34.6) 17 (65.4) p53 intragenic deletion yes 7 6 (85.7) 1 (14.3) 0.018 no 57 22 (38.6) 35 (61.4) 29

  32. MSI Genomic Instability 30

  33. Mismatch recognition (hMSH2) Mismatch repair protein complex formation (hMLH1) Mismatches repaired 31

  34. J. Clin. Invest., 111: 887-895, 2003. PURPOSE To examine the etiological association of mismatch repair gene/protein alterations in lung tumorigenesis, we investigated: • hMLH1, hMSH2mismatch repair gene/protein alteration • Protein expression • mRNA expression • Promoter hypermethylation • Loss of heterozygosity 32

  35. A B C (1) ANALYSIS OF hMLH1 PROTEIN EXPRESSION -- Immunohistochemical analysis AD patient - positive SQ patient - positive SQ patient - negative • Among all the samples analyzed, 51.9% (40/77) of patients had no expression of hMLH1 protein. 33

  36. -- RT-PCR assay Patient no 1 2 3 4 bp 600 400 200 M N T N T N T N T β-actin hMSH2 hMLH1 (2) ANALYSIS OF mRNA EXPRESSION • Among all the samples analyzed, 55.8% (43/77) of patients showed loss mRNA expressionof hMLH1 gene. 34

  37. TF RNAP TF TF m DNA hypermethylation at 5’CpG3’ island TF TF RNAP TF m m m m Gene Promoter m m m m No transcription (3) ANALYSIS OF hMLH1 PROMOTER METHYLATION Normal Gene Promoter Yes transcription mRNA 35

  38. Sample no. n23 n31 n79 n81 Methylation status -- -- -- -- M -- + -- + -- + -- + bp 600 500 hMLH1 IFNβ1 (3) ANALYSIS OF hMLH1 PROMOTER METHYLATION -- Restriction enzyme-based PCR methylation analysis Hpa II: a methylation-sensitive enzyme that cannot digest the 5’CCGG 3’ if methylated Methylated DNA : cannot be digested by HpaII =>PCR/OK Unmethylated DNA : can be digested by HpaII =>PCR/Fail (IFNβ1 is a DNA fragment containing no HpaII site to serve as a control for PCR.) (a). Pre-test of normal lung tissue DNA 36

  39. (b). Methylation status of sputum DNA Patient no. s23 s31 s79 s81 mRNA + -- -- -- Methyl. -- + + + M -- + -- + -- + -- + HpaII bp 600 500 hMLH1 IFNβ1 • A72% (21/29) concordance of sputum samples with matched resected tumors was found. (3) ANALYSIS OF hMLH1 PROMOTER METHYLATION -- Hpa II-based Multiplex PCR methylation analysis (a). Methylation status of tumor DNA Patient no. t23 t31 t79 t81 mRNA + -- -- -- Methyl. -- + + + M -- + -- + -- + -- + HpaII bp 600 500 hMLH1 IFNβ1 • Among all the tumor samples analyzed, 55.8% (43/77) of patients showed promoter methylation of hMLH1 gene. 37

  40. Concordance of protein expression, mRNA expression, and promoter methylation in the hMLH1 gene • The data showed thatloss of hMLH1 protein expression was associated with negative hMLH1 mRNA expression and hMLH1 promoter methylation.(P=0.001) 38

  41. Genetic alterations of hMLH1 gene in relation to the clinicopathological parameters of resected NSCLC tumors 39

  42. CL2 CL1-5-F4 (hMLH1) (hMSH2) -- + -- + 5-Aza-dC U M U M U M U M bp 200 100 CL2 CL1-5-F4 -- + -- + 5-Aza-dC bp 600 400 200 -actin hMSH2 hMLH1 Further evidence that promoter methylation is the predominant mechanism by which hMLH1 (and hMSH2) are silenced in NSCLC Cell line: CL2 (a hMLH1-deficient cell with promoter hypermethylation) CL1-5-F4 (a hMSH2-deficient cell with promoter hypermethylation) 5’-Aza-dC: a de-methylation reagent A. at the DNA level B. at the RNA level 40

  43. CL2 –Aza (hMLH1) CL2 +Aza CL1-5-F4 –Aza (hMSH2) CL1-5-F4 +Aza C. at the protein level 41

  44. SUMMARY & CONCLUSION <hMLH1> -- protein / mRNA / promoter methylation analyses • The overall frequency of alteration: • Loss of protein expression: 51.9% • Loss of mRNA expression: 55.8% • Promoter hypermethylation: 55.8% • The data suggest the importance of the hMLH1 alterations in NSCLC tumorigenesis. 42

  45. hMLH1 promoter methylation • a predominant mechanism leading to a lack of mRNA and protein expression. • Loss of hMLH1 protein expression was correlated with low mRNA expression and resulted from promoter methylation of hMLH1 gene (P = 0.001). [concordant alterations]. • Its frequent occurrence in early-staged tumors and sputum samples indicates its potential use as a diagnostic marker in NSCLC. Hsu et al. Promoter Hypermethylation is the predominant mechanism in hMLH1 and hMSH2 deregulation and is a poor prognostic factor in non-smoking lung cancer. Clin. Cancer Res., 11:5410-5416, 2005. 43

  46. hMLH1 protein expression Characteristics Patient No. Yes (%) No (%) P value Microsatellite Instability MSI + 37 10 (27.0) 27 (73.0)0.025 MSI – 36 19 (52.8) 17 (47.2) Seventy-one patients were analyzed for both MI and hMLH1 protein expression. Among them, 73.0% (27/37) of MSI+ patients showed no expression of hMLH1 protein. • The data suggest that microsatellite instability is associated with the altered expression of hMLH1 mismatch repair protein. 44

  47. Lung Cancer, 55:205-213, 2007 Alteration of DNA methyltransferase expression and its correlation with 5’CpG hypermethylation and clinical implications 45

  48. Our previous studies on 5’CpG hypermethylation at TSGs • Our laboratory has previously shown that many tumor suppressor genes (TSGs) had 5’CpG hypermethylation in their promoter and/or exon1 regionsin lung cancer. • These TSGs are: hMLH1andhMSH2 (J. of Clin. Invest., 2003; Clin Cancer Res, 2005) BRCA1, BRCA2, XRCC5 (Clin. Cancer Res., 2007) p16INK4a(Int. J. Cancer, 2002) p14ARF(Clin. Cancer Res., 2003; J. of Clin, Oncol., 2005) FHIT (Eur. J. Cancer, 2004) AXIN2, -TrCP (Oncogene. 2008) BLU, RASSF1A, RARβ, HIC1, SLIT2, SRGAP1 (manuscripts in preparation) 46

  49. Hypothesis • We proposed that widespread acquired methylation in TSGsmay be caused by (1) increases DNA methylation activity (2) a defect of the protection mechanism againstDNA methylation • DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b are functional DNA methyltransferase enzyme known to contribute to methylation patterns. • The methylation biding protein 2 (MBD2), has the demethylase activity, demethylates both fully or hemi-methylated DNA. 47

  50. MBD 48

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