MDM2 Promoter SNP309 Is Associated with Risk of Occurrence

Imaging, Diagnosis, Prognosis

MDM2 Promoter SNP309 Is Associated with Risk of Occurrence and Advanced Lymph Node Metastasis of Nasopharyngeal Carcinoma in Chinese Population Gangqiao Zhou,1, 2 , 3 Yun Z hai,1, 2 Ying Cui,4 Xiumei Zhang,2 Xiaojia Dong,3 HaoYang,1, 2 Ying He,5 KaitaiYao,5 Hongxing Zhang,1, 2 Lianteng Zhi,1, 2 XiaoyanYuan,1, 2 Wei Qiu,1, 2 Xiaoai Zhang,1, 2 Ya n Sh e n, 3 Boqing Qiang,3 and Fuchu He1, 2 , 3, 6

Abstract Purpose: Mouse double minute 2 (MDM2) is a key negative regulator of the p53 activity. Recently, a polymorphism in the MDM2 intronic promoter, SNP309, was shown to influence MDM2 expression and p53 activity.We examined whether the SNP309 was related to the risk of developing nasopharyngeal carcinoma (NPC) among Chinese populations. Experimental Design: We genotyped the SNP309 in two independent case-control populations in southern China, one is from Guangxi province (including 593 NPC patients and 480 controls) and the other is from Guangdong province (including 239 patients and 286 controls), by PCR direct sequencing. Multivariate logistic regression analysis was used to calculate adjusted odds ratio (OR) and 95% confidence interval (95% CI). Results: We observed that compared with theTT genotype, the genotypes containing G allele (GT + GG genotype) were associated with significant increased susceptibility to NPC in both Guangxi (OR, 1.43; 95% CI, 1.04-1.91) and Guangdong population (OR, 1.53; 95% CI, 1.00-2.36).When these two sample sets were combined, the OR of the GT + GG genotype developing NPC was 1.45 (95% CI, 1.12-1.85) compared with the TT genotype. Furthermore, compared with theTT genotype, the GT + GG genotype was also significantly associated with the advanced lymph node metastasis (OR, 1.84; 95% CI, 1.09-3.05). Conclusions: Our findings suggest that the MDM2 SNP309 may be a risk factor for the occurrence and advanced neck lymph node metastasis of NPC in Chinese population.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy (1). The marked racial and geographic differences in NPC with striking racial and geographic distribution differences (1). susceptibility have been considered as a multifactorial and High incidence rates are observed in the southern Chinese and polygenic event with EBV, environmental, and genetic compo- other individuals of Chinese descent, including Singaporeans, nents (1, 5–9). The identification of susceptibility genes Taiwanese, and Hong Kong Chinese (2–4). These incidence contributing to NPC would assist in predicting individual and rates are f100-fold higher than in the Caucasian populations population risks of NPC development and would help to clarify pathogenesis of this malignancy. Linkage analyses have suggested several chromosomal regions that may harbor NPC susceptibility genes. Lu et al. (10) were the first to map a susceptibility locus to chromosome 1 Authors’ Affiliations: Department of Genomics and Proteomics, Beijing Institute 6p22 in affected sib pairs collected from southern China, of Radiation Medicine; 2Department of Functional Genomics, Beijing Proteome Research Center; 3Chinese National Human Genome Center at Beijing, Beijing, supporting the involvement of the human leukocyte antigens in China; 4Cancer Institute of Guangxi, Nanning, China; 5Institute of Cancer the pathogenesis of NPC. Recently, two genome-wide searches Research, Nanfang Medical University, Guangzhou, China; and 6Institute of carried out in 20 Cantonese-speaking families from the Biomedical Sciences, Fudan University, Shanghai, China Guangdong province and 18 families from the Hunan province Received 9/13/06; revised 1/1/07; accepted 2/16/07. Grant support: Chinese High-tech Program 2001AA224011and 2002BA711A10, in southern China provided support for susceptibility loci on Medicine and Health Research Program 01Z018, Chinese National Science Fund for chromosome 4p15.1-q12 and 3p21.31-21.2, respectively Creative Research Groups 30321003 and 30621063 Chinese ‘‘973’’ Project Program (11, 12). Association studies have also been done, and some 2006 CB910803, and Beijing Science andTechnology NOVAprogram 2006A54. of the identified loci, including the cyclin D1 (CCND1; ref. 13), The costs of publication of this article were defrayed in part by the payment of page DNA repair genes hOGG1 and XRCC1 (14), heat shock protein charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section1734 solely to indicate this fact. 70-2 (HSP70-2; ref. 15), and glutathione S-transferase M1 Note: G. Zhou andY. Zhai contributed equally to this work. (GSTM1; refs. 16, 17), seem to contribute to disease suscepti- Requests for reprints: Fuchu He or Gangqiao Zhou, Department of Genomics bility. Recently, we also reported an association between risk of and Proteomics, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing NPC and the polymorphisms in the promoter of palate, lung 100850, China. Phone/Fax: 86-10-68177417 or 86-10-80705255; E-mail: [email protected] or [email protected]. and nasal epithelial clone (PLUNC) gene, which codes a protein F 2007 American Association for Cancer Research. that may function in the innate immune response in regions of doi:10.1158/1078-0432.CCR-06-2281 mouth and nose (18). Despite these advances, the alleles that

