MDM2 SNP309 promoter polymorphism and p53 mutations in urinary bladder carcinoma stage T1

Urothelial carcinoma of the bladder (UCB) is an unpredictable disease, and this is particularly apparent in patients with stage T1 UCB, who are at high risk of progression (30–50%) [1, 2]. The main treatment for non-muscle-invasive bladder cancer (NMIBC) is transurethral resection (TUR) combined with intravesical instillation of bacillus Calmette-Guérin (BCG). Cystectomy is the treatment of choice in patients with a higher stage of UCB (≥ T2), in spite of cystectomy, the prognosis is poor for these patients [3]. Cystectomy can be considered for NMIBC stage T1, however, performing cystectomy in every new case of stage T1 UCB is overtreatment, and hence it is an essential assignment to identify markers that can assess prognosis and aid individualization of treatment [4, 5].

The molecular mechanism of tumor progression in UCB is poorly understood. Several studies have tried to explain the transformation from normal to malignant urothelium and the progression that is often seen in this disease [6]. It is known that UCB is strongly associated with alterations in the p53 pathway [7]. Mutations in the p53 gene are often correlated with higher tumor grade and more advanced stages, as well as progression of NMIBC to muscle-invasive disease [8].

The murine double minute 2 (MDM2) is a negative regulator of p53. Furthermore, MDM2 SNP 309 (rs2279744) promoter polymorphism has been reported to be a risk modifier in several other malignant neoplasms [9], but few studies have addressed the role of this as such a modifier in UCB, which indicates the need for further research on this subject [10].

MDM2 and p53 play a critical role in carcinogenesis [11, 12]. The latter is encoded by the p53 tumor suppressor gene, and it induces cell cycle arrest, apoptosis, DNA repair, and prevention of angiogenesis [13‐15]. The p53 gene is often mutated in malignancies, which highlights its importance in tumor development and progression. MDM2, on the other hand, is an essential negative regulator of p53, because, when present in excessive amounts, it reduces the activity of p53 via enhanced proteasomal degradation [12, 16]. A single-nucleotide polymorphism (SNP309, rs2279744) located in the first intron of the core promoter region of the MDM2 gene affects binding of the transcription factor Sp1. Sp1 binds with higher affinity to the G allele than to the T allele, which results in increased transcription of the MDM2 gene and higher levels of MDM2 protein, and thereby inhibits the tumor suppressor function of p53 [17]. The interaction between p53 and MDM2 is a target for therapeutic intervention, and several such drugs are under development or in clinical trials [18‐20].

We have previously investigated clinical and histopathological parameters, as well as immunohistochemistry (IHC) for proteins involved in cell cycle regulation ( i.e. p53, p21, pRb, p16, and cyclin D1) and for matrix metalloproteinases [21, 22]. In those studies, we found that normal p53 (cut-off < 10%) was significantly associated with recurrence, and abnormal p16 was associated with progression. It is plausible that the mechanism for the latter relationship is dependent on the step involving phosphorylation of Rb. The importance of these proteins in tumor development has been described by other researchers [23‐25].

In the current study, we examined a population-based cohort of patients with primary stage T1 UCB. We analyzed possible effects of the functional polymorphism SNP309 in the MDM2 gene, and investigated the relationship between that polymorphism and mutations in the p53 gene. We also evaluated the expression of p53, p16, pRb and p21 measured by IHC. All findings were evaluated regarding associations with the risk of progression and recurrence.

SNP309 is a common polymorphism in the regulatory region in intron 1 of MDM2, and it might influence the cellular levels of the p53 protein and thereby promote tumor development and progression [17]. This has been observed in several studies concerning poor outcome of various malignancies, such as lung cancer, breast cancer, gastric carcinoma, and renal cell carcinoma [35‐38]. The role of a single nucleotide polymorphism in MDM2 (i.e., SNP309) has also been evaluated in UCB, but the results have varied [10, 39].

