-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

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.

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.

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.

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)

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.

-1044A>T -641C > G -84T > C

ATG

Drosophila:Mus musculus：Arabidopsis thaliana：

Exon (coding aa 1 ~ 424 for Drosophila, 1 ~397 for Mus musculus, 1 ~366 for Arabidopsis thaliana )

3’UTR

3’UTR

Human:

ATGExon (coding aa 1 ~ 399)

Nkx-2

T

G

A

-1304T>G

T C

TC

G

Fig. 1

GENOTYPING-TagMan assay

Position and base change

Primers Probes PCR products

Genotypes

c.-1304T>G (rs3826392)

aggcccatggaggagaattc (forward)

HEX-ctctccacttaagtacac-MGB (allele T)

131 bp TT (red)

acccaatttcatccatcatcttg (reverse)

FAM-tctccactgaagtacac-

MGB (allele G)

TG (green)

GG (blue)

c.-1044A>T (rs3809728)

ccagttatcctacttcccaaagaatc (forward)

FAM-catggagctggacattttctcccatt-

TAMRA (allele A)

155 bp TT (red)

tttccaacatgctgtgaagaactc (reverse)

HEX-catggagctggacaatttctcccatt-

TAMRA (allele T)

AT (green)

AA (blue)

A. -1304T>G, genotypes of TT were displayed as red, TG as green, and GG as blue.

Fig. 2

B. -1044A>T, genotypes of TT were displayed as red, AT as green, and AA as blue.

Fig. 3

A. MKK4-1304T>G genotyping by direct sequencing: (a). TT genotype; (b). TG genotype; (c). GG genotype.

cba

b

B. MKK4-1044A>T genotyping by direct sequencing: (a). AA genotype; (b). AT genotype; (c). TT genotype.

ca

Table I. Frequency distributions of selected variables in lung cancer cases and controls

Cases (n=1056) Controls (n=1056) Variables

n % n % p a

Age (years) 60 536 (50.8) 534 (50.6) > 60 520 (49.2) 522 (49.4)

0.9306

Sex male 746 (70.6) 746 (70.6) female 310 (29.4) 310 (29.4)

1.0000

Smoking current 394 (37.3) 366 (34.7) former 207 (19.6) 176 (16.7) never 455 (43.1) 514 (48.6)

0.0283

a Two-sided 2 test;

RESULTS

Table I. cont.

Cases (n=1056) Controls (n=1056) Variables

n % n % p a

Drinking current 165 (15.6) 186 (17.6) former 64 (6.1) 41 (3.9) never 827 (78.3) 829 (78.5)

0.0429

Family history

yes 104 (9.9) 103 (9.8) no 952 (90.1) 953 (90.2)

0.9417

BMI <18 134 (12.7) 51 (4.8) 18-25 820 (77.6) 702 (66.5) >25 102 (9.7) 303 (28.7)

<0.0001

Sub-ethnic group TeoChewese(潮汕) 90 (8.5) 81 (7.7) Hakka (客家) 198 (18.8) 195 (18.5) Cantonese（广府） 683 (64.7) 700 (66.3) Other Hans(他省汉) 85 (8.0) 80 (7.5)

0.8358

Lung cancer (1056)

Histological types

Adenocarcinoma 384 36.4

Squamous cell carcinoma 369 34.9

Large cell carcinoma 43 4.1

Small cell lung cancer 128 12.1

Other carcinomas 132 12.5

Stages

I 154 14.5

II 94 8.9

III 333 31.5

IV 475 45.0

Table I. cont.

n %

Table II. MKK4 genotypes and allele frequencies and logistic regression

analysis for associations with lung cancer risk

Cases Controls Genotypes n (%) n (%)

P a Crude OR (95% CI)

Adjusted OR (95% CI) b

-1304T>G TT 692 (65.5) 605 (57.3) 1.00 (ref.) 1.00 (ref.) TG 319 (30.2) 379 (35.9) 0.74 (0.61-0.89) 0.74 (0.61-0.90) GG 45 ( 4.3) 72 ( 6.8)

