TP53 and MDM2 polymorphisms and the risk of endometrial cancer in postmenopausal women

The aim of the study was to determine an association of TP53 codon 72 (Arg72Pro, G>C transversion, rs1042522) and MDM2 SNP309 (T>G change, rs2279744) polymorphisms in endometrial cancer (EC) of postmenopausal women, regarding grading and staging of EC. In the study, endometrial samples from 202 postmenopausal female patients (the study group, n = 152, was women with EC; the control group, n = 50, cancer-free patients) were taken for the evaluation of two gene polymorphisms: TP53 codon 72 and MDM2 SNP309, respectively. Genotypic analyses were performed using the PCR–RFLP technique. There were significant differences in the frequency of TP53 and MDM2 genotypes in EC patients—increased EC occurrence was observed with the presence of MDM2 G/G and TP53 Arg/Arg genotypes, while allele Pro of TP53 decreased cancer risk. Analysis of combined MDM2/TP53 polymorphisms revealed that T/T-Pro/Arg genotype decreased EC risk, whereas G/G-Arg/Arg genotype increased it. Association of these genetic polymorphisms with histological grading showed increased MDM2 G/G homozygote and TP53 Arg/Arg homozygote frequencies in grading 2 as well as allele G overrepresentation in G1 and G3 EC patients. Finally, with clinical FIGO staging under evaluation, an increase in MDM2 G/G and TP53 Arg/Arg homozygote frequencies in staging I and TP53 Arg/Arg homozygote frequencies in staging II were observed. Co-occurrence of some MDM2 SNP309 and TP53 codon 72 polymorphisms seems to influence EC risk, involving grading and staging of this neoplasm at the same time.


Introduction
Genetic base of endometrial cancer (EC), the most frequent neoplasm of sex organs in postmenopausal women, is not fully understood. Among numerous genes postulated to be involved in EC oncogenesis, at least two seem to play here an important role. These genes-MDM2 and TP53-are taking part in cell-cycle regulation. The first one-MDM2-belongs to proto-oncogenes, or proliferation genes responsible for cell growth and differentiation. The second one-TP53-is a suppressor gene preventing genetic anomalies transmission to daughter cells. TP53 in the moment of DNA strand damage inhibits cells in G1 phase and runs repair mechanisms or apoptosis when damage is too much [1,2].
From oncogenic point of view extremely important appears to be that action of these two genes is interrelated: Protein product of TP53 gene-p53 protein-has the ability to activate (a number of genes including) MDM2 gene, resulting in an increased production of mdm2 protein, which in turn inhibits p53 protein activity and by reducing its suppressor properties may initiate the process of carcinogenesis, including EC as well [3]. Any disruption in MDM2/TP53 genes may underlie EC development. Unfortunately, data on the occurrence of specific MDM2/ TP53 mutations or polymorphisms in EC are scant, mostly analyzing each gene separately, not together [4,5].
The aim of this present work was to change the existing situation and to determine the potential relationship between TP53 codon 72 and MDM2 SNP309 polymorphisms in EC of postmenopausal women, regarding histological malignancy (grading) and clinical staging of this neoplasm.

Endometrial cancer patients
A total of 152 patients with histologically proven diagnosis of endometrial cancer were included in the study (Table 1). Paraffin-embedded tumor tissues were obtained from postmenopausal women (aged 60.90 ± 8.96) with endometrial carcinoma treated in Polish Mothers Memorial Hospital, Research Institute (PMMH,RI, Lodz, Poland) between 2004 and 2009 years. All tumors were staged according to the criteria of the International Federation of Gynaecology and Obstetrics (FIGO). DNA from normal endometrial tissue obtained from non-cancer patients (n = 50, aged 53.06 ± 4.75) served as control. Normal endometrial specimens were obtained from patients who had undergone hysterectomy for intramural leiomyomas. The Local Ethic Committee approved the study, and each patient gave a written consent. DNA isolation DNA for analysis was obtained from an archival pathological paraffin-embedded tumor and healthy endometrial samples, which were deparaffinized in xylene and rehydrated in ethanol and distilled water. For tissue deparaffinization, 1,200 ll xylene was added to tissue section and agitated for 5 min, then centrifuged at 12,000 rpm for 10 min. The supernatant was removed and fresh xylene was added, and this step was repeated five times followed by washing with 100 % ethanol for 10 min and centrifuging at 12,000 rpm for 10 min. Then, the tissue pellet was air-dried. 180 ll of DNA extraction buffer solution ATL (Qiagen GmbH, Hilden, Germany) was added to the deparaffinized tissue in a 1.5-ml microcentrifuge tube followed by the DNA extraction step. Genomic DNA was prepared from material by using the commercial QIAmp DNA Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instruction.

