Genetic variations in MDM2, PTEN and P53 might be involved in cancer susceptibility. To assess the contribution of polymorphisms in these three genes to the risk of esophageal squamous cell carcinoma (ESCC) in a Chinese population, we genotyped MDM2 T309G, Del1518, PTEN rs701848, rs2735343 and P53 Arg72Pro polymorphisms using PCR-restriction fragment length polymorphism analysis in 226 ESCC cases and 226 cancer-free controls. Here we showed that the risk of ESCC was elevated in subjects with any of the variant genotypes of PTEN rs2735343 and P53 Arg72Pro polymorphisms, but not any genotype of MDM2 or PTEN rs701848. Moreover, multiplicative interactions were observed between PTEN rs2735343 and P53 Arg72Pro or smoking status on risk of ESCC. Our study firstly indicated that PTEN rs2735343 might be a susceptibility factor for ESCC and reaffirmed the role of P53 Arg72Pro in ESCC in this Chinese population, but did not replicate the positive association between MDM2 T309G and ESCC found previously.
MDM2 is a crucial negative regulator of P53. Besides directly inhibiting the transcriptional activity of P53, MDM2 also stimulates its nuclear export and degradation as an E3 ubiquitin ligase.1, 2 PTEN tumour-suppressor gene is second only to P53 in mutation frequency in human cancers.3 It controls cellular growth mainly by inhibiting phosphoinositide 3-kinase activation, and this restricts MDM2 to the cytoplasm and thus protects P53 from degradation.4
MDM2-PTEN-P53 tumor suppressor–oncoprotein network regulates cell growth and viability.5 Genetic variations in these genes might lead to the disturbance of cell cycle and foster cancer development. One polymorphism in the promoter region of MDM2, T309G (rs2279744), has been reported and cells carrying the GG genotype show high-level expression of MDM2 protein and significant attenuation of the P53 pathway.6 This polymorphism is supposed to be associated with early onset of breast cancer in Li-Fraumeni patients and with risk for gastric carcinoma.7, 8 The Arg72 variant of P53 Arg72Pro (rs1042522) induces apoptosis more effectively than the Pro72 variant.9
Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death worldwide. This disease shows a considerable geographic variation globally, with an exceptionally high incidence in certain areas of China, South Africa and Iran.10 Such an uneven distribution of ESCC suggests a dominant role of environmental factors in its etiology,10 while individual susceptibility to ESCC may be associated with specific genetic variations.11, 12 Till now, the P53 Arg72Pro polymorphism has been well studied in ESCC in Chinese populations.13, 14, 15, 16 But little is known about the association between MDM2 or PTEN polymorphisms and ESCC susceptibility.16, 17
Here we genotyped MDM2 T309G, Del1518 (rs150550023), PTEN rs701848 and rs2735343 and investigated the associations between these polymorphisms and ESCC risk in a case-control study conducted in a high-risk population of China. Though well illustrated, we also included P53 Arg72Pro in our study to replicate previous findings.
Materials and methods
This study included 226 ESCC patients and 226 healthy population controls. Patients were recruited in 2005 at Anyang Cancer Hospital, Henan Province, China, all confirmed by histopathological diagnosis. Patients that received chemotherapy or radiotherapy before surgery were excluded from this study. Healthy controls were randomly selected from a population screening program for risk factors of ESCC in the same regions and 1:1 matched to cases on the basis of age (±2 years) and sex. At recruitment, informed consent was obtained from each subject and detailed personal information on demographic characteristics, smoking and drinking status were collected by interview.
Genomic DNA was isolated from 5 ml peripheral blood using Qiagen DNA Isolation Kit (Qiagen, Dusseldorf, Germany). Genotypes were determined by PCR-restriction fragment length polymorphism assay. Detailed information about primers, restriction enzymes, length of PCR and digest products were summarized in Supplementary Table. Every PCR amplification was performed in a 25-μl reaction mixture containing ∼100 ng template DNA, 0.2 mM dNTP, 0.2 mM each primer, 2.5U of Easy Taq DNA polymerase (5 U μl−1) with 10 × PCR buffer. Reaction was carried out with an initial melting step of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at indicated annealing temperature and 30 s at 72 °C, and a final elongation step of 10 min at 72 °C. PCR products were then digested by indicated restriction enzymes at 37 °C for 8 h. Genotypes were identified by separating digested fragments on 3% agarose gel, whereas MDM2 Del1518 was identified by direct separation of PCR products on 2% agarose gel. Genotypes of 10% randomly selected samples obtained by PCR-restriction fragment length polymorphism were further confirmed by direct sequencing of PCR products.
