Association between MDM2 SNP309 polymorphism and endometrial cancer risk in Polish women

Introduction

Endometrial cancer is the fourth most common carcinoma in women, and due to its growing incidence it is nowadays the leading gynecologic malignancy, especially in well-developed countries [1]. Approximately 150 000 new cases are noted worldwide annually, 80% of which being diagnosed in post menopausal patients. In the past the highest incidence was noted at the age of 57-58 years, however, recent observations confirm that the peak incidence occurs at the 6-7th decade of life. In 2006, endometrial cancer was diagnosed in 4376 Polish women [2] and was indicated as a cause of 814 deaths, presenting as the 12th cause of deaths among women in Poland. The share of endometrial cancer in general morbidity and mortality in our country is estimated at 7.1% and 2%, respectively. Apart from age, there are plenty of recognized risk factors, especially for endometrioid endometrial cancer, including: hyperestrogenism, obesity, diabetes, hypertension, the history of sterility, low parity and late menopause. A growing amount of data seem to support the notion of genetic predisposition to endometrial cancer.

The human MDM2 (murine double minute) oncogene has been mapped to chromosome 12q13-14 and possesses a role of down regulator of p53 suppressor gene. The product of the MDM2 gene promotes a rapid degradation of p53 protein, and through this mechanism protects the cell from p53-mediated growth arrest or apoptosis. It is also capable of inhibiting the p53 transactivation domain which interacts with the transcriptional machinery, resulting in p53 inactivation [3]. Furthermore, MDM2 plays a regulatory role for many tumour-related genes that are important for cell-cycle control. It also contributes to carcinogenesis independently of p53 through interaction with transcriptional factors of the E2F family, inhibition of the Rb growth regulatory function and inhibition of G0/G1-S-phase transition in normal cells [4-6].

In endometrial cancer tissue, p53 and MDM2 levels are correlated, suggesting that p53 is inactivated by MDM2 in endometrial cancer [7, 8]. Furthermore, Stewart et al. sequenced the TP53 gene in a series of endometrial cancer cases overexpressing p53 and found no mutations, suggesting that overexpression was due to another source such as MDM2 abnormalities [9].

A T  G polymorphism found in the promoter region of MDM2 (SNP309) increases MDM2 expression and thereby attenuates p53 activity [10]. The variant allele of SNP309 has been associated with an earlier age at cancer diagnosis [11-14], and an increased risk of both sporadic and hereditary cancers. Terry et al. suggested that SNP309 polymorphism may be associated with the risk of endometrial cancer [15].

In the present study the association between the MDM2 SNP309 polymorphisms and endometrial cancer risk in the Polish population was investigated.

Material and methods

Endometrial cancer patients



152 patients with histologically-proven diagnosis of endometrial cancer were included in the study (Table I). Paraffin-embedded tumour tissues were obtained from postmenopausal women (mean age 64.9 ±8.2) with endometrial carcinoma treated at the Department of Menopausal Diseases, Institute of Polish Mother’s Memorial Hospital in 2004-2009. All tumours were staged according to the criteria of the International Federation of Gynaecology and Obstetrics (FIGO). DNA from normal endometrial tissue (n = 100) served as control (mean age 54.42 ±19.22). The Local Ethic Committee approved the study and each patient gave written consent.

The endometrial tissue samples (cancerous and non-cancerous) were fixed routinely in formaldehyde, embedded in paraffin, cut into thin slices and stained with hematoxylin/eosin for pathological examination. DNA for analysis was obtained from an archival pathological paraffin-embedded tumour and healthy endometrial samples which were deparaffinized in xylene and rehydrated in ethanol and distilled water. In order to ensure that the chosen histological material is representative for cancerous and non-cancerous tissue, every tissue sample qualified for DNA extraction was initially checked by a pathologist. DNA was extracted from material using a commercially available QIAmp Kit (Qiagen GmbH, Hilden, Germany) DNA purification kit according to the manufacturer’s instructions.



Determination of MDM2 genotype



Genotypic analysis of the MDM2 SNP309 polymorphism was determined by the PCR-based restriction fragment length polymorphism (PCR-RFLP) method. Polymorphism SNP309 of the MDM2 gene was determined by PCR-RFLP, using primers 5’-CGCGGGAGTTCAGGGTAAAG-3’ and 5’-AGCTGGAGACAAGTCAGGACTTAAC-3’. The PCR was carried out in a GeneAmp PCR system 9700 (Applied Biosystems) thermal cycler. The reaction mixture contained 5 ng genomic. The 25 l PCR mixture contained about 100 ng of DNA, 12.5 pmol of each primer, 0.2 mmol/l of dNTPs, 2 mmol/l of MgCl2 and 1 U of Taq DNA polymerase (TaKaRa, Japan). The PCR cycle conditions were 94°C for 30 s, 62°C for 30 s, then 72°C for 30 s, repeated for 35 cycles. The 237 bp amplified product was digested overnight with 1 U of MspA1I (BioLaps, New England) at 37°C. The wild-type allele T was identified by the presence of 237 bp band, while the mutant allele G was represented by 189 and 48 bp bands.



Statistical analysis

For each polymorphism, deviation of the genotype frequencies in the controls from those expected under Hardy-Weinberg equilibrium was assessed using the standard c2-test. Genotype frequencies in cases and controls were compared by c2-tests. The genotype-specific risks were estimated as odds ratios (ORs) with associated 95% intervals (CIs) by unconditional logistic regression. P-values < 0.05 were considered to be significant.