Determination of MDM2 genotype
Genotypic analysis of the MDM2 SNP309 (rs2279744) polymorphism was determined by the PCR-based restriction fragment length polymorphism (PCR-RFLP) method. Genome region that includes studied polymorphism was amplified by PCR using primers 5 0 -CGCGGGAGTTCAGG GTAAAG-3 0 and 5 0 -AGCTGGAGACAAGTCAGGACTT AAC-3 0 [6,7]. The PCR (total volume 25 ll) was performed with a mixture containing about 100 ng of DNA, 12.5 pmol of each primer, 0.2 mmol/l of dNTPs, 2 mmol/l of MgCl 2 and 1 U of Taq DNA polymerase (TaKaRa, Japan). PCR conditions were as follows: initial denaturation step at 95°C for 5 min, 35 cycles at 94°C for 30 s and 30 s at the 62°C annealing temperature, and at 72°C for 30 s. The final extension step was performed at 72°C for 5 min. The PCR was carried out in a PTC-100TM (MJ Research, INC, Waltham, MA, USA) thermal cycler. Following PCR, 20-ml aliquots were removed and subjected to restriction digestion with MspA1I (BioLabs, New England, Frankfurt am Main, Germany). The 237-bp amplified product was digested overnight with 1 U of MspA1I at 37°C. The digested products were resolved on a 2 % agarose gel and stained with 0.5 lg/ml ethidium bromide. 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 chisquared test. Genotype frequencies in cases and controls were compared by chi-squared tests. The genotypic-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. Analyses were performed using STATISTICA 10 package (Statsoft, Tulsa, OK, USA).

Results
All the recruited both endometrial cancer (n = 152) and control samples (n = 50) were successfully analyzed for the TP53 and MDM2 genotype. From the PCR analysis, all patients were classified into three genotypes of the MDM2 and TP53 gene: G/G, G/T and T/T and Pro/Pro, Pro/Arg and Arg/Arg, respectively. It can be seen from Table 2 that there are significant differences in the frequency of TP53 and MDM2 genotypes (p \ 0.05). An association was observed between endometrial carcinoma occurrence and the presence of G/G and Arg/Arg genotypes (Tables 3, 4, 5). Variant Pro allele of TP53 decreased cancer risk (Table 3).
We also analyzed combined genotype of all polymorphism pairs. The combined T/T-Pro/Arg genotype decreased the risk of endometrial cancer occurrence (Table 5). Moreover, the combined G/G-Arg/Arg genotype increased the risk of EC ( Table 6).
The observed genotype frequency of TP53 codon 72 (p [ 0.05) as well as MDM2 SNP309 in the controls group was in agreement with Hardy-Weinberg equilibrium (HWE) (p [ 0.05). In case of both investigated genes, the distribution of the genotypes in the patients differed significantly from the one expected from the Hardy-Weinberg equilibrium (p \ 0.05).  It is caused by the very low abundance of the TP53 Pro/ Pro genotype and MDM2 T/T genotype in the examined Polish population.
Because we were interested in the association between the distribution of genotypes and frequencies of alleles of investigated genetic variability on the tumor grade evaluated according to FIGO criteria, these data were also analyzed. Histological grading was evaluated in all the cases (n = 152): grade 1 (G1)-83 cases, grade 2 (G2)-34 cases and grade 3 (G3)-35 cases (see Table 7). Some correlation was observed between the TP53 Pro/Arg and MDM2 G/T genotype and EC invasiveness. A strong increase was observed, regarding G/G homozygotes frequency and Arg/Arg homozygotes in G2 patients. That increase was statistically significant (p \ 0.05). Moreover, EC patients in G1 and G2 had an overrepresentation of G alleles.
Clinical FIGO staging was also related to MDM2 G/T and the TP53 Pro/Arg polymorphisms (Table 8). Staging was evaluated in all the cases (n = 152). An increase was observed, regarding G/G and Arg/Arg homozygotes frequency in staging I (SI) patients, according FIGO classification. That increase was statistically significant (p \ 0.05). Moreover, in case of TP53, an increase was observed, regarding Arg/Arg homozygotes frequency in FIGO staging II (SII). That increase was also statistically significant (p \ 0.05).
Our data did not demonstrate any statistically significant correlation between MDM2 and TP53 polymorphisms and the risk factors for endometrial cancer, such as body mass index, hormone replacement therapy, uterine bleeding, endometrial transvaginal ultrasound, diabetes and hypertension and women with endometrial cancer (data not shown).