The SPSS statistical software package ver.18.0 was used for statistical analysis (IBM, New York, NY, USA). Demographic variables between the study groups were compared by conditional univariate logistic regression. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by conditional multivariate logistic regression as a measure of association with ESCC, adjusted for age, smoking and drinking status. A P-value <0.05 was considered significant and all tests were two-sided. Haplotype frequencies were estimated by Phase2.1.18
Demographic characteristics of the study subjects
Table 1 depicts the distributions of selected demographic characteristics of the study subjects. Though cases and controls were 1:1 matched on the basis of age (±2 years) and sex, there was still a slight difference in age distribution (P=0.045). Smoking and drinking rates in cases were significantly higher than those in controls (41.6% vs 28.8% and 28.3% vs 17.3%, respectively; P<0.01).
Gene polymorphisms and risk of ESCC
Table 2 shows the distributions of the genotypes and ESCC risk. The genotype distribution for each polymorphism was in Hardy–Weinberg equilibrium in controls.
After adjusted for age, smoking, drinking status and other genotypes where appropriate, no association was observed between MDM2 T309G, Del1518 or PTEN rs701848 and ESCC risk. Both variant genotypes (CG and GG) of PTEN rs2735343 were associated with a significantly increased risk of ESCC (OR=2.76 and 4.30, respectively, P<0.005). The frequencies of P53 Arg/Pro and Pro/Pro genotypes among cases were moderately different from those among controls (P=0.032 and 0.012, respectively). Compared with the Arg/Arg genotype, both the Arg/Pro and Pro/Pro genotypes inferred an about two-fold increased risk of ESCC (OR=1.74, 95%CI 1.05–2.89 and OR=2.06, 95%CI 1.17–3.62, respectively).
Haplotype and interaction analyses on risk of ESCC
A total of four PTEN haplotypes were constructed using PHASE software, CC, CT, GC and GT. Each individual had a combination of two most possible haplotypes. Table 3 showed that individuals carrying the homozygote (CT/CT) of haplotype CT had a lower risk of ESCC compared with those without haplotype CT (OR=0.21, 95%CI 0.11–0.42, P<0.001), whereas the heterozygote (- -/GC) of haplotype GC increased the risk of cancer development (OR=2.44, 95%CI 1.52–3.91, P<0.001).
Interaction analyses between genes or gene-smoking on risk of ESCC are shown in Table 4. Given the relative small sample size, detailed stratified analyses were not performed in the present study. The combination of G allele of PTEN rs2735343 and Pro allele of P53 Arg72Pro significantly increased the risk of ESCC (OR=3.21, 95%CI 1.79–5.78, P<0.001). Compared with nonsmokers carrying at least one G allele of rs2735343, smokers with at least one G allele had an OR of 2.25(95%CI 1.16–4.34) for ESCC risk.
In this study, we investigated the associations of genetic polymorphisms in MDM2, PTEN and P53 with ESCC susceptibility in a Chinese population.
Since the identification of MDM2, a variety of case-control studies have been published that investigate the possible association between MDM2 T309G and cancer risk, but the results remain contradictory. A recent report conducted by Hong et al.16 demonstrated that the GG genotype of MDM2 T309G led to a 1.5-fold increased risk of ESCC compared with the TT genotype. The GG genotype frequency was 26.7% and 20.5% in cases and controls, respectively. Here we did not find this association, with a similar GG genotype frequency (25.7%) in cases but much higher in controls (25.7%). We inferred that difference in sample sources between cases and controls might account for this inconsistency. In Hong's study, ESCC patients were enrolled from the Cancer Hospital, Chinese Academy of Medical Sciences, and controls were selected from a community population in Beijing. As far as we know, these patients mostly came from other regions of China. Given that carcinogenesis in diverse genetic backgrounds might be different based on different levels of environmental exposure and there was considerable geographical variation in the incidence of ESCC, this matching mode might lead to poor comparability between cases and controls, and even to a result that varies from the real condition.19 Conversely, cases and controls in our study were mostly from the same region with a high incidence of ESCC. Thus, all the participants were relatively homogeneous with regard to genetic background and environmental risk factors. Previous studies conducted in various tumour types in China showed that the GG genotype frequency ranged from 18 to 30% in controls,16, 20, 21, 22, 23, 24 which indicated that genotype frequency varied in different populations even in the same country. Two of these studies also included MDM2 Del1518 and no association was observed between this polymorphism and cancer risk.20, 21
In spite of the above discrepancy, we observed a similar association between P53 Arg72Pro and ESCC risk compared with Hong's and other previous studies (OR ∼2 for Pro/Pro genotype compared with Arg/Arg genotype, adjusted for age, sex, smoking or drinking status), though sample size, matching mode and study population were quite different from each other.13, 14, 15, 16 We confirm that P53 Arg72Pro polymorphism is a common susceptibility factor for ESCC in Chinese populations.