Here, we studied a large well-characterized group of patients with primary stage T1 UCB, none of whom had received intravesical BCG or chemotherapy before recurrence, a treatment strategy that is mandatory today. Accordingly, these patients exhibited a natural course of stage T1 UCB and were therefore ideal to investigate in the present context. We found that neither MDM2 polymorphism nor p53 gene mutations were associated with tumor progression in this study population. However, there was an association between strong IHC-determined p53 expression and p53 gene mutations. It is debatable whether p53 mutations can be compared with p53 expression measured by IHC. Yemelyanova et al. [40] observed concordance between p53 gene mutations and IHC-detected p53 expression in ovarian carcinoma patients, and those investigators obtained statistically significant results at a cut-off value of 60% positive tumor cells. That value is higher than the cut-off that is usually reported as abnormal for p53 expression, which is most often 10% positive tumor cells [41]. In our investigation, a cut-off value of 50% was found to be associated with mutations, and it may reflect the mutational status of p53 more accurately than the conventional cut-off value used for the p53 protein.

We found an association between abnormal p16 expression and mutations in p53, which may be evidence of linkage between the p53, p16, and pRb proteins. Furthermore, this observation is supported by the results of Kaplan-Meier analysis indicating that the rate of progression and time to progression were significantly shorter in patients with IHC-abnormal p16 and p53 mutations. Moreover, we observed that abnormal expression of p16 alone seems to be associated, with a worse prognosis, which has also been reported previously by us and by other authors [21, 32, 42, 43].

MDM2 polymorphism in the current UCB patients was tested in DNA from tumor tissue, and hence there was a risk of bias due to chromosomal deletions on chromosome 12 (where MDM2 is located). However, chromosomal 12 deletions are not common in this disease, and all of the tissue specimens analyzed in our study contained normal cells, which ensured correct genotyping. Furthermore, the frequency of MDM2 polymorphism in the UCB patients, did not differ from that noted in our reference population, and it was in Hardy-Weinberg equilibrium, which suggests that such polymorphism is stable during tumor development, and consequently there is no difference between benign and malignant cells.

Alhopuro et al. [44] studied patients with leiomyosarcoma, colorectal cancer, and squamous cell carcinoma of the head and neck, and their results indicated essentially the same proportion of individuals with MDM2 polymorphism as noted in our investigation. Alhopuru and coworkers also reported that a healthy reference population and tumor material from their study population had approximately the same frequencies of MDM2 polymorphism. Onat et al. [45] analyzed MDM2 polymorphism in bladder cancer and found an association between development of the disease and MDM2 polymorphism in patients with the G/G genotype. The frequency of MDM2 G/G in their study was somewhat greater in the tumor population than in the control group consisting of healthy individuals, although the values those researchers obtained differ very little from the levels noted in our investigation. In an assessment of B-CLL patients, Willander et al. [46] found that overall survival was poorer in the subjects with one or two G alleles compared to those with T/T. However, the overall frequencies of G/G, T/G, and T/T found by those investigators are equivalent to the corresponding figures recorded in our study, which underlines that the MDM2 gene is probably rather stable throughout tumor development and that one or two G alleles more likely only play a role as an unfavorable genotype.

Several studies have examined p53 and MDM2 polymorphism in UCB. In one of those investigations, Lu et al. [47] found that 61 (44%) of 140 patients with the disease were wild type for p53, whereas the corresponding proportion in our cohort was 62%, and this disparity may reflect difference in T stages between the two studies. A plausible explanation for this is that the tumors investigated by Lu and colleagues were from cystectomy specimens and thus represented higher tumor stages with more p53 mutations (less wild-type p53). Notably, we focused on exons 5–8 in the p53 gene, whereas Lu et al. investigated exons 2–11, although only a small number of the mutations in their study were found outside exons 5–8. Furthermore, Hernandez et al. [48] found a frequency of 65.5% p53 mutations in a group of patients with stage T1 grade 3 UCB, but those mutations were not associated with prognosis; interestingly, exons 4–9 were evaluated in that investigation, but the majority of the mutations were found in exons 5–8.

Polymorphism in MDM2 (SNP309) and mutations in p53 were not associated with worse prognosis in this population-based cohort of patients with stage T1 UCB. Mutations in p53 were associated with an IHC cut-off value of 50% for the p53 protein. Patients with IHC-abnormal p16 and p53 mutations had a higher rate of progression and a shorter time to progression.