Discussion
The authors coping with genetic background of EC found that both MDM2 and TP53 polymorphisms are associated with the risk of this female neoplasm when evaluated separately.
In case of MDM2-EC relationship, an American publication by Walsh et al. [10] found that the presence of   homozygous MDM2 SNP309 G/G genotype is quite frequent-25 % in EC cases (11 % in controls), increasing the EC risk up to 2.76, having no association with EC grading and staging as well as patient's age at diagnosis. Also, another American publication by Terry et al. [11], nested within two large case-control studies-WHS (Women's Health Study) and NHS (Nurses' Health Study) with a total number of 592 EC cases and 1.543 controls, reported increased EC risk (OR = 1.87) in women with G/G genotype of MDM2 SNP309 polymorphism. In a Japanese report by Ueda et al. [12], both G/G genotype and G allele increased the risk of EC development, with OR = 1.91 and 1.20, respectively. In this study, homozygous G/G genotype was positively associated with postmenopausal status and EC type I alike, suggesting that this kind of MDM2 SNP309 polymorphism could be a risk factor for EC type I in Japanese female population [12]. These results are in accordance with our previous reports, indicating that both G/G genotype and G allele are highly associated with increased EC risk in Polish women, OR-3.50 and 2.67, respectively. No relationship of this MDM2 SNP309 polymorphism with EC grading was observed by us in this study [4].
But if it comes to TP53-EC association, high TP53 expression was observed by Kudela et al. [13] in poorly differentiated (G3) EC tumors. According to these Czech researchers, TP53 may serve as one of the genetic markers responsible for differentiation of patients with bad EC prognosis [13]. Having found two meta-analyzes on TP53 polymorphism-EC linkage, the authors of the first one did not observe any correlation between EC risk and TP53 codon 72 polymorphism (although further studies in larger number of patients are recommended) [14], while the second report varies, combining Pro allele and Pro carriers (Pro/Pro, Arg/Pro) with increased EC risk (OR = 1.25 and 1.34, respectively) [15].
From the current point of view, it seems to be more important to analyze the studies focusing on the combined effect of TP53 and MDM2 polymorphisms in EC. Ashton et al. [16] reported that the combination of MDM2 SNP309 and three TP53 codon 72 polymorphisms is associated with a higher grade of EC (OR = 4,15). The Australian researchers found also the association between family history of breast cancer and TP53 polymorphisms, suggesting a low-risk familial cancer grouping in this case   [16]. Nunobiki et al. calculated that homozygous TP53 codon 72 Arg/Arg genotype together with homozygous MDM2 SNP309 G/G polymorphism is responsible for significantly enhanced EC risk (OR = 3.28). The Japanese scientists concluded that these two polymorphisms may cooperatively increase the risk of EC among Japanese women [17]. Similar results were presented by Yoneda et al. [18] founding that the combination of TP53 codon 72 Arg/Arg and MDM2 SNP309 G/G?T/G significantly enhances the risk of EC (OR = 2.53).
Our results not only confirmed the fact that MDM2 SNP309 G/G plus TP53 codon 72 Arg/Arg genotype increases EC risk, but also showed that MDM2 SNP309 T/T plus TP53 codon 72 Pro/Arg genotype could decrease its risk. Additionally, some important connections with grading (GI-GIII) and staging (SI-SII) of EC were presented.

Conclusions
The co-occurrence of selected gene polymorphisms of MDM2 SNP309 and TP53 codon 72 may influence the risk of EC, having also a relationship with grading and staging of this cancer.