www.aacrjournals.orgClin Cancer Res 2007;13(9) May 1,2627 2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis account for most of the genetic susceptibility to NPC remain Materials and Methods undiscovered. It is well known that the p53 pathway plays a key role in Study subjects. This study consisted of two populations of patients preventing carcinogenesis by causing cell cycle arrest or with NPC and controls resided in Guangxi and Guangdong province, apoptosis (19–21). The activity of p53 may be inactivated respectively, both of which located in southern China. The Guangxi through mutations in the p53 gene. It has also been shown population consisted of 593 patients with NPC and 480 controls. All that in cancers lacking p53 mutations, the p53 function is subjects were unrelated ethnic Han Chinese and residents in Nanning abolished or attenuated by other mechanisms, such as the city and the surrounding regions. All patients with NPC were newly overexpressed human homologue of the mouse double minute diagnosed and pathologically confirmed, which were consecutively 2 (MDM2) protein (22). The MDM2 acts as a principal recruited at the Guangxi Cancer Hospital (Nanning, China) between negative regulator of p53 through at least three different ways. September 2003 and July 2005. The response rate for case patients was First, by binding to transactivation domain of p53, it inhibits 95%. Patients that received chemotherapy or radiotherapy before its transcriptional activity (23). Second, MDM2, which acts as surgery or had other type of cancer were excluded from this study. Tumor staging was done according to the tumor-node-metastasis an ubiquitin ligase, promotes p53 degradation (24). Third, on classification by the 1997 American Joint Committee on Cancer system binding to p53, MDM2 favors the export of p53 because it (37). All tumor-node-metastasis classifications were determined by contains a nuclear export signal (25). Additionally, MDM2 also senior pathologists of the hospital based on the postoperative seems to possess significant p53-independent functions, histopathologic examination. The controls were randomly selected through interacting with other cellular proteins that are from a community cancer screening program for early detection of important in cell cycle control, including pRb, E2F/DP1, and cancer conducted in the same regions during the same time period as p19ARF, although some of these functions are less well the NPC cases were collected. The selection criteria for the controls characterized (26, 27). It has been shown that transgenic mice included no individual history of cancer. The response rate for control with overexpression of MDM2 are predisposed to spontaneous subjects was 91%. At recruitment, informed consent was obtained from tumor formation and show both the p53-dependent and the each subject, and personal information on demographic factors, p53-independent tumorigenicity of MDM2 (28). In humans, medical history, and tobacco and alcohol use were collected via the amplification and/or overexpression of the MDM2 have structured questionnaire. The Guangdong population, which contained 239 NPC patients and 286 controls, has been described in detail been extensively described in more than 40 different types of previously (18). As described previously, the data on tumor staging, malignancies (29), and this overexpression has been exten- smoking, and drinking were not available for this sample set. This study sively linked with a worse clinical prognosis, an increased was done with the approval of the Ethical Committee of Chinese metastasis, as well as a decreased response to therapeutic National Human Genome Center. intervention in cancers (29). With regard to NPC, MDM2 is Polymorphism genotyping. We extracted genomic DNA from periph- overexpressed in both EBV-infected cells and 31% to 56% of eral blood leukocytes of 5 mL whole