吕嘉春，刘斌，魏宜胜等 广州医学院 化学致癌研究所、呼吸疾病国家重点实验室 承国家自然科学基金项目 (30200235 、 30371196 、 30671813 、 30872178) 资助

1. -1304T>G variant in the promoter region of MKK4 gene reduces risk of lung cancer and sporadic colorectal cancer in southern Chinese population 吕嘉春，刘斌，魏宜胜等 广州医学院 化学致癌研究所、呼吸疾病国家重点实验室 承国家自然科学基金项目(30200235、30371196、30671813、30872178)资助

2. BACKGROUND • Lung cancer or colorectal cancer (CRC) are leading causes of death in the world, and so does in Guangzhou. • The death rate of lung cancer in Guangzhou city was 11.61/105 during 1970-1972, increased to 32.67/105 during 1980-1982, to 41.54/105 during 1990-1992, and to 48.79/105 during 2000-2002. • The death rate of CRC in Guangzhou city was 4.33/105 during 1970-1972, increased to 6.81 /105 during 1980-1982, to 8.68/105 during 1990-1992, and to 12.13/105 during 2000-2002. • The etiologic factors of both lung cancer and sporadic CRC including some environmental factors, such as cigarette smoking, alcohol consumption and dietary factors. • Usually, the exposures to those environmental factors will be stresses to the cell, and the signals will be transduced by MAPK pathways, which lead to apoptosis, inflammations, and tumorgenesis.

3. BACKGROUND • MKK4, also known as MAP2K4, MEK4, and SEK1, belongs to MAPK pathway, in which MKK4 in turn activates c-JUN NH2-terminal kinases (JNK) and p38,responses to extracellular signals. • The role of MKK4 in tumor is complex. • tumor suppressor? LOH in 24 (86%) of 28 CRC, and homozygous deletions of the MKK4 • pro-oncogene?artificially overexpression of MKK4 in MKK4-negative cancer lines stimulated the cell proliferation and invasion • Several mutations were detected in the exons 4 and 9 of MKK4 gene, however, it is not frequent in LC or CRC. • Based on Genebank dbSNP database, four common SNPs (MAF >5%) locate in the promoter region of MKK4 gene, no common SNP in the exons.

5. HYPOTHESIS The genetic variations in the promoter region of MKK4 gene and their possible interaction with environmental factors may play a role in the risk of lung cancer or sporadic CRC.

6. Study one Liu B, Chen D, Yang L, Li Y, Ling X, Liu L, Ji W, Wei Y, Wang J, Wei Q, Wang L, Lu J*(吕嘉春correspondence), A functional variant (-1304T>G) in the MKK4 promoter contributes to a decreased risk of lung cancer by increasing the promoter activity. Carcinogenesis. 2010 Jun 16. [Epub ahead of print] (SCI IF=4.795)

7. STUDY DESIGN • The studied SNPs were selected based on the dbSNP database and re-sequencing data. • A hospital-based case-control study 1056 newly diagnosed patients with sporadic lung cancer were recruited in Guangzhou. • 1056 cancer-free controls were recruited from healthy subjects in the community health centers. • The cases and controls frequency matched by age (±5 years) and sex • All subjects were Chinese Han.

8. Fig. 1 Exon (coding aa 1 ~ 424 for Drosophila, 1 ~397 for Mus musculus, 1 ~366 for Arabidopsis thaliana ) Drosophila: Mus musculus： Arabidopsis thaliana： 3’UTR ATG Exon (coding aa 1 ~ 399) ATG 3’UTR Human: Nkx-2 A T C T G T C G -641C > G -1044A>T -84T > C -1304T>G

9. GENOTYPING-TagMan assay

10. Fig. 2 A. -1304T>G, genotypes of TT were displayed as red, TG as green, and GG as blue. B. -1044A>T, genotypes of TT were displayed as red, AT as green, and AA as blue.

11. Fig. 3 a c b A. MKK4-1304T>G genotyping by direct sequencing: (a). TT genotype; (b). TG genotype; (c). GG genotype. a b c B. MKK4-1044A>T genotyping by direct sequencing: (a). AA genotype; (b). AT genotype; (c). TT genotype.

12. RESULTS

14. Table I. cont. n %

15. Table II. MKK4 genotypes and allele frequencies and logistic regression analysis for associations with lung cancer risk

16. Table III. Stratification analysis of the MKK4 -1304T>G genotypes by selected variables in lung cancer cases and controls

17. Table III. cont’ a ORs were adjusted by age, sex, smoking, drinking, BMI and family history of cancer b P value of the test for homogeneity between stratum-related ORs for Pin1-842 .

