In a study population of 1,083 SCCHN patients and 1,090 cancer-free controls of Caucasians, although none of the three SNPs in MDM2 and p53 (MDM2 SNP309, A2164G, and p53 codon 72) was alone associated with risk of overall SCCHN, a joint effect of these three SNPs may contribute to risk of SCCHN at non-oropharygenal sites. Furthermore, it appeared that the MDM2 SNP309 G-allele was associated with an early age at onset of non-oropharyngeal cancer in an allele-dose response manner in female patients, and this age effect could be further amplified by the p53 codon 72 C-allele. In contrast, we did not find similar effects for MDM2 A2164G and p53 codon 72, either individually or in combination. Given the role of MDM2 as a key inhibitor of p53, it is biologically plausible that these two SNPs in the MDM2 promoter region and p53 codon 72 SNP may collectively modulate risk of non-oropharyngeal cancer.
It had been demonstrated that the G-allele of SNP309 located in the promoter region of MDM2
increases the affinity of the transcriptional activator Sp1, resulting in high levels of MDM2
mRNA and MDM2 protein, thereby affecting p53 tumor suppressor activity and potentially cancer development in humans. Recently, in mouse models carrying either the polymorphic MDM2SNP309G
], Post et al. found that MDM2SNP309G/G
cells exhibit elevated MDM2 levels, reduced p53 levels, and decreased p53-dependent apoptosis in response to DNA damage; importantly, those mice with two MDM2SNP309G
alleles have an attenuation of the p53 pathway resulting in a decreased latency to tumor formation and decreased survival. In addition, over-expression of MDM2 could be involved in downregulation of other important cellular proteins such as pRB, E2F1, and p19ARF, further promoting carcinogenesis [17
]. Therefore, it is likely that MDM2
SNP309 may be a rate-limiting event in carcinogenesis [16
A number of studies have investigated associations between SNPs of MDM2
SNP309 and p53
codon 72 and SCCHN risk, but the results are controversial [12
]. A recent meta-analysis reported no association between the p53
codon 72 polymorphism and risk of oral carcinoma [22
]. So far, six published studies with relatively small sample sizes have investigated the association between MDM2
SNP309 and the risk or age at onset of SCCHN [28
]. Our mini meta-analysis using these published data and ours in the present study showed that MDM2
SNP309 was not significantly associated with risk of SCCHN. It is possible that the effect of the MDM2
SNP309 on SCCHN risk may be modest and could not be detected in the present study, or the effect can be modified by other SNPs of other genes. Indeed, we did find that those who carried the two to three risk genotypes (i.e., MDM2
SNP309 GT/TT, 2164 AA, and p53
codon72 CC) appeared to have an increased risk of non-oropharyngeal cancer, and this risk was more pronounced among ever smokers and ever drinkers. Human papillomavirus (HPV)-related SCCHN occurs predominantly in the oropharynx, and HPV is an established etiologic agent of oropharyngeal cancer. It has been reported that HPV-
related oropharyngeal cancer is most likely among those who are less smoking and drinking. In contrast, non-HPV related SCCHN (oral cavity, hypopharyngeal, and larynx) is more commonly linked with heavy tobacco and alcohol use [34
]. Hence, our findings in non-oropharyngeal cancer suggest that genetic variants in MDM2
could modulate non-HPV-induced carcinogenesis through gene-environment interaction. However, we did not find statistical evidence for interactions among these variables in the current study, which could be possible that the limited sample size in the group of non-oroparyngeal cancer (530 cases) may not have enough power to detect such interactions. Further studies with large sample size are warranted to confirm our findings in non-oropharygeal cancer.
SNP309 and p53
codon 72 SNPs have been demonstrated to be functional, the function of MDM2
promoter A2164G SNP has not yet been reported. MDM2
A2164G has been predicted to be functional by the computation tool of SNP Function Prediction (FuncPred, http://manticore.niehs.nih.gov/snpfunc.htm
). Hence, based on its putative function and our data reported herein, further functional studies need to be performed on MDM2
A2164G and other untyped putative functional polymorphisms that are in LD with this SNP.
With respect to the association between MDM2
SNP309 and the age at onset of SCCHN in female patients, two published studies found that the GG genotype was associated with an early age at onset of SCCHN in an Asian population. A Japanese case-only study with 119 SCCHN patients found that the SCCHN patients with the GG genotype had a nine-year earlier tumor onset in comparison with those with the TT genotype [29
]. Huang et al.
found that the MDM2
SNP309 G-allele was associated with at least a three-year earlier age of tumor onset in a Taiwanese population [30
]. In contrast, another study showed that patients with the GG genotype showed a five-year later age of tumor onset than those with the TT genotype in women in a Malaysian population [33
]. In the current case-only analysis with 1083 SCCHN patients, we found that the MDM2
SNP309 G-allele associated with an early age at onset of SCCHN at non-oropharygeal sites in female patients, which could be as early as 42.5 years old for women who also carried the p53
codon 72 C-allele.