0.0002

0.55 (0.37-0.81) 0.62 (0.41-0.94) P trend <0.0001 0.0005 TG+GG 364 (34.5) 451 (42.7) 0.71 (0.59-0.84) 0.72 (0.60-0.87) G allele 409 (19.5) 523 (24.8) <0.0001

-1044A>T AA 684 (66.7) 706 (69.2) 1.00 (ref.) 1.00 (ref.) AT 329 (29.6) 307 (27.3) 1.11 (0.92-1.34) 1.08 (0.88-1.32) TT 43 ( 3.7) 43 ( 3.5)

0.5743

1.03 (0.67-1.60) 1.09 (0.69-1.73) P trend 0.396 0.443

CT+TT 372 (33.3) 352 (31.0) 1.10 (0.92-1.31) 1.08 (0.89-1.31)

T allele 416 (19.7) 393 (18.7)

0.3684

a Two-sided 2 test for either genotype distribution or allele frequency. b Adjusted for age, sex, smoking status, and alcohol, family cancer history.

Patients (n = 1056) Controls (n = 1056)Crude OR( 95% CI)

Adjusted OR( 95% CI)a

Phomb

TTn (%)

GG+TGn (%)

TTn (%)

GG+TGn (%)

GG+TG vs TT GG+TG vs TT

Age (years)

60 352(65.7) 184(34.3) 303(56.7) 231(43.3) 0.69(0.54-0.88) 0.70(0.55-0.91) 0.566

> 60 340(65.4) 180(34.6) 302(57.9) 220(42.1) 0.73(0.57-0.93) 0.70(0.57-0.98)

Sex

Male 495(66.4) 251(33.6) 423(56.7) 323(43.3) 0.66(0.54-0.82) 0.71(0.57-0.89) 0.352

Female 197(63.5) 113(36.5) 182(58.7) 128(41.3) 0.82(0.59-1.13) 0.78(0.55-0.99)

Smoking

Ever 405(67.4) 196(32.6) 312(57.6) 230(42.4) 0.66(0.52-0.84) 0.66(0.53-0.85) 0.311

Never 287(63.1) 168(36.9) 293(57.0) 221(43.0) 0.78(0.60-1.00) 0.78(0.59-0.99)

Drinking

Ever 164(71.9) 65(28.4) 138(60.8) 89(39.2) 0.62(0.42-0.91) 0.64(0.42-0.97) 0.564

Never 528(63.8) 299(36.2) 467(56.3) 362(46.7) 0.73(0.60-0.89) 0.73(0.60-0.88)

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

Patients (n = 1056) Controls (n = 1056)Crude OR( 95% CI)

Adjusted OR( 95% CI)a

Phomb

TTn (%)

GG+TGn (%)

TTn (%)

GG+TGn (%)

GG+TG vs TT GG+TG vs TT

Family history of cancer

Yes 72(69.2) 32(30.8) 59(57.3) 44(42.7)

0.57(0.34-1.06) 0.52(0.28-0.98) 0.350

No 620(65.1) 332(34.9) 545(57.2) 407(42.8) 0.72(0.60-0.86) 0.73(0.60-0.88)

BMI

<18 92(68.7) 42(31.3) 19(37.3) 32(62.7) 0.27(0.14-0.53) 0.27(0.13-0.53) 0.003

18-25 540(65.9) 280(34.1) 409(58.3) 293(41.7) 0.72(0.59-0.89) 0.73(0.59-0.90)

>25 60(58.8) 42(41.2) 177(58.4) 126(41.6) 0.98(0.62-1.55) 1.01(0.63-1.59)

Table III. cont’

a ORs were adjusted by age, sex, smoking, drinking, BMI and family history of cancerb P value of the test for homogeneity between stratum-related ORs for Pin1-842 .