blood using standard phenol/ the tumors (30–33). Furthermore, it has been shown that the chloroform protocols. DNA samples were diluted to 10 ng/AL and MDM2 mRNA overexpression was significantly related to neck distributed to 96-well plates; each 96-well plate contained 94 samples lymph node metastasis of NPC (32). Based on above in vitro and 2 no-DNA control water. Then, the MDM2 SNP309 polymorphism and in vivo potential functional relevance of MDM2 in the was genotyped in our case-control populations using PCR direct sequencing. The primers 5¶-GTCCCCTCTATCGCTGGTTC-3¶ and 5¶- pathogenesis of NPC, we hypothesize that the MDM2 may be AGCAAGTCGGTGCTTACCTG-3¶ were used for amplifying and se- an excellent biological candidate susceptibility gene for the quencing the target region containing the SNP309 site. PCR conditions NPC; it is expected that the genetic polymorphisms within were identical to those for the SNP discovery described previously MDM2 could result in genotype-dependent difference in (18, 38) except for an annealing temperature of 56.5jC for the SNP309 susceptibility to NPC. site. Genotyping was done in a blind manner that the performers did Recently, a functional single nucleotide polymorphism not know the subjects’ case and control status. For quality control, (SNP) in the MDM2 intronic promoter (named SNP309) was a 15% masked, random sample of cases and controls was tested twice identified. The cells carrying the SNP309 GG genotype were by different people and all results were 100% concordance. found to increase the binding affinity of the transcriptional Statistical analysis. Genotype and allele frequencies for the SNP309 activator Sp1 and subsequently resulted in higher expression were determined by gene counting, and departures from Hardy- Weinberg equilibrium were tested using the m2 test. Subjects were levels of MDM2 mRNA and protein compared with those considered smokers if they smoked up to 1 year before the date of carrying the SNP309 TT genotype (34). As a consequence, the cancer diagnosis for cases or up to the date of interview for controls. degradation of p53 in cells carrying the GG genotype was Information was collected on the amount of cigarettes smoked per day, enhanced, resulting in a decreased response to DNA-damaging the age at which the subjects started smoking, and the age at which ex- agents and acceleration in tumorigenesis of both sporadic smokers stopped smoking. Lighter or heavier smokers were categorized and hereditary cancers compared with those carrying the TT by the approximated 50th percentile pack-year value among controls genotype (34). Therefore, it is hypothesized that this functional [i.e., V19 or >19 pack-years; (cigarettes per day / 20) Â (years smoked)]. SNP in the MDM2 promoter may contribute to individual’s An alcohol drinker was defined as someone who consumed alcoholic z tumor susceptibility. Indeed, several epidemiologic studies have beverages at least once per week for 6 months. Comparisons of age, shown SNP309 to be associated with the risk of esophageal sex, smoker, smoking level, and drinker distributions between patient and control groups were done by use of the m2 test. Differences of mean squamous cell carcinoma and lung cancer (35, 36). The role of age and mean smoking level between the groups were analyzed by use the SNP309 in NPC, however, has never been specifically of an unpaired t test. Multivariate logistic regression analyses were done investigated. In the present study, we examined whether the to evaluate whether there was association between the MDM2 SNP309 functional polymorphism in MDM2 promoter, SNP309, have and risk and severity of NPC. The P value, odds ratio (OR), and 95% any bearing on the risk or severity of NPC in the Chinese confidence interval (95% CI) were calculated and adjusted for age, population. gender, and tobacco and alcohol use where appropriate. Potential