18. Fig. 4 ATG Chr:17p11.2 Exon (coding aa 1 ~ 399) 3’UTR T A A G T -1304T>G -1044A>T -1304T B Luciferase pGL3 MKK4 promoter region -1304G Luciferase pGL3 MKK4 promoter region C 4.50 T * 4.00 G * 3.50 3.00 * Luciferase activity 2.50 ( Relative level ) 2.00 1.50 1.00 0.50 0.00 16HBE A549 L78

19. Fig. 5 Lane 1 2 4 29 30 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 MKK4 β-actin P=0.017 P=0.513 -1304T>G(rs3826392) -1044A>T(rs3809728)

20. False positive report probability (FPRP) for association between MKK4 -1304 T>G polymorphism with lung cancer risk OR=0.72 (0.60-0.87), Power=0.955 null OR=0.67 (or 1/1.5) Prior probability =0.01 FPRP=0.06 → noteworthy at 0.2 level The finding of this study is unlikely by chance.

21. Conclusion for study one The functional -1304G variant in the MKK4 promoter contributes to a decreased risk of lung cancer by increasing the promoter activity and that the G variant may be a marker for susceptibility to lung cancer.

22. Study two. Wei Y, Wang L, Lan P , Zhao H, Pan Z, Huang J, Lu J*(吕嘉春correspondence) ,Wang J*. The association between -1304T>G polymorphism in the promoter of MKK4 gene and the risk of sporadic colorectal cancer in southern Chinese population. Int J Cancer.2009 May 15;125(8): 1876-1883. (SCI IF=4.734)

23. STUDY DESIGN • The studied SNPs were selected based on the dbSNP database and 30 normal's re-sequencing data. • A hospital-based case-control study 513 newly diagnosed patients with sporadic CRC were recruited in Guangzhou. • 523 cancer-free controls were recruited from healthy subjects in the community health centers. • The cases and controls frequency matched by age (±5 years) and sex • All subjects were Chinese Han.

24. Genomic structure of MKK4 gene Exon (coding aa 1 ~ 424 for Drosophila, 1 ~397 for Mus musculus, 1 ~366 for Arabidopsis thaliana ) Drosophila: Mus musculus： Arabidopsis thaliana： 3’UTR ATG Exon (coding aa 1 ~ 399) ATG 3’UTR Human: Nkx-2 A T C T G C T G -84T > C -641C > G -1044A>T -1304T>G

25. GENOTYPING-PCR-RFLP • The MKK4-1304T>G (rs3826392)PCR primers (386 bp) 5’-GAA GAT ATT GGG CAG CCA AC-3’(FP) 5’-CAG CCT TGC CTA AAA TGG AA-3’(RP) Restriction enzyme:Sml I • The MKK4-1044A>T (rs3809728)PCR primers (222 bp) 5’-TTG GGT TTG GAA CAA GAA GG-3’(FP) 5’-CAG CCT TGC CTA AAA TGG AA-3’(RP) Restriction enzyme: TSP509 I

26. MKK4-1304T>G (rs3826392) MKK4-1044A>T (rs3809728)

29. Table II. MKK4 genotypes and allele frequencies and logistic regression analysis for associations with CRC risk

30. Table V. Stratification analysis of the MKK4 -1304T>G genotypes in CRC cases and controls

32. Correlation between MKK4 protein expression in cancer tissues and MKK4 genotypes Lane 12 2 11 17 13 10 19 22 6 3 4 5 8 9 14 21 24 18 15 16 20 1 7 23 Mkk4 β-actin Relative Protein Expression P = 0.022 P = 0.908 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 T T TG GG AA T T AT -1044A>T (rs3809728) -1304T>G (rs3826392)

33. False positive report probability (FPRP) for association between -1304 T>G SNP with CRC risk OR=0.54 (0.43-0.69), Power=0.99 null OR=0.67 (or 1/1.5) Prior probability =0.0001 FPRP=0.04 → noteworthy at 0.2 level

34. CONCLUSION The carriers of -1304G variant genotypes were associated with decreased risk of sporadic CRC in Chinese population, especially among older than 60 years or ever-drinkers.

35. Limitations • Hospital-based, retrospective study. • Fewer SNPs were genotyped

36. Future… • Validate this find in nasopharyngeal carcinoma (NPC) and breast cancer • Follow up for survival study • Further functional assay for SNPs • Extent to MEKK1/4 or JNK1/2/3

37. Acknowledgement Dr. Lu’s lab staff, GZMC Dr. Wei’s lab staff, UT MDACC

38. Thanks !