Results

Table II shows the genotype distribution of MDM2 SNP309 polymorphisms between endometrial cancer patients and controls. The Table shows that there were significant differences (p < 0.05) between the two investigated groups. Women with endometrial cancer showed an incidence of 16%, 20% and 64%, respectively, for the T/T, T/G, and G/G genotypes of the MDM2 gene, whereas the control group showed 24%, 48%, and 28% for the same genotypes. We observed an association between the endometrial carcinoma occurrence and the presence of the G/G genotypes. Variant G allele of MDM2 increased the cancer risk. In patients, the observed frequencies of the T/T, T/G and G/G genotypes differed significantly (p < 0.05) from the distribution expected from the Hardy-Weinberg equilibrium (Table II).

Because we were interested in the association between the distribution of genotypes and frequencies of alleles of the investigated polymorphism on the tumour grade evaluated according to FIGO criteria, these data were also analyzed. Histological grades were evaluated in all the cases (n = 152); grade I – 83 cases, grade II – 34 cases and grade III – 35 cases. Grades II and III were accounted together for statistical analysis (see Table III).

We did not observe any difference between SNP309 genotype distributions in these groups. There was no correlation between genotypes of the polymorphisms and endometrial cancer invasiveness.

No statistically significant differences were observed in the alleles or in the genotype frequencies of the MDM2 SNP309 gene polymorphisms between risk factors of endometrial cancer such as BMI (body mass index), HRT (hormone replacement therapy), uterine bleeding, endometrial ultrasound transvaginal, diabetes and hypertension and the women with endometrial cancer.

Discussion

The literature data suggest that the identification of new risk factors for endometrial cancer in a population of women is urgently needed, and an analysis of some gene polymorphisms could be an interesting option.

In the present work we investigated the frequencies of the alleles of the MDM2 SNP309 polymorphism in samples from patients with endometrial cancer and from healthy individuals. We investigated the relationship between genotype and the risk of endometrial cancer. In our study we found an association between the endometrial cancer occurrence and SNP309 polymorphism. The MDM2 G/G genotype and G allele were associated with an elevated risk of endometrial cancer in this study population. There was a 3.5-fold higher risk of endometrial carcinoma for the individuals carrying the MDM2-G/G genotype, compared with subjects carrying the MDM2-T/T, G/T genotype, respectively. It is possible that the presence of the G allele is in linkage disequilibrium with another, so far unknown, mutation located outside the coding region in the MDM2 gene, which may be of importance for the MDM2 concentration in plasma.

We also analyzed the distribution of genotypes and frequency of alleles in groups of patients suffering from endometrial cancer according to different cancer grading by FIGO classification. In the present study, the MDM2 SNP309 polymorphism was not related to the cancer grade. The reason for this can be a relatively small group of I, II and III grade cases enrolled in our study.

The effect of MDM2 polymorphism on endometrial cancer occurrence in Poland has not been investigated before.

The presented work was performed on an ethnically homogenous population, which may improve our knowledge as to what extent the genotype-phenotype relationship variations are population-related.

Our results are in line with the data from other reports, introducing an important role of the MDM2 SNP309 polymorphism for endometrial carcinoma occurrence. SNP309 polymorphisms, chosen for the study, have been identified in the 5’ untranslated region of the MDM2 gene and have been shown to influence gene transcription activity [10]. It is known that single nucleotide polymorphism SNP309 in the promoter region of MDM2 gene plays an important role in human tumorigenesis [16-26].

MDM2 gene SNP309 polymorphisms have been studied as a risk factor for endometrial cancer [15, 19, 27-29]. Terry et al. showed that women carrying GG genotype of MDM2 SNP309 polymorphism may be at greater risk of endometrial cancer [15]. Walsh et al. found an association between a functional single nucleotide polymorphism in the MDM2 gene and sporadic endometrial cancer risk [19].

In a large American case-control study of patients with endometrial cancer, the SNP309 G/G variant genotypes of MDM2 were found to be associated with an increased risk of endometrial cancer [15, 19].

Similar results were reported in a Australian case-control study of endometrial cancer [27]. Ashton et al. suggested that the combination of the MDM2 SNP309 and the three TP53 polymorphisms appear to be related to a higher grade of endometrial cancer.

The homozygous variants of wild p53 codon 72 and mutant MDM2 promoter 309 may cooperatively increase the risk of endometrial cancer in a Japanese population [28]. The homozygous GG genotype of SNP309 polymorphism was also associated with the postmenopausal status and type I endometrial cancer in Japanese women [29].

In conclusion, the present study provides evidence for the significance of MDM2 polymorphism in endometrial cancer.

The obtained data suggest that both G allele and homozygous GG genotype of MDM2-SNP309 are associated with the endometrial cancer risk. Finally, it is postulated that the SNP309 polymorphism of the MDM2 gene may be used as predictive factors for endometrial cancer in Poland. Further studies conducted on a larger group are suggested to clarify this point.



The authors declare no conflict of interest.

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Address for correspondence

Beata Smolarz

Laboratory of Molecular Genetics

Department of Pathology

Institute of Polish Mother’s Memorial Hospital

Rzgowska 281/289

93-338 Lodz, Poland

tel. +48 42 271 20 71

e-mail: smolbea@wp.pl