Clin Cancer Res 2007;13(9) May 1, 2007 2628 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. MDM2 SNP309 and NPC modification of the effect of the SNP309 genotypes on risk and severity were presented in the controls compared with cases, whereas of NPC was assessed for the above factors by the addition of interaction more drinkers (m2 = 10.7; P = 0.001) and higher smoker level terms in the logistic model and by separate analyses of subgroups (P = 0.03, t test) were in the cases compared with controls. of subjects determined by these factors. Because age at diagnosis An absolute majority (97.0%) of cases was classified as poorly was normally distributed, t tests were used to examine associations of differentiated squamous cell carcinoma. According to the SNP309 genotypes with age at diagnosis. A P value of <0.05 was used as the criterion of statistical significance, and all statistical tests were two tumor-node-metastasis system, 5.4%, 42.5%, 34.2%, and sided. These analyses were done using SPSS software (version 9.0, SPSS, 17.9% of patients had stage I, II, III, and IV disease, respectively. Inc.). To estimate the contribution of the genotypes containing G allele The genotyping results are shown in Table 2. The observed (GT + GG genotype) to an increase in susceptibility to NPC in the genotype frequencies for the MDM2 SNP309 in both patients population, the population attributable fraction was calculated (39). (P = 0.49) and controls (P = 0.087) in the Guangxi population conformed to the Hardy-Weinberg equilibrium. The frequen- Results cies of the TT, GT, and GG genotype among patients were significantly different from those among controls (m2 = 6.035; Initially, we estimated the effect of SNP309 on NPC df =2;P = 0.049), and this difference was mainly caused by a susceptibility in Guangxi population consisted of 593 patients higher frequency of the GG genotype among patients compared with NPC and 480 controls (Table 1). Overall, controls were with controls (33.3% versus 28.9%). Based on logistic comparable with cases with regard to the status of smoking. regression analysis with adjustment for age, sex, and status However, more men (m2 = 8.8; P = 0.003), ethnic Han Chinese of smoking and drinking, the subjects bearing SNP309 GG (m2 = 4.4; P = 0.04), and higher mean age (P = 0.003, t test) genotype had a significantly increased susceptibility to NPC

