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Keywords:

  • nonsmall cell lung cancer;
  • MDM2;
  • polymorphism;
  • SNP

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Lung cancer is the leading cause of cancer mortality in the world. Although exposure to carcinogens is considered to be the main cause, genetic variation may contribute to lung cancer risk. Murine double minute 2, MDM2, is a key regulator of p53 activity and recently a polymorphism in the promoter region of the MDM2 gene was characterized. This single nucleotide polymorphism, SNP309, was shown to influence MDM2 transcription, MDM2 protein levels and p53 activity. The aim of this study was to investigate whether this functionally important SNP is associated with risk of nonsmall cell lung cancer. The study consisted of 341 nonsmall cell lung cancer cases and 412 healthy controls of Norwegian origin. Our results indicate that the G/G genotype of SNP309 is associated with lung cancer risk with an odds ratio of 1.62 (95% CI: 1.06–2.50). Interestingly, the strongest effect of the polymorphism was seen among women. Females homozygous for SNP309 G/G had associated odds ratio 4.06 (1.29–12.8). We also explored the MDM2 SNP309 in relation to TP53 gene mutations and age at nonsmall cell lung cancer diagnosis. Our results indicate that the G/G genotype of SNP309 is associated with higher age at diagnosis in individuals with TP53 mutations (p = 0.037). © 2006 Wiley-Liss, Inc.

Lung cancer is the leading cause of cancer mortality in the world.1 Although lung cancer has been considered as a disease caused by smoking and environmental/occupational exposure, studies suggest that genetic factors may contribute to risk of the disease.2

Murine double minute 2, MDM2 (or HDM2), is a central regulator of p53 activity through several mechanisms. By binding to p53, MDM2 inhibits p53 transactivation activity and promotes its export from the nucleus.3, 4 MDM2 also acts as an ubiquitin ligase upon p53, thereby promoting rapid degradation of the tumor suppressor.5, 6, 7 In turn, induced transcription of the MDM2 gene by p53 generates an autoregulatory feedback loop.8, 9 P53 independent actions of MDM2 have also been suggested.10 The importance of the p53 pathway in maintaining normal cell growth is underlined by the fact that it is directly inactivated by mutations in the TP53 gene in approximately 50% of many tumors, including lung cancer.11 In addition, overexpression of MDM2 protein has frequently been observed in NSCLC and may be a complementary mechanism to p53 inactivation.12, 13 Together, alterations in p53, MDM2 or p14arf have been found in more than 90% of NSCLC tumors.14

Recently, a SNP located at position 309 in the first intron of the MDM2 gene was characterized.15 This T to G substitution, named SNP309, was shown to lead to higher transcription of the gene through enhanced binding of the SP1 transcription factor. Li-Fraumeni patients carrying either 1 or 2 copies of the G allele also showed significantly lower age at tumor onset. A model was proposed where elevated levels of MDM2 in carriers of the G allele inhibit the p53 apoptotic responses to DNA-damage and thereby allowing mutations to be fixed, leading to lower age at tumor formation.16 In support of this model, it has been shown that overproduction of MDM2 in SNP309 homozygous cells leads to a transcriptionally inactive p53-MDM2 complex.17 In light of these findings, an association of MDM2 SNP309 to lung cancer risk seems plausible.

The aim of this study was to investigate whether this functionally important SNP is associated with risk of nonsmall cell lung cancer in a Norwegian population. To our knowledge, this is the first study investigating the role of the MDM2 SNP309 polymorphism in lung cancer in a Caucasian population.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Study population

The study consisted of 341 NSCLC cases and 412 healthy controls of Norwegian origin. Cases were newly diagnosed lung cancer patients treated by surgery at university hospitals in Oslo and Bergen in the period between 1986 and 2001. Patients were selected consecutively whenever practically feasible. Tumor histology was confirmed by an experienced pathologist, and only NSCLC cases were included in the study. Controls were recruited from the Oslo Health Study 2000–2001 (HUBRO), arranged to evaluate the health status of the general population. About 4100 persons in the age cohorts 59/60 and 75/76 participated. Further selection of the controls was based on the following criteria: age ≥59 years, smoked >5 cigarettes/day, current smoker or quit smoking for less than 5 years. About 950 individuals met these criteria, from which 412 were randomly selected to be part of this study. All controls were individuals without any known history of cancer. Cases and controls were interviewed by a guided questionnaire on demographic and lifestyle factors. The Norwegian population is a homogenous population, and both cases and controls were Caucasians of Norwegian origin. All participating individuals also gave written informed consent to participate in the study and to allow their biological samples to be genetically analyzed. The Regional Committee for Medical Research Ethics gave approval for the study.

