the polymorphisms and haplotypes of pin1 gene are associated with the risk of lung cancer

The polymorphisms and haplotypes of Pin1 gene are associated with the

risk of lung cancer

吕嘉春 , 赵红军，杨磊，刘斌，纪卫东，宾晓农

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

Pin1

Wnt/β-catenin

p53, p63, p73 ras/Neu-Jun

NF-κB

Cancer

Upstream signals

Signaling transduction

Ser/Thr-Pro p Ser/Thr-Pro Ser/Thr-ProKinases

MapksGSKsCDKs

Phosphalases

PP2A

BACKGROUND Lung cancer is one of the 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.

Pro-directed phosphorylation is an important signaling mechanism

controlling diverse cellular processes, including cell-cycle

progression, and cell proliferation and differentiation.

The mechanisms controlling Pro-directed phosphorylation can result

in cell transformation and oncogenesis.

BACKGROUND Pin1 specifically regulates the conformation of Pro-directed

phosphorylation sites.

Pin1 substrates include many essential cancer-related proteins, such

as von Hippel-Lindau tumor suppressor, myc, GSK -3 beta, cyclin D1,

Cdc25, cdc2/cyclin B, p73, and p53.

Aberrant over expressions of Pin1 have been reported in many

cancers, including lung, breast and liver cancers.

Inhibition of Pin1 in cancer cells can trigger apoptosis or suppress

the transformed phenotype. Moreover, the Pin1 knockout -/- female

mice showed impaired growth of mammary cells.

The -842G>C polymorphism has been found to be associated with low risk of

head neck and breast cancer in American white population.

HYPOTHESIS

The genetic variations in the promoter region of Pin1 gene and their possible interaction with environmental factors may play a role in the risk of lung cancer

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.

Genomic structure of PIN1 gene

GENOTYPING-PCR-RFLP

Since the Pin1-842G>C (rs2233678) and -667T>C

(rs2233679) are close in distance, we used one pair of

primers

5’-CGG GCT CTG CAG ACT CTA TT -3’ (FP)

5’-AAA TTT GGC TCC TCC ATC CT -3’(RP)

Restriction enzyme: BanII for -842G>C

SacI for -667T>C

-667TT

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;

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

Table II. PIN1 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

-842G>C GG 948(89.8) 895(84.7) 1.00 (ref.) 1.00 (ref.) GC 103(9.7) 154(14.6) 0.63(0.48-0.83) 0.64(0.48-0.84) CC 5(0.5) 7(0.7)

0.0025

0.68(0.21-2.14) 0.77(0.23-2.52) P trend 0.0008 0.0018 GC+CC 108(10.2) 161(15.3) 0.64(0.49-0.83) 0.64(0.49-0.84) C allele 0.053 0.079 0.0007

-667C>T CC 367 (34.7) 352 (33.3) 1.00 (ref.) 1.00 (ref.) CT 512 (48.5) 522 (49.4) 0.94(0.78-1.14) 0.89(0.73-1.08) TT 177 (16.8) 182 (17.3)

0.5132

0.93(0.72-1.20) 0.83(0.64-1.08) P trend 0.6179 0.6984

CT+TT 689 (65.3) 704 (66.7) 0.94(0.78-1.12) 0.87(0.73-1.06)

T allele 0.410 0.420 0.5322

Haplotypes G-T 863(40.9) 832(39.4) 1.00 (ref) 1.00 (ref) G-C 1136(53.8) 1112(52.6) 0.97(0.75-1.26) 1.11(0.85-1.46) C-T 39(1.8) 54(2.6) 0.50(0.17-1.49) 0.64(0.21-1.95) C-C 74(3.5) 114(5.4)

0.007

0.40(0.20-0.78) 0.41(0.21-0.82) 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

GGn (%)

GC+CCn (%)

GGn (%)

GC+CCn (%)

GC+CC vs GG GC+CC vs GG

Age (years)

60 485(90.5) 51(9.5) 455(85.2) 79(14.8) 0.61(0.42-0.88) 0.65(0.44-0.95) 0.852

> 60 463(89.0) 57(11.0) 440(84.3) 82(15.7) 0.66(0.46-0.95) 0.63(0.43-0.92)

Sex

Male 672(90.1) 74(9.9) 634(85.0) 112(15.0) 0.62(0.46-0.85) 0.62(0.45-0.86) 0.837

Female 276(89.0) 34(11.0) 261(84.2) 49(15.8) 0.66(0.41-1.05) 0.71(0.43-1.16)

Smoking

Ever 540(89.9) 61(10.1) 452(83.4) 90(16.6) 0.57(0.40-0.80) 0.58(0.41-0.83) 0.407

Never 408(89.7) 47(10.3) 443(86.2) 71(13.8) 0.72(0.49-1.06) 0.71(0.47-1.07)

Drinking

Ever 208(90.8) 21(9.2) 187(82.4) 40(17.6) 0.47(0.27-0.83) 0.50(0.28-0.92) 0.366

Never 740(89.5) 87(10.5) 708(85.4) 121(14.6) 0.69(0.51-0.92) 0.69(0.51-0.93)

Table III. Stratification analysis of the PIN1 -842G>C genotypes in lung cancer cases and controls

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

Adjusted OR( 95% CI)a

Phomb

GGn (%)

GC+CCn (%)

GGn (%)

GC+CCn (%)

GC+CC vs GG

GC+CC vs GG

Family history of cancer

Yes 97(93.3) 7(6.7) 81(78.6) 22(21.4) 0.27(0.11-0.65)

0.29(0.11-0.74) 0.047

No 851(89.4) 101(10.6) 814(85.4) 139(14.6)0.70(0.53-0.91)

0.70(0.53-0.93)

BMI

≤23.9 126(94.0) 8(6.0) 44(86.3) 7(13.7)0.40(0.14-1.16)

0.41(0.14-1.23) 0.729

24.0-27.9 730(89.0) 90(11.0) 593(84.5) 109(15.5)0.67(0.50-0.91)

0.66(0.49-0.89)

≥28.0 92(90.2) 10(9.8) 258(85.1) 45(14.9)0.62(0.30-1.29)

0.63(0.31-1.31)

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 .

All P<0.001

G-T

G-C

C-T

C-C

Luciferase assay: Pin1 -842 C variant decrease gene’s transcriptional activity

0

50

100

150

200

250

300

350

A549 L78 16HBE

Luciferase activity

(Relative level)

G-TG-CC-TC-C

Population stratification• Multilevel Logistic regression null model: the sub-ethnic group to the risk of lung cancer

OR=0.971 ， 95%CI=0.862–1.094 ； P=0.6277

• Multilevel Logistic regression 2 level model: the sub-ethnic group as level 2, age, sex, smoking, drinking

and Pin1-842G>C genotypes as level 1, Pin1-842G>C: OR=0.639, 95%C.I.=0.488–0.836 ； P=0.0011.

After controlling the confounding effect of sub-ethnic groups, the main effect of Pin1-842G>C is still significant.

False positive report probability (FPRP) for association between

Pin1 -842 G>C polymorphism with lung cancer risk

OR=0.64 (0.49-0.84), Power=0.927

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 The functional genetic variant -842G>C

of Pin1 gene contributes to decreased

the risk of lung cancer by diminishing

the promoter activity

Limitations

• Hospital-based, retrospective study.

• Fewer SNPs were genotyped

• Restricted to a Chinese Han population

Dr. Lu’s lab staff, GZMC

Dr. Wei’s lab staff, UT MDACC

AcknowledgementAcknowledgement

Thanks for your attention !

谢谢！