Table 1. Selected characteristics of cases and controls in Guangxi and Guangdong population

Characteristics Guangxi population, n (%) Guangdong population,* n (%) Cases Controls Cases Controls (n = 593) (n = 480) (n = 239) (n = 286) Age (y) Mean (SD) 46.6 (11.3) 48.6 (10.0) 46.9 (11.2) 47.2 (12.8) V47 311 (52.4) 229 (47.7) 120 (50.2) 146 (51.0) Men 428 (72.2) 384 (80.0) 163 (68.2) 151 (52.8) Smoker 191 (32.2) 152 (31.7) Smoking level (pack-years) Mean (SD) 22.7 (13.4) 19.1 (15.9) V19 62 (32.5) 88 (57.9) Drinker 236 (39.8) 145 (30.2) Nationality Hanc 409 (69.0) 359 (74.8) Non-Han 184 (31.0) 121 (25.2) Histologic type Poorlyb differentiated squamous cell carcinoma 575 (97.0) 216 (90.4) Others 18 (3.0) 23 (9.6) Clinical stage I 32 (5.4) II 252 (42.5) III 203 (34.2) IV 106 (17.9) Local tumor invasion (T classification)

T1 104 (17.5) T2 311 (52.4) T3 120(20.3) T4 58 (9.8) Lymph node involvement (N classification)

N0 129 (21.8) N1 291 (49.1) N2 129 (21.8) N3 44 (7.3) Distance metastasis (M classification)

M0 578 (97.5) M1 15 (2.5)

*In Guangdong population, the characteristics of cases and controls are derived from our previous study (18). cIn cases of Guangxi population, non-Han includes Zhuang (n = 172), Dong (n = 1), Hui (n = 1), Mulao (n = 3), and Yao (n = 7) nationality; in controls, all the non-Han is Zhuang (n = 121) nationality. bIn Guangxi population, others include vesicular nucleus cell carcinoma (n = 13), poorly differentiated adenocarcinoma (n = 2), and moderate differentiated squamous cell carcinoma (n = 3); in Guangdong population, the others include poorly differentiated adenocarcinoma (n = 8), higher differentiated squamous cell carcinoma (n = 6), and undifferentiated cancer (n = 9).