Fig. 4

A

B

C

-1304T

MKK4 promoter region pGL3 Luciferase

MKK4 promoter region pGL3 Luciferase

-1304G

3’UTR

ATG

-1044A>T

Chr:17p11.2 Exon (coding aa 1 ~ 399)

T

G

A

-1304T>GT

0.000.501.001.502.002.503.003.504.004.50

16HBE A549 L78

Luci

fera

se a

ctiv

ity

( R

elat

ive

leve

l )

T

G

*

*

*

Lane

MKK4

β-actin

Fig. 5

52 3 41 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

-1304T>G(rs3826392) -1044A>T(rs3809728)

AA AT TT

Genotype

0.50

0.60

0.70

0.80

0.90

TT TG GG

Genotype

0.50

0.60

0.70

0.80

0.90

Rel

ativ

e p

rote

in e

xpre

ssio

n

P=0.017 P=0.513

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.

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.

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)

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.

-1044A>T -641C > G-84T > C

ATG

Drosophila:Mus musculus：Arabidopsis thaliana：

Exon (coding aa 1 ~ 424 for Drosophila, 1 ~397 for Mus musculus, 1 ~366 for Arabidopsis thaliana ) 3’UTR

3’UTRHuman:

ATGExon (coding aa 1 ~ 399)

Nkx-2T

G

A

-1304T>G

T C

TC

G

Genomic structure of MKK4 gene

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

MKK4-1304T>G (rs3826392)

MKK4-1044A>T (rs3809728)

Table I. Frequency distributions of selected variables in colorectal cancer cases and controls

Cases (n=513) Controls (n=523) Variables

n % n % p a

Age (years) 49 120 (23.4) 122 (23.3)

50- 60 146 (28.5) 149 (28.5) >60 247 (48.2) 252 (48.2)

1.000

Sex

male 300 (58.5) 300 (57.4) female 213 (41.5) 223 (42.6)

0.716

Smoking current 228 (44.4) 142 (27.2)

former 59 (11.5) 79 (15.1) never 226 (44.1) 302 (57.7)

<0.0001

a Two-sided 2 test;

Table I. cnt’

Cases (n=513) Controls (n=523) Variables n % n %

p a

Drinking current 275 (53.6) 83 (15.9) former 14 (2.7) 25 (4.8) never 224 (43.7) 415 (79.4)

<0.0001

Family history

yes 71 (13.8) 57 (10.9) no 442 (86.2) 466 (89.1)

0.150

BMI

＜24 297 (57.9) 346 (66.2) 24- 167 (32.9) 139 (26.6) 28- 47 ( 9.2) 38 (7.3)

0.023

Menopause not yet 43 (20.2) 41 (18.4)

yes 170 (79.8) 182 (81.6)

0.633

Table II. MKK4 genotypes and allele frequencies and logistic regression

analysis for associations with CRC risk

Cases Controls Genotypes n (%) n (%)

P a Crude OR (95% CI)

Adjusted OR (95% CI) b

-1304 T>G TT 317 (61.8) 279 (53.4) 1.00 (ref.) 1.00 (ref.) TG 174 (33.9) 214 (40.9) 0.72 (0.55-0.93) 0.63 (0.47-0.84) GG 22 ( 4.3) 30 ( 5.7) 0.65 (0.36-1.15) 0.46 (0.24-0.88)

P trend

0.022 0.007 0.003

TG+GG 196(38.2) 244(46.7) 0.71 (0.55-0.91) 0.61 (0.46-0.80) G allele 0.212 0.262 0.073

-1044 A>T AA 342 (66.7) 362 (69.2) 1.00 (ref.) 1.00 (ref.) AT 152 (29.6) 143 (27.3) 1.13 (0.86-1.48) 1.09 (0.80-1.48) TT 19 (3.7) 18 (3.5) 1.12 (0.58-2.17) 1.03 (0.49-2.17)

P trend

0.116

0.410 0.646

AT+TT 171 (33.3) 161 (30.8) 1.12 (0.87-1.46) 1.08 (0.81-1.45) C allele 0.185 0.171

0.403

a Two-sided 2 test for either genotype distribution or allele frequency. b Adjusted for age, sex, smoking status, and alcohol, family cancer history in a logistic regression

model.