Because Mitsuo et al.25 reported that nuclear PTEN expression might be a biologic marker of ESCC and Chang et al.26 found that PTEN might have an important role in carcinogenesis and progression of ESCC, two polymorphisms, −9C/G and IVS4 (−/+) (a 5′-IndexTermACTAA-3′ deletion/insertion polymorphism), have been genotyped in a Chinese population with a high incidence of ESCC.17 The authors found that only IVS4+/+ genotype was associated with a decreased risk of ESCC. Here we evaluated the possible roles of the rs701848 and rs2735343 polymorphisms in ESCC susceptibility for the first time. These two polymorphisms have been studied in endometriosis, breast and prostate cancer, colon cancer and hepatocellular carcinoma but no association is observed.27, 28, 29, 30 We found that the variant genotypes of rs2735343 were associated with a significantly increased risk of ESCC. The conflicting results could be attributable to the different ethnicities and tumorigenesis of different cancers.
It should be noted that several limitations existed in our study. First, our small sample size or inadequate adjustment for confounding factors could also cause the inconsistent results. Second, cases and controls recruited in this study were from a high-risk area of ESCC and thus might not be representative of the general Chinese populations.
In conclusion, our data firstly indicate that PTEN rs2735343 might be a susceptibility factor for ESCC and reaffirm the role of P53 Arg72Pro in ESCC risk in this Chinese population, but do not replicate the positive association between MDM2 T309G and ESCC found previously. Further investigations would be warranted to confirm these results in larger and different ethnic populations, and the functional analysis of PTEN rs2735343 should also be addressed.
Momand, J., Zambetti, G. P., Olson, D. C., George, D. & Levine, A. J. The mdm2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 69, 1237–1245 (1992).
Haupt, Y., Maya, R., Kazaz, A. & Oren, M. Mdm2 promotes the rapid degradation of p53. Nature. 387, 296–299 (1997).
Yin, Y. & Shen, W. H. PTEN: a new guardian of the genome. Oncogene. 27, 5443–5453 (2008).
Lindsey, D. M., Jack, E. D., Donald, L. D., Nickolas, K. T. & David, B. D. PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy. J. Biol. Chem. 277, 5484–5489 (2002).
Lindsey, D. M. & David, B. D. The PTEN, MDM2, P53 tumor suppressor oncoprotein network. Trends. Biochem. Sci. 27, 9 (2002).
Bond, G. L., Hu, W., Bond, E. E., Robins, H., Lutzker, S. G., Arva, N. C. et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 119, 591–602 (2004).
Bond, G. L., Hu, W. & Levine, A. A single nucleotide polymorphism in the MDM2 gene: from a molecular and cellular explanation to clinical effect. Cancer. Res. 65, 5481–5484 (2005).
Ohmiya, N ., Taguchi, A ., Mabuchi, N ., Itoh, A., Hirooka, Y., Niwa, Y. et al. MDM2 promoter polymorphism is associated with both an increased susceptibility to gastric carcinoma and poor prognosis. J. Clin. Oncol. 24, 4434–4440 (2006).
Dumont, P., Leu, J. I., Della, A. C., George, D. L. & Murphy, M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat. Genet. 33, 357–365 (2003).
Chang, F., Syrjänen, S., Wang, L. & Syrjänen, K. Infectious agents in the etiology of esophageal cancer. Gastroenterology 103, 1136–1148 (1992).
Hiyama, T., Yoshihara, M., Tanaka, S. & Chayama, K. Genetic polymorphisms and esophageal cancer risk. Int. J. Cancer. 121, 1643–1658 (2007).
Xing, D., Tan, W. & Lin, D. Genetic polymorphisms and susceptibility to esophageal cancer among Chinese population (review). Oncol. Rep. 10, 1615–1623 (2003).
Lee, J.M., Lee, Y.C., Yang, S. Y., Shi, W. L., Lee, C.J., Luh, S.P. et al. Genetic polymorphisms of p53 and GSTP1, but not NAT2, are associated with susceptibility to squamous cell carcinoma of the esophagus. Int. J. Cancer. 89, 458–464 (2000).
Cai, L ., Mu, L.N., Lu, H ., Lu, Q.Y., You, Y ., Yu, S.Z. et al. Dietary selenium intake and genetic polymorphisms of the GSTP1 and p53 genes on the risk of esophageal squamous cell carcinoma. Cancer. Epidemiol. Biomarkers. Prev. 15, 294–300 (2006).
Zhang, L ., Xing, D ., He, Z . & Lin, D . P53 gene codon 72 polymorphism and susceptibility to esophageal squamous cell carcinoma in a Chinese population. Zhonghua. Yi. Xue. Yi. Chuan. Xue. Za. Zhi. 19, 10–13 (2002).