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Table 2. The genotype frequencies of SNP309 in cases and controls of Guangxi and Guangdong population

Genotypes Cases, n (%) Controls, n (%) OR (95% CI)* P * Guangxi population (n = 580) (n = 478) TT (reference) 111 (19.1) 120(25.1) 1.00 GT 276 (47.6) 220 (46.0) 1.36 (0.99-1.86) 0.056 GG 193 (33.3) 138 (28.9) 1.51 (1.08-2.12) 0.016 GT + GG 469 (80.9) 358 (74.9) 1.43 (1.04-1.91) 0.019 Guangdong population (n = 223) (n = 285) TT (reference) 40(17.9) 71 (24.9) 1.00 GT 98 (43.9) 128 (44.9) 1.37 (0.84-2.19) 0.20 GG 85 (38.2) 86 (30.2) 1.76 (1.06-2.88) 0.024 GT + GG 183 (82.1) 214 (75.1) 1.53 (1.00-2.36) 0.049

NOTE: Due to genotyping failure, the actual sample size was 580and 478 for the cases and controls, respectively, in Guangxi population and 223 and 285 for the cases and controls, respectively, in Guangdong population. *In Guangxi population, the ORs and P values were adjusted for age, sex, smoking status, and alcohol consumption; in Guangdong group, the ORs and P values were adjusted for age and sex. compared with ones bearing TT genotype (OR, 1.51; 95% CI, status, and smoking level (Table 3). Although the susceptibility 1.08-2.12; P = 0.016). The heterozygous GT genotype also to NPC associated with the SNP309 GT + GG genotype seemed presented a higher risk for the NPC (OR, 1.36; 95% CI, 0.99- to be more pronounced in subjects who were males, older (>47 1.86), although the association was not statistically significant years), drinkers, smokers, and heavier smokers (>19 pack- (P = 0.056). When the GT genotype was combined with the GG years), these differences could be attributed to chance (all genotype, the subjects bearing the SNP309 G allele (GT + GG P > 0.30, test for homogeneity), indicating that these potential genotype) had a significantly increased susceptibility to NPC confounding factors had no modification effect on the risk of compared with ones bearing TT genotype (OR, 1.43; 95% CI, NPC related to SNP309 genotypes. 1.04-1.91; P = 0.019). When cases were limited to those with We then confirmed this finding (i.e., increased susceptibility poorly differentiated squamous cell carcinoma (n = 575), the of SNP309 GT + GG genotype to NPC) in a further sample set, general pattern of results was similar (data not shown). the Guangdong population (Table 1). This population The associations between the SNP309 and susceptibility to contained 239 cases and 286 controls, and as described NPC in the Guangxi population were further examined with previously (18), there was no significant difference between stratification by age, sex, nationality, smoking and drinking patients and control subjects in terms of mean age distribution.

Table 3. Risk of NPC associated with SNP309 by potential risk factors in Guangxi and Guangdong population

Category Guangxi population Guangdong population c b c b * * * TT GT + GG OR (95% CI) P homogeneity TT* GT + GG OR (95% CI) P homogeneity Sex Male 76/94 341/288 1.47 (1.02-2.08) 0.76 29/43 123/97 1.87 (1.08-3.25) 0.23 Female 35/26 128/70 1.33 (0.75-2.45) 11/28 60/117 1.30 (0.59-2.82) Age (y) V47 58/53 248/174 1.31 (0.85-2.00) 0.64 20/32 91/114 1.27 (0.67-2.41) 0.47 >47 53/67 221/184 1.53 (1.00-2.31) 20/39 92/100 1.78 (0.98-3.31) Smoking status Nonsmoker 74/76 324/250 1.32 (0.92-1.93) 0.89 Smoker 37/44 145/108 1.58 (0.95-2.66) Smoking level (pack-years) V19 9/20 52/68 1.69 (0.71-4.05) 0.32 >19 28/24 93/401.98 (1.02-3.87) Drinking status Nondrinker 65/79 284/255 1.34 (0.92-1.99) 0.88 Drinker 46/41 185/103 1.59 (0.98-2.62) Nationality Han 81/93 322/264 1.39 (0.99-1.98) 0.94 Non-Han 30/27 147/94 1.40 (0.78-2.54)