Patients (n = 513) Controls (n = 523)Crude OR( 95% CI)

Adjusted OR( 95% CI)a

Pintb

TTn (%)

TG+GGn (%)

TTn (%)

TG+GGn (%)

TT vs TG+GG

TT vs TG+GG

Age (years)

60 161(60.5) 105(39.5) 150(55.4) 121(44.7)0.81(0.57-1.14)

0.73(0.50-1.09) 0.026

> 60 156(63.2) 91 (36.8) 129 (51.2) 123(48.8)0.61(0.43-0.88)

0.49(0.32-0.74)

Sex

Male 170(56.7） 130(43.3） 142(47.3) 158(52.7)

0.69(0.50-0.95)

0.51(0.33-0.77) 0.484

Female 147(69.0） 66(31.0） 137 (61.4) 86 (38.6)0.72(0.48-1.06)

0.71(0.48-1.06)

Smoking

Ever 164(57.1） 123(42.9） 95(43.0) 126(57.0)

0.57(0.40-0.81)

0.44(0.29-0.69) 0.138

Never 153(67.7） 73(32.3） 184(60.9) 118(39.1)0.74(0.52-1.07)

0.76(0.52-1.11)

Drinking

Ever 166(57.4） 123(42.6） 41(38.0） 67(62.0） 0.45(0.29-

0.71)0.42(0.26-0.67) 0.045

Never 151(67.4） 73(32.6） 238(57.4）

177(42.7）

0.65(0.46-0.91)

0.78(0.54-1.12)

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

Patients (n = 513) Controls (n = 523)Crude OR( 95% CI)

Adjusted OR( 95% CI)a

Pinteraction

b

TTn (%)

TG+GGn (%)

TTn (%)

TG+GGn (%)

TT vs TG+GG

TT vs TG+GG

Tumor site

Colon cancer 148(58.5) 105(41.5)

279(53.4) 244(46.7)

0.81(0.60-1.10)

0.65(0.46-0.92)

―

Rectal cancer 169(65.0) 86(35.0)0.62(0.45-0.84)

0.51(0.36-0.72)

Family history of cancer

Yes 40(56.3) 31(43.7) 27(47.4) 30(52.6)0.70(0.35-1.41)

0.48(0.20-1.15)

0.782

No 277(62.7) 165(37.3) 252(54.1) 214(45.9)0.70(0.54-0.91)

0.61(0.45-0.83)

BMI

≤23.9188(63.3) 109(36.7) 187(54.1) 159(46.0)

0.68(0.50-0.94)

0.61(0.42-0.86)

0.989

24.0-27.9100(59.2) 69(40.8) 71(51.1) 68(48.9)

0.72(0.46-1.33)

0.58(0.34-0.99)

≥28.029(61.7) 18(38.3) 21(55.3) 17(44.7)

0.77(0.32-1.83)

0.53(0.19-1.47)

0.7

0.6

0.5

0.4

0.3

0.2T T TG GG

1 112 3 4 5 6 7 8 9 10 12 13 1514 16

β-actin

Mkk4

Lane 17 18 19 20 21 22 23 24

0.2

0.3

0.4

0.5

0.6

0.7

AA

Rel

ativ

e P

rote

in E

xpre

ssio

n

T T

-1304T>G (rs3826392) -1044A>T (rs3809728)AT

Correlation between MKK4 protein expression in cancer tissues and MKK4 genotypes

P = 0.022 P = 0.908

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

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.

Limitations

• Hospital-based, retrospective study.

• Fewer SNPs were genotyped

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

Dr. Lu’s lab staff, GZMC

Dr. Wei’s lab staff, UT MDACC

AcknowledgementAcknowledgement

Thanks !