Genotyping

DNA was extracted from whole blood samples or normal lung tissue, using standard methods. Genotyping of the MDM2 SNP309 (rs2279744) was carried out using a nested TaqMan assay. First, primers (5′-ttg cgg agg ttt tgt tgg a-3′ and 5′-gct caa gag gaa aag ctg agt ca-3′) flanking the SNP were used in PCR amplification. Approximately 10 ng genomic DNA was amplified in a 7.5 μl reaction mixture containing 5 pmol of each primer, 2.5 mM MgCl2, 200 μM of each dNTP and 1 unit HotFirePol DNA polymerase in 1× reaction buffer, as recommended by the supplier (Solis Biodyne, Tartu, Estonia). After an initial denaturation at 95°C for 15 min, the reaction mixture was subjected to 30 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec, followed by a final extension at 72°C for 7 min. Next, 0.5 μl PCR product diluted 1:1000 was used as template in a TaqMan reaction of 5 μl containing 4.5 pmol of each primer (5′-gtc tcc gcg gga gtt ca-3′ and 5′-tgc gat cat ccg gac ct-3′), 1 pmol of each MGB-probe (6-FAM-ccg ctt cgg cgc g-MGB and VIC-ccg ctg cgg gcg-MGB) in 1× TaqMan universal PCR mastermix. The reactions were performed on an ABI 7900HT sequence detection system as recommended by the supplier (Applied Biosystems, Foster City, CA). An equal number of cases and controls were analyzed simultaneously, and negative controls containing water, instead of DNA, were included in every run. Genotypes were determined by automatic scoring in the SDS 2.2 software (Applied Biosystems).

TP53 mutational analysis

Mutational analysis of the TP53 gene in tumors from NSCLC cases was performed either by DNA sequencing or by capillary denaturant gradient gel electrophoresis, as described elsewhere.18, 19, 20 Tumor DNA from 222 NSCLC cases was available for TP53 mutation analysis.

Statistical analysis

Hardy–Weinberg equilibrium was tested by the χ2 test in the controls. Genotype distributions were compared and associated risk calculated by unconditional logistic regression analysis in SPSS (version 12.0.1). Mann-Whitney nonparametric tests were performed in SPSS, and the power of the study was evaluated using StatXact-4 (version 4.0.1). Given a minor allele frequency of approximately 30%, this study had more than 80% power to detect an odds ratio of 1.6.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we investigated the MDM2 SNP309 polymorphism in 341 NSCLC cases and 412 healthy controls of Norwegian origin. Characteristics of the subjects are given in Table I. The alleles were in Hardy–Weinberg equilibrium in the controls (χ2 = 3.56, p = 0.06), with frequencies similar to previously reported studies.15, 21

Table I. Characteristics of NSCLC Cases and Healthy Controls
 Cases (n = 341)Controls (n = 412)
  • 1

    Mean ± SD.

Male/female258/83315/97
Age163.3 ± 10.263.5 ± 7.1
Smoking
 Cigarettes/day115.5 ± 8.314.8 ± 6.3
 Smoking years140.5 ± 12.242.3 ± 8.4
 Pack-years131.1 ± 17.831.7 ± 15.1

The MDM2 SNP309 G/G genotype was more frequently observed among the lung cancer cases than the controls. Subjects carrying the G/G genotype had elevated odds ratio of 1.62 (1.06–2.50) compared to T/T and T/G carriers (Table II). Subgroup analysis revealed that the effect was higher among women. Female patients, heterozygous T/G and homozygous G/G carriers, had odds ratios of 1.7 (0.86–3.29) and 4.1 (1.29–12.8) respectively (Table III).

Table II. Distribution of MDM2 SNP309 Genotypes in NSCLC Cases and Healthy Controls and Associated Odds Ratios
GenotypeCases n (%)Controls n (%)OR1 (95% CI)
  • OR, odds ratio; CI, confidence interval.

  • 1

    Adjusted by age, sex and smoking (pack-years).

T/T130 (38.1)161 (39.1)1.0
T/G156 (45.7)207 (50.2)0.93 (0.68–1.27)
G/G55 (16.1)44 (10.7)1.57 (0.99–2.48)
T/T+T/G286 (83.9)368 (89.3)1.0
G/G55 (16.1)44 (10.7)1.62 (1.06–2.50)
Table III. Distribution of MDM2 SNP309 Genotypes, and Associated Odds Ratios in Relation to Sex
GenotypeCases n (%)Controls n (%)OR1 (95% CI)
  • OR, odds ratio; CI, confidence interval.

  • 1

    Adjusted by age and smoking (pack-years).

Male
 T/T106 (41.1)121 (38.4)1.0
 T/G109 (42.2)156 (49.5)0.80 (0.56–1.14)
 G/G43 (16.7)38 (12.1)1.25 (0.75–2.09)
Female
 T/T24 (28.9)40 (41.2)1.0
 T/G47 (56.6)51 (52.6)1.69 (0.86–3.29)
 G/G12 (14.5)6 (6.2)4.06 (1.29–12.8)

Mutational status of the TP53 gene was analyzed in 222 NSCLC cases. Genotype distributions were 34.3% T/T, 45.2% T/G and 20.4% G/G in the cases having mutated TP53 gene (n = 137) and 40.0% T/T, 45.9% T/G and 14.1% G/G in wild type tumors (n = 85).