NOTE: Due to genotyping failure, the actual sample size was 580and 478 for the cases and controls, respectively, in Guangxi population and 223 and 285 for the cases and controls, respectively, in Guangdong population. *Number of genotype in cases/number of genotype in controls. cORs were calculated by logistic regression with the TT genotype as the reference group and adjusted for age, sex, and smoking and drinking status where appropriate within the strata. bFor difference in ORs within each stratum.

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However, more men were presented in the cases compared with and these differences were not significant (P = 0.23 for GG controls (68.2% versus 52.8%; m2 = 12.2; P < 0.01). Overall, versus TT and P = 0.36 for GT versus TT). Similarly, in 239 NPC the SNP309 genotypic frequencies observed in this group were patients of Guangdong population, the average age at diagnosis very comparable with those from the Guangxi population and were 49.3 F 11.0, 45.8 F 11.6, and 46.4 F 10.5 years, conformed to the Hardy-Weinberg equilibrium (P = 0.21 in respectively, for subjects with the GG, GT, and TT genotype, patients and P = 0.094 in controls; Table 2). Again, there was an respectively, and these differences were also not statistically excess of GT + GG genotype in patients than in controls (82.1% significant (P = 0.19 for GG versus TT and P = 0.71 for GT versus 75.1%; OR, 1.53; 95% CI, 1.00-2.36; P = 0.049). versus TT). Similarly, in the stratification analyses, sex and age had no modification effect on the risk of NPC related to SNP309 Discussion genotypes (Table 3). When Guangxi and Guangdong population were combined, the OR for SNP309 GT + GG genotype In this study, we investigated the associations of the developing NPC was 1.45 (95% CI, 1.12-1.85; P = 0.0028) functional SNP309 in the intronic promoter of MDM2 with compared with TT genotype. By Adams’s formula (39), risk of occurrence and progression of NPC in populations in the population attributable fraction calculated by relative risk southern China. We found that the SNP309 GT + GG genotypes (OR, 1.45; 95% CI, 1.12-1.85) combined with the SNP309 were associated with a significantly increased risk of occurrence GT + GG genotype frequency in the overall population (78.2%; of NPC in both independent populations. We also observed Table 2) indicates that 26.0% (95% CI, 8.6-39.9%) of elevation that this polymorphism was significantly associated with the in risk of NPC can be attributed to the susceptible effect of the advanced neck lymph node metastasis of NPC in Guangxi SNP309 GT + GG genotype. population. Our data, together with the earlier described We also assessed the effect of SNP309 GT + GG genotype on functional significance of the SNP309 (34) and the recent severity of NPC (as measured by tumor-node-metastasis staging evidence for association of SNP309 with the risk of esophageal system) in the Guangxi population. The distributions of the squamous cell carcinoma (35) and lung cancer (36), suggest SNP309 genotypes were not statistically significantly different that the SNP309 GT + GG genotypes were potent genetic risk among the subgroups with different clinical stage, or different factor for both onset and advanced neck lymph node metastasis T and M classification of the cancer (data not shown). However, of NPC. Given the role of MDM2 in the development of we did note a trend toward SNP309 GT + GG genotype in cancers, one might expect individuals who carry the SNP309 the patients with more frequent involvement of lymph node GT + GG genotypes, and thus have increased expression of (P = 0.030, test for trend; Table 4). After adjustment for age, sex, MDM2 and subsequently attenuated p53 function over a and status of smoking and drinking, multivariate regression lifetime, may be at a higher susceptibility to developing NPC analyses revealed that patients with the GT + GG genotype, and a higher risk of advanced lymph node metastasis after compared with the TT genotype, had an OR of 1.84 (95% CI, establishment of this malignancy. To our knowledge, this is the 1.09-3.05; P = 0.019) for being more frequent involvement of first report of the genetic associations between MDM2 and lymph node (N2 +N3 versus N0 +N1; Table 4). In the occurrence and progression of NPC, confirming the initial stratification analyses, sex, age, and smoking and drinking had hypothesis that the MDM2 oncoprotein may play a role in the no modification effect on the risk of more frequent involve- pathogenesis of this malignancy. ment of lymph node related to the GT + GG genotype (all The genetic association between MDM2 SNP309 and P > 0.20, test for homogeneity within each strata). The effect of occurrence of NPC is biologically plausible. It is well known SNP309 genotypes on severity of NPC was not reassessed in the that the p53 pathway plays a key role in preventing Guangdong population because the tumor-node-metastasis carcinogenesis (19–21). The p53 function can be abolished staging data were not available in this sample set. or attenuated by overexpressed MDM2 protein (22). Transgenic In 593 NPC patients of Guangxi population, the average age mice with overexpression of MDM2 are predisposed to at diagnosis (FSD, years) were 47.1 F 10.7 years for subjects spontaneous tumor formation and show both the p53- with the GG genotype, 45.9 F 11.9 years for those with the GT dependent and the p53-independent tumorigenicity of genotype, and 47.3 F 10.9 years for those with the TT genotype MDM2 (28). Furthermore, amplification and/or overexpression