It is likely that primarily female-specific hormones, such as estrogen, could allow for the SNP309 G-allele to accelerate tumor formation in women [37
]. More recent studies reported that estrogen receptors were expressed in the majority of SCCHN tumors and cell lines [39
]. It had been shown that estrogen signaling could regulate MDM2 expression levels [42
]. SNP309 is located only 10 bp away from the potential estrogen receptor responsive region of the MDM2
promoter. Since SNP309 enhances the binding affinity of Sp1, a co-transcriptional activator of hormone receptor, it may affect the transcriptional regulation of MDM2
by estrogen signaling. Indeed, Hu et al.
demonstrated that estrogen preferentially stimulated transcription of the MDM2
gene from the SNP309 G-allele and increased the levels of MDM2 protein in estrogen-responsive cells homozygous for SNP 309 (G/G), leading to further attenuation of the p53 pathway [42
Although our results are somewhat inconsistent with those previously published findings on SCCHN [31
], our findings are consistent with the biological function of the MDM2
SNP309 SNP and similar to previous reports on other cancer types including colorectal cancer, diffuse large B-cell lymphoma, soft tissue sarcoma, breast cancer and ovarian cancer, in which acceleration of tumor formation was shown to be associated with the G-allele, particularly in female patients [26
]. For example, Bond G. et al.
also demonstrated that the GG genotype was associated with a 13-year, 14-year, and a 10-year earlier age at onset of diffuse large B-cell lymphoma, soft tissue sarcomas and colorectal cancer, respectively, only in women [37
]. A Finland study found that GG carriers showed a 2.7-year earlier age at onset of colorectal cancer than TT carries only in women but not in men [32
]; however, that study found that MDM2
SNP309 was neither associated with risk nor with the age at onset of SCCHN, and further stratified analysis by sex was not reported in that study because of it small sample size (157 SCCHN patients and 185 controls). The discrepancies between our present study and those reported studies could be due to differences in ethnicity and study designs. Indeed, genotype distributions of MDM
2 SNP309 may vary by ethnicity. For example, the frequencies of the GG, TG, and TT genotypes of MDM2
SNP309 among our Caucasians population were 11.9%, 43.3%, and 44.8%, respectively, compared with 27.5%, 41.7%, and 30.8% of 120 Japanese subjects in the study by Nakashima et al.
], and 27.1%, 51.3%, and 21.5% of 1,272 Taiwanese subjects in the study by Huang et al
]. Therefore, these ethnic variations in genotype distributions and their influence on the risk of cancer need to be further investigated.
To the best of our knowledge, the present study is the first one using a large patient cohort to investigate the association between the risk and age at onset of SCCHN and MDM2 promoter SNPs, individually and in combination with p53 codon 72 SNP. Furthermore, our study population is ethnically homogeneous for Caucasians, decreasing possible biases from population stratification. Nevertheless, some potential limitations in the current study should be considered. Firstly, three SNPs were included in the analyses and the possibility of false-positive associations could not be ruled out because of multiple tests. The FPRP analysis showed that some possible biases may exist in our findings. Secondly, although our sample size was relatively large, the small sample size in subgroup analyses may have limited statistical power. Hence, our findings need to be confirmed by studies with larger sample sizes. Finally, HPV is another etiologic factor, in addition to tobacco and alcohol, for SCCHN, particularly for oropharyngeal cancer. However, when we evaluated the modification effects of polynorphisms in MDM2 and p53 on the risk of SCCHN, HPV status was not taken into account because only a small subset of SCCHN patients included in this study had available data on HPV infection. This limitation should be overcome in our future studies. Despite these limitations, our findings are biologically plausible. Our results provide some novel clues for the role of MDM2 and p53 SNPs in the development of non-orophryngeal cancer and age at onset of the disease.
In summary, we found that the combined risk genotypes of three SNPs in MDM2 and p53 (i.e., MDM2 SNP309 GT+GG, 2164AA, and p53 codon 72 CC) were significantly associated with risk of non-oropharyngeal cancer. We also found that the MDM2 SNP309 G-allele was associated with an earlier age at onset of non-oropharyngeal cancer in women but not in men, and this age effect could be amplified by the p53 codon 72 C-allele. Our findings indicate that MDM2 promoter SNPs, i.e., MDM2 SNP309 and MDM2 A2164G, as well as p53 codon 72 SNP may collectively contribute to the development of non-oropharyngeal cancer and that MDM2 SNP309 individually or in combination with p53 codon 72 may accelerate non-oropharyngeal cancer development in women. Further independent studies with large sample sizes and relative functional studies are needed to validate these findings.