Interestingly, the group of cases carrying the G/G genotype had a significantly higher age at NSCLC diagnosis (median 67 years) compared to the T/T group (median 64 years, p = 0.016, Table IV). This effect was only observed in the group of cases having mutated TP53 gene (median age 70 years vs. 64 years, p = 0.034) and not observed in wild type tumors (median age 63.5 years vs. 64 years, p = 0.5, Table IV).

Table IV. MDM2 SNP309 Genotypes and Age at NSCLC Diagnosis in Relation to TP53 Mutations
GenotypeMedian age at diagnosis
All cases (n = 341)TP53 wild type (n = 851)TP53 mutated (n = 1371)
  • 1

    Mutational status analyzed in tumors from 222 cases.

  • 2

    Median (min–max).

  • 3

    Mann-Whitney test T/G vs. T/T.

  • 4

    Mann-Whitney test G/G vs. T/T.

T/T64.0 (36–85)264.0 (39–82)64.0 (36–79)
T/G66.0 (31–82) p = 0.22364.0 (33–79) p = 0.95368.0 (42–82) p = 0.223
G/G67.0 (46–79) p = 0.016463.5 (46–79) p = 0.50470.0 (46–79) p = 0.0374

The subjects were divided into light (PY ≤ 20), medium (20 >PY ≤ 35) and heavy (PY > 35) smokers. No differences in associated odds ratios were seen among any of these groups (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our results indicate that the G/G genotype is more frequently found among NSCLC cases than healthy controls. This finding seems to be in line with the well-documented functional relevance of this SNP. It has recently been shown that the G-allele of SNP309 promotes higher transcription of the MDM2 gene, resulting in a transcriptionally inactive p53-MDM2 complex.15, 17 Attenuation of the p53 pathway results in increased lung tumor multiplicity in p53 mutated mice exposed to tobacco smoke.22, 23 Predisposition to cancer has been demonstrated in MDM2 overproducing mice.24 In addition, recent findings showed that the G/G genotype of SNP309 was associated with risk of esophageal squamous cell carcinoma.25

Subgroup analysis revealed that the effect of the SNP309 polymorphism was more evident among women. In a recent study, association of SNP309 and lowered age at diagnosis of colorectal cancer was seen only among women. In addition, genotype distributions differed between male and female cases, indicating a sex difference.21 Involvement of estrogen receptor alpha (ERα) in regulation of MDM2 expression, in some cell types, has been suggested.26, 27 We have previously demonstrated that ER genes are expressed in normal lung tissue and cell lines.28 Ligand-activated estrogen receptors were reported to interact with the transcription factor SP1, thereby inducing SP1 responsive promoters.29, 30 Higher MDM2 gene expression due to the T to G substitution of SNP309 has been shown to be caused by enhanced binding of SP1.15 Whether effects of hormones and their receptors on MDM2 functions exist in the lung remains to be explored.

MDM2 overexpression has been proposed as a mechanism complementary to p53 inactivation by mutations in the TP53 gene. Thus, complete inactivation of the p53 pathway by SNP309 would result in lower burden of TP53 mutations in SNP309 carriers. We found, however, no relationship between the distribution of SNP309 genotypes and TP53 status in the NSCLC cases.

Previous studies have shown the SNP309 to be associated with lower age at tumor formation.15 Our results indicate different roles of SNP309 depending on the TP53 status. In cases having tumors harboring mutations in the TP53 gene, the G allele of SNP309 was associated with higher age of NSCLC diagnosis. Conversely, no such effect on age was seen in cases without mutations in TP53. Even though regulation of p53 is considered the main task of MDM2, additional cell cycle regulatory functions have been suggested.31 MDM2 has been shown to promote cell cycle arrest and inhibit cell proliferation in some cell types.32, 33

The data presented here are in line with a recently published case–control study, involving Chinese lung cancer patients.34 The investigators found that the G allele of SNP309 was associated with elevated lung cancer risk. In contrast, no association was found in another Chinese study.35 It is worth noting that the minor allele seems to be more frequent in the Chinese population than in Caucasians.

In conclusion, our results indicate an association between the SNP309 and risk of nonsmall cell lung cancer, especially among women, in a Norwegian population. Relations to TP53 mutations and age at diagnosis were also explored and suggest different roles of SNP309 in tumors having mutated or wild type TP53 gene. However, the number of females in this study is low and larger studies are needed to replicate our results. It is also possible that the observed association is caused by a functional polymorphism located in MDM2 or nearby genes in linkage disequilibrium with SNP309.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors acknowledge Dr. Lodve Stangeland, Haukeland University Hospital, Bergen, for recruiting patients and supplying lung tissue specimens. We also thank David Ryberg for involvement in sample collection and preparation, and Tove Andreassen and Erik B. Eide for technical assistance. The National Health Service (Norway) is acknowledged for collecting materials from the controls. This project was supported by the Norwegian Research Council and the Norwegian Cancer Society.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References