Table 4. Association between SNP309 genotypes and lymph node metastasis in NPC patients in Guangxi population

Genotypes N classification, n (%) OR (95% CI)* P *

N0 (n = 124) N1 (n = 288) N2 (n = 125) N3 (n =43) TT (reference) 27 (21.8) 62 (21.5) 17 (13.6) 5 (11.6) 1.00 GT 60 (48.4) 136 (47.2) 60 (48.0) 20 (46.5) 1.65 (0.97-2.82) 0.064 GG 37 (29.8) 90 (31.3) 48 (38.4) 18 (41.9) 2.10 (1.21-3.66) 0.0080 GT + GG 97 (78.2) 226 (78.5) 108 (86.4) 38 (88.4) 1.84 (1.09-3.05) 0.019

NOTE: Due to genotyping failure, the actual sample size was 580for the cases in Guangxi population. Abbreviation: N, lymph node involvement. * ORs and P values were calculated for N2 +N3 versus N0 +N1 and adjusted for age, sex, smoking status, and alcohol consumption.

www.aacrjournals.orgClin Cancer Res 2007;13(9) May 1,2631 2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis of the MDM2 gene have been extensively described in several NPC associated with the GT + GG genotypes seemed to be more forms of human cancer (29). With regard to NPC, it has been pronounced in subjects who were smokers and heavier smokers shown that MDM2 is overexpressed in both EBV-infected cells with >19 pack-years (Table 3), suggesting that smoking may not and 31% to 56% of the tumors (30–33). have modification effect on the onset and advanced lymph It is also biologically plausible for the association between node metastasis of NPC related to SNP309 genotypes. This MDM2 SNP309 and advanced neck lymph node metastasis of discrepancy may be the result of different molecular mecha- NPC. Accumulating evidence shows that the overexpression of nisms of NPC and other types of cancers. Alternatively, the MDM2 is associated with increased metastasis, decreased limited power of statistics due to the small sample size of response to therapy, and poor prognosis (29). With regard to smokers in the present study might also account for the null NPC, it has been shown that the MDM2 mRNA overexpression interaction between smoking and SNP309. Additional studies was significantly related to neck lymph node metastasis but not investigating the interaction between the smoking and SNP309 to T stage of NPC (32). A recent report proposes a mechanism should be required in the future. for the increase in metastasis. Hypoxia, which is common in Recently, the MDM2 SNP309 has been extensively studied in many tumors, up-regulates MDM2 in a p53-independent many cancers but the results are conflicting. Several studies manner. This overexpression leads to an increased metastatic have reported an association of SNP309 G allele with the efficiency in cell lines, perhaps by rendering tumor cells less increased risk of esophageal squamous cell carcinoma and lung sensitive to stress-induced cell death (40). Additionally, MDM2 cancer (35, 36). In contrast, some reports showed no increases levels of vascular endothelial growth factor (41), association between SNP309 and risk of breast and lung cancer which facilitates extravasation of tumor cells and can lead to (46, 47); a very recent study revealed reverse association about growth of metastases (42). MDM2 expression also increases the risk allele (i.e., TT genotype was reported to be associated basement membrane degradation in urothelial carcinoma (43), with lung cancer risk; ref. 48). The conflicting results could be allowing the cancer to spread. Thus, MDM2 expression attributable to the different ethnicities of study populations increases the risk of metastasis, contributing to the overall and/or different tumorigenesis of different cancers. Addition- poorer prognosis for cancers expressing MDM2. However, the ally, other factors in the studies, such as small sample size or role of MDM2 in cancer metastasis and its underlying inadequate adjustment for confounding factors, could also mechanisms are not fully understood, and more investigations cause the inconsistent results. Consequently, additional well- with larger sample sizes and longer durations of follow-up are designed case-control studies in a wide spectrum of cancers needed. with ethnically diverse populations are warranted to under- Given the functional relevance of SNP309 in modulation of stand the roles of MDM2 polymorphism in the etiology of MDM2 expression level and subsequent efficiency of the p53 cancers. pathway, it would be expected that this polymorphism has a In reviewing the results of this study, one must also keep phenotype that affects the age at onset of cancers. Indeed, Bond several potential limits in mind. First, as a hospital-based study, et al. (34) reported that individuals with the Li-Fraumeni our NPC cases were enrolled from the hospitals and the syndrome with SNP309 GG genotype developed cancers some controls were selected from the community population; 10 to 12 years earlier than those individuals with a TT genotype. inherent selection bias cannot be completely excluded. However, contrary to the expectation, we failed to find evidence However, by further adjustment and stratification in data of association between SNP309 genotype and age of NPC onset analyses, the potential confounding factors might have been in patients from both Guangdong and Guangxi populations. minimized. Second, several association studies have addressed Consistent with our results, very recent studies in several tumor to identify the genes that may relate to the susceptibility to NPC types, including colorectal cancer, uterine leiomyosarcoma, (13–18). Most of the results, however, could not be replicated and squamous cell carcinoma of the head and neck (44, 45), in subsequent studies in other populations. Although the also showed no association of age of tumor onset with SNP309 highly significant association between MDM2 and onset and genotype. Because of limited statistical power, subtle changes metastasis of NPC derived from a biologically based a priori may have been missed. In addition, these findings could be the hypothesis, our initial findings should be independently result of the different tumorigenesis mechanisms of different verified in other populations with high incidence rate of tumor types. Consequently, we urge that the role of SNP309 in NPC, such as other southern Chinese, Singaporeans, and age of tumor onset be investigated in additional studies with Taiwanese. larger sample sizes. In conclusion, our data provide strong evidence that the Previous studies revealed that the long-term cigarette MDM2 SNP309 GT + GG genotypes may be genetic risk factor smoking was associated with the risk of NPC (1, 6). for the occurrence and progression of NPC in Chinese patients. Furthermore, in the recent two reports, a significant interaction If confirmed by other studies, knowledge of genetic factor between the MDM2 SNP309 and smoking was observed contributing to the pathogenesis of the NPC as presented here (35, 36). In the present study, however, we did not find that may have implications for the cancer screening and treatment the SNP309 interact with tobacco smoking, although the risk of of this disorder.

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www.aacrjournals.orgClin Cancer Res 2007;13(9) May 1,2633 2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. MDM2 Promoter SNP309 Is Associated with Risk of Occurrence and Advanced Lymph Node Metastasis of Nasopharyngeal Carcinoma in Chinese Population

Gangqiao Zhou, Yun Zhai, Ying Cui, et al.

Clin Cancer Res 2007;13:2627-2633.

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