Hong, Y ., Miao, X.P., Zhang, X.M., Ding, F ., Luo, A.P., Guo, Y.L. et al. The role of P53 and MDM2 polymorphisms in the risk of esophageal squamous cell carcinoma. Cancer. Res. 65, 9582–9587 (2005).
Ge, H., Cao, Y. Y., Chen, L. Q., Wang, Y. M., Chen, Z. F., Wen, D. G. et al. PTEN polymorphisms and the risk of esophageal carcinoma and gastric cardiac carcinoma in a high incidence region of China. Dis. Esophagus. 21, 409–415 (2008).
Stephens, M., Smith, N. J. & Donnelly, P. A new statistical method for haplotype reconstruction from population data. Am. J. Hum. Genet. 68, 978–989 (2001).
Sholom, W., Debra, T. S., Joseph, K. M. & Jack, S. M. Selection of Controls in Case-Control Studies. Am. J. Epidemiol. 135, 1029–1041 (1992).
Hu, Z. B., Ma, H. X., Lu, D. R., Qian, J., Zhou, J. N., Chen, Y. J. et al. Genetic variants in the MDM2 promoter and lung cancer risk in a Chinese population. Int. J. Cancer. 118, 1275–1278 (2006).
Ma, H. X., Hu, Z. B., Zhai, X. J., Wang, S., Wang, X. C., Qin, J. W. et al. Polymorphisms in the MDM2 promoter and risk of breast cancer: a case-control analysis in a Chinese population. Cancer. Lett. 240, 261–267 (2006).
Zhang, X. M., Miao, X. P., Guo, Y. L., Tan, W., Zhou, Y. F., Sun, T. et al. Genetic polymorphisms in cell cycle regulatory genes MDM2 and TP53 are associated with susceptibility to lung cancer. Hum. Mutat. 27, 110–117 (2006).
Xiao, M., Zhang, L., Zhu, W. H., Huang, J., Jiang, H. F., Hu, S. H. et al. Genetic polymorphisms of MDM2 and TP53 genes are associated with risk of nasopharyngeal carcinoma in a Chinese population. BMC. Cancer. 10, 147 (2010).
Zhou, G. Q., Zhai, Y., Cui, Y., Zhang, X. M., Dong, X. J., Yang, H. et al. MDM2 promoter SNP309 is associated with risk of occurrence and advanced lymph node metastasis of nasopharyngeal carcinoma in Chinese population. Clin. Cancer. Res. 13, 2627–2633 (2007).
Mitsuo, T., Muneaki, S. & Naofumi, N. Expression and prognostic significance of PTEN product protein in patients with esophageal squamous cell carcinoma. Cancer. 94, 1955–1960 (2002).
Chang, D., Wang, T. Y., Li, H. C., Wei, J. C. & Song, J. X. Prognostic significance of PTEN expression in esophageal squamous cell carcinoma from Linzhou City, a high incidence area of northern China. Dis. Esophagus. 20, 491–496 (2007).
Susan, A. T., Zhao, Z. Z., Lien, L., Krina, T. Z., Nicholas, G. M., Stephen, K. et al. Variants in EMX2 and PTEN do not contribute to risk of endometriosis. Mol. Hum. Reprod. 13, 587–594 (2007).
Christopher, A. H., Daniel, O. S., Iona, C ., Elena, E.G., Loreall, P ., Kathryn, P . et al. Common genetic variation at PTEN and risk of sporadic breast and prostate cancer. Cancer. Epidemiol. Biomarkers. Prev. 15, 1021–1024 (2006).
Lynette, S. P., Cheryl, L. T., Alona, M., Sarah, J. P., Thomas, C. T., Graham, C. et al. No association between phosphatase and tensin homolog genetic polymorphisms and colon cancer. World. J. Gastroenterol. 15, 3771–3775 (2009).
Ding, J., Gao, Y. Z., Liu, R. Y., Xu, F. & Liu, H. Y. Association of PTEN polymorphisms with susceptibility to hepatocellular carcinoma in a han Chinese population. DNA. Cell. Biol. 30, 229–234 (2011).
This paper was supported by the Program of Shandong Province's Pharmaceutical Health and Technology Development (No HZ068).
Supplementary Information accompanies the paper on Journal of Human Genetics website
About this article
Cite this article
Ma, J., Zhang, J., Ning, T. et al. Association of genetic polymorphisms in MDM2, PTEN and P53 with risk of esophageal squamous cell carcinoma. J Hum Genet 57, 261–264 (2012). https://doi.org/10.1038/jhg.2012.15
- esophageal squamous cell carcinoma
MDM2 promoter polymorphism del1518 (rs3730485) and its impact on endometrial and ovarian cancer risk
BMC Cancer (2017)
High-incidence of PTEN mutations in Chinese patients with primary small cell carcinoma of the esophagus
BMC Cancer (2014)